Natural killer cells with enhanced anticancer activity and immunotherapeutic use thereof

Abstract

To provide a natural killer cell having increased anticancer activity, and to provide an immunotherapeutic use thereof.SOLUTION: (a) the relative MFI value of NKG2D is increased by 1.2 times to 12 times, (b) the relative MFI value of NKp30 is increased by 1.5 times to 15 times, (c) the relative MFI value of NKp44 is increased by 12 times to 22 times, (d) the relative MFI value of ITGA1 is increased by 1.8 times to 25 times, and (e) the relative MFI value of ITGA2 is increased by 1.5 times to 25 times, compared to those on day 0 of the culture of PBMCs; Wherein the relative MFI is defined by receptor MFI / isotype MFI, wherein the MFI is increased between 1.4 fold and 6 fold.SELECTED DRAWING: Figure 9

Description

[Technical Field]

[0001] The present invention relates to natural killer cells with enhanced anti-cancer activity and their immunotherapeutic use. [Background technology]

[0002] Natural killer cells (NK cells), which are used in immune cell therapy, are morphologically cells with large granules in their cytoplasm and account for approximately 5-15% of blood lymphocytes. The main functions of NK cells that have been elucidated so far include the ability to kill tumor cells, cytotoxicity against virus-infected cells, and the ability to kill bacteria and fungi. Therefore, NK cells are expected to play important roles in anti-tumor immunity and protective immunity against microorganisms.

[0003] Natural killer cells (NKCs) possess surface receptors as immune receptors. Because NKCs lack dominant receptors, such as the BCR of B cells or the TCR (T cell receptor) of T cells, they are predicted to have a distinct activation mechanism. Furthermore, NKCs are known to have the ability to nonspecifically kill cancer cells. Their killing ability, along with lymphokine-activated killer cells (LAKs) and tumor infiltrating lymphocytes (TILs), is utilized in the treatment of solid tumors. Furthermore, donor lymphocyte infusion-mediated immunotherapy (Tilden, AB et al., J. Immunol., 136) has been applied to novel cell therapy to prevent rejection during bone marrow or organ transplants.

[0004] Meanwhile, glioma accounts for 60% of primary brain tumors and is a malignant tumor for which there is currently no specific treatment other than radiation therapy. Furthermore, glioblastoma, classified as a malignant tumor, is highly resistant to radiation and anticancer drug treatment compared to other cancers, and once diagnosed, the expected survival time is only one year. Malignant glioblastoma accounts for 12-15% of all brain tumors and is the most common single tumor that occurs in the brain.

[0005] Despite existing treatment options for cancer patients, including glioblastoma, therapies that can improve efficacy and overcome self-tolerance are being investigated, necessitating the development of natural killer cells with increased activity of specific immune receptors. Summary of the Invention [Problem to be solved by the invention]

[0006] One embodiment provides natural killer cells with enhanced anti-cancer activity that have receptor expression characteristics with a specific range of relative MFI (mean fluorescence intensity) values.

[0007] Another aspect provides a cell therapy agent or pharmaceutical composition comprising the natural killer cells or a population thereof as an active ingredient.

[0008] Yet another embodiment provides a method of treating cancer comprising administering to an individual in need thereof an effective amount of the natural killer cells or population thereof. [Means for solving the problem]

[0009] One embodiment provides natural killer cells with enhanced anti-cancer activity that have receptor expression characteristics with a specific range of relative MFI (mean fluorescence intensity) values.

[0010] As used herein, the term "natural killer cells" or "NK cells" refers to cytotoxic lymphocytes that constitute a major component of the innate immune system. They are defined as large granular lymphocytes (LGLs) and constitute a third cell type differentiated from B lymphocytes and T lymphocytes produced by common lymphoid progenitors (CLPs). The term "natural killer cells" or "NK cells" includes natural killer cells derived from any tissue source without further transformation, and may include not only mature natural killer cells but also natural killer precursor cells. Natural killer cells are activated in response to interferon or macrophage-derived cytokines, and contain two types of surface receptors, labeled "activating receptors" and "inhibitory receptors," that control the cytotoxic activity of the cells. Natural killer cells can also be generated from hematopoietic cells from any source, such as placental tissue, placental permeate, umbilical cord blood, placental blood, peripheral blood, spleen, liver, etc., such as hematopoietic stem cells or hematopoietic progenitor cells, placenta- or umbilical cord-derived stem cells, induced pluripotent stem cells, or cells differentiated therefrom.

[0011] The natural killer cells can also be derived from peripheral blood mononuclear cells, for example. The term "peripheral blood mononuclear cells (PBMCs)" refers to mononuclear cells isolated from mammalian, preferably human, peripheral blood, and includes immune cells such as B cells, T cells, and natural killer cells; and granulocytes such as basophils, eosinophils, and neutrophils. The peripheral blood mononuclear cells (PBMCs) can be prepared from peripheral blood collected from a living body by a conventional manufacturing method. Preferably, the PBMCs can be isolated from peripheral blood by specific gravity centrifugation using Ficoll.

[0012] Natural killer cells can also be obtained from the products of leukapheresis, a method of removing red blood cells, by separating white blood cells from blood.

[0013] In one embodiment, the natural killer cells herein also have one or more properties selected from the following (a) through (e): (a) The relative MFI value of NKG2D increased by 1.2-fold to 12-fold, 2-fold to 12-fold, 4-fold to 12-fold, 2-fold to 10-fold, 2-fold to 8-fold, 4-fold to 8-fold, 3-fold to 6-fold, or 3.5-fold to 4.5-fold compared to day 0 of PBMC culture; (b) the relative MFI value of NKp30 increased by 1.5-fold to 15-fold, 1.5-fold to 12-fold, 2-fold to 12-fold, 4-fold to 10-fold, 2-fold to 10-fold, 4-fold to 8-fold, 3-fold to 6-fold, or 4-fold to 6-fold compared to day 0 of PBMC culture; (c) a 12-fold to 22-fold, 12-fold to 20-fold, 14-fold to 22-fold, 16-fold to 22-fold, 16-fold to 20-fold, 12-fold to 18-fold, 14-fold to 18-fold, or 16-fold to 18-fold increase in the relative MFI value of NKp44 on day 14 of culture compared to day 0 of PBMC culture; (d) the relative MFI value of ITGA1 increased by 1.8 to 25 times, 2 to 25 times, 4 to 22 times, 4 to 18 times, 6 to 16 times, 6 to 10 times, or 6 to 8.5 times compared to day 0 of PBMC culture; and (e) The relative MFI value of ITGA2 is increased by 1.4 to 6 times, 1.8 to 6 times, 1.8 to 5 times, 2 to 5.5 times, 2 to 5 times, 2 to 4 times, or 2.5 to 3.5 times compared to day 0 of PBMC culture.

[0014] The relative MFI values ​​are also relative MFI values ​​on day 14 of PBMC culture compared to day 0 of culture.

[0015] As used herein, the term relative MFI refers to the expression intensity value of positive cells relative to the isotype, and is defined by the following Equation 1:

[0016]

number

[0017] The relative MFI is a different concept from the expression ratio, which measures the expression ratio of positive cells compared to isotype. Even if the expression ratio is the same percentage, the strength of each receptor function varies depending on the MFI value, and it is understood that only a high relative MFI value indicates an actual increase in function.

[0018] The present specification provides novel natural killer cells that have been treated with a novel substance, such as PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD, or PDGF-AB, during natural killer cell culture from PBMCs. The novel natural killer cells are produced by a method different from other known natural killer cells, and the relative MFI values ​​of NKG2D, NKp30, NKp44, ITGA1, and ITGA2, which are factors involved in anti-cancer activity and natural killer cell activation, are increased by at least 1.2-fold and up to 30-fold compared to before PBMC culture.

[0019] In other embodiments, the natural killer cells herein are selected from the group consisting of CD16 with an MFI value of 8 to 140, 10 to 140, 8 to 40, 15 to 30, 80 to 140, 100 to 140, or 110 to 130; LFA-1 with an MFI value of 20 to 160, 30 to 160, 30 to 150, 20 to 60, 25 to 50, 120 to 160, or 135 to 150; NKG2D with an MFI of 2 to 25, 5 to 25, 5 to 20, 2 to 14, 7 to 20, or 10 to 18; NKp30 with an MFI of 10 to 40, 12 to 30, 12 to 25, 14 to 22, 12 to 18, or 18 to 22; ITGA1 with an MFI of 2 to 30, 4 to 25, 4 to 22, 2 to 16, 4 to 12, 10 to 25, or 14 to 25; ITGA2 with an MFI of 1.5 to 10, 2 to 10, 1.6 to 4, 2 to 8, 4 to 8, or 4 to 6; CD2 with an MFI of 20 to 180; CD27 with an MFI of 0.1 to 1.5; CD69 with an MFI of 1 to 10; CD226 with an MFI of 2 to 12; NKp46 with an MFI of 2 to 8; CD160 with an MFI of 0.1 to 4; KIR2DL1 with an MFI of 0.1 to 4; KR2DL3 with an MFI of 0.1 to 5; and ITGB7 with an MFI value of 1 to 16.

[0020] In other embodiments, the natural killer cells further have the following characteristic (f): (f) The expression level of the KIR2DS4 gene on day 14 of PBMC culture is at least 10 times higher, specifically 10 to 60 times, 10 to 50 times, 20 to 40 times, 20 to 45 times, or 25 to 40 times higher, compared to day 0 of culture.

[0021] In other embodiments, the natural killer cells further have the following characteristic (g) or (h): (g) a 0.02-fold to 0.85-fold, 0.04-fold to 0.8-fold, 0.08-fold to 0.8-fold, 0.1-fold to 0.6-fold, 0.1-fold to 0.4-fold, or 0.12-fold to 0.3-fold decrease in the relative MFI value of CD16 on day 14 of culture compared to day 0 of culture of PBMCs; or (h) The relative MFI value of LFA-1 was reduced by 0.08 to 0.8 times, 0.1 to 0.8 times, 0.1 to 0.7 times, 0.1 to 0.6 times, 0.2 to 0.6 times, 0.4 to 0.6 times, or 0.2 to 0.5 times on day 14 of PBMC culture compared to day 0 of culture.

[0022] In another embodiment, the natural killer cells also express any one receptor selected from NKG2D, NKp30, NKp44, CD16, LFA-1, ITGA1, ITGA2, KIR2DS1, KIR2DS2, KIR2DS3, KIRDS4, CXCR1, CXCR2, CXCR3, CCR3, CCR5, CCR6, PSA-NCAM, nestin, tyrosine hydroxylase, CD147, CD127, CD15, CD31, CD146, CD49c, CD107a, NKG2A, CD45, CD140a, and CD11b.

[0023] The activating receptors of natural killer cells primarily recognize ligands whose expression increases when target cells are in an abnormal state, and cause cytotoxicity to eliminate the target cells.

[0024] PSA-NCAM is a marker of neural cell differentiation and a factor involved in neuronal development and synapse formation in the nervous system during embryonic development. Tyrosine hydroxylase is an enzyme required for the synthesis of neurotransmitter hormones. CD147 is a factor involved in embryonic brain development and has an integrin-mediated adhesion function in the brain endothelium. S100B also enhances blood-brain barrier permeability. CD15 plays a role in chemotaxis, phagocytosis, and / or bactericidal activity. CD31 binds to CD38 and is involved in wound healing, angiogenesis, and cell migration. CD146 is a cell surface marker expressed on activated T cells, mesenchymal stem cells, etc., and is involved in the extravasation of leukocytes. CD49c is involved in neural migration and plays a role in cell-to-cell and cell-to-substrate adhesion.

[0025] NKG2D senses UL16-binding proteins (ULBPs), MIC A / B, RAE1, H60, and MULT1, which are intracellular molecules whose expression is increased during DNA damage, carcinogenesis, and viral infection, and provides cytotoxic activity.

[0026] NKp30 is a receptor activated by the binding of extracellular ligands, including BAG6, NCR3LG1, and B7-H6, and stimulates cytotoxicity upon binding to these ligands.

[0027] NKp44 recognizes ligands such as cell surface glycoproteins and proteoglycans, nuclear proteins that may be exposed to the cell exterior, and molecules that are released into the extracellular space or transported in extracellular vesicles. Recently, NKp44 has been reported to recognize extracellular matrix (ECM)-derived glycoproteins and soluble plasma proteins such as growth factors (e.g., PDGF-DD) (Cell. 2018 January 25; 172(3): 534-548. e19., Front Immunol. 2019; 10: 719).

[0028] KIR2DS4 is involved in numerous diseases, including cancer, pregnancy disorders, and resistance to HIV. Although the exact ligand has not been defined, it recognizes peptides presented by HLA-C*05:01 (e.g., recombinant peptide:HLA-C complexes) and activates NK cells, which then produce TNF-alpha and IFN-gamma and degranulate. Therefore, KIR2DS4 is a highly peptide-specific activating receptor and plays a significant role in immune defense (J Immunol May 1, 2019, 202 (1 Supplement) 177.24).

[0029] ITGA1 is a receptor for laminin and collagen, and is involved in cell-cell adhesion. It recognizes a partial sequence of collagen and is involved in negative regulation of EGF-stimulated cell growth.

[0030] ITGA2 is a receptor for laminin, collagen, collagen C-propeptide, fibronectin, and E-cadherin, and is involved in adhesion to platelets and other cells, collagen regulation, and organization of the synthesized extracellular matrix.

[0031] The natural killer cells may comprise at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or about 99%, or 50% to 100%, 50% to 90%, 60% to 90%, 60% to 80%, or 60% to 70% of the cell population expressing one of NKG2D, NKp30, NKp44, CD16, LFA-1, ITGA1, ITG A2, KIR2DS1, KIR2DS2, KIR2DS3, KIRDS4, CXCR1, CXCR2, CXCR3, CCR3, CCR5, CCR6, PSA-NCAM, nestin, tyrosine hydroxylase, CD147, CD127, CD15, CD31, CD146, CD49c, CD107a, NKG2A, CD45, CD140a, or CD11b.

[0032] The natural killer cells may also be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or about 99% of the cell population, or 50% to 100%, 50% to 90%, 60% to 90%, 60% to 80%, or 60% to 70% of the cell population expressing KIR2DS1. + , KIR2DS2 + , KIR2DS3 + , KIR2DS4 + , CXCR1 + , CXCR2 + , CXCR3 + , CCR3 + , CCR5 + , CCR6 + , PSA-NCAM + , Nestin + , CD127 + , CD15 + , CD31 + , CD146 + , CD49c + , CD107a + , NKG2A + , CD45 + , CD140a + and CD11b + It also indicates any one characteristic selected from the above.

[0033] Also, natural killer cells may be present in a population of cells in which at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or about 99%, or 50% to 100%, 50% to 90%, 60% to 90%, or 60% to 80% of the cell population is CD87 - , CD10 - and CD80 - It also indicates any one characteristic selected from the group consisting of:

[0034] As used herein, the term "positive or +" can refer to a cellular marker and can mean that the marker is present in a greater amount or at a higher concentration than other cells in a reference population. That is, a cell is positive for a marker if the marker is present inside or on the cell, allowing the cell to be distinguished from one or more other cell types using the marker. It can also mean that the cell has enough of the marker to produce a signal, e.g., a signal from a cell measuring device, that is greater than background. For example, cells can be detectably labeled with an antibody specific for NKp44, and if the signal from the antibody is detectably greater than a control (e.g., background), the cell is said to be "NKp44 positive." + As used herein, the term "negative or -" means that an antibody specific for a particular cell surface marker cannot be used to detect that marker compared to background values. For example, CD87 - If the cells cannot be detectably labeled with antibodies specific for "CD87", then the cells are "CD87 - "

[0035] In one embodiment, the natural killer cells may refer to cells in which the cytotoxicity or innate immunoregulatory ability of natural killer cells is activated or the expression of the aforementioned immune receptors is increased compared to parent cells, such as hematopoietic cells or natural killer progenitor cells. In a specific embodiment, the natural killer cells are CD3-CD56+. In a specific embodiment, the activated natural killer cells are CD3-CD56+CD16+. In another specific embodiment, the activated natural killer cells are further CD94+CD117+. In another specific embodiment, the activated natural killer cells are further CD161-. In another specific embodiment, the activated natural killer cells are further NKG2D+. In another specific embodiment, the activated natural killer cells are further NKp46+. In another specific embodiment, the activated natural killer cells are further CD226+. In specific embodiments, greater than 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, or 98% of the activated natural killer cells are CD56+ and CD16-. In other embodiments, at least 50%, 60%, 70%, 80%, 82%, 84%, 86%, 88%, or 90% of the activated natural killer cells are CD3- and CD56+. In other embodiments, at least 50%, 52%, 54%, 56%, 58%, or 60% of the activated natural killer cells are NKG2D+. In other embodiments, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3% of the cells are NKB1+. In certain other embodiments, less than 30%, 20%, 10%, 8%, 6%, 4%, or 2% of the activated natural killer cells are NKAT2+. In more specific embodiments, at least 10%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the CD3-,CD56+ activated natural killer cells are NKp46+.In other more specific embodiments, at least 10%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the CD3-, CD56+ activated natural killer cells are CD117+. In other more specific embodiments, at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the CD3-, CD56+ activated natural killer cells are CD94+. In other more specific embodiments, at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the CD3-, CD56+ activated natural killer cells are CD161-. In other more specific embodiments, at least 10%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 95% of the CD3-, CD56+ activated natural killer cells are CD226+. In other more specific embodiments, at least 20%, 25%, 30%, 35%, or 40% of the CD3-, CD56+ activated natural killer cells are CD7+. In even more specific embodiments, at least 30%, 35%, 40%, 45%, 50%, 55%, or 60% of the CD3-, CD56+ activated natural killer cells are CD5+.

[0036] In one embodiment, activated natural killer cells, or an enriched population of activated natural killer cells, are also assessed by detecting one or more functionally related markers, such as CD94, CD161, DNAM-1, 2B4, NKp46, KIR, and the NKG2 family of activating receptors (e.g., NKG2D).

[0037] In one embodiment, activated natural killer cells are also generated from the aforementioned hematopoietic cells. In a specific embodiment, activated natural killer cells are also obtained from expanded hematopoietic cells, e.g., hematopoietic stem cells and / or hematopoietic progenitor cells. In a specific embodiment, the hematopoietic cells are continuously expanded and differentiated in a first medium without the use of nutrient cells. The cells are then cultured in a second medium in the presence of nutrient cells. Such isolation, expansion, and differentiation are performed at a central facility, which provides expanded hematopoietic cells for expansion and differentiation at the point of use, e.g., a hospital.

[0038] In one embodiment, generating activated natural killer cells includes expanding a population of hematopoietic cells, during which a plurality of hematopoietic cells within the hematopoietic cell population differentiate into natural killer cells.

[0039] As used herein, the terms "natural killer progenitor cells" or "NK progenitor cells," or cell populations thereof, can refer to cells or populations thereof that include cells of the natural killer cell lineage that have not yet developed into mature natural killer cells, as indicated, for example, by the expression levels of one or more phenotypic markers, e.g., CD56, CD16, and KIR. In one embodiment, the natural killer progenitor cell population comprises cells with low CD16 and high CD56. For example, the natural killer progenitor cell population comprises approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD3-CD56+ cells. In another specific embodiment, the natural killer progenitor cell population comprises no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD3-CD56+ cells. In other specific embodiments, the natural killer progenitor cell population comprises 0% to 5%, 5% to 10%, 10% to 15%, 15% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, or 45% to 50% CD3-CD56+ cells.

[0040] In one embodiment, the CD3-CD56+ cells in the natural killer progenitor cell population are further CD117+. In a specific embodiment, about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the CD3-CD56+ cells in the natural killer progenitor cell population are CD117+. In another specific embodiment, more than 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the CD3-CD56+ cells in the natural killer progenitor cell population are CD117+. In other specific embodiments, in the natural killer progenitor cell population, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99% of the CD3-CD56+ cells are CD117+.

[0041] In another embodiment, the CD3-CD56+ cells in the natural killer progenitor cell population are further CD161+. In a specific embodiment, about 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the CD3-CD56+ cells in the natural killer progenitor cell population are CD161+. In another specific embodiment, more than 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the CD3-CD56+ cells in the natural killer progenitor cell population are CD161+. In other specific embodiments, in the natural killer progenitor cell population, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, or 70% to 75% of the CD3-CD56+ cells are CD161+.

[0042] In yet another embodiment, the CD3-CD56+ cells in the natural killer progenitor cell population are further NKp46+. In a specific embodiment, about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more of the CD3-CD56+ cells in the natural killer progenitor cell population are NKp46+. In another specific embodiment, about 25%, 30%, 35%, 40%, 45%, 50%, or 55% of the CD3-CD56+ cells in the natural killer progenitor cell population are NKp46+. In other specific embodiments, 25%, 30%, 35%, 40%, 45%, 50%, or 55% or less of the CD3-CD56+ cells in the natural killer progenitor cell population are NKp46+. In other specific embodiments, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90% or more of the CD3-CD56+ cells in the natural killer progenitor cell population are NKp46+. In more specific embodiments, in the natural killer progenitor cell population, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, or 50% to 55% of the CD3-CD56+ cells are NKp46+.

[0043] Furthermore, for example, the natural killer progenitor cell population is CD52+, CD16+, CD244+CD94+, or CD94+, as described above.

[0044] As used herein, the natural killer cells are those that have been cultured or genetically engineered to express the aforementioned receptors or to have their expression or activity increased.

[0045] As used herein, the term "culture" may refer to hematopoietic cells, such as hematopoietic stem cells or hematopoietic progenitor cells, cultured from any source, such as placental tissue, placental permeate, umbilical cord blood, placental blood, peripheral blood, spleen, liver, etc., so as to increase the expression or activity of the receptors described above.

[0046] As used herein, "genetic engineering" or "genetically engineered" refers to the act of introducing one or more genetic modifications into a cell, or the cell produced thereby.

[0047] As used herein, the term "increased activity" or "increased activity" can refer to a detectable increase in the activity of a protein or enzyme. "Increased activity" or "increased activity" refers to a higher level of protein or enzyme activity compared to a given parent cell, wild-type cell, or pre-cultured cell (e.g., PBMC).

[0048] In another embodiment, the natural killer cells are genetically modified or engineered. The natural killer cells are genetically modified to enhance targeting and / or homing specificity.

[0049] In other embodiments, the natural killer cells also secrete perforin, granzymes, or interferons.

[0050] The granzyme may be one or more selected from the group consisting of granzyme A, granzyme B, granzyme H, granzyme K, and granzyme M.

[0051] The interferon may be a type 1 interferon (eg, interferon α, interferon β, interferon κ, interferon ω), a type 2 interferon (eg, interferon γ) or a type 3 interferon.

[0052] In another aspect, there is provided a cell therapy agent or pharmaceutical composition comprising the immune cells or cell populations thereof as an active ingredient.

[0053] The cell therapy agent or the pharmaceutical composition may also be used for the prevention or treatment of cancer or infectious diseases.

[0054] Yet another aspect provides a use of the natural killer cells or cell populations thereof for the manufacture of a medicine.

[0055] Yet another aspect provides a method of treating a disease comprising administering the natural killer cells or cell populations thereof to an individual.

[0056] As used herein, the term "disease" may refer to a pathological condition, in particular cancer, infectious disease, inflammatory disease, metabolic disease, autoimmune disorder, degenerative disease, cell death-related disease and graft rejection.

[0057] As used herein, the term "treatment" refers to or includes the alleviation, slowing down of progression, or prevention of a disease, disorder, or condition, or one or more symptoms thereof, and "active ingredient" or "pharmaceutically effective amount" can refer to any amount of a composition utilized in the course of practicing the inventions provided herein sufficient to alleviate, slow down or prevent a disease, disorder, or condition, or one or more symptoms thereof.

[0058] As used herein, the terms "administer," "introduce," and "implant" are used interchangeably and may refer to the placement of a composition according to an embodiment into an individual by a method or route that results in at least partial localization of the composition according to an embodiment at a desired site. Administration may also be by any suitable route that delivers at least a portion of the cells or cellular components of a composition according to an embodiment to a desired location within a living individual. After administration to an individual, the survival period of the cells may be as short as a few hours, e.g., 24 hours to several days, or as long as several years.

[0059] The administration may be in combination with an additional anti-cancer agent. Examples of the additional anti-cancer agent may include alkylating agents, antimetabolites, spindle inhibitors, plant alkaloids, cytotoxic / anti-tumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Examples of the anti-cancer agent may include compounds used in targeted therapy and conventional chemotherapy. Further, examples of the antibody include alemtuzumab, apolizumab, aselizumab, atlizumab, bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidopcitusumab, cidutuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, eculizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motavizumab, natalizumab, and nimotuzumab. , norobizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resivizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, trastuzumab, tucotuzumab celmoleukin, tuxituzumab, umavizumab, urtoxazumab, and visilizumab.

[0060] As used herein, the term "isolated cells," eg, "isolated natural killer cells," refers to cells that have been substantially separated from the tissue from which they originate, eg, peripheral blood.

[0061] The compositions of the present invention can be used to treat or prevent tumors or cancers derived from neoplasms. The neoplasms can be malignant or positive, the cancers can be primary or metastatic, and the neoplasms or cancers can be early or late stage. Non-limiting examples of neoplasms or cancers that can be cured include one or more selected from the group consisting of lung cancer, laryngeal cancer, gastric cancer, colon cancer, rectal cancer, liver cancer, gallbladder cancer, pancreatic cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, prostate cancer, kidney cancer, skin cancer, bone cancer, muscle cancer, fat cancer, fibrocytic cancer, blood cancer, leukemia, lymphoma, multiple myeloma, and glioma.

[0062] The glioma may also be an astrocytic tumor, an oligodendroglial tumor, a mixed glioma, or an ependymal tumor. More particularly, the astrocytoma may be a glioblastoma, an anticancer drug-resistant glioblastoma, or a recurrent glioblastoma.

[0063] Without being limited to a particular theory, the interaction between platelet-derived growth factor-DD (PDGF-DD) secreted by glioblastoma and NKp44 on natural killer cells has been reported. Also, without being limited to a particular theory, the interaction between MHC class I polypeptide-related sequence A (MICA) expressed in glioblastoma and NKG2D on natural killer cells has been reported. Also, without being limited to a particular theory, the association between KIR2DS2-positive natural killer cells and glioblastoma has been reported.

[0064] Therefore, natural killer cells according to one embodiment are also effective against cancers that secrete or express MICA or PDFG-DD (eg, glioma, glioblastoma, etc.).

[0065] Furthermore, natural killer cells according to one embodiment can suppress self-tolerance. Thus, natural killer cells according to one embodiment can also suppress immune activity, blood-brain barrier penetration, cell migration promotion, or self-tolerance.

[0066] The method of administration of an embodiment of a pharmaceutical composition is not particularly limited, and may be parenteral or oral, such as intravenous, subcutaneous, intraperitoneal, inhalation, or topical administration, depending on the intended method. The dosage range varies depending on the patient's weight, age, sex, and health condition, diet, administration time, administration method, excretion rate, and disease severity. A daily dosage refers to the amount of a therapeutic substance in one embodiment sufficient to treat a disease state alleviated by administration to an individual in need of treatment. The effective amount of a therapeutic substance varies depending on the specific compound, the disease state and its severity, and the individual in need of treatment, and can be generally determined by one skilled in the art. As a non-limiting example, the dosage of a composition in one embodiment administered to a human body may vary depending on the patient's age, weight, sex, administration method, health condition, and disease severity. For example, based on an adult patient weighing 70 kg, the dose is about 1,000 to 10,000 cells / injection, 1,000 to 100,000 cells / injection, 1,000 to 1,000,000 cells / injection, 1,000 to 10,000,000 cells / injection, 1,000 to 100,000,000 cells / injection, 1,000 to 1,000,000,000 cells / injection, 1,000 to 10,000,000,000 cells / injection, or 1,000 to 100,000,000,000 cells / injection, and can be administered once or several times a day at regular intervals.

[0067] "Individual" means a subject in need of treatment for a disease, and more specifically means a mammal, such as a human or non-human primate, mouse, rat, dog, cat, horse, or cow.

[0068] The pharmaceutical composition according to one embodiment may contain pharmaceutically acceptable carriers and / or additives, such as sterile water, physiological saline, conventional buffers (such as phosphate, citric acid, and other organic acids), stabilizers, salts, antioxidants (such as ascorbic acid), surfactants, suspending agents, isotonicity agents, or preservatives. For local administration, the composition may also contain organic materials such as biopolymers, inorganic materials such as hydroxyapatite, specifically, collagen matrices, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers, and chemical derivatives thereof. When the pharmaceutical composition according to one embodiment is formulated into a dosage form suitable for injection, the immune cells or the substance that enhances their activity may be dissolved in a pharmaceutically acceptable carrier or frozen in a dissolved solution.

[0069] Depending on the administration method and dosage form, the pharmaceutical composition according to one embodiment may contain, as needed, suspending agents, solubilizers, stabilizers, isotonicity adjusting agents, preservatives, anti-adsorption agents, surfactants, diluents, excipients, pH adjusters, soothing agents, buffers, reducing agents, antioxidants, and the like. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995. The pharmaceutical composition according to one embodiment may be formulated using pharmaceutically acceptable carriers and / or excipients in a unit dose form or in a multi-dose container, using a method readily accessible to those skilled in the art. In this case, the dosage form may be in the form of a solution, suspension, or emulsion in an oily or aqueous medium, or in the form of a powder, granules, tablet, or capsule. [Effects of the Invention]

[0070] In one embodiment, natural killer cells have the effect of increasing the relative MFI of a significant specific receptor against cancers including glioblastoma, thereby enabling effective anti-cancer immunotherapy. [Brief explanation of the drawings]

[0071] [Figure 1A] 1 is a dot plot showing phenotypic analysis of natural killer cells according to culture period (CD3CD56). [Figure 1B] 1 is a dot plot showing phenotypic analysis of natural killer cells according to culture period (CD3CD19, CD14SSC). [Figure 1C] 1 is a dot plot showing phenotypic analysis of natural killer cells according to the culture period (CD3SSC, CD4CD8). [Figure 2A] Figure 1A shows a graph of the culture evaluation results for seven individuals (proportions of CD3+CD56+, CD3-CD56+, and CD3+CD56- cells). [Figure 2B] Figure 1B shows a graph of the culture evaluation results for seven patients (b: percentage of CD19+ cells). [Figure 2C] Figure 1B shows a graph of the culture evaluation results of seven patients (proportion of CD14+ cells). [Figure 2D] Figure 1C shows a graph of the culture evaluation results for seven patients (proportions of CD4+ and CD8+ cells). [Figure 2E] 1 is a graph showing cell viability and fold expansion of natural killer cells (percent expansion of natural killer cells). [Figure 2F] 1 is a graph showing cell viability and fold proliferation of natural killer cells (cell viability). [Figure 3] 1 shows dot plot results of changes in expression of immune receptors in natural killer cells and PBMCs before culture according to one embodiment. [Figure 4A] 1 is a graph showing changes in the expression of immune receptors of natural killer cells and PBMCs before culture according to one embodiment (NKG2D+, DNAM1+, CD69+, NKp30+). [Figure 4B] 1 is a graph showing changes in expression of immune receptors of natural killer cells and PBMCs before culture (NKp44+, NKp46+, CD2+, CD16+) according to one embodiment. [Figure 4C] 1 is a graph showing changes in expression of immune receptors in natural killer cells and PBMCs before culture according to one embodiment (KIR2DL1+, KIR2DL3+, KIRDL1+, NKG2A+). [Figure 5] 1 shows the results of a flow cytometer analysis using dot plots and graphs of the expression of natural killer cells and anti-cancer substances (granzyme B, perforin, and interferon gamma) in PBMCs before culture according to one embodiment. [Figure 6] 1 shows dot plots and graphs illustrating expression of natural killer cells and degranulation of CD107a in pre-culture PBMCs according to one embodiment. [Figure 7A] 1 shows a dot plot of ligand expression of natural killer cells on a glioblastoma cell line (T98G cells) according to one embodiment. [Figure 7B] 1 shows a dot plot of ligand expression of natural killer cells on a glioblastoma cell line (U-87MG cells) according to one embodiment. [Figure 7C] 1 shows the results of a dot plot of natural killer cell ligand expression on a glioblastoma cell line (A172 cells) according to one embodiment. [Figure 7D] 1 shows a dot plot of ligand expression of natural killer cells on a glioblastoma cell line (U-373MG cells) according to one embodiment. [Figure 8] 1 shows the results of cell-killing ability of natural killer cells against blood cancer cell line K562 at different E:T ratios according to one embodiment. [Figure 9] 1 shows the results of cell killing ability of natural killer cells against glioblastoma cell lines A172, U-87MG, U-373MG, and T98G at different E:T ratios according to one embodiment. [Figure 10]1 is a graph showing the cytotoxicity of natural killer cells against cancer cell lines after blocking specific receptors in the natural killer cells according to one embodiment. [Figure 11] 1 is a graph showing the observed decrease in tumor weight after natural killer cells according to one embodiment are administered to an animal model of ovarian cancer (arrows indicate the time of drug administration). [Figure 12] 1 is a graph showing the observed decrease in tumor weight after natural killer cells according to one embodiment are administered to an animal model of stomach cancer (arrows indicate the time of drug administration). [Figure 13] 1 is a graph showing the observation of tumor weight reduction after administering natural killer cells according to one embodiment to an animal model of glioblastoma. DETAILED DESCRIPTION OF THE INVENTION

[0072] The present invention will be described in more detail below through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0073] Example: Production of natural killer cells with increased immune receptor activity To prepare natural killer cells expressing significant immune receptors for glioblastoma, natural killer cells were cultured as follows.

[0074] 1. Peripheral Blood Mononuclear Cell Preparation 1.1. Peripheral blood mononuclear cells (PMBCs) and plasma isolation from blood Blood was prepared by venous collection from a normal subject. Blood was collected using heparinized blood collection tubes. 30 ml of blood was carefully transferred into two tubes (#352070, BD, or equivalent) containing Ficoll (#17-1440-02, GE Healthcare, or equivalent). The tubes containing the blood were centrifuged at 2,500 rpm for 10 minutes with break-off, and the upper plasma layer was transferred to a new tube. The transferred plasma was inactivated in a heat block for 30 minutes and then centrifuged at 4,000 rpm for 5 minutes. The supernatant was transferred from the centrifuged tube to a new tube, labeled as plasma, and stored at 2–8°C.

[0075] After centrifuging the blood and Ficoll, the plasma was collected from the tube, and the remaining pale yellow layer was carefully transferred to a new tube, taking care not to mix it with the red blood cell layer. Ca / Mg-free Dulbecco's Phosphate-Buffered Saline (DPBS) (#14190, Gibco) was then added. The tube was then centrifuged at 1,500 rpm for 5 minutes, and the supernatant was removed. The remaining precipitated cells were resuspended in 5 ml of RBC lysis buffer (#158904, Qiagen). The cell suspension was then centrifuged at 1,500 rpm for 5 minutes, and the supernatant was removed. Ca / Mg-free DPBS was then added to the tube, and the tube was centrifuged at 1,500 rpm for another 5 minutes. The supernatant was removed and the remaining precipitated cells were suspended in 1 ml of Alys505NK-EX (#01410P10, CSTI) medium.

[0076] A small amount of the cell suspension suspended in Alys505NK-EX was taken and diluted 100 times with Ca / Mg-free DPBS. A small amount of the diluted solution was mixed with the same volume of trypan blue and placed on a hemocytometer to measure the cell count and viability.

[0077] Freezing PBMCs All cell suspensions obtained in Example 1.1 above were centrifuged at 1,500 rpm for 5 minutes, and the supernatant was then removed. The cells were suspended in a Cryostor CS10 or ALyS505NK-EX + albumin + DMSO mixture stored at 2°C to 8°C, and the cell count was adjusted to 1-100x10. 6 The suspended cells were dispensed into 2 ml cryogenic vials at 1 ml each and frozen using controlled rate freezers (CRFs) at 0°C for 10-15 minutes, -12°C for 5-10 minutes, and -42°C for 0.5-1 minute. After the first freezing step, the cells were frozen at -25°C for 1-3 minutes and -15°C for 1-3 minutes. After the second freezing step, the cells were frozen at -42°C for 20-40 minutes and -120°C for 20-50 minutes. Alternatively, the cells were frozen at 4 to -40°C at 3°C / m, followed by a second freezing step at -40 to -90°C at 5°C / m, followed by a second freezing step at -90 to -120°C at 5°C / m. The frozen cells were transferred to an LN2 tank and stored (below -130°C).

[0078] 1.3. Thawing frozen PBMCs After setting the heat block to 37°C, the culture medium supplemented with 10% plasma was added to a T-flask. The volume of the culture medium can be adjusted to various values, such as 4 ml, 6 ml, 8 ml, or 10 ml, depending on the cell concentration. The cryovials frozen in Example 2.2 were placed in a heat block to thaw the frozen PBMCs. When the frozen PBMCs were about half thawed, they were transferred to a T-flask containing culture medium. The PBMCs were then cultured in a 37°C, 5% CO2 incubator for one day. The cultured PBMCs were collected in a tube, added with Ca / Mg-free DPBS, and centrifuged at 1,500 rpm for 5 minutes, after which the supernatant was removed. The cells separated by centrifugation were suspended in a small amount of culture medium and counted. The post-thaw viability of the cryopreserved cells is shown in Table 1. As can be seen from Table 1, more than 93% of the PBMCs thawed after cryopreservation according to the present invention remained viable, confirming that a high viability was maintained.

[0079] [Table 1]

[0080] 2. Natural Killer Cell Culturing 2.1. Preparation of Fibronectin and γ-Gamma Globulin Coated Culture Flasks (Primary) A 15 ml tube was charged with 0.01 ml of fibronectin (#FC-010, Millipore) and 0.121 ml of gamma globulin (#020A1004, Green Cross) solution, followed by the addition of 9.859 ml of Ca / Mg-free DPBS. The resulting coating solution was pipetted into a T75 flask (#156499, Nunc) and incubated at 2-8°C for at least 16 hours. Before cell culture, residual coating solution was removed by washing with Ca / Mg-free DPBS.

[0081] 2.2. Primary culture of natural killer cells The cell suspension prepared in Example 1 was placed in the coated flask prepared in Example 2.1. 1.5 ml of autologous plasma, 0.075 ml of IL-18 (#B003-2, R&D), 0.075 ml of PDGF-DD (#1159-SB, R&D), 0.03 ml of anti-NKp46 (#MAB1850, R&D), and 13.4625 ml of Alys505NK-EX (#01410P10, CSTI) were added and the cells were cultured in a CO2 incubator for 2-3 days. Then, 1.5 ml of autologous plasma and 13.5 ml of Alys505NK-EX were added to the flask and the cells were cultured in a CO2 incubator for 1-2 days.

[0082] 2.3. Preparation of Fibronectin and γ-globulin Coated Culture Flasks (Secondary) A 50 ml tube was charged with 0.025 ml of fibronectin (#FC-010, Millipore) and 0.303 ml of gamma globulin (#020A1004, Green Cross) solution, followed by 24.647 ml of Ca / Mg-free DPBS. The resulting coating solution was pipetted into a T175 flask (#159910, Nunc) and incubated at 2-8°C for at least 16 hours. Before cell culture, residual coating solution was removed by washing with Ca / Mg-free DPBS.

[0083] 2.4. Secondary culture of natural killer cells and treatment with novel substances for functional enhancement After the primary culture in Example 2.2, the T75 flask containing the cells was removed from the incubator, collected, and transferred to a T175 flask (#159910, Nunc). 3 ml of plasma, 0.06 ml of anti-NKp46 (#MAB1850, R&D), and 27 ml of Alys505NK-EX (#01410P10, CSTI) were added to the T175 flask and cultured in a CO2 incubator for 1-2 days. The remaining plasma, 0.12 ml of anti-NKp46 solution, 0.03 ml of IL-18, 0.03 ml of PDGF-DD (1159-SB, R&D), 53.85 ml of Alys505NK-EX (#01410P10, CSTI), and one or more of the following functional enhancement novel substances were added: PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD (1159-SB, R&D), and PDGF-AB (50 ng / ml), and the cells were then further cultured in a CO2 incubator for 1 to 2 days.

[0084] 2.5. Tertiary culture of natural killer cells The cells and plasma from the T175 flask cultured in Example 2.4 above were placed in a culture medium containing 2000 IU / ml IL-2 and cultured in a CO2 incubator. After 2-3 days, the same volume of fresh culture medium (containing 2000 IU / ml IL-2) was mixed with the cell suspension and cultured in a CO2 incubator.

[0085] In the above culture (any of the primary culture, secondary culture, and tertiary culture), instead of the culture medium to which IL-2 is added, a predetermined amount of IL-2 can be added to an immune cell culture medium to which IL-2 is not added.

[0086] 3. Phenotypic analysis of natural killer cells according to culture period The activated natural killer cells cultured by the culture method according to the above-mentioned example were analyzed for cell characteristics on days 0, 6, 10, and 14 during the culture period from pre-culture PBMCs to differentiation and expansion into natural killer cells.

[0087] To analyze cell characteristics, CD3, CD56, CD19, CD16, CD14, CD4, and CD8 were used as primary markers. The proportion of CD3-CD56+ NK cells increased during the culture period, and by day 14, NK cells accounted for 85.5% (4.92%) of the total. Other CD3+CD56- T cells decreased to 10% by day 14, while CD3+CD56+ NKT cells maintained a constant proportion of 5%. Monocytes and B cells were absent, accounting for 0% of the total. Furthermore, the proportion of CD8+ cells was higher than that of CD4+ cells in T cells after culture.

[0088] Figures 1A to 1C show that the cells were distributed as highly purified natural killer cells compared to before culture.

[0089] 2A to 2D are graphs showing the results of the culture evaluation of the seven subjects related to FIGS. 1A to 1C.

[0090] As shown in Figures 2E and 2F, a high viability of over 90% was maintained in both cultures on day 14, and natural killer cell proliferation was increased by 1,259-fold.

[0091] 4. Relative MFI values ​​of NK cell receptor expression before culture (D0) and after culture (D14) The relative MFI values ​​of activated natural killer cells cultured by the culture method according to the above example were measured.

[0092] The relative MFI means the expression intensity value of positive cells relative to the isotype, and is defined by the following mathematical formula 1.

[0093] [Number 1] JPEG2026016520000004.jpg8148

[0094] Relative MFI is a different concept from expression ratio, which measures the expression ratio of positive cells compared to isotype. Even if the expression ratio is the same, the strength of each receptor function varies depending on the MFI value, and it can be understood that only a high relative MFI value indicates an actual increase in function.

[0095] In this example, as described above in 2.5.8, novel natural killer cells with enhanced function were produced by treating them with novel substances, and the MFI values ​​of their specific receptors were measured to define novel natural killer cell characteristics.

[0096] To measure the relative MFI, cells were first harvested before and after incubation and then diluted to 1 x 10 7 The cells were prepared and centrifuged at 1,500 rpm for 5 minutes, after which the supernatant was removed and diluted to 2 ml with FACS buffer (PBS containing 2% FBS). For the substances listed in Table 2 below, antibodies containing fluorescent dyes were placed in 5 ml FACS tubes according to the conditions, and 100 μl of the diluted cell solution was dispensed into aliquots and stained in a refrigerator for 30 minutes. After staining, 500 μl of PBS was added to each tube, and the tubes were centrifuged at 3,200 rpm for 3 minutes, after which the supernatant was removed. The stained cell pellets were fixed in 500 μl of 1% PFA and then analyzed for cellular immune receptor expression using a flow cytometer (Beckman Coulter, USA). The relative MFI value was calculated using Equation 1.

[0097] The relative MFI values ​​are shown in Table 2 below.

[0098] [Table 2]

[0099] As shown in Table 2 above, the relative MFI values ​​of NKG2D, NKp30, NKp44, ITGA1, and ITGA2, which are factors involved in anti-cancer activity and natural killer cell activation, were found to be increased by at least 1.5-fold to as much as 25-fold. These results indicate that natural killer cells treated with a novel substance according to one embodiment are novel natural killer cells with specific MFI values, and that the anti-cancer activity and activity of the cells themselves are enhanced.

[0100] 5. Comparative analysis of immune receptor expression The expression of immune receptors was compared between activated natural killer cells cultured by the culture method according to the above-mentioned embodiment and PBMCs before culture.

[0101] Specifically, using the same method as in 4. above, the expression of cellular immune receptors was analyzed for the substances listed in Table 3 below using antibodies containing fluorescent substances, and the results are shown in Figure 3 and Figures 4A to 4C.

[0102] [Table 3]

[0103] Figure 3 is a dot plot comparing immune receptor expression on natural killer cells according to one embodiment and on PBMCs before culture. Figures 4A to 4C are graphs showing changes in immune receptor expression on natural killer cells according to one embodiment and on PBMCs before culture.

[0104] As shown in Figures 3 and 4A to 4C, natural killer cells according to one embodiment showed increased expression of NKG2D, DMAN-1, CD69, CD2, NKp30, NKG2A and NKp44, while the expression of NKp46, CD16, KIR2DL1, KIR2DL2 / 3 and KIR3DL1 showed almost no change compared to PMBCs before culture.

[0105] 6. Analysis of the expression of activated natural killer cells in brain tissue, blood-brain barrier permeability-related, or cell migration-promoting factors The activated natural killer cells cultured by the culture method according to the above-mentioned embodiment were subjected to comparative analysis of brain tissue, blood-brain barrier permeability, and expression of factors involved in promoting cell migration.

[0106] Specifically, for the substances listed in Table 4 below, the same method as in 4. above was used, except that antibodies containing fluorescent substances were used, and the expression of factors related to brain tissue, blood-brain barrier permeability, or cell migration promotion in cells was analyzed, and the results are shown in Table 4.

[0107] [Table 4] JPEG2026016520000008.jpg28154

[0108] 7. Expression Analysis of KIR2DS4 on Activated Natural Killer Cells The KIR2DS4 mRNA expression pattern of activated natural killer cells cultured by the culture method according to the above example was compared with that of PBMCs before culture.

[0109] Specifically, RNA was extracted from the cells before and after culture using the Trizol separation method, and total RNA sequencing was performed to confirm the mRNA expression pattern. The results are shown in Table 5 below.

[0110] [Table 5]

[0111] As shown in Table 5 above, natural killer cells according to one embodiment were found to have approximately 32-fold increased KIR2DS4 expression compared to PBMCs before culture.

[0112] Experimental Example 1. Expression analysis of anticancer substances The expression of anti-cancer substances (granzyme B, perforin, interferon-γ, and CD107a) in activated natural killer cells cultured by the culture method according to the above example was compared with that in PBMCs before culture.

[0113] First, granzyme B and perforin were analyzed as follows.

[0114] 5x10 for each sample 5 The cells were prepared and centrifuged at 1,500 rpm for 5 minutes, and the supernatant was removed to obtain cell pellets. The cell pellets were diluted in 100 μl portions with FACS buffer and stained for surface antigens with anti-IgG1k-FITC (eBioscience, 11-4714-42), anti-IgG1k-APC (eBioscience, 17-4714-42), anti-CD3-FITC (eBioscience, 11-0038-42), and anti-CD56-APC (eBioscience, 17-0567-42) antibodies at room temperature for 15 minutes. Then, 500 μl of PBS was added to each portion and centrifuged at 6,000 rpm for 3 minutes. For intracellular staining, cells were incubated in a refrigerator for 20 minutes using a Fixation / Permeabilization Solution Kit (BD, 554714). The cells were then incubated in a refrigerator for 20 minutes with the Fixation / Permeabilization solution, followed by two centrifugations at 6,000 rpm for 3 minutes each. The supernatant was removed and the resulting cell pellets were diluted with 100 μl of Permeabilization / Wash buffer. Anti-IgG1k-PE (eBioscience, 12-4714-42), anti-perforin-PE (eBioscience, 12-9994-42), or anti-granzyme B-PE (eBioscience, 12-8899-41) antibodies were added and incubated in a refrigerator for 30 minutes for intracellular staining. After staining, the cells were added to 500 μl of PBS, centrifuged, fixed with 1% PFA, and analyzed using a flow cytometer.

[0115] Interferon-γ was prepared by the same method as described above, followed by dilution with phenol red-free RPMI medium supplemented with 10% FBS and 1% penicillin. 500 μl of the diluted medium was then dispensed into a 24-well plate. 0.5 μl of PMA / ionomycin (Biolegend) and GolgiPlug were added. TM The cells were treated with 0.5 μl of BD Bioscience, USA, and incubated for 4 hours in a 37°C, 5% CO2 incubator. After 4 hours, the cells were harvested and centrifuged at 6,000 rpm for 3 minutes, and the supernatant was removed. The cell pellet was stained for surface antigens using anti-IgG1k-FITC (eBioscience, 11-4714-42), anti-IgG1k-APC (eBioscience, 17-4714-42), anti-CD3-FITC (eBioscience, 11-0038-42), and anti-CD56-APC (eBioscience, 17-0567-42), followed by intracellular staining. Intracellular staining was performed using anti-IgG1k-PE (eBioscience, 12-4714-42) and anti-IFN-γ-PE (eBioscience, 12-8899-41) antibodies, followed by fixation with 1% PFA and analysis using a flow cytometer.

[0116] The results for granzyme B, perforin, and interferon γ are shown in FIG.

[0117] FIG. 5 shows the results of a flow cytometer analysis using dot plots and graphs of the expression of natural killer cells and anti-cancer substances (granzyme B, perforin, and interferon gamma) in PBMCs before culture according to one embodiment.

[0118] As shown in Figure 5, natural killer cells according to one embodiment were found to express at least 80% or more of the anti-cancer substances granzyme B, perforin, and interferon gamma, which is at least four times higher than the levels of PBMCs before culture.

[0119] In addition, to compare the expression of CD107a degranulation between natural killer cells and PBMCs before culture, K562 lymphoblasts extracted from the bone marrow of a patient with chronic myeloid leukemia were used as target cells and the level of CD107a expression was analyzed.

[0120] Specifically, 1x105 target K562 cells were prepared per condition, centrifuged at 1,500 rpm for 5 minutes, and the supernatant was removed to obtain a pellet. The cell pellet was diluted with 250 μl of culture medium containing phenol red-free RPMI medium supplemented with 10% FBS and 1% penicillin-streptomycin (10,000 U / mL) (Gibco, 15140122). Natural killer cells were prepared at a 5:1 ratio between activated natural killer cells and target cells, with 5x105 cells per condition. 5 The cells were prepared using a 24-well plate. After centrifugation, the supernatant was removed and the target cells were suspended in 250 μL of the same culture medium used to dilute the cells. The prepared activated natural killer cells and target cells were then placed in a 24-well plate at a 5:1 ratio, and anti-IgG1k-PE (eBioscience) and anti-CD107a-PE (eBioscience) antibodies were added. The cells were incubated for 4 hours at 37°C in 5% CO2. After the 4-hour incubation, the cells were harvested and stained with fluorescent antibodies: anti-IgG1k-FITC (eBioscience), anti-IgG1k-APC (eBioscience), anti-CD3-FITC (eBioscience), and anti-CD56-APC (eBioscience) to isolate natural killer cells. After staining, the cells were washed with 500 μL of PBS and centrifuged. Then, they were fixed with 1% PFA and analyzed using a flow cytometer. The results are shown in Figure 6.

[0121] FIG. 6 shows the results of dot plots and graphs illustrating the expression of CD107a degranulation in natural killer cells and PBMCs before culture according to one embodiment.

[0122] As shown in Figure 6, natural killer cells according to one embodiment were found to express CD107a by at least 60%, which is at least three times higher than that of PBMCs before culture.

[0123] Experimental Example 2. Analysis of the interaction between ligands in glioblastoma cell lines and activated natural killer cells The interaction of the ligands with glioblastoma cell lines and, in one embodiment, natural killer cells was analyzed.

[0124] First, glioblastoma cell lines U-87MG and T98G cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin (10,000 U / mL), while other glioblastoma cell lines U-373MG and A172 cells were cultured in RPMI medium supplemented with 10% FBS and 1% penicillin-streptomycin (10,000 U / mL). Subsequently, the glioblastoma cell line T98G and the glioblastoma cell lines U-87MG, A172, and U-373MG were stained with antibodies to natural killer cell ligands HLA-ABC, HLA-E, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, PVR, ICAM-1, ICAM-2, ICAM-3, LFA-3, B7-H6, PVR, and Necin-2, and the expression levels of the cells were analyzed.

[0125] Specifically, 0.25% trypsin-EDTA (1X) and phenol red were added to each cancer cell line cultured in a T75 flask, and the cells were incubated for 3 to 5 minutes in a 37°C, 5% CO2 incubator. After incubation, the cells were suspended and the enzyme was inactivated in a culture medium supplemented with 10% FBS. The cells were then harvested. The harvested cells were counted and found to be 8.5 x 10 6The cells were prepared and centrifuged at 1,500 rpm for 5 minutes. The supernatant was removed and diluted to 1.7 ml with FACS buffer (PBS containing 2% FBS). Next, antibodies containing fluorescent substances listed in Table 6 below were added to 5 ml FACS tubes according to the conditions, and 100 μl of the diluted cell solution was dispensed into each tube and stained at room temperature for 15 minutes. The stained cells were fixed in 500 μl of 1% PFA and then analyzed using a flow cytometer (Beckman Coulter, USA). The results are shown in Figure 7.

[0126] [Table 6]

[0127] 7A to 7D are dot plots showing the expression of ligands of natural killer cells to glioblastoma cell lines according to one embodiment.

[0128] As shown in Figures 7A-7D, T98G is a temozolomide-resistant glioblastoma cell line, and U-373MG and U-87MG are grade 3 and 4 glioblastoma cell lines, respectively. Analysis of cell ligands suggests that killing activity is mediated by interaction with the primary NK cell receptor. Significant results are expected in T98G (rMFI 24.8) and U-87MG (rMFI 30.5), which have low HLA-ABC expression and relatively high NK ligand expression.

[0129] Experimental Example 3: Confirmation of cell killing ability against glioblastoma cell lines In one embodiment, to confirm the direct cell-killing ability of natural killer cells, the cell-killing ability was evaluated against the blood cancer cell line K562, which is highly sensitive to natural killer cells, and the glioblastoma cell lines A172, U-87MG, U-373MG, and T98G.

[0130] Target cancer cells (K562, U-87MG, U-373MG, A172, T98G) were harvested and centrifuged at 1,500 rpm for 5 minutes, and the supernatant was removed. Then, the cells were diluted with DPBS and washed. The washed cell pellet was suspended in phenol red-free RPMI medium supplemented with 10% FBS. 1x10 cells per condition were cultured. 5 Cells were prepared and stained with CFSE (Life Technologies) at a concentration of 5 μM for 10 minutes in a 5% CO2 incubator. After washing twice with DPBS, the cells were diluted with phenol red-free RPMI medium supplemented with 10% FBS. Activated natural killer cells were prepared at different E:T ratios with target cells (1:1, 1.25:1, 2.5:1, 5:1, 10:1, and 20:1) and mixed with target cells in a 24-well plate. The reaction was allowed to proceed for 4 hours, and 20 minutes before the end of the reaction, the cells were treated with 7-aminoactinomycin D (7AAD). After the reaction, the cells were collected in 5 ml FACS tubes and analyzed for cell killing activity using a flow cytometer. The results are shown in Figures 8 and 9.

[0131] FIG. 8 shows the results of cell killing ability of natural killer cells against blood cancer cell line K562 according to an embodiment of the present invention at different E:T ratios.

[0132] FIG. 9 shows the cell-killing ability of natural killer cells against glioblastoma cell lines A172, U-87MG, U-373MG, and T98G at different E:T ratios according to one embodiment.

[0133] As shown in Figures 8 and 9, while pre-culture PBMCs did not have significant anti-cancer activity against glioblastoma, natural killer cells according to one embodiment were found to have significant anti-cancer activity in blood cancer cell lines and glioblastoma cell lines.

[0134] The above results indicate that the novel natural killer cells of one embodiment not only express significant immune receptors against glioblastoma, but also have the effect of being useful in the treatment of blood cancers, glioblastoma, and the like, as cells that express immune receptors that can overcome self-tolerance.

[0135] Experimental Example 4: Natural Killer Cell Blocking Assay According to one embodiment, it was confirmed whether the cytotoxicity of natural killer cells against cancer cells is suppressed when the expression of a specific factor is suppressed.

[0136] Specifically, natural killer cells in which the activity of NKp30-, NKp44-, and NKG2D-receptor antibodies was blocked were prepared. Then, the cytotoxicity of these cells against U-87MG, U-373MG, A172, and T98G was examined using the same method as in Experimental Example 3, and the results are shown in Figure 10.

[0137] FIG. 10 is a graph showing the cytotoxicity of natural killer cells against cancer cell lines after blocking specific receptors in the natural killer cells according to one embodiment.

[0138] As shown in Figure 10, in natural killer cells according to one embodiment, inhibition of NKp30, NKp44, or NKG2D activity significantly reduced cytotoxicity. In particular, inhibition of NKG2D activity or inhibition of all three receptors significantly reduced cytotoxicity.

[0139] Such results indicate that in natural killer cells according to one embodiment, the activity of NKp30, NKp44 and / or NKG2D is the main factor in cytotoxicity, and that natural killer cells according to one embodiment having NKp30, NKp44 and / or NKG2D with a particular MFI value are cells with significantly enhanced anti-cancer activity.

[0140] Experimental Example 5. Anti-cancer activity analysis of natural killer cells 5.1. Anticancer activity analysis in ovarian cancer animal models According to one embodiment, the anti-cancer activity of natural killer cells was confirmed in vivo.

[0141] First, NOD-SCID mice were inoculated with 1x10 OVCAR3 ovarian cancer cell line. 7 Cells were subcutaneously administered per animal to prepare xenograft animal models, and then test groups were established as shown in Table 7 below.

[0142] [Table 7]

[0143] The negative control group (G1 vehicle group) was prepared with 5% albumin and dextran at a ratio of 1:1 and administered intravenously in 100 μl. The positive control group (G2 cisplatin group) was administered with 1.5 mg / kg cisplatin. The natural killer cell administration groups (G3 fresh NK group and G4 frozen NK group) were the difference between fresh and frozen cells. The G4 frozen NK group was thawed frozen NK cells and administered at 1 x 10 7 For the G3 fresh NK group, cells were collected during culture and administered at 1x10 7 Cells were administered per mouse. Each treatment group received six doses, twice weekly. The mice were monitored for survival, tumor size, and symptoms over the course of the study period. After 78 days, the animals were sacrificed and tumors were extracted and weighed. The results are shown in Table 8 and Figure 11.

[0144] [Table 8]

[0145] FIG. 11 is a graph showing the observed decrease in tumor weight after natural killer cells according to one embodiment were administered to an animal model of ovarian cancer (arrows indicate the time of drug administration).

[0146] As shown in Table 8 and Figure 11, administration of natural killer cells according to one embodiment reduced tumor weight by approximately 50% to 60% compared to the positive control group. In particular, while the positive control group showed accelerated tumor growth and rapid tumor volume growth after 50 days, the group administered natural killer cells according to one embodiment showed a significant slowdown in tumor growth over the monitoring period.

[0147] 5.2. Anticancer activity analysis in gastric cancer animal models According to one embodiment, the anti-cancer activity of natural killer cells was confirmed in vivo.

[0148] First, NOD-SCID mice were inoculated with gastric cancer cell line NCI-N87 at 1x10 6 Cells / animal were administered to prepare xenograft animal models. Six days after tumor inoculation, the test groups shown in Table 9 below were established.

[0149] [Table 9]

[0150] The negative control group (G1 vehicle group) was prepared with 5% albumin:dextran injection at a ratio of 1:1 and administered intravenously in 200 μl. The positive control group (G2HER2 group) was administered Herceptin at 1 mg / kg intravenously twice a week for a total of six doses. The G3 fresh NK group and the G4 frozen NK group were administered natural killer cells alone and administered 1x10 7 Cells were administered intravenously twice a week per animal for a total of six times. G3 fresh NK group received cells collected during culture, and G4 frozen NK group received 1x10 thawed frozen NK cells. 7 Cells were administered per mouse. The G5 NKL + Herceptin group and the G6 NKF + Herceptin group, which were administered in combination with NK cells and Herceptin, were the same groups as the NK cell single administration group, but in combination with 1 mg / kg of Herceptin, and were administered intravenously twice a week for a total of six times.

[0151] During the test period, the mice were monitored for survival rate, tumor size, and symptoms for 52 days. After 52 days, the animals were sacrificed and the tumors were extracted and weighed. The results are shown in Table 10 and Figure 12.

[0152] [Table 10]

[0153] FIG. 12 is a graph showing the observed reduction in tumor weight after natural killer cells according to one embodiment were administered to an animal model of stomach cancer.

[0154] As shown in Table 10 and Figure 12, administration of natural killer cells according to one embodiment reduced tumor weight by approximately 60% to 70% compared to the positive control group. In particular, while tumor growth accelerated and tumor volume increased rapidly in the positive control group after 28 days, tumor growth in the group administered with natural killer cells according to one embodiment was significantly slowed during the monitoring period.

[0155] Additionally, it was confirmed that tumor weight was reduced by approximately 17% to 20% in the group administered the combination of Herceptin and natural killer cells compared to the group administered natural killer cells alone. This means that when applying ADCC (antibody-dependent cell cytotoxicity) through the combined administration of natural killer cells and Herceptin, a high tumor growth suppression effect and a long-lasting anti-cancer efficacy can be achieved.

[0156] 5.2. Anticancer activity analysis in glioblastoma animal models According to one embodiment, the anti-cancer activity of natural killer cells was confirmed in vivo.

[0157] First, 7-week-old NOG female mice were injected with 1 × 10 U87MG-luci cells, a human glioblastoma-derived cell line, transduced with the luciferase gene. 4Each cell was transplanted into the right brain cerebral hemisphere skull of a mouse model to establish an orthotopic transplant animal model. After a certain period of time had passed since cell transplantation, the bioluminescence (BLI) of the tumor was measured using an IVIS bioimaging device (PerkinElmer, USA). Seven days after transplantation, animals with an average BLI value were selected. The selected animals were divided into test groups as shown in Table 11 below.

[0158] [Table 11]

[0159] The negative control group G1 (vehicle group) was prepared with 5% albumin and dextran at a ratio of 1:1 and administered intravenously in an amount of 200 μl per mouse. The test group G2 (NK cell group) contained 1x10 natural killer cells. 6 Cells were administered intravenously per mouse twice weekly for a total of six doses. During the monitoring period, tumor volume was measured twice weekly using an IVIS bioimaging system (PerkinElmer, USA). To measure tumor volume, 150 mg / kg of luciferin (Promega) was intraperitoneally administered at 10 mL / kg. Ten minutes later, each mouse was anesthetized using an inhalation anesthesia machine. Once fully anesthetized, tumors were sequentially measured using the IVIS system. The measured region of interest (ROI) values ​​were analyzed according to the internal threshold established by Woojung Bio Co., Ltd. (Gyeonggi-do, South Korea), a contract research institute. The results are shown in Table 12 and Figure 13.

[0160] [Table 12]

[0161] FIG. 13 is a graph showing the observed reduction in tumor weight after natural killer cells according to one embodiment were administered to an animal model of stomach cancer.

[0162] As shown in Table 12 and Figure 13 above, in the negative control group (G1), the change in tumor volume up to 16 days after administration was 4.28E+08p / sec / cm 2 / sr to 1.09.E+10p / sec / cm 2 A sustained increase in p / sec / cm was observed over the course of the experiment. In contrast, when natural killer cells were administered according to one embodiment, the p / sec / cm was 3.19.E+08. 2 / sr to 1.78.E+09p / sec / cm 2 The tumor weight increased slightly during the experimental period, and by day 16, it was about five times higher than that of the negative control group. This indicates that natural killer cells according to one embodiment have a significant tumor growth inhibitory effect on glioblastoma.

Claims

1. An isolated natural killer cell having one or more characteristics selected from the following (a) to (e): (a) The relative MFI value of NKG2D increased by 1.2 to 12 times compared to day 0 of PBMC culture; (b) The relative MFI value of NKp30 increased by 1.5 to 15 times compared to day 0 of PBMC culture; (c) The relative MFI value of NKp44 increased by 12 to 22 times compared to day 0 of PBMC culture. (d) The relative MFI value of ITGA1 increased by 1.8 to 25 times compared to day 0 of PBMC culture; and (e) The relative MFI value of ITGA2 increased by 1.4 to 6 times compared to day 0 of PBMC culture; The relative MFI is defined by the following equation: [Equation 1] 。

2. the relative MFI value of (a) is increased by 3 to 6 times; the relative MFI value of (b) is increased by 3 to 6 times; the relative MFI value of (c) is increased by 12 to 18 times; The relative MFI value of (d) is increased by 6 to 10 times; and The natural killer cells according to claim 1, wherein the relative MFI value of (e) is increased by 2 to 4 times.

3. The natural killer cells of claim 1, having one or more characteristics selected from CD16 with an MFI value of 10 to 140, LFA-1 with an MFI value of 20 to 160, NKG2D with an MFI value of 5 to 25, NKp30 with an MFI value of 5 to 20, NKp44 with an MFI value of 12 to 25, ITGA1 with an MFI value of 4 to 25, and ITGA2 with an MFI value of 2 to 10.

4. CD2 with an MFI value of 20 to 180, CD27 with an MFI value of 0.1 to 1.5, CD69 with an MFI value of 1 to 10, CD226 with an MFI value of 2 to 12, NKp46 with an MFI value of 2 to 8, CD160 with an MFI value of 0.1 to 4, KIR2DL1 with an MFI value of 0.1 to 4, KR2DL3 with an MFI value of 0.1 to 5, KIR3DL1 with an MFI value of 0.1 to 4, NKG2A with an MFI value of 0.4 to 16, The natural killer cells of claim 1, further having one or more characteristics selected from CD161 with an MFI value of 0.2 to 12, CCR3 with an MFI value of 0.3 to 3, CCR5 with an MFI value of 0.5 to 4, CCR6 with an MFI value of 0.8 to 6, CXCR3 with an MFI value of 0.4 to 5, CXCR1 with an MFI value of 0.4 to 5, CXCR2 with an MFI value of 0.1 to 3, and ITGB7 with an MFI value of 1 to 16.

5. The natural killer cells of claim 1 further have the following characteristic (f): (f) The expression level of KIR2DS4 gene is 10 to 50 times higher on day 14 of PBMC culture compared to day 0 of culture.

6. KIR2DS1 ＋ , KIR2DS2 ＋ , KIR2DS3 ＋ , KIRDS4 ＋ , CXCR1 ＋ , CXCR2 ＋ , CXCR3 ＋ , CCR3 ＋ , CCR5 ＋ , CCR6 ＋ , PSA-NCAM ＋ , Nestin ＋ , tyrosine hydroxylase ＋ , CD147 ＋ , CD127 ＋ , CD15 ＋ , CD31 ＋ , CD146 ＋ , CD49c+, CD107a ＋ , NKG2A ＋ , CD45 ＋ , CD44 － , CD140a ＋ , CD87 － , CD11b ＋ , CD10 － and CD80 － The natural killer cell according to claim 1, having any one of the following characteristics:

7. The natural killer cells of claim 1, wherein 50% to 90% of the cell population expresses NKp44, 50% to 90% of the cell population expresses KIR2DS2, or 60% to 100% of the cell population expresses NKG2D.

8. The natural killer cells of claim 1, wherein the natural killer cells were treated with PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD, or PDGF-AB during culture from PBMCs.

9. A pharmaceutical composition for preventing or treating cancer, comprising, as an active ingredient, the natural killer cells or cell population thereof according to any one of claims 1 to 8.

10. The pharmaceutical composition of claim 9, wherein the natural killer cells suppress immune activity, blood-brain barrier penetration, cell migration promotion, or self-tolerance.

11. 10. The pharmaceutical composition according to claim 9, wherein the cancer is one or more selected from the group consisting of lung cancer, laryngeal cancer, gastric cancer, colon cancer, rectal cancer, liver cancer, gallbladder cancer, pancreatic cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, prostate cancer, kidney cancer, skin cancer, bone cancer, muscle cancer, fat cancer, fibrocytic cancer, blood cancer, leukemia, lymphoma, multiple myeloma, and glioma.

12. 12. The pharmaceutical composition of claim 11, wherein the glioma is an astrocytoma, oligodendroglioma, mixed glioma, or ependymoma.

13. The pharmaceutical composition of claim 12, wherein the astrocytoma is glioblastoma, anti-cancer drug resistant glioblastoma, or recurrent glioblastoma.

14. The pharmaceutical composition according to claim 9, wherein the cancer secretes or expresses MHC class I polypeptide-related sequence A (MICA) or platelet-derived growth factor-DD (PDFG-DD).

15. 10. A method for treating cancer, comprising administering to an individual in need thereof an effective amount of natural killer cells or a population thereof according to any one of claims 1 to 8.

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