Universal antigen-presenting cells and their use

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 of related applications) This application relates to the benefits of U.S. Provisional Patent Application No. 62 / 633,587, filed on February 21, 2018. This assertion is incorporated herein by reference in its entirety.

[0002] This is KB (measured on Microsoft Windows), as of February 2, 2019. A file named "UTFCP1355WO_ST25.txt" was created on the 1st. The sequence listings included herein were filed electronically together with this specification and are referred to herein by reference. It will be used in the context of the project. [Background technology]

[0003] This invention generally relates to the fields of medicine and immunology. More specifically, this invention relates to antigen preparation. This relates to indicator cells and their use, for example, to the expansion of natural killer (NK) cells.

[0004] Natural killer (NK) cells, in contrast to T cells, require antigens to be activated in advance. Because it does not require exposure to the substance, it is highly effective in immune surveillance against tumors. Activation of is not necessary, but in order to prevent unintentional cytotoxicity and autoimmunity, N The tremendous killing power of K cells requires strict control. Clinically sufficient quantity Furthermore, another obstacle is creating NK cells that function optimally. Therefore, in order to design helper cells that improve NK cell-mediated cancer immunotherapy, The molecular mechanism governing the function of NK cells that distinguish between "anti-" and "sensitive" target cells. It is necessary to pay attention to this. [Overview of the Initiative]

[0005] Thus, certain embodiments of the present disclosure relate to methods and compositions for the manufacture, expansion, quality control, and functional characterization of clinical-grade NK cells for cell therapy and

[0006] immunotherapy. In a first embodiment, a universal antigen-presenting cell (UAPC) engineered to express (1) CD48 and / or CS1 (CD319), (2) membrane-bound interleukin-21 (mbIL-21), and (3) 4-1BB ligand (4-1BBL) is provided. In some embodiments, the UAPC expresses CD48. In other embodiments, the UAPC expresses CS1. In certain embodiments, the UAPC expresses

[0007] both CD48 and CS1. In some embodiments, the UAPC essentially does not express endogenous HLA class I, II, or CD1d molecules. In certain embodiments, the

[0008] UAPC expresses ICAM-1 (CD54) and LFA-3 (CD58). In certain embodiments, the UAPC is further defined as an aAPC derived from leukemia cells. In some embodiments, the UAPC derived

[0009] from leukemia cells is further defined as a K562 cell. In some embodiments, the engineering is further defined as retroviral transduction. In some embodiments, the retroviral transduction is further defined as the introduction of viral constructs

[0010] of SEQ ID NO: 1 and / or SEQ ID NO:In a further embodiment, a method for expanding immune cells is provided, the method comprising culturing the immune cells in the presence of an effective amount of the UAPC of the above embodiment (e.g., (1) CD48 and / or CS1 (CD319), (2) membrane-bound interleukin-21 (mbIL-21), and (3) 41BB ligand (41BBL)-expressing engineered universal antigen-presenting cells (UAPCs)). In some embodiments, the immune cells and the UAPCs are cultured at a ratio of 3:1 to 1:3, such as 3:1, 3:2, 1:1, 1 :2 or 1:3. In certain embodiments, the immune cells and the UAPCs are cultured at a ratio of 1 :2.

[0011] In some embodiments, the expansion is an expansion in the presence of IL-2. Specifically, in certain embodiments, IL-2 is present at a concentration of 10 to 500 U / mL (e.g., 10 to 25, 25 to 50, 5 0 to 75, 75 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 3 00, 300 to 350, 350 to 400 or 400 to 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 some embodiments, IL-2 is recombinant human IL-2. Specifically, in certain embodiments, IL-2 is replenished every 2 to 3 days (e.g., every 2 days or every 3 days). In certain embodiments, the UAPC is added at least twice. In some embodiments, the immune cells are NK cells or T cells. In certain embodiments, the immune cells are

[0012] NK cells.

[0013]

[0013] ​ In certain embodiments, immune cells are derived from umbilical cord blood (CB), peripheral blood (PB), stem cells, or bone. They originate from the pulp. In a specific embodiment, the stem cells are induced pluripotent stem cells. Several In this embodiment, immune cells are obtained from CBs. In a particular embodiment, CBs are 2 units or less It is pooled from the individual umbilical cord blood units above. In a specific embodiment, CB is 3, 4, 5, It is pooled from 6, 7, or 8 individual umbilical cord blood units.

[0014] In some aspects, NK cells are CB mononuclear cells (CBMCs). In this embodiment, NK cells further include CD56 + These are defined as NK cells.

[0015] In a particular embodiment, the above method is carried out in serum-free medium.

[0016] In a further embodiment, immune cells express chimeric antigen receptors (CARs). It is operated in some embodiments, CAR is CD19, CD123, Mesothe Linn, CD5, CD47, CLL-1, CD33, CD99, U5snRNP200, C Contains antigen-binding domains of D200, CS1, BAFF-R, ROR-1, or BCMA In some embodiments, CAR is a humanized CAR. In some embodiments, CAR contains IL-15. In certain embodiments, CAR contains a suicide gene. In some embodiments, the suicide gene is CD20, CD52, EGFRv3 or induction It is sex caspase 9.

[0017] In the above embodiment (for example, the immune cells are given an effective amount of the above embodiment's UAPC (for example, (1) CD48 and / or CS1 (CD319), (2) Membrane-bound interleukins -21(mbIL-21), and (3) expressing 41BB ligand (41BBL) Culturing in the presence of manipulated universal antigen-presenting cells (UAPCs) A population of immune cells prepared and expanded according to (and) is further provided herein.

[0018] In another embodiment, an expanded population of immune cells and pharmaceutically acceptable A pharmaceutical composition containing a carrier is provided for the treatment of a disease or disorder in a subject. A composition comprising an effective amount of the enlarged immune cells of the above embodiment for use in the following: Further details are provided herein.

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

[0020] In some embodiments, the above-mentioned disease or disorder is cancer, inflammation, graft-versus-host disease, graft Rejection, autoimmune disorder, immunodeficiency, B-cell malignancy, or infection. And cancer is leukemia. In some aspects, leukemia is acute lymphoblastic leukemia. Disease (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), or chronic Myeloid leukemia (CML). In certain embodiments, the above disorder is graft-versus-host disease (G VHD). In some embodiments, the above disorder is multiple sclerosis, inflammatory bowel disease, Rheumatoid arthritis, type 1 diabetes, systemic lupus erythematosus, contact hypersensitivity, asthma, or Sjögren's disease. This is Lenn syndrome. In some aspects, the subject is human.

[0021] In certain aspects, immune cells are homogeneous. In some aspects, Epidemic cells are autologous. In some embodiments, immune cells are NK cells or T cells. That is the case.

[0022] In a further embodiment, the method includes the step of administering at least one second therapeutic agent. Furthermore, it includes, in some embodiments, at least one second therapeutic agent is therapeutically effective. A quantity of anticancer agent, immunomodulator, or immunosuppressant. In a particular embodiment, the anticancer agent This includes chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenic therapy, or immunotherapy. It is an epidemic treatment.

[0023] In some aspects, immunosuppressants include calcineurin inhibitors, mTOR inhibitors, Antibodies, chemotherapy irradiation, chemokines, interleukins, or chemokines or in It is a tarleukin inhibitor.

[0024] In a further embodiment, immune cells and / or at least one second therapeutic agent are static Intravascular, intraperitoneal, intratracheal, intratumoral, intramuscular, endoscopically, intralesionally, percutaneously, It is administered subcutaneously, regionally, or by direct injection or perfusion. In several embodiments In this context, the second therapeutic agent is an antibody. In a particular embodiment, the antibody is a monoclonal The antibody is a bispecific antibody, a bispecific antibody, or a trispecific antibody. In some embodiments, the antibody is It is rituximab.

[0025] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, those skilled in the art will see that various changes and modifications will not exceed the spirit and scope of the present invention. As will become clear from the detailed description, the detailed description and specific examples are preferred embodiments of the present invention. It should be noted that while the form is shown, it is given merely as an example.

[0026] The following drawings form part of this specification and are included to further illustrate certain aspects of this disclosure. This disclosure, combined with a detailed description of the specific embodiments presented herein, is applicable. Furthermore, this can be better understood by referring to one or more of these drawings. [Brief explanation of the drawing]

[0027] [Figure 1A] A zipper model of the interaction between receptors on human natural killer cells and ligands on target cells. Human NK cells interact with target cells via many receptors, including killer immunoglobulin receptors (KIRs), innate cytotoxic 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]A zipper model of the interaction between receptors on human natural killer cells and ligands on target cells. Human NK cells interact with target cells via many receptors, including killer immunoglobulin receptors (KIRs), innate cytotoxic 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] A zipper model of the interaction between receptors on human natural killer cells and ligands on target cells. Human NK cells interact with target cells via many receptors, including killer immunoglobulin receptors (KIRs), innate cytotoxic 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] Magnification of fresh or frozen NK cells when using IL-2 or APC. [Figure 2B]Magnification of fresh or frozen NK cells when using IL-2 or APC. [Figure 2C] Flow cytometry of CD3 and CD56 expression in NK cells on day 0, day 7, or day 14. [Figure 2D] The number of NT-NK cells or SG4-NK cells stimulated by APC. [Figure 2E] Proliferative dynamics of NK cells stimulated with clone 9APC or UAPC.

[0029] [Figure 3A] Flow cytometry of parental K562 cells for the listed markers. Expression of mb-IL21, 41BBL, CD48, and SLAMF7(CS1) was not observed. [Figure 3B] Flow cytometry analysis of APC after clone 46 introduction (mbIL-21, 41BBL). [Figure 3C] Flow cytometry analysis of APCs after introduction of a uAPC construct for CD48 expression (nmIL-21, 41BBL, and CD48). [Figure 3D] Flow cytometry analysis of APCs after introduction of the uAPC2 construct for CS1 expression (mbIL-21, 41BBL, and CS1).

[0030] [Figure 4A] Maps and annotations of MMLV retrovirus-introduced constructs. Retroviral transfer vector for mb-IL21. [Figure 4B] Maps and annotations of MMLV retrovirus-introduced constructs. Retrovirus transfer vectors for 41BBL. [Figure 4C] Maps and annotations of MMLV retrovirus-introduced constructs. Retroviral transfer vector for CD48-Katushka. [Figure 4D]Maps and annotations of MMLV retrovirus-introduced constructs. Retroviral transfer vector for CS1-EGFP. [Modes for carrying out the invention]

[0031] In this study, NK cells were initially found to be different from other components of the innate immune compartment. The behavior of NK cells was characterized by their antitumor function and anti-allogeneic cell lysis function. The countless immune receptors that exert control are illustrated in Figure 1 as a "shipper" graph. Gunal signaling molecules can be broadly classified into two main groups: activating and inhibitory. Based on their structure and mode of signaling, these categories further relate to immune receptors. They can be divided into families. The membrane proteome of NK cells is not yet fully understood. Functional redundancy within the NK cell "zipper" is a complex and dynamic interaction between complementary and antagonistic pathways. It was used to regulate cell lysis while maintaining a delicate balance.

[0032] Therefore, certain embodiments of this disclosure are intended for clinical applications of cell therapy and immunotherapy. Methods for the production, expansion, quality control, and functional characterization of bed-grade NK cells and The composition is provided. A clinically reasonable number of units for injection into a patient while meeting time constraints. Proliferating and forming NK cells is difficult even under ideal conditions. In this disclosure, the methods and compositions are technical processes for producing NK cells, and the achievable N Detailed description and dynamics of K cell expansion, as well as molecular characterization to verify the success of cell formation. This will be explained in detail.

[0033] In further embodiments, human natural killer (NK) cells are conditioned against tumors. , specifically as universal antigen-presenting cells (UAPCs) for formation and weaponization A robust platform technology that will be implemented is provided herein. "UAPC" is In this specification, the following are described as being designed for the optimized expansion of immune cells such as NK cells. This refers to antigen-presenting cells. This UAPC is used to overcome inhibitory signals, and NK cells To induce optimal and specific cell-killing function, a unique combination of co-stimulatory molecules is used. Therefore, it was created. From large-scale trials, UAPC was found to improve the mechanism of NK cells and tumors. It was shown that the disappearance could be fine-tuned. This UAPC is used in NK cell-mediated cancer immunotherapy. It is possible.

[0034] An example APC is the NK cell-sensitive K562 antigen-presenting cell line (APC) (clone 46 (referred to as) membrane-bound interleukin 21 (mbIL-21) and 4-1B It is produced by forced expression of B ligand. In another embodiment, in K562 cells m By forcibly expressing bIL-21, 4-1BB ligand, and CD48, UAP C was fabricated (called Universal APC (UAPC)). In another embodiment, K5 Forced expression of mbIL-21, 4-1BB ligand, and CS1 in 62 cells. They created a UAPC (called UAPC2) using mbI. L-21, 41BBL, and NK cell-specific antigens (e.g., SLAM family antigens (Table) 1)) can be produced to express

[0035] The UAPC platform uses T cells (e.g., alpha-beta and gamma-del). It can also be applied to expand other immune effectors, including T cells. These immune cells (e.g., NK cells and T cells) are found in peripheral blood, umbilical cord blood, bone marrow, or stem cells. These immune cells may originate from (including induced pluripotent stem cells). These immune cells are used in clinical applications such as cancer immunotherapy. The amount of UAPC required for cultivation to reach a meaningful number for a suitable application It can be used for expansion and activation in xobiotics.

[0036] Therefore, this disclosure is conditional on human immune cells (e.g., NK cells) for cancer immunotherapy. Designed to attach, adjust, prime, and enlarge. We provide a series of manufactured helper cell lines. I. Definition

[0037] When used herein, “essentially does not include” with respect to a particular component, The absence of certain components in a composition and / or impurities In this specification, the term is used to mean that something exists only as a small amount or in trace amounts. Therefore, the total amount of a particular component of a composition due to any unintended contamination of that composition , well below 0.05%, preferably below 0.01%. Its particular component Compositions in which the amount of the substance cannot be detected by standard analytical methods are most preferable.

[0038] As used herein, "a" or "an" may mean one or more. When used in a clause, the word "a" or "an" is used with the word "include". When used, it can mean one or more than one.

[0039] The use of the term "or" in a claim is prohibited unless explicitly indicated to refer only to the alternative. , or used to mean "and / or" unless the options are mutually exclusive. However, this disclosure supports the definition of "and / or" as referring only to options. When used in detail, "another" means at least one second or more. It can mean that. The terms "about," "substantially," and "approximately" generally refer to the stated value. It means plus or minus 5%.

[0040] "Immune disorder," "immune-related disorder," or "immune-mediated disorder" refers to a condition in which the immune response is impaired in the development of disease. This refers to disorders that play a significant role in life or progression. Immune-mediated disorders include: Autoimmune disorders, allograft rejection, graft-versus-host disease, and inflammatory and allergic conditions The condition can be described as follows.

[0041] "Immune response" refers to the response of immune system cells to a stimulus (e.g., B cells or T cells or natural This is a response of immune cells. In one embodiment, the response is specific to a particular antigen. Yes ("antigen-specific response").

[0042] An "autoimmune disease" is a condition in which the immune system reacts to antigens that are part of a normal host (i.e., autoantigens). ) triggers an immune response (e.g., B cell response or T cell response) against the tissue This refers to a disease that causes damage. Autoantigens can originate from host cells, or are usually derived from host cells. It originates from symbiotic organisms such as microorganisms that form colonies on the mucous membrane surface (known as symbiotic organisms). obtain.

[0043] To “treat” a disease or condition, or such treatment, means treating the signs or symptoms of that disease. The implementation of a protocol that may include administering one or more drugs to a patient for the purpose of alleviating symptoms. This refers to the following. The desired effects of the treatment include slowing the progression of the disease and improving the disease state. This includes reducing or alleviating symptoms, and achieving remission or improving the prognosis. Reducing symptoms means... It can occur before the onset of signs or symptoms of a disease or condition, and after they have appeared. Therefore, “to treat” or “treatment” means “to prevent” a disease or undesirable condition. " or may include the "prevention" of those. Furthermore, "to treat" or "treatment" may include signs Furthermore, complete relief of symptoms is not required, nor is a cure necessary; specifically, the patient has limitations. Includes protocols that only produce results.

[0044] The terms "therapeutic effect" or "therapeutically effective" are used throughout this application to mean this state This refers to any medical procedure that promotes or improves the health of the subject. This includes, but is not limited to, a decrease in the frequency or severity of signs or symptoms of the disease. No. For example, cancer treatment can reduce tumor size, decrease tumor invasiveness, and reduce cancer growth. This may include slowing the rate of progression or preventing metastasis. Cancer treatment refers to the survival time of a subject with cancer. It can also refer to an extension of that.

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

[0046] The phrase "pharmaceutically or pharmacologically acceptable" means that it can be administered to animals such as humans as necessary. When used, the molecular entities do not cause side effects, allergic reactions, or other adverse reactions. This refers to the composition. The preparation of a pharmaceutical composition containing antibodies or further active ingredients is the process described above. In light of the indication, this is common knowledge to those skilled in the art. Furthermore, in the case of administration to animals (e.g., humans), The product must comply with the FDA Office of Biological Standards. It must meet the required standards for sterility, pyrogenicity, overall safety, and purity. This is understood.

[0047] As used herein, “pharmaceutically acceptable carrier” means “a carrier known to those skilled in the art.” Any and all aqueous solvents (e.g., water, alcohol solution / water-soluble) may be used. Liquid, saline solution, parenteral vehicle, e.g., sodium chloride, Ringer's dextrose (and), non-aqueous solvents (for example, propylene glycol, polyethylene glycol, vegetable oil and Injectable organic esters (e.g., ethyl oleate), dispersion media, coatings, interfaces Activators, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents) (and inert gases), isotonic agents, absorption retarders, salts, drugs, drug stabilizers, gels, binders, excipients Agents, disintegrants, lubricants, sweeteners, flavorings, colorants, fluids and nutritional supplements, and the like. This includes various materials and combinations. The pH and accuracy of various components in the pharmaceutical composition. The concentration is adjusted according to well-known parameters.

[0048] The term "haplotyping or tissue typing" refers, for example, to the lymphatic system of a specific subject. To determine which HLA loci (or multiple loci) are expressed on the sphere. The method used to identify the haplotype or tissue type of a subject. This refers to the HLA gene, which is located in the major histocompatibility complex, a region on the short arm of chromosome 6. They are located in the body (MHC) and are involved in intercellular interactions, immune responses, organ transplantation, cancer development, and It is related to susceptibility to disease. There are six important gene loci in transplantation, including HLA-A. These were named HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ. Each gene locus can contain one of several different alleles.

[0049] A widely used method for haplotyping is polymerase chain reaction (PCR). Using this method, we compare known segments of the gene encoding the MHC antigen with the DNA of the subject. The diversity in these regions of those genes indicates the tissue type or haplogroup of the subject. Determine the prognosis. Serological methods are also used to detect serologically defined antigens on the cell surface. It is used for the following purposes. HLA-A, -B, and -C determinants are measured by known serological methods. It is possible. In short, lymphocytes derived from the subject (isolated from fresh peripheral blood) are known to be used. Incubate these cells with antiserum that recognizes all HLA antigens. Spread the cells in a tray with microwells containing various types of antiserum. Incubate the cells for 30 minutes. After incubation, the incubation is supplemented for another 60 minutes. Lymphocytes are then subjected to anti- If a lymphocyte has an antigen on its surface that is recognized by antibodies in the serum, that lymphocyte is lysed. By adding a dye, changes in cell membrane permeability and cell death can be observed. The pattern of cells destroyed by the solution indicates the degree of histological incompatibility. For example, HL Lymphocytes derived from a certain individual being tested for A-A3 have shown anti-hemorrhagic effects against HLA-A3. If the antigen is destroyed in a well containing the solution, the test will be positive for this group of antigens.

[0050] The term "antigen-presenting cell (APC)" refers to a cell recognized by specific effector cells of the immune system. It is possible to present one or more antigens in the form of a peptide-MHC complex, and therefore This means that the cell can induce an effective cellular immune response to the presented antigen. This refers to a whole cell (e.g., macrophage). The term "APC" refers to an intact whole cell (e.g., macrophage). (B cells, endothelial cells, activated T cells and dendritic cells), or cells that can present antigens. These are naturally occurring or synthetic molecules (for example, complexed with y2-microglobulin). It includes purified MHC class I molecules. II. Universal antigen-presenting cells

[0051] Some embodiments of this disclosure relate to the production of universal antigen-presenting cells (UAPCs) and Regarding use: UAPC is for the expansion of immune cells (e.g., NK cells and T cells). It can be used. UAPC is membrane-bound IL-21 (mbIL-21) and 41BBL( It can be manipulated to express the CD137 ligand.

[0052] UAPC expresses CD137 ligand and / or membrane-bound cytokines. It can be manipulated. The membrane-bound cytokine can be mRNA-21 or mRNA-15. In certain embodiments, UAPC expresses CD137 ligand and mIL-21. It is manipulated to do so. APCs can originate from cancer cells such as leukemia cells. APCs are internal None of the resident HLA class I, II, or CD1d molecules may be expressed. AP C may express ICAM-1 (CD54) and LFA-3 (CD58). In particular, A PCs are designed to express K562 cells (for example, CD137 ligand and mRNA-21). (K562 cells that have been manipulated) APC may be irradiated. The above manipulation is performed in the said part This could be any method known in the field, such as the introduction of a retrovirus.

[0053] Cytokines exert very potent regulatory effects on all classes of immune cells (including NK cells). It exerts its influence on cell fate, activity, and the effectiveness of cellular responses. It activates NK cells. The power of cytokine stimulation lies in the fact that these "natural" effector functions are highly sensitive to environmental interventions. Being receptive and priming can control the behavior of NK cells in vivo. This supports the point.

[0054] To address the problem of obtaining a clinically valid amount of NK cells, this method is used in this U Interlo is used as a driving substance for NK cell expansion in APC platform technology. Ikin (IL-21) can be used. In humans, NK cells are induced by IL-10 and IL-21. Cellular stimulation induces NKG2D expression in a STAT3-dependent manner. Cytokine receptor However, many immune cells share similar cellular signaling components, but NK cells Specific signaling is dependent on the IL-21 receptor-STAT3 relationship for proliferation. To exist.

[0055] Cytokine signaling is crucial for maintaining lymphocyte survival and proliferation. In vivo, IL-2 administration is the only treatment approved by the FDA to enlarge NK cells. This is the method. Another potent NK cell activator, IL-15, is a promising alternative to IL-2. Although it is currently undergoing Phase I clinical trials as an alternative, it has shown significant toxicity after systemic administration. In addition to significant toxicity, these cytokines limit the persistence of NK cells, It also induces T cell proliferation. Despite sharing the same receptor for signal transduction, IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 receptors This has different and specific effects on various types of cells. A. Membrane-bound IL-21

[0056] In a specific embodiment, this UAPC is created to specifically activate NK cells. IL-21 may be used for this purpose. The IL-21 receptor (IL21R) is common. Cytokine receptor gamma chain (transmits signals through dimerization with gamma(c)) It has a close relationship with the IL-2 receptor beta chain, which can activate human NK cells. Type I cytokines are upregulated in cells that are ready to be stimulated. IL-21 can regulate the functions of T cells, B cells, and NK cells, but this study found that Only human NK cells show significant expansion through activation of the IL21 receptor signaling pathway. They found that this caused (i.e., a 1000-fold increase over 21 days). Conversely, IL21 CD8 + It plays an important role in the reduction of T cells.

[0057] IL21R signaling is primarily driven by STAT3, a highly potent cell proliferation activator. This is promoted by the IL21R-STAT3 relationship, which was investigated using an anti-phosphotyrosine antibody. GAS and cis-inducible markers are verified by immunoprecipitation and Western blotting. Driven by IL-21-inducible STAT3 DNA binding to the element. IL21 The molecular basis of the proliferation mediated by IL-21 is STAT1 and It is tyrosine 510 (Y510) on IL21R that mediates the phosphorylation of STAT33. This can be specifically identified. This mechanism is Stat1 / Stat3 double knockout. In mice, this is clearly demonstrated by a weakened IL-21 response.

[0058] IL21R signaling is important for NK cell toxicity. Human IL21R deficiency This is associated with impaired cytolysis of 51Cr-labeled K562 target cells, but is antibody-dependent. Cell damage is unaffected. Monogenic non-embryonic lethal deletion (IL21R loss-of-function mutation) This provides an excellent opportunity to clarify the innate NK cell lysis response, and therefore the innate N It is useful for modeling, enhancing, and shaping K cell lysis reactions.

[0059] CD56 dim Cell populations and CD56 bright Both cell populations have a similar number of surface ILs. Even if 21R is present, IL-21 signaling selectively targets a subset of NK cells. Forms IL-21 induction of STAT1 and STAT3 phosphorylation, CD56dimN CD56brightNK cells show higher levels compared to K cells. In contrast, IL-21 This does not affect STAT5 activation, which is an IL-2 activation pathway that also drives T cell expansion. In addition to STAT3 activation, IL-21 signaling involves the MAPK and PI3K pathways. This involves the innate immune response genes (IFN-gamma, T-bet, IL) in NK cells. It induces the expression of (including IL-12R beta-2 and IL-18R) to kill tumor cells. Priming sea urchin NK cells.

[0060] To efficiently utilize IL-21, we use mbIL-21 expression (Figure 4A) to N The trans interaction with IL21R on K cells may be concentrated and localized. Since 21 is in close proximity to the membrane, it can be readily available when needed. Optimal cell proliferation can be maintained without the need for a large and high-concentration supply of IL-21 from an external source. Co-culture with injected K562-mb15-41BBL revealed CD56+CD in peripheral blood. A median 21.6-fold expansion of 3-NK cells was induced. This expansion was due to IL-2, IL- Derived from the covalent γc family, including 12, IL-15, and IL-21 individually or in combination. This is greater than stimulation by simple soluble cytokines. In comparison, this UAPC This can enlarge NK cells by at least 1000 times (3-log) over 14-21 days. The expression of mbIL21 on UAPC does not require 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 activating molecules also enhances the cell proliferation response. CD137(4 -1BB) is a member of the tumor necrosis receptor (TNF-R) gene family, It mediates cell proliferation, differentiation, and programmed cell death (apoptosis). Mouse receptor After the first homolog was characterized, the human homolog was characterized, and it was found to be 6 amino acid level. They share 0% identity, and their cytoplasmic / signaling domains are significantly conserved. CD1 37 is primarily expressed in activated T cells and activated NK cells, as well as in thymocytes and myeloid cells. It is expressed at various detectable levels in cells and endothelial cells at inflammatory sites. (Physiology) Typical CD137 signaling involves 1) NF- that promotes survival through Bcl-XL activation. κB, and 2) the PI3K / ERK1 / 2 pathway which specifically drives cell cycle progression. It is mediated by.

[0062] In activated NK cells, CD137 is a cytokine-inducing co-stimulatory molecule, and this co The stimulating molecule increases cell proliferation and IFN-γ secretion, thereby affecting NK cells. This drives the antitumor response. Studies using CD137L- / - knockout mice have shown that The importance of CD137 / CD137L signaling in the development of anti-tumor immune cells has been elucidated. CD137- / - knockout mice showed a higher tumor metastasis compared to control mice. The frequency of migration was four times higher.

[0063] CD137 ligan, a 34kDa glycoprotein member of the TNF superfamily CD137L (4-1BB ligand) is primarily used in B cells, macrophages, and dendritic cells. It was detected on activated antigen-presenting cells (APCs), including T cells, and was also detected at low levels on activated T cells. It is expressed transiently. Human CD137L is only 36% homologous to its mouse counterpart. Not possible. Consistent with the antitumor efficacy of activating CD137 antibodies, the binding of CD137L is consistent with CT It has been shown to induce L and antitumor activity.

[0064] Therefore, this UAPC is a physiological counterreceptor for CD137. The 4-1BB ligand can be manipulated to express it for stimulation. C.SLAM / CD48

[0065] In addition to cytokine conditioning and 4-1BBL co-stimulation, NK cells and target Direct physical interaction with target cells also influences the cellular response, i.e., the killing of target cells. A signaling lymphocyte-activating molecule that is widely expressed in the immune system and plays a crucial role. SLAM (formerly known as the CD2 Superfamily) Family Homologousity Epidemic globulin receptors are particularly important in relation to intercellular interactions.

[0066] Therefore, the UAPCs of this disclosure may express one or more SLAM family antigens. As members of the SLAM family, CD2, CD48, CD58 (LFA-3), CD244 (2B4), CD229 (Ly9), CD319 (CS1 (CD2 subset) 1); CRACC (CD2-like receptor-activated cytotoxic cells) and CD352 (N TB-A (NK-TB antigen) is one example. In a particular embodiment, UAPC is CD NK cells express 48 and / or CS1. NK cells belong to the SLAM family (SLAMF). It expresses at least three members of the 2B4, NK cells, T cells, and B cells. Cellular antigen (NTB-A), and CD2-like receptor-activated cytotoxic cells (CRAC) C) (These are the respective ligands on target cells and possibly other NK cells.) It recognizes CD48, NTB-A and CRACC. SLAMF1, 3, 5, 6, 7, 8, and 9 are homoaffinity (self-ligand) receptors, but SLAMF2 And SLAMF4 are counterreceptors (heteroaffinity) to each other. The affinity of knowledge (dissociation constant Kd <1 μM to 200 μM) is wide. The fact is that the signal transduction mechanisms for SLAM glycoproteins are overlapping but different. This suggests the foundation of the mechanism by which things become what they are.

[0067] Table 1. Binding affinity of SLAM family members. [Table 1]

[0068] In certain embodiments, the UAPC supports intercellular interactions and enhances NK cell responses. CD48 is manipulated to express CD48. CD48 is expressed by NK cells, T cells, monocytes, etc. Glycosylphosphatidylinositol anchored tannins are found on the surface of basophils. These are proteins (GPI-APs) involved in adhesion and activation pathways in these cells. Despite lacking an intracellular domain, CD48 stimulation is effective in lipid rafts. This involves rearranging signaling factors, inducing Lck-kinase activity, and tyrosine phosphorylation. As an adhesion-costimulatory molecule, CD48 is involved in the activity of B lymphocytes and T lymphocytes, NK cells, and mass cells. It induces numerous effects in NK cells and eosinophils. In human NK cells, CD48 It is a counterreceptor for 2B426, an important activator of NK cells. Heteroaffinity interactions compete for the interaction between MHC-I and CD244. It is thought that the 2B4 / CD48 interaction is activated in human NK cells. While it induces Gnar, mouse NK cells send inhibitory signals.

[0069] 2B4-CD48 interactions between cells of the same population, i.e., interactions between NK cells, The interaction between T cells is APC (for example, K562 myeloid cells that normally lack CD48). By expressing CD48 on the (series), activation is enhanced, but this UAPC is NK This can transform the 2B4 signaling pathway in cells and turn it on. D.SLAM / CS1

[0070] In some embodiments, the UAPC switches on the killing power of NK cells. As a co-stimulatory molecule, express CS1, another member of the SLAM family. It is manipulated. Unlike CD48 which interbonds with 2B4, the CS1 interaction is homoffinity This can be characterized in a situation that contrasts cis-interactions with trans-interactions. K562 cells, which normally do not contain CS1, are manipulated to express CS1. E. Nucleic acid delivery

[0071] mbIL-21, 41BBL, and the co-stimulatory antigens are used in any of the methods known in the art. Therefore, it can be manipulated. Nucleic acids are usually in the form of expression vectors, such as viral expression vectors. It is administered. 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 above delivery is one or more vectors, one or more of which This is due to the delivery of the copy and / or one or more proteins transcribed therefrom. It is then delivered to the aforementioned cells.

[0072] Methods for introducing polynucleotide constructs into animal cells are well known, and as As a non-limiting example, polynucleotide constructs are integrated into the cell genome. A stable transformation method in which polynucleotide constructs are integrated into the cell genome. Examples include transient transformation methods and virus-mediated methods. Several implementations In this state, polynucleotides are, for example, recombinant viral vectors (e.g., retrograde viral vectors). It can be introduced into cells by viruses (such as adenoviruses), liposomes, etc. For example, In some embodiments, transient transformation methods include microinjection, electro Examples include oxyporation or particle guns. In some embodiments, polynucleation Considering that rheotide is expressed in cells, the vector, more specifically, plus It may be included in the Mido or Virus category.

[0073] Nonviral delivery methods for nucleic acids include lipofection, nucleofection, Microinjection, microparticle gun, virosome, liposome, immunoliposome, poly Cation or lipid: nucleic acid conjugates, naked DNA, artificial virions, and DNA One example is agent-enhanced uptake. Lipofection is (for example, U.S. Patent No. 5,049,386, and No. 4,946) As described in No. 787; and No. 4,897,355, lipofection reagents These are commercially available (for example, Transfectam TM and Lipofectin TM ). Cations suitable for efficient receptor recognition lipofection of polynucleotides. As for sexual and neutral lipids, Feigner's lipids, WO91117424;WO9 1116024 is an example. Delivery is delivery to cells (e.g., in vitro administration or e- This may involve xenovivo administration or delivery to target tissue (e.g., in vivo administration).

[0074] In some embodiments, delivery is performed by an RNA virus or D for delivery of nucleic acids. Delivery is by using a system based on the NA virus. The viral vector is used in several forms. In this context, it may be administered directly to the patient (in vivo), or using a viral vector. Cells may be treated in vitro or ex vivo and then administered to the patient. The virus-based system in the embodiment is a retroviral vector for gene transfer. Trench virus vectors, adenovirus vectors, adeno-associated virus vectors Examples include herpes simplex virus vectors. III. Immune cells

[0075] Some embodiments of this disclosure relate to immune cells (e.g., NK cells) used in cancer immunotherapy. This concerns the isolation and expansion of T cells.

[0076] In a particular embodiment, immune cells are used in a manner well known in the art to treat human peripheral tissue. Blood mononuclear cells (PBMCs), unstimulated leukocyte transfer products (PBSCs), human embryonic stem cells (hES) C) Obtained from induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood. For details, see Immunology. Cells can be isolated from umbilical cord blood (CB), peripheral blood (PB), bone marrow, or stem cells. In this embodiment, immune cells are isolated from pooled CBs. CBs are 2, 3, 4 They can be pooled from 5, 6, 7, 8, 10 or more units. Immune cells are autologous Alternatively, they may be allogeneic. Isolated immune cells are haplogroups with the subjects receiving cell therapy. The types may match. NK cells have specific surface markers (e.g., CD16 in humans). It can be detected by CD56 and CD8).

[0077] In a certain aspect, NK cells are previously expanded ex vivo. The reported methods (Spanholtz et al., 2011; Shah et al ., 2013) are isolated. In this method, CB mononuclear cells are isolated by ficoll density gradient centrifugation. The cell culture may be depleted of any cells expressing CD3, and the percentage of CD56 / CD3 + cells or NK cells can be characterized for measurement - . In other methods, NK cells are obtained by isolation of CD34 cells using umbilical CB. This method may include depletion of CD3, CD14, and / or C + ells D19 positive cells.

[0078] The isolated immune cells can be expanded in the presence of this UAPC. The expansion can be for about 2 to 30 days (e.g., 3 to 20 days, particularly 12 to 16 days, e.g., 12, 13, 14, 15, 16, 17, or 19 days, specifically about 14 days). The immune cells and UAPC can be present in a ratio of about 3:1 to 1:3 (e.g., 2:1, 1:1, 1:2, specifically about 1:2 ). The expansion culture may further contain cytokines that promote expansion (e.g., IL-2) . IL-2 can be present at a concentration of about 10 to 500 U / mL (e.g., 100 to 300 U / mL, particularly about 200 U / mL). IL-2 can be replenished in the expansion culture, for example , every 2 to 3 days. UAPC can be added to the culture at least twice (e.g., about 7 days after expansion ).

[0079] The immune cells may be injected immediately after expansion or stored by cryopreservation or the like . In certain embodiments, the cells are within about 1, 2, 3, 4, 5 days, within a few days They can be grown as a bulk population in exovivo for several weeks or months.

[0080] Expanded NK cells activate both innate and adaptive immune cells through type I cytotoxicity. In (for example, interferon-γ, tumor necrosis factor-α, and granulocyte macrophage γ) It secretes Ronnie's stimulating factor (GM-CSF), as well as other cytokines and chemokines. It is possible to measure the activation state of NK cells by using the measurement of these cytokines. It is possible to measure NK cell activation using other methods known in the field. The method may be used for characterizing the NK cells of this disclosure. IV. Chimeric Antigen Receptors

[0081] In a particular embodiment, these immune cells (e.g., T cells or NK cells) are used to control skin texture It is genetically modified to express the RA antigen receptor (CAR). Several embodiments In this context, CAR comprises a) an intracellular signaling domain, b) a transmembrane domain, and c ) Includes an extracellular domain containing an antigen-binding region.

[0082] CAR recognizes cell surface tumor-associated antigens unrelated to human leukocyte antigens (HLA), and one Using the above signaling molecules, genetically engineered to kill, proliferate, and produce cytokines. It activates NK cells that have been modified. In a particular embodiment, these NK cells are (i ) Nonviral treatment using an electroporation device (e.g., a nucleofector) (ii) gene transfer for saturation, (ii) endodomain (e.g., CD28 / CD3-ζ, CD13 CARs that signal via 7 / CD3-ζ or other combinations, (iii) Anti CARs having extracellular domains of various lengths connecting the original recognition domain to the cell surface, and In some cases, (iv)CAR + K can reliably and numerically increase NK cells. Artificial antigen-presenting cells (aAPCs) derived from 562 (Singh et al., 2011) It can be genetically modified by methods including ).

[0083] Embodiments of this disclosure include an intracellular signaling domain, a transmembrane domain, and one or more Contains an extracellular domain containing a signaling motif, which reduces immunogenicity in humans. Contains nucleic acids encoding antigen-specific CAR polypeptides, including conditioned CARs (hCARs). Regarding the use of nucleic acids. In a particular embodiment, the CAR is one or more antigens It is possible to recognize an epitope that includes a space shared between them. In a particular embodiment, The binding region is the complementarity-determining region of the monoclonal antibody, the variable region of the monoclonal antibody, and It may contain a vi / or its antigen-binding fragment. In another embodiment, its specificity is It originates from peptides (e.g., cytokines) that bind to receptors.

[0084] Human CAR nucleic acids are used to improve cell immunotherapy for human patients. It is intended to be a gene. In a specific embodiment, this method uses the full length CA Use RcDNA or coding region. The antigen-binding region or antigen-binding domain is specific. Human monoclonal antibodies (e.g., U.S.7, as incorporated herein by reference) Single-stranded variable fragments (scFv) derived from those described in publications 109 and 304. )'s V H Chain and V L It may contain chain fragments. These fragments are human antigen-specific. This could also be any number of different antigen-binding domains of the target antibody. In more specific embodiments... And that fragment is the most suitable for human codon usage for expression in human cells. It is an antigen-specific scFv encoded by an optimized sequence.

[0085] The configuration can be a multimer, for example, a diabody or multimer. A multimer is, Typically, the variable parts of the light and heavy chains form pairs with each other to create a diamond body. It is formed by... The hinge portion of the structure has several options, and these include... It is completely removed, the first cysteine ​​is retained, and proline is used instead of serine. This includes substitution and cleavage down to the first cysteine. The Fc portion is shaved. Any protein that is stable and / or dimerizes can be eliminated. It is possible. One of the Fc domains, for example, the CH2 or CH3 domain derived from human immunoglobulin. Either of the main ones may be used. Human immunoglobulins modified to improve dimerization. The hinge, CH2 and CH3 regions of the brin may be used. In other embodiments, simply immunoglobulin may be used. The hinge portion of Roblin or a part of CD8α may be used.

[0086] In some embodiments, CAR nucleic acids interact with other co-stimulatory receptors (e.g., transmembrane receptors). Includes sequences encoding the domain and modified CD28 intracellular signaling domain. Hmm. Other co-stimulatory receptors include CD28, CD27, OX-40 (CD134), One or more of the following are listed: DAP10 and 4-1BB (CD137). Not limited. In addition to the primary signal induced by CD3ζ, the human CAR is inserted Further signals provided by human co-stimulatory receptors enable the full activation of NK cells. It is important for sexualization, improving residual disease in vivo and the success of adoptive immunotherapy treatment. It can help improve the situation.

[0087] The intracellular signaling domain of the chimeric antigen receptor is located where the chimeric antigen receptor is positioned. It is involved in the activation of at least one of the normal effector functions of the immune cells. The effector function is a special function of differentiated cells such as NK cells. In CAR, the intracellular receptor signaling domain contains T cell antigen receptors. The intracellular receptor signaling domain of the receptor complex (e.g., the zeta chain of CD3, FcγRIII co-stimulatory signaling domain, CD28, CD27, DAP10, CD This includes 137, OX40, and CD2 (either individually or in conjunction with, for example, CD3 zeta). In a specific embodiment, the intracellular domain (which may be referred to as the cytoplasmic domain) is the TCR Zeta chain, CD28, CD27, OX40 / CD134, 4-1BB / CD137, Fc εRIγ, ICOS / CD278, IL-2R Beta / CD122, IL-2R Alpha / Includes one or more parts or all of CD132, DAP10, DAP12, and CD40 Any part of the endogenous T cell receptor complex is used in the intracellular domain. To obtain. So-called third-generation CARs are fused together for additive or synergistic effects, for example. Since it has at least two or three signaling domains, one or more The cytoplasmic domain may also be used.

[0088] In a particular embodiment of CAR, the antigen-specific portion (antigen-binding region) of the receptor is The extracellular domain (which may be referred to as the extracellular domain) is a tumor-associated antigen or pathogen-specific antigen-binding domain. It contains. The antigen is recognized by pattern recognition receptors such as Dectin-1. It contains glycosylated antigens. Tumor-associated antigens are optional as long as they are expressed on the cell surface of tumor cells. It may be of any type. The tumor antigen binding domain may be CD19, CD20, carcinoembryonic antigen, or Fafetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma-associated antigen, Mutation p53, mutation ras, HER2 / Neu, ERBB2, folate-binding protein, HIV HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp4 1, GD2, CD123, CD23, CD30, CD56, c-Met, Mesotheline, G D3, HERV-K, IL-11R Alpha, Copper Chain, Lambda Chain, CSPG4, ER It may be, but is not limited to, BB2, EGFRvIII, or VEGFR2. AR may include humanized scFv (e.g., humanized CD19 or CD123).

[0089] In a particular embodiment, when the amount of tumor-associated antigen is low, the persistence of CAR is improved. To improve performance, it can be co-expressed with cytokines. For example, CAR can be co-expressed with IL-15. It is possible.

[0090] The sequence of the open reading frame that codes for the chimeric receptor is genomic DNA. Origin, can it be obtained from cDNA origin, or can it be synthesized (e.g., PCR) It may be via, or a combination thereof. Introns stabilize mRNA. Since this has been found, depending on the size of the genomic DNA and the number of introns, cDN It may be preferable to use A or a combination thereof. Also, to stabilize mRNA Using intrinsic or extrinsic non-coding domains to refine the process is also beneficial. obtain.

[0091] The chimeric construct can be introduced into NK cells as naked DNA or in the form of a suitable vector. The aim is to stabilize cells by electroporation using naked DNA. Methods for transfecting are publicly known in the art. For example, U.S. Patent No. 6,410 See issue 319. Naked DNA generally refers to plasmin in an orientation suitable for expression. This refers to the DNA that codes for a chimeric receptor, which is included in a DNA expression vector.

[0092] Alternatively, viral vectors (e.g., retroviral vectors, adenovirus vectors) By using a vector (adeno-associated virus vector or lentiviral vector), The chimeric construct can then be introduced into NK cells. Suitable vectors are non-replicating in NK cells. Numerous virus-based vectors. It is known that the number of viral copies maintained within a cell is essential for maintaining the cell's survival. Low enough to be effective, for example, based on HIV, SV40, EBV, HSV, or BPV. He is a ctor.

[0093] CARs are suicide genes (e.g., CD20, CD52, EGFRv3, or inducible CARs). It may express Pase 9).

[0094] CAR may contain a tumor antigen-binding domain. This tumor antigen-binding domain may contain CD19 CD20, fetal carcinoma antigen, alpha-fetoprotein, CA-125, MUC-1, epithelium Sex tumor antigen, melanoma-associated antigen, p53 mutation, ras mutation, HER2 / Neu, ERBB2 , folate-binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 Glucoprotein gp41, GD2, CD123, CD23, CD30, CD56 c-Met, Mesothelin, GD3, HERV-K, IL-11R Alpha, Copper Chain It could be a lambda chain, CSPG4, ERBB2, EGFRvIII, or VEGFR2. However, it is not limited to these. CARs include humanized scFv, for example, humanized CD19, CD 123, Mesotheline, 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 this disclosure describe the transfer of an immune cell population that induces an immune response to internal medicine diseases. Immune cells provided herein for treating or preventing disease or disorder, e.g., N Methods for using K or T cells (e.g., those expanded by this UAPC) The method involves administering a therapeutically effective dose of expanded immune cells to the subject. , including steps to treat or prevent injury in the subject. A specific embodiment of the present disclosure In this condition, cancer or infectious diseases can be treated by introducing a population of immune cells that induce an immune response. They are placed. Immune cells, due to the release of pro-inflammatory cytokines, are associated with auxiliary immune cells. By promoting differentiation, activation, and / or recruitment to malignant disease sites, anti-inflammatory This can reverse the tumor microenvironment and enhance the adaptive immune response.

[0096] This treatment method is useful for any type of malignant cell, such as solid tumors or hematopoietic tumors. Examples of fluid tumors include those found in liquid tumors. Exemplary solid tumors include those of the pancreas, colon, and cecum. Stomach, brain, head, neck, ovaries, kidneys, larynx, sarcoma, lungs, bladder, melanoma, prostate and breast Examples include, but are not limited to, tumors of organs selected from the following groups. Hematological malignancies include bone marrow tumors, T or B cell malignancies, leukemia, lymphoma, blastoma, and migraines. Examples include eloma. Further cancers that can be treated using the methods provided herein 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 tract cancer and digestive cancer) (Including stromal carcinoma), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, rectal cancer Bowel cancer, endometrial or uterine cancer, salivary gland cancer, kidney or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer This includes, but is not limited to, adenocarcinoma, various types of head and neck cancer, and melanoma. .

[0097] Cancer can be, but is not limited to, the following histological types: neoplasms, malignant cancers. Carcinoma; carcinoma, undifferentiated type; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma Cancer; lymphoid epithelial carcinoma; basal cell carcinoma; pilomatric carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastri Norma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed type of hepatocellular carcinoma and cholangiocarcinoma; cord-like adenocarcinoma; adenoid cystic carcinoma; Adenocarcinoma within adenomatous polyps; 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 ; Endometroid carcinoma; Skin appendage carcinoma; Apocrine adenocarcinoma; Sebaceous gland carcinoma; Ceruminous gland adenocarcinoma; 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; Lobular cell carcinoma; Adenoid cystic carcinoma ; Adenocarcinoma with squamous metaplasia; Thymoma, malignant; Ovarian stromal tumor, malignant; Capsular 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; Carcinoid ; Glomus angiosarcoma; Malignant melanoma; Amelanotic melanoma; Superficial spreading melanoma; Lentigo maligna ; Melanoma; Acral lentiginous melanoma; Nodular melanoma; Malignant melanoma in giant congenital nevus; Epithelioid melanoma ; Blue nevus, malignant; Sarcoma; Fibrosarcoma; Fibrous histiocytoma, malignant; Myxosarcoma; Liposarcoma ; Leiomyosarcoma; Rhabdomyosarcoma; Fetal rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Mixed tumor, malignant ; Müllerian duct mixed tumor; Wilms tumor; Hepatoblastoma; Carcinosarcoma; Mesenchymal tumor, malignant; Brenner tumor, malignant ; Phyllodes tumor, malignant; Synovial sarcoma; Mesothelioma, malignant; Undifferentiated embryonal cell tumor; Fetal carcinoma; Teratoma, malignant ; Ovarian teratoma, malignant; Choriocarcinoma; Mesonephroma, malignant; Angiosarcoma; Angioendothelioma, malignant; Kaposi sarcoma ; Perivascular cell tumor, malignant; Lymphangiosarcoma; Osteosarcoma; Juxtacortical osteosarcoma; Chondrosarcoma; Chondroblastoma, malignant ; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing sarcoma; Odontogenic tumor, malignant ; Ameloblastic odontogenic sarcoma; Ameloblastoma, malignant; Ameloblastic odontogenic sarcoma; Pinealoma, malignant ; Chordoma; Glioma, malignant; Ependymoma; Astrocytoma; Protoplasmic astrocytoma ; Fibrous astrocytoma; Glioblastoma; Oligodendroglioma; Oligodendroblastoma; Primitive neuroectoderm; Cerebellar sarcoma; Ganglioneuroblastoma; Neuroblastoma; Retinoblastoma; Olfactory neurogenic tumor; Myeloma ; Medulloblastoma; Neuroepithelioma Malignant neoplasm of the meninges; Neurofibrosarcoma; Malignant schwannoma; Malignant granular cell tumor; Malignant lymphoma; Hodgkin disease; Hodgkin lymphoma; Granuloma faciale; Malignant lymphoma, small lymphocytic; Malignant lymphoma, large cell type, diffuse; Malignant lymphoma, follicular; Mycosis fungoides; Other specified non-Hodgkin lymphoma; B-cell lymphoma; Low-grade / follicular non-Hodgkin 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 non-cleaved cell NHL; Large lesion (bulky disease) NHL; Mantle cell lymphoma; AIDS-related lymphoma; Waldenström macroglobulinemia; Malignant histiocytosis; Multiple myeloma; Mast cell sarcoma; Immunoproliferative small intestinal disease; Leukemia; Lymphocytic leukemia; Plasma cell leukemia; Erythroleukemia; Lymphosarcoma cell leukemia; Myelogenous leukemia; Basophilic leukemia; Eosinophilic leukemia; Monocytic leukemia; Mast cell leukemia; Megakaryoblastoid leukemia; Myelosarcoma; Hairy cell leukemia; Chronic lymphocytic leukemia (CLL); Acute lymphoblastic leukemia (ALL); Acute myelogenous leukemia (AML); And chronic myeloblastic leukemia.

[0098]

[0099] Certain embodiments relate to a method of treating leukemia. Leukemia is a cancer of the blood or bone marrow characterized by abnormal proliferation (production by mitotic proliferation) 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 encompassing a range of diseases. Leukemia is clinically and pathologically divided into acute and chronic forms. Acute leukemia is characterized by rapid proliferation of immature blood cells. This crowding causes the bone marrow to The body becomes unable to produce healthy blood cells. Acute leukemia occurs in children and young adults. It is possible. In acute leukemia, there is rapid progression and accumulation of malignant cells, which then flood the bloodstream. Because it can spread to other organs in the body, prompt treatment is necessary. Central nervous system (C Although involvement of NS is rare, this disease can sometimes cause cranial nerve palsy. Chronic leukemia is It is distinguished by an excessive accumulation of relatively mature but still abnormal blood cells. Over the course of several months to several years, cells are produced at a much faster rate than normal cells. This results in the production of many abnormal white blood cells in the blood. Chronic leukemia usually occurs in the elderly. Theoretically, it can occur in any age group. Acute leukemia must be treated immediately. In contrast, for chronic cases, to ensure that the effectiveness of treatment is maximized, pre-treatment is sometimes performed. You will be under surveillance for a while.

[0100] Furthermore, the above diseases are lymphocytic or lymphoblastic (normally, lymphocytes continue to form (This indicates that cancer has occurred in a certain type of bone marrow cell) and myeloid or myeloid ( A certain type of bone that normally continues to form red blood cells, several types of white blood cells, and platelets. This is classified as a condition indicating that cancer has occurred in the medullary cells.

[0101] Acute lymphoblastic leukemia (also known as acute lymphoblastic leukemia, or ALL) is It is the most common type of leukemia in young children. This disease is also common in adults, especially 6. It can develop even in people over 5 years old. Chronic lymphocytic leukemia (CLL) almost always occurs in people over 55 years old. It develops in adults. Chronic lymphocytic leukemia (CLL) occasionally occurs in younger adults. However, it rarely occurs in children. Acute myelogenous leukemia (also known as acute myeloid leukemia, or AML) usually occurs in adults rather than children. This type of leukemia was previously called acute non-lymphocytic leukemia. Chronic myelogenous leukemia (CML) mainly occurs in adults. Lymphoma is a type of cancer that originates 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 about 5 percent of all cancer cases in the United States, and particularly Hodgkin lymphoma accounts for less than 1 percent of all cancer cases in the United States. Since the lymphatic system is part of the body's immune system, patients with a weakened immune system due to HIV infection or certain drugs or drug therapies also have a high incidence of lymphoma. In certain embodiments of the present disclosure, immune cells are delivered to an individual who needs them (e.g., an individual with cancer or an infectious disease). Then, those cells enhance the individual's immune system to attack each cancer cell or pathogenic cell. In some cases, the individual is provided with immune cells more than once. When the individual is provided with immune cells more than once, the time between administrations should be sufficient for the time for propagation to elapse in that individual. In a specific embodiment, the time between administrations is 1, 2, 3, 4, 5, 6, 7 days or more. The origin of the pre-activated and expanded immune cells can be of any type, but specific

[0102]

[0103]

[0104] ​ In embodiments, the cells are, for example, umbilical cord blood, peripheral blood, human embryonic stem cells, or induced polycystic cells. It is obtained from a potent stem cell bank. A suitable dose for therapeutic effect is preferably a series of doses. In a given cycle, for example, at least 10 per dose 5 or about 10 5 ~about 10 10 It may be cells. An exemplary dosing regimen is at least about 10 on day 0. 5 Starting from cells For example, within a few weeks of starting an intra-patient dose escalation scheme, approximately 10 10 Cells as the target dose This consists of four weekly dose escalation cycles, gradually increasing the dosage. The methods of administration include intravenous, subcutaneous, intracavitary (e.g., via a reservoir access device), and intraperitoneal. Other methods include direct injection into the tumor.

[0105] In one exemplary method, NK cells are found in biological samples such as one or more units of umbilical cord blood. It may originate from the cord. The cord blood units may be frozen cord blood units. Thaw the cord blood units, By subjecting them to a Ficol gradient, mononuclear cells can be obtained. These mononuclear cells are then used in Clin iMAS and other factors are causing depletion of CD3, CD14, and CD19-positive cells. Yes. Then, negatively selected NK cells are APC and IL-2 (e.g., 20 They can be cultured in the presence of (0 U / mL). Then, the NK cells express CAR. The retroviral supernatant can be transduced and, together with gamma-irradiated APC and IL-2 It can be cultured. Finally, the cells will exhibit positive expression of CARs (e.g., CD56, CD16, It can be sorted by CD3, CD19, CD14, or CD45.

[0106] The immune cells produced according to this method have many applications, including experimental and therapeutic uses. There are many potential uses. In particular, such cell populations can be used to eliminate undesirable or inappropriate immunological conditions. It is expected to be extremely useful in suppressing the epidemic response. In such a method, a small number of Immune cells are extracted from the patient, then manipulated and enlarged using exovivo, and then returned to the patient. Reinject. Examples of diseases that can be treated in this way include, for example, immunotherapy for allograft tolerance. Inhibition of activity is desirable in autoimmune diseases and autoimmune states. Therapeutic methods include suckling The process of preparing materials, the process of obtaining immune cells from the mammal; the method of this method as described above. The process of ex vivo expanding the immune cells according to the procedure; and the process of treating the expanded immune cells This may include the step of administering the substance to a mammal.

[0107] The pharmaceutical compositions of this disclosure may be used alone or in combination with other established agents useful for the treatment of cancer. They can be used in combination. They can be delivered alone or in combination with other active ingredients. Regardless of the cause, the pharmaceutical compositions of this disclosure are effective in mammals, particularly humans, through various pathways. By being delivered to various parts of the body, specific effects can be achieved. Those skilled in the art will know that 2 Although more than one route can be used for administration, a particular route is faster-acting and You will recognize that it can provide an effective response. For example, in the case of treating melanoma, inhalation Intradermal delivery may be advantageously used. Local or systemic delivery is also possible for the application of the formulation to body cavities. Alternatively, by drip infusion, aerosol inhalation or gas injection, or intramuscular, intravenous, or portal vein injection. This can be achieved by administration including parenteral induction, such as intrahepatic, peritoneal, subcutaneous, or intradermal administration. .

[0108] Certain embodiments of this disclosure provide methods for treating or preventing immune-mediated disorders. Provided. In one embodiment, the subject has an autoimmune disease. Limited examples include alopecia areata, ankylosing spondylitis, antiphospholipid antibody syndrome, and autoimmune diseases. Dison's disease, adrenal autoimmune disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oocyte 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, scarring pemphigoid, C REST syndrome, cold agglutinin disease, Crohn's disease, lupus discoid, essential mixed cryoglobulin disease Fibromyalgia-fibromyitis, glomerulonephritis, Graves' disease, Guillain-Barré disease, Hashimoto's thyroiditis 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., micro (Variant group, focal segmental glomerulosclerosis or membranous nephropathy), pemphigus vulgaris, pernicious anemia, polyamorticoid syndrome Pulsitis, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, Primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's disease Elephant, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, Generalized rigidity syndrome, systemic lupus erythematosus, lupus erythematosus, ulcerative colitis, uvea Inflammation, vasculitis (e.g., polyarteritis nodosa, Takayasu's arteritis, temporal arteritis / giant cell arteritis) These include herpetiform dermatitis (vasculitis), vitiligo, and Wegener's granulomatosis. Examples of autoimmune diseases that can be treated using the methods disclosed herein include For example, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes, Crohn's disease Examples include ulcerative colitis, myasthenia gravis, glomerulonephritis, ankylosing spondylitis, vasculitis, or psoriasis. However, this is not limited to these. The subjects may also have allergic disorders such as asthma.

[0109] In yet another embodiment, the subject is a recipient of organ transplantation or stem cell transplantation. Yes, immune cells are used to prevent and / or treat rejection. In certain embodiments... In this study, the subjects were squirrels that had graft-versus-host disease or were prone to developing graft-versus-host disease. There is a risk. GVHD can be treated by using stem cells from related or unrelated donors or It is a complication that can occur with any transplant, including GVHD. There are two types of GVHD: acute and chronic. Sexual GVHD appears within the first three months after transplantation. Signs of acute GVHD include hand and One symptom is a reddish rash on the feet, which can spread and become more severe. This is accompanied by skin peeling or blister formation. Acute GVHD can also affect the stomach and intestines. In that case, muscle spasms, nausea, and diarrhea may occur. Yellowing of the skin and eyes (jaundice) is acute. This indicates that GVHD is affecting the liver. Chronic GVHD is diagnosed based on its severity. Grade 1 indicates mild symptoms; Grade 4 indicates severe symptoms. Chronic GVHD develops 3 months or later after transplantation. Symptoms of chronic GVHD include: The symptoms are similar to those of acute GVHD, but chronic GVHD also involves the mucous glands of the eyes, the salivary glands of the mouth, and so on. Furthermore, it can also affect the glands that lubricate the inner wall of the stomach and the intestines. Disclosed herein Any population of immune cells can be used. Examples of organs that can be transplanted include parenchymal organs. Planting (e.g., kidney, liver, skin, pancreas, lung and / or heart) or cell transplantation (e.g., Examples include pancreatic islets, hepatocytes, myoblasts, bone marrow, or hematopoietic stem cells or other stem cells. The transplant may be a compound transplant (e.g., facial tissue). Immune cells are used before the transplant. It can be administered simultaneously with or after transplantation. In some embodiments, immune cells are Before transplantation (for example, at least 1 hour, at least 12 hours, at least 1 day before transplantation) Previously, at least 2 days ago, at least 3 days ago, at least 4 days ago, at least 5 days ago, less At least 6 days ago, at least 1 week ago, at least 2 weeks ago, at least 3 weeks ago, It is administered 4 weeks or at least 1 month prior. In one non-specific example, The administration of a therapeutically effective dose of immune cells is performed 3 to 5 days before transplantation.

[0110] In a particular embodiment, immune cells are administered in combination with a second therapeutic agent. For example, a second therapeutic agent could be T cells, immunomodulators, monoclonal antibodies, or chemotherapy. Examples include immunomodulators. In a non-limiting example, an immunomodulator is lenolidomide, and mono Clonal antibodies are rituximab, ofatumab, or lumiliximab, and chemotherapy The drugs are fludarabine or cyclophosphamide.

[0111] The compositions of this disclosure may be provided in unit dosage forms, where each dosage unit, for example, an injectable preparation, The composition comprises a predetermined amount of the composition alone or in appropriate combination with other active substances. The term "unit dosage form" as used herein refers to a single dosage form for human and animal subjects. This refers to physically discontinuous units suitable as dosage units, where each unit has the desired effect. A predetermined amount of the composition of the present invention, calculated as a sufficient amount for the purpose, is used alone or with other active ingredients. Combined with the active substance, a pharmaceutically acceptable diluent, carrier, or It is included together with the vehicle. The specifications for the novel unit dosage form of the present invention are for specific subjects. It depends on the specific pharmacodynamics related to the pharmaceutical composition.

[0112] Ideally, a long-term specific antitumor response is established, resulting in a reduction in tumor size, or Or, in order to prevent tumor growth or regrowth that may occur if such treatment is not performed, An effective amount or sufficient number of isolated and introduced immune cells are present in the composition, and the subject It is introduced into the body. Preferably, the amount of immune cells that are reintroduced into the subject is otherwise the same. Compared to the condition (absence of immune cells), tumor size was reduced by 10%, 20%, 30%, and 40%. Reduce by 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 a sufficient number The amount should be such that immune cells are introduced to achieve the desired therapeutic response. The amount of each active substance included in the composition described herein (for example, The amount per cell touched or per a certain body weight may vary depending on the application. Generally, the concentration of immune cells is preferably at least approximately in the treated subject. 1 x 10 6 ~Approx. 1×10 9 Immune cells, and more preferably, about 1 × 10⁻⁶ 7 ~Approx. 5×10 8It should be sufficient to provide immune cells, but not in excess, for example, 5 × 10 8 An amount exceeding or falling below the number of cells, for example, 1 × 10⁻⁶ 7 A quantity less than a cell, Any suitable dose can be used. The administration schedule is based on established cell-based therapies. (e.g., Topalian and Rosenberg, 1987; U.S. Patent) (See Issue No. 4,690,915) or using an alternative sustained infusion strategy. It is also possible to do so.

[0114] These values ​​are used by practitioners when optimizing the method of the present invention to carry out the present invention. This provides general guidance on the range of immune cells. Details of such ranges described herein are Never use more or less of a component than what may be guaranteed for a particular application. Do not exclude. For example, the exact dosage and schedule may vary depending on the composition and other pharmaceutical compositions. Depending on whether they are administered together or not, or depending on individual differences in pharmacokinetics, drug disposition, and metabolism Depending on the circumstances, it may vary. A person skilled in the art can make any necessary adjustments in response to an emergency in a particular situation. It can be done using the I Ching. VI. Kit

[0115] In some embodiments, for example, to produce UAPCs and / or immune cells A kit is provided which may contain one or more culture media and components. Such a formulation is 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 in an aqueous medium or in a lyophilized form, as needed. The container means for the kit generally includes a container in which the components can be arranged, preferably one that is appropriately divided. Note: At least one vial, test tube, flask, bottle, syringe or other Examples of container means include: If a kit has two or more components, the kit is generally This may also include a second, third, or further container in which additional components can be placed separately. However, various combinations of components may be included in a single vial. The components of the kit may be supplied as dry powders. Reagents and / or components may be dry When provided as a dry powder, the powder is reconstituted by adding a suitable solvent. The solvent may be provided in a separate container. The kit is Typically, this involves tightly sealing the components of a kit for commercial sale. Steps may also be included. Such a container may be injection molded or in which the desired vial is held. Examples include blow-molded plastic containers. The kit includes instructions for use (e.g., For example, this may include printed or electronic formats (e.g., digital format). [Examples]

[0116] VII. Examples The following examples are included to illustrate preferred embodiments of the present invention. The disclosed methods are those that the inventors have found to work well in carrying out the present invention. Therefore, these methods can be considered to constitute a preferred form for implementation. This should be recognized by those skilled in the art. However, those skilled in the art will see this disclosure In the specific embodiments disclosed, many things can be done without departing from the spirit and scope of the present invention. It should be recognized that changes may be made, and that similar or comparable results can still be obtained.

[0117] Example 1 - Combination of mbIL21, 4-1BBL, and SLAM (CD48 / CS1) Wase Along with either CD48-Katushka or CS1-EGFP, membrane-bound I Retroviral constructs against L-21 and 41BBL (CD137 ligand) are K5 Universal antigen-presenting cells (UAPCs) were created by transduction into 62 cells. The maps and annotations of the MMLV retrovirus constructs are shown in Figures 4A-4D. NK cell sensitive K562 (HLA-A - HLA-B - ) In APC (Figure 3A) Clone 46 (Figure 3B) was generated from the forced expression of mbIL-21 and 41BBL. By forcing the expression of mbIL-1, 41BBL, and CD48 in K562 cells, U APC was produced (Figure 3C). mbIL-21, 41BBL and UAPC2 was synthesized by forcibly expressing CS1 (Figure 3D).

[0118] Numerical expansion of NK cells is achieved by activation using clone 46, UAPC, or UAPC2. The proliferation and expansion of NK cells by APC co-culture was previously achieved with clone 46. Compared to the published clone 9 (Figure 2B), the transduced NK cells (NT-NK) ) excels at expanding both CAR-transduced NK cells (SG4-NK). It showed that they were there.

[0119] The expansion of NK cells through co-culture with UAPC was previously reported as clone 9 (33 hours). Compared to the doubling time of NK cells, it has superior ability to shorten the doubling time of NK cells to 31.38 hours. This was shown to be beneficial, resulting in a growth rate of 44% in two weeks (in the case of UAPC, (1671x magnification for clone 9, compared to 1161x magnification) (Figure 2E). The relationship between the interval, magnification, and initial cell population can be simplified by the following equation:

number

[0120] In the formula, N f This is the final cell number, N b This is the initial cell, and e=2.718 28 (a mathematical constant also known as Euler's number), where T is the incubation time in units of hours. It is a range, T D This is the doubling time, measured in hours. The doubling time is the final number of cells. Because it has a numerical effect, it further enhances the proliferative advantage over the specified cell culture period. This allows for the collection of a clinically reasonable number of cells within a compressed timeframe. Therefore, this method enables the efficient production of NK cells through co-culture with UAPC.

[0121] This platform technology utilizes specific therapeutic combination pathways to advance cancer immunotherapy. The objective was to produce a large number of clinical-grade, highly functional, and cytotoxic NK cells. This provides a practical method for this purpose. The enlarged cells are 100% NK cells, and T cells are detected. Because it is impossible to obtain, unintended graft pairs are common in many immunotherapy preparations. Host side effects are avoided.

[0122] All methods disclosed and claimed herein are, in view of this disclosure, excessive. The present invention can be carried out and implemented without conducting experiments. The compositions and methods of the present invention are preferred Although embodiments have been described, this does not deviate from the concept, spirit, and scope of the present invention. Changes may be made to the methods and steps or sequence of steps described herein. This will be obvious to those skilled in the art. More specifically, certain chemical and physiologically related special Certain active substances may be used in place of the active substances described herein, but the same It will be clear that similar results can be achieved. All substitutions and modifications are in line with the spirit, scope, and nature of the invention as defined by the attached claims. It is considered not to go beyond the concept.

[0123] The following references provide exemplary procedures or other details that supplement those described herein. To the extent that it provides, it is 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. A composition for expanding immune cells by intercellular contact culture for use in cell therapy or immunotherapy, comprising modified cells, The modified cells are leukemia cells, cancer cells, macrophages, B cells, endothelial cells, activated T cells, or dendritic cells that are stably transformed and manipulated to express (1) CD48 and / or CS1 (CD319), (2) membrane-bound interleukin-21 (mbIL-21), and (3) 41BB ligand (41BBL) on their cell surface. The composition comprises immune cells including NK cells.

2. The composition according to claim 1, wherein the modified cells express CD48.

3. The composition according to claim 1, wherein the modified cells express CS1.

4. The composition according to claim 1, wherein the modified cells express CD48 and CS1.

5. The composition according to claim 1, wherein less than 0.05% of the modified cells express an endogenous HLA class I, II, or CD1d molecule.

6. The composition according to claim 1, wherein the modified cells express ICAM-1 (CD54) and LFA-3 (CD58).

7. The composition according to claim 1, wherein the leukemia cells are further defined as K562 cells.

8. The composition according to claim 1, wherein the modified cells are operated by a nonviral delivery method.

9. The composition according to claim 1, wherein the modified cells are manipulated by the introduction of a virus.

10. The composition according to claim 1, wherein the modified cells are manipulated by retrovirus introduction.

11. The composition according to claim 10, wherein the retrovirus introduction is further defined as an introduction using the viral construct of sequence number 1 and / or sequence number 2 in the sequence listing.

12. The composition according to claim 1, wherein the modified cells are irradiated with radiation.

13. The composition according to claim 1, wherein the modified cells are irradiated with gamma rays.

14. Furthermore, the composition according to any one of claims 1 to 13, further comprising IL-2.

15. The composition according to claim 14, wherein IL-2 is present at a concentration of 10 to 500 U / mL.

16. Furthermore, the composition according to any one of claims 1 to 15, further comprising NK cells.

17. The composition according to any one of claims 1 to 16, wherein the NK cells are manipulated to express a chimeric antigen receptor (CAR).

18. The composition according to any one of claims 1 to 17, wherein the NK cells are derived from umbilical cord blood.

19. A method for expanding immune cells, The method involves culturing immune cells in the presence of an effective amount of leukemia cells, cancer cells, macrophages, B cells, endothelial cells, activated T cells, or dendritic cells that are stably transformed and express (1) CD48 and / or CS1 (CD319), (2) membrane-bound interleukin-21 (mbIL-21), and (3) 41BB ligand (41BBL) on their cell surface. A method wherein the immune cells include NK cells.

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