Composition for prevention or treatment of fibrosis, comprising natural killer (NK) cells

Abstract

The present invention relates to a pharmaceutical composition for prevention or treatment of fibrosis, comprising directly reprogrammed natural killer (drNK) cells prepared through the reprogramming of isolated cells, and a cell therapy product, and specifically relates to: a pharmaceutical composition for prevention or treatment of fibrotic disease, comprising drNK cells expressing any one or more selected from the group consisting of CD56superbright, CD16superbright, and a combination thereof; and a cell therapy product.

Description

Composition for preventing or treating fibrosis containing natural killer (NK) cells

[0001] The present invention relates to a pharmaceutical composition and cell therapy agent for preventing or treating fibrosis, comprising directly reprogrammed natural killer (drNK) cells produced through direct reprogramming of isolated cells, and specifically, the induced natural killer cells of the present invention are characterized by expressing CD56superbright, CD16superbright, or both.

[0002]

[0003] Fibrosis is a pathological condition characterized by the excessive accumulation of fibrous connective tissue, particularly collagen, in organs or tissues, resulting in thickening and stiffness. This process is caused by chronic inflammation or injury, which replaces normal tissue with scar tissue and impairs the structure and function of the affected organ. Fibrosis can occur in various organs, including the lungs, liver, kidneys, heart, and skin, and is often associated with progressive, irreversible damage, contributing to the development of chronic diseases.

[0004] In the lungs, conditions such as idiopathic pulmonary fibrosis (IPF) cause thickening and scarring of lung tissue, leading to progressive respiratory failure. The liver can also be affected, as seen in liver cirrhosis and non-alcoholic steatohepatitis (NASH), both of which cause extensive scarring due to chronic damage. In the kidneys, chronic kidney disease (CKD) and glomerulonephritis can cause fibrosis, which can impair kidney function and potentially lead to kidney failure. Cardiac fibrosis often occurs after a heart attack or chronic hypertension, causing stiffening and impaired function of the heart muscle. Systemic sclerosis, or scleroderma, is an autoimmune disease that causes extensive fibrosis, affecting the skin and internal organs. Chronic inflammatory conditions, such as Crohn's disease, can lead to fibrous strictures in the intestines, which can lead to intestinal obstruction. Cystic fibrosis is a genetic disorder that primarily affects various organs, including the lungs, liver, pancreas, urinary and reproductive systems, and sweat glands. It causes thick mucus to form, leading to persistent infections and fibrosis. Dupuytren's contracture is a condition in which the fibrous tissue between the skin and tendons of the palm becomes abnormally thickened, preventing the fingers from straightening. Fibrosis can also affect the eyes, as seen in retinal fibrosis and conjunctival fibrosis, often caused by chronic inflammation or injury. In the skin, fibrotic conditions such as keloids and hypertrophic scars can develop after injury, causing thickened, raised scars.Chronic pancreatitis can cause fibrosis in the pancreas, impairing its function and leading to complications such as diabetes. Myelofibrosis, which occurs spontaneously in conditions such as primary myelofibrosis (PMF), caused by certain genetic mutations, replaces healthy bone marrow tissue with fibrous tissue, affecting blood cell production.

[0005] Many existing treatments target symptoms or underlying conditions rather than directly addressing fibrosis itself, resulting in limited efficacy and primarily slowing disease progression rather than reversing the fibrotic process. These treatments can have significant side effects, and given the complex and multifactorial nature of fibrosis, they may not be effective for all types of fibrotic diseases or all patients. Furthermore, the lack of biomarkers for early detection and monitoring hinders timely and accurate treatment and effectively manages the disease. Currently, active development is underway to target key pathways associated with fibrosis, including pathway-specific inhibitors (e.g., TGF-β and Wnt signaling), antifibrotic small molecules and biologics, cell-based therapies such as mesenchymal stem cells, and gene therapies. However, there is a growing need for more targeted, safer, and effective therapies that can halt or even reverse fibrosis in various organs.

[0006] Myofibroblasts, known as fibrogenic cells in various tissues and organs, are important for wound healing and tissue repair, and in the context of fibrosis, their overactivation leads to excessive production of extracellular matrix (ECM) components including collagen, accumulation of fibrous tissue, and consequently, sclerosis and dysfunction of the affected organs. Myofibroblasts are known to be derived from various precursor cells, such as fibroblasts, pericytes, hepatic stellate cells, and epithelial cells, by differentiation into them in response to pro-fibrotic stimuli, such as activation of TGF-β signaling, mechanical stress, and inflammatory cytokines. In animal models of fibrosis, the presence of myofibroblasts in fibrotic lesions is associated with the development of active fibrosis, and their persistence and localization to fibrotic lesions in human disease are known to be associated with disease progression (Kuhn C. et al. Am J Pathol. 1991; 138(5): 1257-65). Therefore, there is growing interest in developing anti-fibrotic therapies that target myofibroblasts, such as inhibiting their activation and differentiation or promoting their apoptosis, as a strategy to interfere with the fibrotic process and reduce the burden of fibrotic diseases, in order to develop more effective and targeted anti-fibrotic therapies.

[0007] Meanwhile, natural killer (NK) cells, which possess the ability to eliminate activated myofibroblasts, are emerging as promising research targets for the development of fibrosis treatments. Studies have reported using cytokines such as IL-2, IL-15, and IL-18 to enhance the cytotoxic activity of existing NK cells against myofibroblasts. However, these cells still have limited antifibrotic effects and difficulties in producing therapeutic NK cells, which remain a challenge.

[0008] Recently, with the advancement of direct reprogramming technology, efforts are actively underway to directly produce functional cells with high clinical utility, without going through stem cell production or differentiation processes. Functional cells produced through direct reprogramming technology have significant technological advantages, including a lower risk of epigenetic remodeling and tumorigenesis, and the simplified cell production process facilitates increased safety, reliability, and efficiency. These characteristics are expected to ultimately contribute to dramatically reducing the time and cost required for therapeutic development, thereby removing barriers to commercialization. Therefore, R&D efforts to secure raw materials for cell therapy for various diseases using direct reprogramming technology are continuously increasing.

[0009] To date, no reports have been published on the preventive, therapeutic, or ameliorating effects of NK cells produced by direct reprogramming on fibrosis.

[0010]

[0011] The present inventors have completed the present invention by producing induced natural killer (drNK) cells directly through reprogramming from somatic cells that are easy to obtain without going through the above process, differentiating from the existing method of obtaining NK cells through stem cell production and differentiation processes, and confirming that these can be usefully used in the prevention or treatment of fibrotic diseases.

[0012]

[0013] One object of the present invention is to provide a pharmaceutical composition for preventing or treating a fibrotic disease, comprising directly reprogrammed natural killer (drNK) cells expressing at least one selected from the group consisting of CD56superbright, CD16superbright, and combinations thereof.

[0014] Another object of the present invention is to provide a cell therapy composition for preventing or treating fibrosis, comprising directly reprogrammed natural killer (drNK) cells expressing at least one selected from the group consisting of CD56superbright, CD16superbright, and combinations thereof.

[0015] Another object of the present invention is to provide a method for preventing or treating fibrosis, comprising administering to a subject other than a human a pharmaceutical composition for preventing or treating fibrosis, the pharmaceutical composition comprising directly reprogrammed natural killer (drNK) cells expressing at least one selected from the group consisting of CD56superbright, CD16superbright, and combinations thereof, to a subject suspected of having fibrosis.

[0016]

[0017] When the directly reprogrammed natural killer (drNK) cells expressing at least one selected from the group consisting of CD56superbright, CD16superbright, and a combination thereof of the present invention were treated with hepatic stellate cells (liver fibrosis cell model), alveolar epithelial cells (lung fibrosis cell model), cardiac cells (cardiac fibrosis cell model), skin cells (skin fibrosis cell model), and kidney cells (kidney fibrosis cell model), which are fibrosis origin cells associated with the induction of fibrosis response in each tissue, the direct killing ability was superior to that of the previously known control natural killer cells, and further, when the conditioned medium for the induced natural killer cells was treated to the fibrosis cell model, the effect of inhibiting fibrosis was confirmed by significantly reducing collagen production, activity, and viability compared to the previously known control natural killer cells. Therefore, the drNK cells can be usefully used for the prevention or treatment of fibrosis.

[0018]

[0019] Figure 1 is a schematic diagram illustrating the antifibrotic effect of induced natural killer (drNK) cells and conditioned medium (CM) obtained by culturing them.

[0020] Figure 2 shows representative types of NK cell types showing different CD56 and CD16 expression patterns that were comparatively analyzed to identify natural killer cell types effective in the antifibrotic activity of the present invention. Figure 2A shows the main cell types of 1. PBMC-derived NK cells (pNK) showing different CD56 [dim (low), bright (high), superbright (superhigh)], CD16 CD56 [dim (low), bright (high), superbright (superhigh)] expression patterns. dim CD16 bright pNK (CD56dim pNK), 2. IL-2 / IL-15 cytokine activation CD56 bright CD16 bright pNK (CD56 bright pNK), 3. CD56 bright CD16 dim Immortalized NK cell line NK92, 4. CD56 induced differentiation from iPSCs bright CD16 dim iPS-NK, 5. CD56superbrightCD16superbright-induced natural killer cells (drNK) produced through direct reprogramming of the present invention are shown. Figure 2B shows the results of confirming the CD56 and CD16 expression patterns of each NK cell in Figure 2A using flow cytometry.

[0021] Figure 3 shows the killing ability of drNK on human hepatic stellate cells (HSCs) that induce liver fibrosis. Figure 3A shows the results of immunohistochemical staining in activated HSCs (aHSCs) treated with TGF-β, confirming the increase in type 1 collagen and alpha-SMA protein expression compared to quiescent HSCs (qHSCs). Figure 3B shows the results of Picro sirius red staining in aHSCs confirming the increase in collagen deposition. Figure 3C shows the results of control (CD56 dim pNK, CD56 bright This is a result confirming the superior aHSC killing ability of drNK compared to pNK, NK92, and iPS-NK.

[0022] Figure 4 shows the antifibrotic effect of drNK dependent on CD56 and CD16 expression. Figure 4A shows a schematic diagram of aHSC cytotoxicity assay using anti-CD56 and anti-CD16 antibodies to analyze the correlation between aHSC cytotoxicity and CD56 and CD16 expression. Figure 4B shows the results of the anti-CD56 antibody blocking assay of Figure 4A, which showed that the control group (CD56 dim pNK, CD56 bright The results confirm that the high anti-fibrotic activity of drNK is associated with CD56 expression by confirming that aHSC cytotoxicity is most significantly inhibited by CD56 blocking in drNK of the present invention compared to pNK, NK92, and iPS-NK. Figure 4C shows the results of the anti-CD16 antibody blocking assay of Figure 4A compared to the control group (CD56 dim pNK, CD56 bright By confirming that aHSC cytotoxicity was most significantly suppressed by CD16 blockade in drNK of the present invention compared to pNK, NK92, and iPS-NK, it was confirmed that the high anti-fibrotic activity of drNK is associated with CD16 expression.

[0023] Figure 5 shows the anti-fibrotic effect of drNK dependent on NKG2D expression. Figure 5A shows the results confirming that drNK of the present invention exhibits relatively high NKG2D expression characteristics compared to control NK [peripheral blood (pNK), placental (UCB-NK), embryonic stem cell- (ESC-NK), induced pluripotent stem cell-differentiated (iPS-NK)] (Figure 5A, left), and relatively high NKG2D ligand MICA expression characteristics in aHSC compared to control qHSC (Figure 5A, right). Figure 5B shows a schematic diagram of an aHSC cytotoxicity assay using an anti-NKG2D antibody to analyze the correlation between aHSC cytotoxicity and NKG2D expression. Figure 5C shows the results of the anti-NKG2D antibody blocking assay of Figure 5B, which showed that the control (CD56 dimpNK, CD56 bright By confirming that the cytotoxicity of drNK, which has relatively high NKG2D expression compared to pNK, NK92, and iPS-NK, was most significantly suppressed by NKG2D blockade, the results confirmed that the high anti-fibrotic activity of drNK is associated with NKG2D expression.

[0024] Figure 6 shows the anti-fibrotic effect of drNK that is dependent on CCR5 expression. Figure 6 shows the results of confirming the relatively high CCR5 mRNA (Figure 6A) and protein expression (Figure 6B) of drNK compared to conventional NK (pNK, iPS-NK). Figure 6C shows a schematic diagram of an aHSC cytotoxicity assay using an anti-CCR5 antibody to analyze the correlation between aHSC cytotoxicity and CCR5 expression. Figure 6D shows the results of the anti-CCR5 antibody blocking assay of Figure 6C, confirming that the cytotoxicity of drNK was most significantly suppressed by CCR5 blocking compared to the control group (pNK, NK92, iPS-NK), confirming that the high anti-fibrotic activity of drNK with relatively high CCR5 expression is associated with CCR5 expression. Figure 6E shows the results of confirming the high CCL5 expression pattern in the liver of fibrosis-induced mice. Figure 6F shows the results of verifying the usefulness of transplanted drNK as an anti-fibrotic cell therapy agent by confirming that the remaining NK cells in the liver were higher than those in the pNK when the liver and spleen were extracted after intravenous injection of pNK and drNK into mice, respectively, and the remaining NK cells were analyzed using a flow cytometer with human-specific CD45 antibodies.

[0025] Figure 7 shows the superior anti-fibrotic effect of drNK in a fibrotic liver organoid model. Figure 7A shows the results of producing a multicellular liver organoid model from iPS and confirming the expression of hepatic differentiation characteristic markers. Figure 7B shows the results of confirming the superior anti-fibrotic effect of drNK compared to pNK in an oleic acid (sodium oleate; oleic acid, OA)-mediated fibrosis-induced liver organoid model, as confirmed by analysis of cells positive for the lipophilic fluorescent dye BODIPY493 / 503, collagen (Anti-COL1A1), and fibrotic extracellular matrix (Sirius red).

[0026] Figure 8 shows the body weight and liver weight reduction inhibitory effect of drNK in a methionine-choline deficient diet (MCD-diet) mouse model induced with nonalcoholic steatohepatitis (NASH). Figure 8A is a schematic diagram of the animal experiment method for the NASH-induced MCD-diet mouse model. Figure 8B is the result confirming the superior body weight reduction inhibitory effect compared to the control group (pNK, Poly IC) 9 weeks after administration of the induced natural killer cells in Figure 8A. Figure 8C is the result confirming the superior liver tissue weight reduction inhibitory effect compared to the control group (pNK, Poly IC) 9 weeks after administration of the induced natural killer cells in Figure 8A.

[0027] Figure 9 shows the anti-fibrotic effect of drNK through inhibition of liver damage indicators and steatosis in the NASH-induced MCD-diet mouse model. Figure 9A shows the results confirming the excellent effect of drNK on the increase of liver damage indicators (ALT, AST) compared to the control group (pNK, Poly IC) 9 weeks after cell transplantation in Figure 8A. Figure 9B shows the results of measuring the degree of steatosis (Steatosis score) using Oil Red O staining and the degree of collagen development (Fibrosis score) using Masson's Trichrome staining in Figure 8A by Hematoxylin & Eosin staining to determine whether there was structural loss in the liver tissue. 9 weeks after cell transplantation, drNK showed excellent recovery of structural loss (Figure 9B), inhibition of steatosis increase (Figures 9B and 9C), and inhibition of collagen tissue development (Figures 9B and 9D) compared to the control group (pNK, Poly IC).

[0028] Figure 10 shows the anti-fibrotic effect of drNK through suppression of fibrosis marker expression in the NASH-induced MCD-diet mouse model. As shown in Figure 8A, 9 weeks after cell transplantation, the expression of fibrosis markers (α-SMA, TGF-β, TIMP-1, COL1A1, COL3A1) and extracellular matrix degradation (MMP-1, MMP-2) genes was analyzed via RT-qPCR. Compared to the control group (pNK, Poly IC), drNK significantly suppressed the expression of fibrosis markers and enhanced the expression of extracellular matrix degradation markers MMP-1 and MMP-2.

[0029] Figure 11 shows the anti-fibrotic effect of drNK through enhancing the expression of anti-inflammatory markers and suppressing the expression of inflammatory markers in the NASH-induced MCD-diet mouse model. As shown in Figure 8A, the gene expression of anti-inflammatory markers (IL-10, IL-11, IL-4) and inflammatory markers (IL-1β, IL-6, TNF-α) was analyzed using RT-qPCR 9 weeks after cell transplantation. Compared to the control group (pNK, Poly IC), drNK significantly enhanced the expression of anti-inflammatory markers and suppressed the expression of inflammatory markers.

[0030] Figure 12 shows the inhibitory effect of drNK-conditioned medium on the pro-fibrotic activity of human hepatic stellate cells. Figure 12A shows the results of analyzing the gene expression of fibrosis markers LOXL2, COL1A1, and ACTA2 through RT-qPCR in human hepatic stellate cells cultured in the presence (+ / -) of TGF-β or NK-CM. It was confirmed that the gene expression of fibrosis markers LOXL2, COL1A1, and ACTA2 was significantly reduced in the group treated with drNK-conditioned medium (CM-drNK) compared to the control group (pNK-CM). Figure 12B shows the results of confirming the superior inhibitory effect of CM-drNK on type 1 collagen and α-SMA protein expression compared to the control group (CM-pNK) in human hepatic stellate cells cultured in the presence (+ / -) of TGF-β or NK-CM by immunohistochemical staining (cell fluorescence staining images and quantitative graphs). Figure 12C shows the results of Sirius red collagen staining in human hepatic stellate cells cultured in the presence or absence (+ / -) of TGF-β or NK-CM, confirming the superior collagen deposition / fibrosis inhibition effect of CM-drNK compared to the control group (pNK-CM). Figure 12D shows the results of confirming the superior aHSC cell proliferation inhibition effect of CM-drNK compared to the control group (pNK-CM).

[0031] Figure 13 shows the killing ability of drNK on human lung fibroblasts (MRCs) that induce pulmonary fibrosis. Figure 13A shows the results of RT-qPCR confirming increased COL1A1 and COL3A1 gene expression in activated human lung fibroblasts (aMRCs) treated with TGF-β compared to inactivated quiescent MRCs (qMRCs). Figure 13B shows the results of Picro sirius red staining confirming increased collagen deposition in aMRCs. Figure 13C shows the results of immunohistochemical staining confirming increased expression of type 1 collagen and alpha-SMA protein in aMRCs. Figure 13D shows the results of drNK confirming the superior killing ability of aMRC compared to control NK92 cells.

[0032] Figure 14 shows the inhibitory effect of drNK-conditioned medium on the pro-fibrotic activity of human lung fibroblasts. Figure 14A shows the results of analyzing the gene expression of fibrosis markers COL1A1, COL3A1, ACTA2, MYH9, MYH10, MYH14, and MMP2 in human lung fibroblasts (MRC-5) cultured in the presence or absence (+ / -) of TGF-β or NK-CM. It was confirmed that the gene expression of COL1A1, COL3A1, ACTA2, MYH9, MYH10, MYH14, and MMP2 was significantly reduced in the group treated with drNK-conditioned medium (CM-drNK) compared to the control group (CM-pNK). Figure 14B shows the results of immunohistochemical staining in human lung fibroblasts (MRC-5) cultured in the presence (+ / -) of TGF-β or NK-CM, confirming the superior inhibitory effect of CM-drNK on type 1 collagen and alpha-SMA protein expression compared to the control group (pNK-CM). Figure 14C shows the results of Sirius red collagen staining in human lung fibroblasts cultured in the presence (+ / -) of TGF-β or NK-CM, confirming the superior inhibitory effect of CM-drNK on collagen deposition / fibrosis compared to the control group (pNK-CM). Figure 14D shows the results of confirming the superior inhibitory effect of CM-drNK on aMRC-5 cell proliferation compared to the control group (pNK-CM).

[0033] Figure 15 shows the killing ability of drNK in human cardiac fibroblasts (IM-HCFs) that induce cardiac fibrosis. Figure 15A shows the results of RT-qPCR confirming the increased expression of fibrosis markers COL1A1, TIMP1, and ACTA2 mRNA in human cardiac fibroblasts (aIM-HCFs) activated by TGF-β treatment compared to inactivated quiescent IM-HCFs (qIM-HCFs). Figure 15B shows the results of Picro sirius red staining confirming the increased collagen deposition in aIM-HCFs. Figure 15C shows the results of immunohistochemical staining confirming the increased expression of type 1 collagen and alpha-SMA protein in aIM-HCFs. Figure 15D shows the results of confirming the superior killing ability of drNK compared to control NK92 cells in aIM-HCFs.

[0034] Figure 16 shows the inhibitory effect of drNK-conditioned medium on the pro-fibrotic activity of human cardiac fibroblasts. Figure 16A shows the results of analyzing the gene expression of fibrosis markers COL1A1, CILP1, THBS4, VEGF-A, and MMP9 through RT-qPCR in human cardiac fibroblasts (IM-HCF) cultured in the presence (+ / -) of TGF-β or NK-CM. It was confirmed that the gene expression of COL1A1, CILP1, THBS4, VEGF-A, and MMP9 was significantly reduced in the group treated with drNK-conditioned medium (CM-drNK) compared to the control group (CM-pNK). Figure 16B shows the results of confirming the superior inhibitory effect of CM-drNK on type 1 collagen and alpha-SMA protein expression compared to the control group (pNK-CM) by immunohistochemical staining in human cardiac fibroblasts (IM-HCF) cultured in the presence (+ / -) of TGF-β or NK-CM. Figure 16C shows the results of Picro sirius red collagen staining in human cardiac fibroblasts (IM-HCF) cultured in the presence or absence (+ / -) of TGF-β or NK-CM, confirming the superior collagen deposition / fibrosis inhibition effect of CM-drNK compared to the control group (pNK-CM). Figure 16D shows the results of confirming the superior aIM-HCF cell proliferation inhibition effect of CM-drNK compared to the control group (pNK-CM).

[0035] Figure 17 shows the killing ability of drNK to induce skin fibrosis in human dermal fibroblasts. Figure 17A shows the results of RT-qPCR confirming increased expression of fibrosis markers COL1A1, COL3A1, and ACTA2 in human dermal fibroblasts (aHDFs) activated by TGF-β treatment compared to control quiescent HDFs (qHDFs). Figure 17B shows the results of Picro sirius red staining confirming increased collagen deposition in aHDFs. Figure 17C shows the results of immunohistochemical staining confirming increased expression of type 1 collagen and alpha-SMA protein in aHDFs. Figure 17D shows the results of confirming the superior killing ability of drNK compared to control NK92 cells in aHDFs.

[0036] Figure 18 shows the inhibitory effect of drNK-conditioned medium on pro-fibrotic activity in human dermal fibroblasts (HDFs). Figure 18A shows the results of RT-qPCR analysis of the gene expression of fibrosis markers COL1A1, COL3A1, and ACTA2 in human dermal fibroblasts (HDFs) cultured in the presence (+ / -) of TGF-β or NK-CM. It was confirmed that the gene expression of fibrosis markers COL1A1, COL3A1, and ACTA2 was significantly reduced in the group treated with drNK-conditioned medium (CM-drNK) compared to the control group (CM-pNK). Figure 18B shows the results of immunohistochemical staining in aHDFs, confirming the excellent inhibitory effect of CM-drNK on type 1 collagen and alpha-SMA protein expression compared to the control group (pNK-CM). Figure 18C shows the results of Picro sirius red collagen staining in human dermal fibroblasts (HDFs) cultured in the presence or absence (+ / -) of TGF-β or NK-CM, confirming the superior collagen deposition / fibrosis inhibition effect of CM-drNK compared to the control group (pNK-CM). Figure 18D shows the results of confirming the superior aHDF cell proliferation inhibition effect of CM-drNK compared to the control group (pNK-CM).

[0037] Figure 19 shows the killing ability of drNK on human renal tubular epithelial cells that induce renal fibrosis. Figure 19A shows the results of RT-qPCR confirming an increase in the expression of fibrosis markers COL1A1, COL3A1, and ACTA2 in human renal tubular epithelial cells (aHK-2) activated by TGF-β treatment compared to the control quiescent HK-2 (qHK-2). Figure 19B shows the results of Picro sirius red staining confirming an increase in collagen deposition in aHK-2. Figure 19C shows the results of immunohistochemical staining confirming an increase in the expression of type 1 collagen and alpha-SMA protein in aHK-2. Figure 19D shows the results of confirming the superior killing ability of drNK in aHK-2 compared to the control NK92 cells.

[0038] Figure 20 shows the inhibitory effect of drNK-conditioned medium on pro-fibrotic activity in human renal tubular epithelial cells (aHK-2). Figure 20A shows the results of RT-qPCR analysis of gene expression of fibrosis markers COL3A1, ACTA2, and PAI-1 in HK-2 cultured in the presence (+ / -) of TGF-β or NK-CM. It was confirmed that the gene expression of fibrosis markers COL3A1, ACTA2, and PAI-1 was significantly reduced in the group treated with drNK-conditioned medium (CM-drNK) compared to the control group (CM-pNK). Figure 20B shows the results of immunohistochemical staining in aHK-2 confirming the excellent inhibitory effect of CM-drNK on type 1 collagen and alpha-SMA protein expression compared to the control group (pNK-CM). Figure 20C shows the results of Picro sirius red collagen staining in HK-2 cultured in the presence or absence (+ / -) of TGF-β or NK-CM, confirming the superior collagen deposition / fibrosis inhibition effect of CM-drNK compared to the control group (pNK-CM). Figure 20D shows the results of confirming the superior a HK-2 cell proliferation inhibition effect of CM-drNK compared to the control group (pNK-CM).

[0039] Figure 21 shows the results of quantitative comparative analysis of cytokine gene expression levels in NK-92, iPS-NK, and drNK using qPCR. Ten cytokines (CCL5, IFN-γ, CXCL11, CXCL12, GDNF, VEGF, XCL1, IL16, LIF, LTB) that were highly expressed in drNK compared to the control group NK-92 and iPS-NK were identified.

[0040] Figure 22 is CD56 dim The results of a comparative analysis of the cytokine profiles in conditioned media of pNK and drNK cells using a human proteome cytokine array are shown. Figure 22A is a graph showing 56 secreted proteins identified in the conditioned media of drNK cells. Figure 22B shows CD56. dim This graph shows 28 secreted proteins that were increased in drNK conditioned medium compared to pNK cell conditioned medium. Figure 22C shows the results of confirming the proteins DPP4, M-CSF, and BDNF that were specifically identified in drNK cell conditioned medium.

[0041]

[0042] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below.

[0043]

[0044] One aspect of the present invention for achieving the above object provides a pharmaceutical composition for preventing or treating fibrosis, comprising directly reprogrammed natural killer (drNK) cells expressing at least one selected from the group consisting of CD56superbright, CD16superbright, and combinations thereof.

[0045] The present inventors have first demonstrated that the composition has an excellent effect in inhibiting fibrosis by significantly reducing collagen production, activity, and viability when applied to a fibrotic cell model.

[0046] In particular, the induced natural killer (drNK) cells of the present invention have higher expression of NK cell activating receptors such as CD69, NKG2D, DNAM-1, and NKp46 compared to the control NK, and 10 types of cytokine / chemokine genes overexpressed in the drNK of the present invention were confirmed, and 3 types of proteins specifically identified only in drNK were confirmed.

[0047] Furthermore, when directly reprogrammed natural killer (drNK) cells expressing at least one selected from the group consisting of CD56superbright, CD16superbright, and a combination thereof of the present invention were treated with hepatic stellate cells (liver fibrosis cell model), alveolar epithelial cells (lung fibrosis cell model), cardiac cells (cardiac fibrosis cell model), skin cells (skin fibrosis cell model), and kidney cells (kidney fibrosis cell model), which are fibrosis origin cells associated with the induction of fibrosis response in each tissue, the direct killing ability was superior to that of the previously known control natural killer (NK) cells, and further, when a conditioned medium (CM) for the drNK cells was treated to the fibrosis cell model, the effect of inhibiting fibrosis was confirmed by significantly reducing collagen production, activity, and viability compared to the previously known control natural killer (NK) cells.

[0048] This suggests that the composition of the present invention is useful for preventing or treating various fibrotic diseases when treating drNK of the present invention compared to previously known NK cells.

[0049]

[0050] Specifically, the induced natural killer cells of the present invention may be produced by direct reprogramming of isolated cells.

[0051] As a specific example, the induced natural killer cells may be produced by a method including, but not limited to, (a) a step of introducing a reprogramming factor into isolated cells; (b) a step of culturing the cells of step (a) in a first medium containing i) cytokines, growth factors, and GSK3β (Glycogen synthase kinase 3β) inhibitors to increase the efficiency of direct reprogramming starting from the next day after the introduction of the reprogramming factors; and (ii) a step of culturing the cells in a second medium containing cytokines, growth factors, and AHR (Aryl hydrocarbon receptor) inhibitors to promote the production of natural killer cells.

[0052] In the present invention, step (a) is a step of introducing one or more reprogramming factors into (i) separated cells.

[0053] The term "isolated cell" in the present invention has no particular limitations, but specifically may be a cell whose lineage has already been determined, such as a germ cell, somatic cell, or progenitor cell. For example, it may be a cell derived from a human, but cells derived from various individuals also fall within the scope of the present invention.

[0054] The isolated cells may be derived from humans, but are not limited thereto, and cells derived from various organisms may also fall within the scope of the present invention. Furthermore, the isolated cells of the present invention may include both in vivo and ex vivo cells.

[0055] Additionally, the specifically separated cells may be somatic cells other than natural killer cells.

[0056] In the present invention, the term "reprogramming factor" refers to a gene (or a polynucleotide encoding the same) or protein that can be introduced into a cell to induce reprogramming. The reprogramming factor may vary depending on the target cell in which reprogramming is to be induced and the type of isolated cell in which reprogramming is to be induced. For example, in the case of producing natural killer cells, the reprogramming factor may include one or more factors selected from the group consisting of Lin28, Asc11, Pitx3, Nurr1, Lmx1a, Nanog, Oct3, Oct4, Sox2, Klf4, and Myc, and may also include any factor known in the art to be capable of producing natural killer cells. In addition, the reprogramming factor can be used to directly induce reprogramming into natural killer cells. In the direct reprogramming methodology, there is a method of using a reprogramming genetic factor, and the vector of the present invention can be utilized for such purposes. Therefore, a person skilled in the art can select an appropriate factor depending on the type of the target cell and the cell before reprogramming, and all factors within the range known in the art are included in the scope of the present invention, and the type is not particularly limited. Since reprogramming using a reprogramming genetic factor regulates the entire gene expression pattern of a cell to induce conversion into a target cell, the initial cell can be reprogrammed into a target cell having a gene expression pattern of a target type of cell by introducing the reprogramming genetic factor into the cell and culturing the cell for a certain period of time. In the present invention, the "reprogramming factor" may be used interchangeably with "direct reprogramming induction factor", "direct cell transformation induction factor", and "cell transformation induction factor".

[0057] In the present invention, the "step of introducing a reprogramming factor" may be a method of increasing the expression level of a reprogramming factor present in a cell, particularly, Oct4, Sox2, Klf4, and c-Myc genes; or a method of increasing the expression level of a reprogramming factor in a cell through an expression vector, genetic modification, introduction of an exogenous expression gene, treatment with a substance having an expression-inducing effect, etc., but is not limited as long as the expression level of the reprogramming factor is increased. In particular, the step of introducing a reprogramming factor may be a method of inducing the expression of the reprogramming factor under a desired time and condition.

[0058] Specifically, the method for introducing the reprogramming factor of step (a) into a cell can be used without limitation in any method commonly used in the art for providing a nucleic acid molecule (DNA or RNA) or protein to a cell. For example, a method of administering the reprogramming factor to a cell culture medium, a method of directly injecting the reprogramming factor into a cell, or a method of transforming a cell using an expression vector containing the gene of the reprogramming factor can be used.

[0059] Any method known in the art can be selected and used for the method of directly injecting the above reprogramming factor into cells, and is not limited thereto, but may be appropriately selected and applied from among microinjection, electroporation, particle bombardment, direct muscle injection, insulator, and transposon-using methods.

[0060] The term "expression vector" of the present invention refers to a genetic construct that is capable of expressing a target protein in a suitable host cell and includes essential regulatory elements operably linked to enable expression of a gene insert.

[0061] The expression vector of the present invention comprises, in addition to expression control elements such as a promoter, operator, initiation codon, termination codon, polyadenylation signal, and enhancer, a signal sequence or leader sequence for membrane targeting or secretion, and can be manufactured in various ways depending on the purpose. The promoter of the expression vector may be constitutive or inducible. In addition, the expression vector comprises a selectable marker for selecting a host cell containing the vector, and, if it is a replicable expression vector, comprises an origin of replication. The expression vector can be self-replicating or can be integrated into host DNA.

[0062] The above expression vector may include, but is not limited to, a viral vector, an episomal vector, a plasmid vector, a cosmid vector, etc.

[0063] Specifically, the viral vector may include a vector derived from a lentivirus, a retrovirus, such as human immunodeficiency virus (HIV), murineleukemia virus (MLV), avian sarcoma / leukosis virus (ASLV), spleen necrosis virus (SNV), rous sarcoma virus (RSV), mouse mammary tumor virus (MMTV), adenovirus, adeno-associated virus, herpes simplex virus, etc. In addition, it may be an RNA-based viral vector more specifically, but is not limited thereto.

[0064] In addition, the episomal vector of the present invention is a non-viral, non-inserting vector known to have the property of being able to express a gene contained in the vector without being integrated into a chromosome. For the purposes of the present invention, a cell containing an episomal vector includes both cases in which the episomal vector is integrated into the genome or exists within the cell without being integrated into the genome. In addition, the episomal vector may include one or more reprogramming factors.

[0065] The term "operably linked" in the present invention refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein so that the nucleic acid expression control sequence performs a general function. The functional linkage with a recombinant vector can be produced using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes generally known in the art.

[0066] Next, step (b) is a step of directly reprogramming the cells of step (a) into natural killer (NK) cells by culturing them in i) a first medium containing cytokines, growth factors, and GSK3β inhibitors, and ii) a second medium containing cytokines and growth factors.

[0067] The term "cytokine," as used herein, refers to a variety of relatively small proteins produced by cells and used in cell signaling, which can affect other cells, including the cell itself. It is generally associated with, but not limited to, immune responses to inflammation or infection. Specifically, the cytokines may include, but are not limited to, IL-2, IL-3, IL-5, IL-6, IL-7, IL-11, IL-15, BMP4, Acivin A, Notch ligand, G-CSF, and SDF-1.

[0068] In the present invention, the term "growth factor" means a polypeptide that promotes division, growth, and differentiation of various cells, and includes, but is not limited to, epidermal growth factor (EGF), platelet-derived growth factor-AA (PDGF-AA), insulin-like growth factor-1 (IGF-1), transforming growth factor-β (TGF-β), or fibroblast growth factor (FGF).

[0069] For the purposes of the present invention, cytokines and growth factors are included in a medium that directly reprograms isolated cells into lineage-converted cells, and are not limited to the types of cytokines and growth factors as long as they are used for direct reprogramming.

[0070] In the present invention, the term "GSK3β inhibitor" refers to a substance that inhibits or suppresses the activity of GSK3β, one of the two isoforms of glycogen synthase kinase-3 (GSK3), and may include lithium, SB216763, CHIR-98014, TWS119, AR-A014418, etc., and may be specifically CHIR99021, but is not limited thereto.

[0071] The term "cultivation" in the present invention refers to growing cells under appropriately controlled environmental conditions. The culturing process of the present invention can be performed using appropriate media and culture conditions known in the art. These culturing processes can be easily adjusted and used by those skilled in the art depending on the selected cells.

[0072] In particular, since step (b) is a process of culturing cells into which reprogramming factors have been introduced in a first medium and a second medium, the composition of the first medium and the second medium for culturing cells has a composition suitable for directly reprogramming cells into which reprogramming factors have been introduced into natural killer cells, and specifically, the first medium may include cytokines, growth factors, and GSK3β inhibitors, and the second medium may include cytokines and growth factors, but is not limited thereto.

[0073] The term "medium" in the present invention means a known medium used in culturing cells, and encompasses all known cell culture media or modified media thereof.

[0074] The term "StemRegenin I" in the above second medium refers to (4-(2-(2-(Benzo[b]thiphen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol hydrochloride) as an aryl hydrocarbon receptor inhibitor. The above StemRegenin I can be additionally included together with interleukin 7 and interleukin 15 in addition to the cytokines, growth factors, and GSK3β inhibitors of the first medium to increase the efficiency of direct reprogramming.

[0075] Specifically, the first medium and the second medium increase the efficiency of direct reprogramming to promote natural killer cell production. The first medium may include cytokines, growth factors, and GSK3β inhibitors, specifically, but not limited to, FBS, CHIR99021, IL-3, IL-6, SCF, FLT3L, and TPO. In addition, the second medium may include cytokines, growth factors, and StemRegenin I, specifically, but not limited to, FBS, IL-2, IL-7, IL-15, SCF, FLT3L, and StemRegenin I. However, the medium composition is not limited thereto as long as it increases the efficiency of direct reprogramming.

[0076]

[0077] In the present invention, the term "reprogramming" refers to a method of converting a specific cell into a desired cell by controlling the global gene expression pattern, etc. thereof. In other words, reprogramming in the present invention refers to a method of converting a cell into a cell with completely different characteristics by artificially manipulating the fate of a cell, and for the purpose of the present invention, the reprogramming may be performed by introducing a vector containing a foreign gene or DNA into the cell. For example, reprogramming may include, but is not limited to, cell dedifferentiation, direct reprogramming (direct conversion), or direct transdifferentiation.

[0078] In the present invention, the term "direct reprogramming" is differentiated from the technology of producing induced pluripotent stem cells with pluripotency through a reprogramming process, and is a technology that directly induces conversion into the desired target cell through reprogramming culture. In order to produce the target cell, a natural killer cell, using the existing induced pluripotent stem cell reprogramming technology, induced pluripotent stem cells must first be produced from isolated somatic cells, and then hematopoietic stem (progenitor) cells, which are intermediates, must be differentiated and produced. This complex production and culture process, in which hematopoietic stem (progenitor) cells, which are differentiated from induced pluripotent stem cells, must then be differentiated and produced into the final target cell, a natural killer cell, must be sequentially produced, has the disadvantages of low production efficiency and high time and cost consumption. Furthermore, because it is produced via pluripotent stem cells, the presence of undifferentiated cells and ensuring safety are important issues that must be verified. However, the present invention is expected to provide an alternative that can overcome the problems of the above technology, such as production time, cost, efficiency, and safety, by directly producing target cells, natural killer cells, from initial cells through direct reprogramming technology. For the purposes of the present invention, direct reprogramming may be used interchangeably with direct dedifferentiation, direct differentiation, direct conversion, direct cross-differentiation, and cross-differentiation. In the present invention, direct reprogramming may specifically mean direct dedifferentiation or cross-differentiation into natural killer cells.

[0079] As used herein, the term "differentiated cell" refers to a cell with a specialized structure or function. In other words, it refers to a state in which cells, tissues, etc. of a living organism have changed into a form and function suitable for performing their respective roles. For example, ectodermal, mesodermal, and endoderm cells derived from pluripotent stem cells, such as embryonic stem cells, are differentiated cells, and more specifically, erythrocytes, leukocytes, and platelets derived from hematopoietic stem cells are also differentiated cells.

[0080] In the present invention, the term "intermediate cell" or "natural killer precursor cell" refers to a cell at a stage prior to differentiation into a natural killer cell, which may be a lymphoid stem cell, but is not limited thereto as long as it has the potential to differentiate into a natural killer cell.

[0081] The term "natural killer (NK) cell" of the present invention is a key innate immune cell that immediately recognizes and eliminates infections by viruses, bacteria, fungi, and parasites, as well as abnormal self-cells. Unlike T cells that recognize target cells by expressing antigen-specific receptors, NK cells recognize abnormal changes in target cells (particularly, cancer cells and infected cells), such as the balance of inhibitory or activating receptors such as killer immunoglobulin receptors (KIR), natural cytotoxicity receptors (NCR), DNAM-1 (DNAX accessory molecule-1), and NKG2D (NK group 2 member D), and the loss of surface MHC (Major histocompatibility complex) class I antigens, without specificity for antigens and human leukocyte antigen (HLA) matching, and exhibit contact-dependent cytotoxicity through various mechanisms. Unlike T cells, which can cause graft-versus-host disease (GVHD) against non-self allogeneic cells with mismatched human leukocyte antigen (HLA), allogeneic NK cells have been shown to have little side effects of graft-versus-host disease and rather a strong therapeutic effect.

[0082] For the purpose of the present invention, natural killer cells are those obtained through direct reprogramming, and can be used interchangeably with induced natural killer (drNK) cells.

[0083] Another specific aspect of the present invention provides induced natural killer (drNK) cells produced by the above method. The induced natural killer cells produced by the method of the present invention may express at least one selected from the group consisting of CD56superbright, CD16superbright, and combinations thereof, but are not limited thereto.

[0084] In another specific example, the induced natural killer (drNK) cells may overexpress any one or more genes selected from the group consisting of CCL5, IFN-γ, CXCL11, CXCL12, GDNF, VEGF, XCL1, IL16, LIF, and LTB compared to a control, but are not limited thereto.

[0085] In another specific example, the induced natural killer (drNK) cells may express one or more proteins selected from the group consisting of, but not limited to, DDP4, M-CSF, and BDNF.

[0086] Additionally, specifically, the composition may further comprise NKG2D or CCR5, which are substances secreted from induced natural killer (drNK) cells.

[0087] In one specific example of the present invention, to compare the drNK of the present invention with the existing NK, fresh primary NK (pNK) cells isolated from PBMC, pNK cells activated with IL-2 and IL-15 (ApNK) cells, or NK cell line (NK92, ATCC) were used as controls. According to the expression of CD56 and CD16 markers, the main cell populations were drNK: CD56 superbright CD16 superbright, NK92: CD56 bright CD16 dim , pNK(CD56 dim pNK): CD56 dim CD16 bright , ApNK(CD56 bright pNK): CD56 bright CD16 bright NK cell phenotypes were distinguished.

[0088]

[0089] In another specific example of the present invention, the gene expression of cytokines / chemokines expressed in the drNK of the present invention was compared with that of the control NK, and as a result, 10 types (CCL5, IFN-γ, CXCL11, CXCL12, GDNF, VEGF, XCL1, IL16, LIF, LTB) that were overexpressed were confirmed, and 3 types of proteins (DDP4, M-CSF, BDNF) that were confirmed to be drNK-specific were identified.

[0090]

[0091] The term "fibrosis" of the present invention refers to the development of excessive fibrous connective tissue within an organ or tissue. Fibrosis is characterized by the accumulation and remodeling of the extracellular matrix (ECM). Despite clear etiological and clinical differences, most chronic fibrotic disorders share a persistent stimulus that sustains the production of growth factors, proteolytic enzymes, angiogenic factors, and fibrogenic cytokines. These factors, together, stimulate the deposition of connective tissue elements, particularly collagen and proteoglycans, resulting in the ongoing remodeling and destruction of normal tissue architecture.

[0092] In the present invention, the fibrotic disease may specifically occur in one or more selected from the group consisting of the lungs, kidneys, liver, heart, brain, blood vessels, joints, intestines, skin, soft tissue, bone marrow, penis, peritoneum, muscles, spine, testes, ovaries, breasts, thyroid, eardrums, pancreas, gallbladder, bladder, prostate, esophagus, stomach, uterus, spleen, lymph nodes, salivary glands, and nervous system, but is not limited thereto.

[0093] The term "prevention" of the present invention means any act of inhibiting or delaying the occurrence of fibrosis by using a pharmaceutical composition for preventing or treating fibrosis, which includes induced natural killer cells expressing at least one selected from the group consisting of CD56superbright, CD16superbright, and combinations thereof.

[0094] The term "treatment" of the present invention means any act of controlling or alleviating symptoms of fibrosis by using a pharmaceutical composition for preventing or treating fibrosis, which includes induced natural killer cells expressing at least one selected from the group consisting of CD56superbright, CD16superbright, and combinations thereof.

[0095] The term "pharmaceutical composition" of the present invention may include a pharmaceutically acceptable carrier, and may be formulated in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods.

[0096] The pharmaceutically acceptable carrier may include, but is not limited to, those commonly used in the art, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, the pharmaceutical composition of the present invention may include diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants, and other pharmaceutically acceptable additives.

[0097] When the pharmaceutical composition of the present invention is formulated as an oral solid preparation, it may include tablets, pills, powders, granules, capsules, etc., and such solid preparation may include at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., and may include a lubricant, such as magnesium stearate or talc, but is not limited thereto.

[0098] When the pharmaceutical composition of the present invention is formulated as an oral liquid, it may include a suspension, a solution, an emulsion, a syrup, etc., and may include a diluent such as water or liquid paraffin, a wetting agent, a sweetener, a fragrance, a preservative, etc., but is not limited thereto.

[0099] When the pharmaceutical composition of the present invention is formulated for parenteral use, it may include a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, or a suppository. Non-aqueous solvents and suspensions include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include, but are not limited to, witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

[0100] In the present invention, the dosage of the pharmaceutical composition varies depending on the patient's condition, weight, age, degree of disease, drug form, administration route, and period, but can be appropriately selected by a person skilled in the art.

[0101] The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans by various routes, for example, orally, intraperitoneally, intravenously, intramuscularly, subcutaneously, intrauterinely, or intracerebrovascularly.

[0102] In one specific example of the present invention, the anti-fibrotic effect of the drNK of the present invention was confirmed in a NASH-induced MCD feeding mouse model. As a result, it was confirmed that compared to the previously known NK, body weight increased normally, liver weight loss was suppressed, and liver damage indicators and steatosis alleviation effects were excellent. In addition, it was confirmed that compared to the previously known NK, the expression of liver fibrosis markers was suppressed, the expression of inflammatory markers was suppressed, and the expression of anti-inflammatory markers was enhanced. Through this, it can be seen that the composition including the drNK of the present invention is effective in preventing or treating liver fibrosis.

[0103] In another specific embodiment of the present invention, the anti-fibrotic effect of the drNK of the present invention was confirmed in human lung, heart, skin, or kidney fibrosis cell models, and it was confirmed that the drNK of the present invention exhibited superior direct killing ability compared to previously known NKs and suppressed the expression of fibrosis markers. This indicates that the composition comprising the drNK of the present invention is effective in the prevention or treatment of lung, heart, skin, or kidney fibrosis.

[0104] This suggests that the induced natural killer (drNK) cells of the present invention are effective in treating fibrotic diseases.

[0105]

[0106] Another aspect of the present invention for achieving the above object provides a cell therapy for preventing or treating fibrosis, comprising induced natural killer (drNK) cells expressing at least one selected from the group consisting of CD56superbright, CD16superbright, and combinations thereof.

[0107] The terms used herein are as described above.

[0108] The term "cell therapy product" of the present invention refers to a medicine (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention by separating, culturing, and manufacturing cells and tissues from an individual through special manipulation, and by proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro to restore the function of cells or tissues, or by changing the biological characteristics of cells through other methods.

[0109] The above cell therapy composition may have an effect of preventing or treating fibrosis by including induced natural killer cells prepared according to the method of the present invention.

[0110] The above cell therapy composition contains 1.0X10 to 1.0X10 of the induced natural killer cells based on the total weight of the composition. 10 Dog cells / ml, specifically 1.0X10 6 1.0X10 9 May include, but is not limited to, dog cells / ml.

[0111] The above cell therapy composition can be formulated into a unit dosage form pharmaceutical preparation suitable for administration into the patient's body according to a conventional method in the pharmaceutical field, and the preparation can contain an effective dosage amount through one or multiple administrations. Suitable dosage forms for this purpose include parenteral administration preparations such as injections such as injection ampoules, infusions such as infusion bags, and sprays such as aerosol preparations. The injection ampoules can be mixed and prepared with an injection solution immediately before use, and the injection solution can be physiological saline, glucose, mannitol, Ringer's solution, etc. In addition, the infusion bag can be made of polyvinyl chloride or polyethylene, and examples thereof include infusion bags from Baxter, Becton Dickinson, Medcep, National Hospital Products, and Terumo.

[0112] In addition to the active ingredient, the above pharmaceutical preparation may further include one or more pharmaceutically acceptable conventional inert carriers, for example, in the case of injections, a preservative, analgesic, solubilizer, or stabilizer, and in the case of topical administration preparations, a base, excipient, lubricant, or preservative.

[0113] The cell therapy composition of the present invention manufactured in this way or the pharmaceutical preparation thereof can be administered together with other cells used for the treatment of fibrosis or in the form of a mixture with such cells using an administration method commonly used in the art, and specifically, it can be directly engrafted or transplanted into the diseased site of a patient requiring treatment, or directly transplanted or injected into the abdominal cavity, but is not limited thereto. In addition, the administration can be both non-surgical administration using a catheter and surgical administration methods such as injection or transplantation after incision of the diseased site. In addition to parenteral administration according to a conventional method, for example, direct administration to the lesion, transplantation by intravascular injection is also possible.

[0114] The above cell therapy composition may be administered at a dosage of 0.0001 to 1,000 mg / kg per day, specifically 0.01 to 100 mg / kg, and the administration may be administered once a day or in several divided doses. However, it should be understood that the actual dosage of the active ingredient should be determined in light of various related factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age, and sex, and therefore, the above dosage does not limit the scope of the present invention in any way.

[0115]

[0116] One aspect of the present invention for achieving the above object provides a method for preventing or treating fibrosis, comprising administering to a subject other than a human suspected of having fibrosis a pharmaceutical composition for preventing or treating fibrosis comprising drNK (Directly reprogrammed natural killer) cells expressing at least one selected from the group consisting of CD56superbright, CD16superbright, and combinations thereof.

[0117] The terms used herein are as described above.

[0118] The above "administration" means introducing the composition of the present invention into a subject by any suitable method, and the route of administration of the composition may be through any common route as long as it can reach the target tissue. It may be intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, or intranasal administration, but is not limited thereto.

[0119] The above "subject" refers to any animal, including monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs, excluding humans, that has developed or may develop fibrosis. The type of subject is not limited as long as the disease can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the subject.

[0120]

[0121] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and are not to be construed as limiting the scope of the present invention.

[0122]

[0123] Example 1. Conceptual diagram of the antifibrotic effect of induced natural killer (drNK) cells.

[0124] By comparing and analyzing the antifibrotic effects of the induced natural killer cells and the induced natural killer cell conditioned medium prepared through reprogramming culture from the isolated somatic cells of the present invention and the existing NK cells and the existing NK cell conditioned medium using human tissue-specific fibrotic cell models (1. hepatic stellate cell-based hepatic fibrotic cell model, 2. alveolar epithelial cell-based pulmonary fibrotic cell model, 3. cardiac cell-based cardiac fibrotic cell model, 4. skin fibroblast-based skin fibrotic cell model, 5. renal tubular epithelial cell-based renal fibrotic cell model), the excellent antifibrotic effect of the induced natural killer cells of the present invention was confirmed.

[0125]

[0126] Example 2. Cell phenotypic characteristics of the induced natural killer cells of the present invention

[0127] To obtain drNK cells, reprogramming factor OSKM was introduced into PBMCs separated from blood by Ficoll gradient, and then PBMC cells and polybrene (4 μg / ml) were cultured together for 1 day. The next day, 3x10 5 The transformed cells were cultured for an additional 5 days in culture medium RIM (StemSpan SFEM II containing 10% FBS, 1% Penicillin / Streptomycin, 5 μM CHIR99021, 20 ng / ml Human IL-3, 20 ng / ml Human IL-6, 20 ng / ml Human SCF, 20 ng / ml Human FLT3L, 20 ng / ml Human TPO). The cells were cultured for 18-40 days in culture medium RMM (StemSpan SFEM II containing 10% FBS, 1% Penicillin-Streptomycin, 200 IU / ml Human IL-2, 20 ng / ml Human IL-7, 20 ng / ml Human IL-15, 20 ng / ml Human SCF, 20 ng / ml Human FLT3L, 2 uM StemRegenin I). To confirm whether drNK cells were produced through the above direct reprogramming, the cells were stained with anti-CD56-APC (Biolegend) antibody and anti-CD16-PE (Biolegend) antibody as NK cell markers, and then NK cells (CD56) were analyzed using flow cytometry. + and CD16 + ) group was analyzed.

[0128] As control cells, fresh primary NK (pNK; CD56) isolated from PBMC dimpNK) cells, and pNK cells activated with 200 IU / ml Human IL-2 and 20 ng / ml Human IL-15 for 4–14 days (Cytokine activated pNK; CD56 bright To produce pNK cells, NK cell line (NK92, ATCC), and iPS-NK (iPS-NK) cells, iPS cells were dissociated into single cells by ReLeSR (Stem cell technologies) treatment and added to STEMdiff APEL2 medium (Stem cell technologies), a medium composition for spindle embryoid bodies, containing 1x Penicillin-Streptomycin (Invitrogen), 40 ng / ml SCF (Invitrogen), 20 ng / ml VEGF (R&D), and 20 ng / ml BMP4 (R&D), and 3 X 10 4Cells suspended in cells / ml were seeded at 3,000 cells per well in a round-bottom 96-well plate, centrifuged at 1,500 rpm for 4 minutes, and cultured in a 37°C incubator for 3 to 4 days. Afterwards, half of the culture medium was replaced with new culture medium and cultured for 9 to 11 days. On day 9 to 11 of the differentiation of the spindle embryoid bodies, the spindle embryoid bodies from 6 to 8 wells of a 96-well plate were transferred to 1 well of a 24-well plate coated with 2% gelatin, and the differentiation culture medium was 85% DMEM / F12 (GIBCO), 15% FBS (GIBCO), 5 ng / ml Sodium selenite (Sigma), 50 μM Ethanolamine (Sigma), 20 μg / ml Ascorbic acid (Sigma), 25 μM β-mercaptoethanol (GIBCO), 1x Glutamax (GIBCO), 1% Penicillin-Streptomycin (GIBCO), and 5 ng / ml IL-3 (Peprotech), 10 ng / ml IL-15 (Peprotech), 20 ng / ml IL-7 (Peprotech), 20 ng / ml SCF (Invitrogen), 10 ng / ml Flt3L. (Peprotech) cytokines were added and cultured, and the cells cultured for 28 days with NK differentiation medium supplemented with cytokines except IL-3 every 5 to 7 days were differentiated iPS-NK. Pure iPS-NK was isolated using NK isolation kit (Miltenyi Biotec), and iPS-NK cultured in culture medium containing 90% RPMI 1640, 10% FBS, 1% Penicillin-Streptomycin, 20 ng / ml IL-15, and 20 ng / ml IL-2 was used. The main cell groups were divided into drNK: CD56 superbright CD16 superbright, NK92: CD56 according to the expression of CD56 and CD16 markers, respectively. brightCD16 dim , pNK (CD56 dim pNK): CD56 dim CD16 bright , Cytokine activated pNK (CD56 bright pNK): CD56 bright CD16 bright , iPS-NK: CD56 bright CD16 dim It was confirmed that NK cell phenotypes were distinguished (Figures 2A and 2B). Here, the most commonly used pNK (CD56 dim Based on the intensity of CD56 and CD16 fluorescence of pNK (dim (10 4 below), bright (10 4 -10 5 ), superbright (10 5 It is indicated as (above).

[0129]

[0130] Example 3. drNK's ability to kill hepatic stellate cells, which cause liver fibrosis

[0131] The LX-2 human hepatic stellate cell line was used as a cell model. Quiescent LX-2 cells (qHSC) were cultured in DMEM medium containing 2% FBS and treated with 20 ng / ml recombinant human TGF-β1 protein (rh TGF-β1 protein) for 48 hours to induce an activated state, producing activated HSC (aHSC). As fibrosis progressed in aHSC, high intracellular protein expression levels of collagen type I alpha 1 (COL1A1) and α-smooth muscle actin (α-SMA), which develop in the developed extracellular matrix (ECM), were confirmed using immunocytochemistry (ICC). When the results were quantified by converting them into the density of fluorescence expression (Integrated density, IntDen) using the Image J (NIH) program, the expression of COL1A1 and α-SMA increased 5.77-fold and 8.83-fold, respectively, in aHSCs compared to qHSCs, verifying their significance as a liver fibrosis cell model (Fig. 3A).

[0132] In addition, the degree of collagen development was confirmed using a Picro sirius red staining kit (Abcam), and the concentration of red staining was darker compared to qHSC, confirming that the degree of development into collagen connective tissue was high (Fig. 3B).

[0133] To determine the killing activity of NK cells against activated human hepatic stellate cells (aHSCs) treated with TGF-β, aHSCs, drNKs, and control CD56 were cultured in 24-well plates. dim pNK, CD56 bright pNK, NK92, and iPS-NK cells were cultured at a cell number of 2 x 10 5) were reacted 1:1 and after 5 hours of reaction, Propidium iodide (PI) was stained and analyzed by flow cytometry. As a result, the control group (CD56 dim pNK, CD56 bright The killing capacity of induced natural killer cells (pNK, NK92, iPS-NK) compared to CD56 dim 3.63 times higher than pNK, CD56 bright The excellent direct killing ability of induced natural killer cells was confirmed to be 2.98 times higher than pNK, 2.60 times higher than NK92, and 3.19 times higher than iPS-NK (Fig. 3C).

[0134]

[0135] Example 4. Antifibrotic effect of drNK

[0136] 4-1. Antifibrotic effect of drNK dependent on CD56 and CD16 expression

[0137] To analyze the antifibrotic effect of drNK cells dependent on CD56 and CD16 expression, cytotoxicity assays using anti-CD56 and anti-CD16 antibodies were performed (Fig. 4A). To analyze the direct correlation between aHSC cytotoxicity and CD56 expression, 2 × 10 5 After reacting 1 μg / ml antibody-CD56 on aHSC cells for 30 minutes, the reacted aHSCs were washed with culture medium at least once to prepare. 2 x 10 5 Cell counts of drNK and control CD56 dim pNK, CD56 bright pNK, NK92, and iPS-NK cells were reacted 1:1 for 5 hours, stained with Propidium iodide (PI), and analyzed for killing capacity using flow cytometry. As a result, the killing capacity of anti-CD56-treated aHSCs was significantly higher than that of the anti-CD56-untreated control group (100%) for each cell. dim pNK (88.0%), CD56bright It was confirmed that the aHSC killing ability of induced natural killer cells was most dependently related to CD56 expression (Fig. 4B).

[0138] To analyze the direct correlation between aHSC cytotoxicity and CD16 expression, 2 x 10 5 After reacting 1 μg / ml antibody-CD16 with aHSC cells for 30 minutes, the reacted aHSCs were washed with culture medium at least once to prepare. 2 x 10 5 Cell counts of drNK and control CD56 dim pNK, CD56 bright pNK, NK92, and iPS-NK cells were reacted 1:1 for 5 hours, stained with Propidium iodide (PI), and analyzed for killing capacity using flow cytometry. As a result, the killing capacity of anti-CD16-treated aHSCs was significantly higher than that of the anti-CD16-untreated control group (100%) for each cell type. dim pNK (86.21%), CD56 bright It was confirmed that the aHSC killing ability of induced natural killer cells was most dependently related to CD16 expression (Fig. 4C).

[0139]

[0140] 4-2. Antifibrotic effects of drNK dependent on NKG2D expression

[0141] As a result of analyzing the NKG2D expression level by flow cytometry, it was confirmed that drNK of the present invention (86.20%) showed relatively high NKG2D expression characteristics compared to the existing known NK cells such as peripheral blood (pNK, 40.12%), placental (UCB-NK, 55.33%), embryonic stem cell- (ESC-NK, 31.67%), and induced pluripotent stem cell-differentiated (iPS-NK, 36.67%) (Fig. 5A, left). In addition, higher expression of NKG2D ligand MICA was confirmed in activated liver fibrosis-inducing cells aHSC (54.5%) compared to the control inactivated qHSC (6.4%) (Fig. 5A, right). To analyze the antifibrotic effect of drNK cells dependent on NKG2D expression, a cytotoxicity assay using an NKG2D antibody was performed by utilizing the mechanism in which the NKG2D receptor of NK cells binds to the MICA ligand of aHSCs (Fig. 5B). To analyze the direct correlation between aHSC cytotoxicity and NKG2D expression, 2 × 10 5 After reacting 1 μg / ml antibody-NKG2D with aHSC cells for 30 minutes, the reacted aHSCs were washed with culture medium at least once to prepare. 2 x 10 5 Cell counts of drNK and control CD56 dim pNK, CD56 bright After reacting pNK, NK92, and iPS-NK cells at a 1:1 ratio for 5 hours, they were stained with Propidium iodide (PI) and the killing ability was analyzed using flow cytometry.

[0142] As a result, the aHSC killing ability was CD56 in anti-NKG2D treated conditions compared to the anti-NKG2D untreated control (100%) for each cell. dim pNK (88.74%), CD56 brightBy confirming that the superior aHSC killing ability of induced natural killer cells was most dependently related to NKG2D expression, it was confirmed that the aHSC killing ability was reduced to pNK (83.93%), NK92 (93.48%), iPS-NK (99.46%), and drNK (73.33%) (Fig. 5C).

[0143]

[0144] 4-3. Antifibrotic effect of drNK dependent on CCR5 expression

[0145] RT-qPCR was performed to quantitatively analyze the expression of CCR5, a receptor for CCL5 [chemokine (C-C motif) receptor 5], which is known to be increased in relation to liver fibrosis in the drNK cells, compared to the control NK cells (NK-92, pNK, and iPS-NK cells). As a result, when the CCR5 gene expression level in NK-92 cells was set to control group 1, it was confirmed that CCR5 gene expression was relatively higher in drNK (3.75) compared to pNK (0.29) and iPS-NK groups (0.20) (Fig. 6A). In addition, when CCR5 antibody was labeled and analyzed using flow cytometry, it was confirmed that CCR5 was expressed more highly in drNK (93.4%) than in the control pNK (21.9%) (Fig. 6B).

[0146] To analyze the antifibrotic effect of drNK cells dependent on CCR5 expression, a cytotoxicity assay using a CCR5 antibody was performed by utilizing the mechanism in which the CCR5 receptor of NK cells binds to the CCL5 ligand of aHSC, a liver fibrogenic cell (Fig. 6C). To analyze the direct relationship between aHSC cytotoxicity and CCR5 expression, 2 × 10 5 After reacting 1 μg / ml antibody-CCR5 with aHSC cells for 30 minutes, the reacted aHSCs were washed with culture medium at least once to prepare. 2 x 10 5 Cell counts of drNK and control CD56dim pNK, CD56 bright pNK, NK92, and iPS-NK cells were reacted 1:1 for 5 hours, stained with Propidium iodide (PI), and analyzed for killing capacity using flow cytometry. As a result, aHSC killing capacity under anti-CCR5 treatment was significantly higher than that under anti-CCR5 untreated control (100%) for each cell type. dim pNK (94.01%), CD56 bright By confirming that the superior aHSC killing ability of induced natural killer cells was most dependently related to CCR5 expression, it was confirmed that the aHSC killing ability was reduced to pNK (82.13%), NK92 (90.99%), iPS-NK (92.21%), and drNK (86.45%) (Fig. 6D).

[0147] Blood and tissue analyses were performed to analyze the antifibrotic effect of drNK and the association with CCL5-CCR5 in a methionine-choline deficient (MCD) diet-induced NASH (Non-alcoholic steatohepatitis) mouse model of Example 8. The experiment was conducted for a total of 9 weeks, and drNK 1x10 7 The experiment was conducted by administering a dose of 100 mg intravenously (IV). To analyze the cell distribution in the mouse blood, blood was collected from the retro-orbital plexus. The liver and spleen of MCD mice were extracted, and the tissues were lysed with a homogenizer. RNA was extracted and the mRNA expression levels in each tissue were analyzed. When the expression level in the normal group (Control) was set to 1, the expression levels of CCL5 in the liver were Control (1), Fibrosis (2.4), and in the spleen, Control (1), Fibrosis (1.12) were confirmed (Fig. 6E).

[0148] In addition, to confirm the residual pNK and drNK cells in each tissue, the liver and spleen of MCD mice were extracted, and the tissues were lysed using a homogenizer, suspended in DPBS containing 1% BSA, and passed through a cell strainer to prepare a cell suspension. The cell suspension was reacted with ACK lysis buffer at 4°C for 5 minutes, centrifuged at 800 G for 5 minutes, and then suspended in DPBS containing 0.5% BSA and 2 mM EDTA, stained with CD45 antibody, and analyzed using a flow cytometer. As a result, drNK was found to be 1.28 times more residual than pNK in the liver, and drNK was found to be 0.78 times less residual than pNK in the spleen (Fig. 6F). Through this, we confirmed that the infiltration of drNK into liver fibrosis tissue with increased CCL5 expression was increased compared to pNK, and that this was associated with the high CCR5 expression level of drNK (Fig. 6F).

[0149]

[0150] Example 5. Effect of treatment on liver fibrosis

[0151] 5-1. Verification of the superior anti-fibrotic effect of drNK in a fibrotic liver organoid model.

[0152] To analyze the antifibrotic effects of drNK, a human iPSC-derived multicellular liver organoid model mimicking steatohepatitis was prepared. First, human iPSCs were differentiated into definitive endoderm in RPMI 1640 (Gibco) culture medium containing 50 ng / mL recombinant human bone morphogenetic protein-4 (hBMP-4) and 100 ng / mL recombinant human activin A. During this process, the serum concentration was gradually increased from D1: 0%, D2: 0.2%, and D3: 2%, and the culture was continued for 3 days. Foregut spheroids were then formed by culturing for an additional 3 days in Advanced DMEM culture medium containing 500 ng / mL recombinant human fibroblast growth factor-4 (hFGF-4) and 3 μM CHIR99021. Successful induction of the expression of FOXA2 (1:100, anti-FOXA2, #07-633, Millipore) and SOX17 (1:50, anti-SOX17, #MAB1924, R&D) markers was confirmed by immunocytochemistry (Fig. 7A, left / upper). On day 6 of differentiation, Matrigel domes were created and foregut spheroids were embedded, and then 2 μM RA (Retinoic acid) was added to Advanced DMEM culture medium and cultured for up to 10 days to mature the organoids. On day 10 of differentiation, organoids were cultured in William's E basal medium containing 10 ng / mL Hepatocyte growth factor (HGF) and 20 ng / mL Oncostatin M (OSM) and Hepatocyte maintenance supplements (cat. no. CM4000), and the medium was replaced every 3 days until day 20 of differentiation (Fig. 7A, left / bottom).Immunocytochemistry confirmed the expression of markers HNF-4α (1:300, #3113s, cell signaling), α-SMA (1:20, #A5228, sigma), Cytokeratin 18 (1:20, #M7010, DAKO), Desmin (1:50, #AB907, chemicon), and E-cadherin (1:300, #3195, cell signaling), and confirmed the formation of multicellular liver organoids composed of hepatocytes, hepatic epithelial tissue, and stellate cells with a 3D structure of approximately 100 μm in size (Fig. 7A, right).

[0153] Additionally, a fatty liver fibrosis model with fatty acid accumulation (Control) was created by treating the prepared liver organoids with 500 μM sodium oleate for 3 to 5 days. Afterwards, the organoids were co-cultured with NK cells (direct reaction for 24 hours at a ratio of 1 organoid to 100 NK cells) to analyze the antifibrotic effect of drNK. After harvesting the liver organoids cultured under NK cell (+ / -) conditions, they were fixed with 4% paraformaldehyde (PFA) for 2 hours at room temperature, washed with 1X DPBS, and centrifuged at 400 G for 3 minutes. After that, the organoids fixed in 30% sucrose were infiltrated at 4°C for 1 day, frozen blocks were created with a frozen tissue embedding agent (OCT compound), and 15 μm-thick sections were prepared using a cryosectioner (Sakura, Tissue-Tek TEC) for analysis. Fat endoplasmic reticulum was stained with BODIPY 493 / 503 fluorescent dye, cell nuclei were stained with Hoechst 33342, collagen type 1 cell protein was stained with COL1A1 antibody through immunocytochemistry, and cell nuclei were stained with DAPI. In addition, collagen tissue was stained using a Picro sirius red staining kit. As a result, compared to the organoids that induced liver fibrosis (Control), the group reacted with pNK and drNK cells showed a decrease in the expression level of BODIPY, an indicator of liver fibrosis, and a decrease in collagen staining, confirming the anti-fibrotic effect of NK cells (Fig. 7B). In particular, the effect of recovery to normal levels was more remarkable in the group reacted with drNK cells, confirming the excellent anti-fibrotic effect of drNK (Fig. 7B).

[0154]

[0155] 5-2. Antifibrotic effects of drNK in the MCD-fed mouse model (body weight, liver weight)

[0156] The anti-fibrotic effect of natural killer cells was confirmed for a total of 9 weeks in a NASH-induced MCD feeding mouse model. C57BL / 6J mice (average weight 20-25 g) were used, and the experimental groups consisted of a total of 6 groups: Control, MCD only group, MCD+pNK, MCD+drNK, MCD+PolyIC, and MCD+drNK+PolyIC (Fig. 8A). At this time, pNK and drNK were each administered at 1x10 7 Intravenous injection (IV) was performed in a volume of cell count.

[0157] As a result of measuring the body weight and liver weight of each group of mice for 9 weeks, the normal group (Control, approximately 22 g) showed normal body weight increase, and the mice that received the MCD diet showed an alleviation of body weight loss in the order of MCD+drNK+PolyIC (approximately 19 g), MCD+PolyIC (approximately 18 g), MCD+drNK (approximately 19 g), and MCD+pNK (approximately 18 g) compared to the MCD control group (approximately 16 g) (Fig. 8B). This confirmed that in the drNK cell group compared to pNK, the drNK and Poly IC combined transplantation was more effective in suppressing body weight loss than the single transplantation (Fig. 8B). In terms of liver weight, it was analyzed as Control (approximately 1.7 g), MCD (approximately 0.51 g), MCD+pNK (approximately 0.7 g), MCD+drNK (approximately 0.85 g), MCD+PolyIC (approximately 0.78 g), and MCD+drNK+PolyIC (approximately 1.05 g). Compared to pNK, the drNK cell group suppressed liver weight loss more effectively, and drNK and Poly IC complex transplantation showed more effective results than single transplantation (Fig. 8C).

[0158]

[0159] 5-3. Antifibrotic effects of drNK in the MCD-fed mouse model (liver damage indicators, steatosis)

[0160] To confirm the antifibrotic effect of drNK through changes in liver damage markers (ALT, AST) levels in the NASH-induced MCD diet mouse model, blood samples were obtained through orbital blood collection from each group of mice and then analyzed. In the MCD diet group, it was confirmed that ALT and AST levels increased compared to the normal group (Control), and in the group administered NK cells and Poly IC, it was confirmed that liver values ​​decreased relatively compared to the MCD only diet group [ALT group: Control (approximately 10 U / L), MCD only (approximately 407 U / L), M+pNK (approximately 303.3 U / L), M+drNK (approximately 193 U / L), M+PolyIC (approximately 248.3 U / L), M+drNK+PolyIC (204.7 U / L), AST group: Control (approximately 11.33 U / L), MCD only (approximately 344.3 U / L), M+pNK (approximately 253.7 U / L), M+drNK (approximately 161.7 U / L), M+PolyIC (approximately 208.7 U / L), M+drNK+PolyIC (approximately 144.7 U / L)] (Fig. 9A). In particular, drNK was more effective in suppressing liver damage indicators than conventional pNK, and the effect was further enhanced when drNK and Poly IC were administered in combination (Fig. 9A). For histomorphometric analysis, hematoxylin & eosin (H&E), Oil red O, and Masson's trichrome staining were performed (Fig. 9B).

[0161] H&E staining results showed that the control group maintained the structure of the liver lobule well, and there were no morphological changes such as necrosis or inflammation. However, in the MCD diet group, the liver structure was lost and many necrotic forms were observed. This phenomenon was alleviated in the group administered NK cells and Poly IC, and in particular, the group administered drNK cells showed a better effect in suppressing the loss of structure than the existing pNK, and the effect was confirmed to be further increased when administered in combination with drNK cells and Poly IC.

[0162] As a result of Oil red O staining to confirm the degree of intracellular steatosis, fat accumulation was observed in the MCD diet group compared to the control group (red series), and similar to the H&E results, the effect of inhibiting fat accumulation in the drNK cell transplantation group was superior to that of the existing pNK, and the effect was further increased when drNK cells and Poly IC were administered together. The degree of intracellular steatosis was evaluated on a scale of 0 to 3, with 0 being asymptomatic, 1 being mild fat accumulation, 2 being moderate, and 3 being severe, and the scores for each group were added up [control group (0 points), MCD alone (3.8 points), MCD+pNK (2.2 points), MCD+drNK (1.4 points), MCD+PolyIC (1.8 points), MCD+drNK+PolyIC (0.6 points)] (Fig. 9C). In addition, when collagen development was measured (Fibrosis score) through Masson's trichrome staining, drNK was confirmed to have a better effect on inhibiting liver structure loss and steatosis than the control group, pNK, and Poly IC. After staining, the Fibrosis score (%) was measured by converting the area of ​​collagen development into a ratio to the total image area through the image results of each group [Control group (7.33%), MCD alone (54.9%), MCD+pNK (44.2%), MCD+drNK (38%), MCD+PolyIC (40.9%), MCD+drNK+PolyIC (25.2%)] (Fig. 9D). Therefore, it was confirmed that drNK cells showed a better effect than pNK in the anti-fibrotic effect, such as the above liver damage index and steatosis alleviation, and that the combined administration of drNK and Poly IC showed a synergistic effect.

[0163]

[0164] 5-4. Antifibrotic effect of drNK in the MCD-fed mouse model (fibrosis markers)

[0165] The antifibrotic effect of drNK was comparatively analyzed in a NASH-induced MCD diet mouse model through the expression levels of fibrosis markers and extracellular matrix degrading enzymes. Nine weeks after NK cell administration, RNA was extracted from the liver tissues of mice in each group and RT-qPCR was performed. As a result, when the control group was used as the standard (1), α-SMA was increased by MCD alone (4.71 times), M+pNK (3.55 times), M+drNK (1.96 times), M+PolyIC (2.77 times), M+drNK+PolyIC (1.80 times), TGF-β was increased by MCD alone (2.73 times), M+pNK (2.58 times), M+drNK (2.20 times), M+PolyIC (2.07 times), M+drNK+PolyIC (1.95 times), TIMP-1 was increased by MCD alone (7.24 times), M+pNK (5.56 times), M+drNK (4.30 times), M+PolyIC (5.24 times), M+drNK+PolyIC (3.99 times), COL1A1 was increased by MCD. Alone (14.39 times), M+pNK (10.56 times), M+drNK (6.31 times), M+PolyIC (9.10 times), M+drNK+PolyIC (5.98 times), COL3A1 is MCD alone (18.00 times), M+pNK (14.04 times), M+drNK (8.99 times), M+PolyIC (12.69 times), M+drNK+PolyIC (7.39 times), MMP-1 is MCD alone (1.36 times), M+pNK (1.65 times), M+drNK (2.30 times), M+PolyIC (1.71 times), M+drNK+PolyIC (2.42 times), MMP-2 is MCD alone (1.91 times), M+pNK (2.50 times), M+drNK (3.20 times), M+PolyIC (2.41 times), It was confirmed as M+drNK+PolyIC (3.40 times) (Fig. 10). Through this, it was verified that drNK showed a high antifibrotic effect by more effectively suppressing the expression of fibrosis markers (α-SMA, TGF-β, TIMP-1, COL1A1, COL3A1) and promoting the expression of extracellular matrix degrading enzymes (MMP-1, MMP-2) compared to pNK and PolyIC (Fig. 10).

[0166]

[0167] 5-5. Anti-fibrotic effect (inflammation) of drNK in the MCD-fed mouse model

[0168] The anti-fibrotic effect of drNK was analyzed in a NASH-induced MCD diet mouse model through the expression of anti-inflammatory and inflammatory markers. Nine weeks after NK cell administration, RNA was extracted from the liver tissue of each group of mice and RT-qPCR was performed. As a result, when the control group was used as the standard (1), IL-10 was increased by MCD alone (0.25 times), M+pNK (1.30 times), M+drNK (2.55 times), M+PolyIC (0.94 times), M+drNK+PolyIC (2.02 times), IL-11 was increased by MCD alone (1.36 times), M+pNK (2.12 times), M+drNK (2.29 times), M+PolyIC (1.98 times), M+drNK+PolyIC (2.62 times), IL-4 was increased by MCD alone (1.30 times), M+pNK (2.41 times), M+drNK (2.67 times), M+PolyIC (2.07 times), M+drNK+PolyIC (2.88 times), IL-1β was increased by MCD alone. (1.77 times), M+pNK (1.35 times), M+drNK (0.95 times), M+PolyIC (0.91 times), M+drNK+PolyIC (0.77 times), IL-6 was confirmed as MCD alone (2.40 times), M+pNK (1.61 times), M+drNK (1.40 times), M+PolyIC (1.41 times), M+drNK+PolyIC (1.22 times), TNF-α was confirmed as MCD alone (3.22 times), M+pNK (2.28 times), M+drNK (1.49 times), M+PolyIC (1.71 times), M+drNK+PolyIC (1.30 times) (Fig. 11). Through this, it was confirmed that drNK more effectively promoted the expression of anti-inflammatory markers (IL-10, IL-11, IL-4) and had an excellent effect in suppressing the expression of inflammatory markers (IL-1β, IL-6, TNF-α) compared to the control group pNK and Poly IC (Fig. 11).

[0169]

[0170] 5-6. Inhibitory effect of drNK conditioned medium on the activation of fibrotic human hepatic stellate cells

[0171] To confirm the anti-fibrotic effects and actions of various proteins and cytokines secreted from NK cells, NK cells were cultured for 48 hours (2 x 10 6 After harvesting the conditioned medium (CM) after incubation (cells / ml), the effect on TGF-β1-mediated hepatic stellate cell activation was analyzed. When 5% NK-CM was added during the TGF-β1-mediated hepatic stellate cell activation culture process, it was confirmed through real-time polymerase chain reaction that the gene expression of fibrosis markers LOXL2 (CM-pNK: 43.72% and CM-drNK: 32.58%), COL1A1 (CM-pNK: 12.04% and CM-drNK: 9.76%), and ACTA2 (CM-pNK: 60.93% and CM-drNK: 4.34%) was significantly reduced compared to 100% expression in aHSC (Con), and it was confirmed that the effect of the CM-drNK of the present invention was significantly higher than that of CM-pNK (Fig. 12A).

[0172] Immunocytochemistry confirmed that the protein expression of fibrosis markers COL1A1 (CM-pNK: 9.26% and CM-drNK: 6.45%) and a-SMA (CM-pNK: 21.24% and CM-drNK: 9.45%) was reduced to the level of qHSCs compared to 100% expression in aHSCs (Con) (Fig. 12B). Picro sirius red collagen staining confirmed that the degree of red staining was lower in CM-drNK than in aHSCs (Con), verifying that fibrosis was significantly inhibited (Fig. 12C). EZ-Cytox kit (DoGenBio) was used to verify the viability (%) of fibrotic cells in the conditioned medium. In the TGF-β1-mediated HSC fibrosis process, tetrazolium was treated under conditions in which CM-pNK or CM-drNK was treated, and the absorbance at 450 nm, which reacts with dehydrogenase in living cells, was measured. When qHSC was set as 100%, it was confirmed that the viability was further reduced in the CM-drNK (73.6%) treatment condition compared to the control CM-pNK (99.0%) compared to aHSC (115.5%), confirming that CM-drNK significantly inhibited the transformation into fibrogenic cells (Fig. 12D).

[0173]

[0174] Example 6. Effect of treatment on pulmonary fibrosis

[0175] 6-1. Superior fibrogenic cell-killing ability of drNK in a human lung fibrosis cell model

[0176] Human fetal lung fibroblast cells (MRC-5) were used as a cell model. Quiescent MRC-5 (qMRC-5) cells were cultured in MEM medium containing 0.5% FBS, and activated MRC-5 (aMRC-5) cells were produced by treating 2 ng / ml recombinant human TGF-β1 protein (rh TGF-β1 protein) for 48 hours. Real-time polymerase chain reaction confirmed that the gene expression of fibrosis markers COL1A1 (2.98-fold) and COL3A1 (1.38-fold) was significantly increased in aMRC-5 compared to qMRC-5 (Fig. 13A).

[0177] In addition, the degree of collagen development was confirmed using a Picro sirius red staining kit (Abcam), and the concentration of red staining was darker compared to qMRC-5, confirming a high degree of development into collagen connective tissue (Fig. 13B). Immunocytochemistry was performed to confirm that the expression levels of collagen (COL1A1) and α-SMA proteins increased as fibrosis progressed in aMRC-5. The result of quantifying the density of fluorescence expression (integrated density, IntDen) using the Image J (NIH) program showed that the expression of COL1A1 increased 10.60-fold and α-SMA increased 11.01-fold in aMRC-5 compared to qMRC-5, verifying its significance as a lung fibrosis cell model (Fig. 13C).

[0178] To determine the killing activity of NK cells against activated human lung fibroblasts (aMRC-5) treated with TGF-β, aMRC-5, drNK, and control NK92 cells were seeded in 24-well plates at a cell number of 2 × 10 5) and stained with Propidium Iodide (PI) after 5 hours of reaction. As a result of analysis using flow cytometry, the direct killing ability was confirmed to be 4.8 times increased with 17.9% killing ability of induced natural killer cells, while the control group NK92 cells showed 3.7% killing ability (Fig. 13D).

[0179]

[0180] 6-2. Inhibitory effect of drNK conditioned medium on the activation of fibrotic human lung fibroblasts

[0181] To confirm the anti-fibrotic effects and actions of various proteins and cytokines secreted from NK cells, NK cells were cultured for 48 hours (2 x 10 6After harvesting the conditioned medium (CM) (cells / ml), the effect on TGF-β1-mediated lung fibroblast (MRC-5) activation was analyzed. When 5% NK-CM was added to each culture during TGF-β1-mediated MRC-5 activation culture, real-time polymerase chain reaction showed that 100% expression of aMRC-5 (Con) was compared to the expression of fibrosis markers COL1A1 (CM-pNK: 65.04% and CM-drNK: 18.08%), COL3A1 (CM-pNK: 29.93% and CM-drNK: 16.55%), ACTA2 (CM-pNK: 29.29% and CM-drNK: 16.02%), MYH9 (CM-pNK: 27.72% and CM-drNK: 5.04%), MYH10 (CM-pNK: 61.19% and CM-drNK: 1.67%), MYH14 (CM-pNK: 53.03% and CM-drNK: It was confirmed that the gene expression of MMP2 (CM-pNK: 73.88% and CM-drNK: 21.67%) was significantly reduced, and the effect of the CM-drNK of the present invention was significantly higher than that of CM-pNK (Fig. 14A). Through immunocytochemistry, it was confirmed that the protein expression of the fibrosis markers COL1A1 (CM-pNK: 40.99% and CM-drNK: 16.00%) and a-SMA (CM-pNK: 42.54% and CM-drNK: 12.62%) was reduced to the level of qMRC-5, compared to 100% expression of aMRC-5 (Con) (Fig. 14B).

[0182] Picro sirius red collagen staining confirmed that the degree of red staining was lower in CM-drNK than in CM-pNK compared to aMRC-5 (Con), demonstrating that fibrosis was significantly inhibited (Fig. 14C). To verify the viability (%) of fibrogenic cells in conditioned media, the EZ-Cytox kit (DoGenBio) was used. The absorbance at 450 nm, which reacts with dehydrogenase in living cells, was measured under conditions in which CM-pNK or CM-drNK was treated with tetrazolium during the TGF-β1-mediated MRC-5 fibrosis process. When qMRC-5 was taken as 100%, compared to aMRC-5 (121.0%), the viability was confirmed to be more reduced in the CM-drNK (59.8%) treatment condition than in the control CM-pNK (87.0%), confirming that CM-drNK significantly inhibits the transformation into fibrogenic cells (Fig. 14D).

[0183]

[0184] Example 7. Effect of treatment on cardiac fibrosis

[0185] 7-1. Antifibrotic effect of drNK in a cardiac cell model

[0186] Immunized human cardiac fibroblasts (IM-HCF; P10453-IM) were used as a cell model. Quiescent IM-HCF (qIM-HCF) cells were cultured in Fibroblast medium plus (FM-Plus) containing 5% FBS, and fibrogenic activated IM-HCF (aIM-HCF) was produced by treating with 10 ng / ml recombinant human TGF-β1 protein (rh TGF-β1 protein) for 24 hours. Real-time polymerase chain reaction demonstrated that the gene expression of fibrosis markers COL1A1 (2.72-fold), TIMP1 (1.14-fold), and ACTA2 (3.39-fold) was significantly increased in aIM-HCF compared to qIM-HCF (Fig. 15A). In addition, the degree of collagen development was confirmed using a Picro sirius red staining kit (abcam), and the concentration of red staining was darker compared to qIM-HCF, confirming that the degree of collagen connective tissue development was high (Fig. 15B).

[0187] As fibrosis progressed in aIM-HCF, the expression levels of collagen (COL1A1) and α-SMA proteins increased, as confirmed by immunocytochemistry. The fluorescence expression level was quantified as the integrated density (IntDen) using the Image J (NIH) program. As a result, the expression of COL1A1 increased 7.83-fold and α-SMA increased 5.94-fold in aIM-HCF compared to qIM-HCF, verifying its significance as a cardiac fibrosis cell model (Fig. 15C). In addition, to confirm the killing activity of NK cells against TGF-β-activated human cardiac fibroblasts (aIM-HCF), 2 × 10 aIM-HCF, drNK, and control NK92 cells were seeded in 24-well plates, respectively. 5After reacting with cells at a 1:1 ratio for 5 hours, the cells were stained with Propidium Iodide (PI) and analyzed by flow cytometry. As a result, it was confirmed that induced natural killer cells (drNK) (52.3%) exhibited excellent killing ability, 6.8 times higher than that of control NK92 cells (7.6%) (Fig. 15D).

[0188]

[0189] 7-2. Inhibitory effect of drNK conditioned medium on the activation of fibrotic human cardiac fibroblasts

[0190] To confirm the anti-fibrotic effects and actions of various proteins and cytokines secreted from NK cells, NK cells were cultured for 48 hours (2 x 10 6 After harvesting the conditioned medium (CM) (cells / ml), the effect on TGF-β1-mediated activation of cardiac fibroblasts (IM-HCF) was analyzed. When 5% NK-CM was added during the TGF-β1-mediated IM-HCF activation culture process, it was confirmed through real-time polymerase chain reaction that the gene expression of fibrosis markers COL1A1 (CM-pNK: 67.19% and CM-drNK: 51.35%), CILP1 (CM-pNK: 77.12% and CM-drNK: 18.47%), THBS4 (CM-pNK: 56.06% and CM-drNK: 48.72%), VEGF-A (CM-pNK: 52.78% and CM-drNK: 50.72%), and MMP9 (CM-pNK: 63.18% and CM-drNK: 46.75%) was significantly reduced compared to 100% expression of aIM-HCF (Con), and the effect of CM-drNK of the present invention was significantly higher than that of CM-pNK. It was confirmed (Fig. 16A).

[0191] Immunocytochemistry confirmed that the protein expression of fibrosis markers COL1A1 (CM-pNK: 25.54% and CM-drNK: 9.60%) and a-SMA (CM-pNK: 27.88% and CM-drNK: 11.40%) was reduced to the level of qIM-HCF compared to 100% expression in aIM-HCF (Con) (Fig. 16B). Picro sirius red collagen staining confirmed that fibrosis was significantly suppressed as the degree of red staining in CM-drNK was lower than that in aIM-HCF (Con) (Fig. 16C). EZ-Cytox kit (DoGenBio) was used to verify the viability (%) of fibrogenic cells in the conditioned medium. In the TGF-β1-mediated IM-HCF fibrosis process, tetrazolium was treated under conditions in which CM-pNK or CM-drNK was treated, and the absorbance at 450 nm, which reacts with dehydrogenase in living cells, was measured. When qIM-HCF was set as 100%, it was confirmed that viability was further reduced in the CM-drNK (80.5%) treatment condition compared to the control CM-pNK (108.2%) compared to aIM-HCF (133.5%), confirming that CM-drNK significantly inhibits the transformation into fibrogenic cells (Fig. 16D).

[0192]

[0193] Example 8. Effect of treatment on skin fibrosis

[0194] 8-1. Antifibrotic effect of drNK in a skin cell model

[0195] Human foreskin dermal fibroblasts (HDF) cell line (CRL2097) were used as a cell model. Quiescent HDF (qHDF) cells were cultured in MEM medium containing 10% FBS, and fibrogenic activated HDF (aHDF) were produced by treating with 20 ng / ml recombinant human TGF-β1 protein (rh TGF-β1 protein) for 48 hours. Real-time polymerase chain reaction confirmed that the gene expression of fibrosis markers COL1A1 (2.57-fold), COL3A1 (1.64-fold), and ACTA2 (2.59-fold) was significantly increased in aHDF compared to qHDF (Fig. 17A). In addition, the degree of collagen development was confirmed using a Picro sirius red staining kit (Abcam), and the concentration of red staining was darker compared to qHDF, confirming that the degree of development into collagen connective tissue was high (Fig. 17B).

[0196] As fibrosis progressed in aHDF, we confirmed that the expression levels of collagen (COL1A1) and α-SMA proteins increased using immunocytochemistry. The fluorescence expression density (Integrated density, IntDen) was quantified using the Image J (NIH) program. As a result, the expression of COL1A1 increased 13.89-fold and α-SMA increased 6.18-fold in aHDF compared to qHDF, verifying its significance as a skin fibrosis cell model (Fig. 17C).

[0197] To determine the killing activity of NK cells against activated human dermal fibroblasts (aHDFs) treated with TGF-β, 2 x 10 aHDFs, drNK, and control NK92 cells were seeded in 24-well plates. 5After reacting with cells at a 1:1 ratio for 5 hours, they were stained with Propidium Iodide (PI) and analyzed by flow cytometry. As a result, it was confirmed that induced natural killer cells (drNK) (37.1%) exhibited excellent killing ability, 1.6 times higher than that of control NK92 cells (22.3%) (Fig. 17D).

[0198]

[0199] 8-2. Inhibitory effect of drNK conditioned medium on the activity of human dermal fibroblasts

[0200] To confirm the anti-fibrotic effects and actions of various proteins and cytokines secreted from NK cells, NK cells were cultured for 48 hours (2 x 10 6 After harvesting the conditioned medium (CM) after incubation (cells / ml), the effect on TGF-β1-mediated skin fibroblast (HDF) activation was analyzed. When 5% NK-CM was added and cultured during the TGF-β1-mediated skin HDF activation culture process, it was confirmed through real-time polymerase chain reaction that the gene expression of fibrosis markers COL1A1 (CM-pNK: 90.66% and CM-drNK: 69.55%), COL3A1 (CM-pNK: 50.67% and CM-drNK: 43.20%), and ACTA2 (CM-pNK: 114.89% and CM-drNK: 70.24%) was significantly reduced compared to 100% expression of aHDF (Con), and it was confirmed that the effect of the CM-drNK of the present invention was significantly higher than that of CM-pNK (Fig. 18A).

[0201] Through immunocytochemistry, it was confirmed that the protein expression of fibrosis markers COL1A1 (CM-pNK: 41.44% and CM-drNK: 26.43%) and a-SMA (CM-pNK: 58.48% and CM-drNK: 18.69%) was reduced to the level of qHDF compared to 100% expression of aHDF (Con) (Fig. 18B).

[0202] Picro sirius red collagen staining confirmed that the degree of red staining was lower in CM-drNK than in CM-pNK compared to aHDF (Con), verifying that fibrosis was significantly inhibited (Fig. 18C). To verify the viability (%) of fibrogenic cells in the conditioned medium, the EZ-Cytox kit (DoGenBio) was used. The absorbance at 450 nm, which reacts with dehydrogenase in living cells, was measured by treating tetrazolium under conditions in which CM-pNK or CM-drNK was treated during the TGF-β1-mediated HDF fibrosis process. When qHDF was set as 100%, it was confirmed that the viability was further reduced in the CM-drNK (73.5%) treatment condition compared to the control CM-pNK (106.1%) compared to aHDF (113.6%), confirming that CM-drNK significantly inhibits the transformation into fibrogenic cells (Fig. 18D).

[0203]

[0204] Example 9. Effect of treatment on renal fibrosis

[0205] 9-1. Antifibrotic effect of drNK in a renal cell model

[0206] Human immortalized proximal tubule epithelial cells (HK-2) were used as a cell model. Quiescent HK-2 (qHK-2) cells were cultured in RPMI 1640 medium containing 10% FBS, and activated HK-2 (aHK-2) cells were produced by treating 10 ng / ml recombinant human TGF-β1 protein (rh TGF-β1 protein) for 48 hours. Real-time polymerase chain reaction confirmed that the gene expression of fibrosis markers COL1A1 (8.06-fold), COL3A1 (5.54-fold), and ACTA2 (4.76-fold) was significantly increased in aHK-2 compared to qHK-2, and aHK-2 cells were established as activated fibrosis cells (Fig. 19A). In addition, the degree of collagen development was confirmed using a Picro sirius red staining kit (abcam), and the concentration of red staining was darker compared to qHK-2, confirming that the degree of collagen connective tissue development was high (Fig. 19B).

[0207] As fibrosis progressed in aHK-2, increased expression of collagen (COL1A1) and α-SMA proteins was confirmed using immunocytochemistry. Quantification of fluorescence expression density (integrated density, IntDen) using the Image J (NIH) program revealed an 8.94-fold increase in COL1A1 expression and a 3.77-fold increase in α-SMA expression in aHK-2 compared to qHK-2, verifying its significance as a renal fibrosis cell model (Fig. 19C).

[0208] To determine the killing activity of NK cells against activated human renal fibroblasts (aHK-2) treated with TGF-β, aHK-2, drNK, and control NK92 cells were seeded in 24-well plates at a cell number of 2 x 10 5) and stained with Propidium Iodide (PI) after 5 hours of reaction. As a result of analysis using flow cytometry, the direct killing ability was confirmed to be 1.6 times increased, with 27.9% killing ability of induced natural killer cells, while 17.1% killing ability of control NK92 cells was observed (Fig. 19D).

[0209]

[0210] 9-2. Inhibitory effect of drNK conditioned medium on the activation of fibrotic human renal tubular epithelial cells

[0211] To confirm the anti-fibrotic effects and actions of various proteins and cytokines secreted from NK cells, NK cells were cultured for 48 hours (2 x 10 6 After harvesting the conditioned medium (CM) after incubation (cells / ml), the effect on TGF-β1-mediated renal tubular epithelial cell (HK-2) activation was analyzed. When 5% NK-CM was added during the TGF-β1-mediated renal HK-2 activation culture process, it was confirmed through real-time polymerase chain reaction that the gene expression of fibrosis markers COL3A1 (CM-pNK: 65.26% and CM-drNK: 41.74%), ACTA2 (CM-pNK: 25.48% and CM-drNK: 15.33%), and PAI-1 (CM-pNK: 82.27% and CM-drNK: 39.72%) was significantly reduced compared to 100% expression of aHK-2 (Con), and it was confirmed that the effect of the CM-drNK of the present invention was significantly higher than that of CM-pNK (Fig. 20A).

[0212] Through immunocytochemistry, it was confirmed that the protein expression of fibrosis markers COL1A1 (CM-pNK: 66.69% and CM-drNK: 17.41%) and a-SMA (CM-pNK: 61.15% and CM-drNK: 26.65%) was reduced to the level of qHK-2 compared to 100% expression of aHK-2 (Con) (Fig. 20B).

[0213] Picro sirius red collagen staining confirmed that the degree of red staining was lower in CM-drNK than in CM-pNK compared to aHK-2 (Con), demonstrating that fibrosis was significantly inhibited (Fig. 20C). To verify the viability (%) of fibrogenic cells in conditioned media, the EZ-Cytox kit (DoGenBio) was used. In the TGF-β1-mediated HK-2 fibrosis process, tetrazolium was treated under conditions in which CM-pNK or CM-drNK was treated, and the absorbance at 450 nm, which reacts with dehydrogenase in living cells, was measured. When qHK-2 was taken as 100%, it was confirmed that viability was further reduced in the CM-drNK (97.9%) treatment condition compared to the control CM-pNK (110.0%) compared to aHK-2 (130.7%), thereby confirming that CM-drNK significantly inhibited transformation into fibrogenic cells (Fig. 20D).

[0214]

[0215] Example 10. Quantitative analysis of drNK expression cytokine / chemokine gene levels

[0216] qRT-PCR was performed to identify genes showing differential expression patterns compared to control NK (NK92 and iPS-NK cells) targeting the cytokine / chemokine gene group.

[0217] As a result, 10 gene groups (CCL5, IFN-γ, CXCL11, CXCL12, GDNF, VEGF, XCL1, IL16, LIF, LTB) whose expression was relatively increased in drNK compared to the NK92 and iPS-NK groups were identified. When the amount expressed in NK92 is set as 1, the fold expression in iPS-NK and drNK is expressed as CCL5 is NK92 (1), iPS-NK (0.85), drNK (3.64), IFN-γ is NK92 (1), iPS-NK (0.1), drNK (1.9), CXCL11 is NK92 (1), iPS-NK (0.01), drNK (2.93), CXCL12 is NK92 (1), iPS-NK (1.82), drNK (5.73), GDNF is NK92 (1), iPS-NK (0.03), drNK (5.69), VEGF is NK92 (1), iPS-NK (1.29), drNK (2.3), XCL1 is NK92 (1), iPS-NK (0.18), drNK (2.15), IL16 is NK92 (1), iPS-NK (0.98), drNK (4.35), LIF was confirmed as NK92 (1), iPS-NK (1.78), drNK (8.5), LTB was confirmed as NK92 (1), iPS-NK (1.02), drNK (2.01) (Fig. 21).

[0218]

[0219] Example 11. Identification of drNK secretion factors using human proteome cytokine arrays.

[0220] To identify cytokines, particularly cytokines, that may influence the antifibrotic effects of drNK, we performed a drNK conditioned medium analysis. Specifically, to obtain conditioned medium (CM) of NK cells, drNK cells and CD56 dim 10 pNK cells each 6The cells were cultured at a density of 10 cells / ml. After 24 hours, the cultured medium was filtered through a 0.22 μm filter (Millipore). CM-drNK and CM-CD56 dim To measure cytokine levels in pNK, a proteome profiler array (Proteome Profiler Human XL Cytokine Array Kit, ARY022B; R&D systems) was used according to the manufacturer's instructions, and quantitative analysis of the final images was performed using ImageJ software.

[0221] Using ImageJ, 56 drNK secreted proteins with a fluorescence expression level (Mean Pixel Density, MPD) of 1,000 or more were identified. The major proteins were RANTES (average 81,174 MPD), DPPIV (average 78,046 MPD), CD31 (average 33,901 MPD), TIM-3 (average 31,688 MPD), Emmprin (average 29,204 MPD), MIP-1α / MIP-1ß (average 27,937 MPD), GM-CSF (average 22,299 MPD), Fas Ligand (average 21,602 MPD), MIF (average 19,398 MPD), IL-16 (average 16,867 MPD), IL-17A (average 82,49 MPD), Flt-3 Ligand (average 5,834 MPD), ICAM-1 (average 4,532 MPD), M-CSF (average 2,867 MPD), FGF-19 (average 2,481 MPD), Serpin E1, MIP-3ß (average 2,165 MPD), IL-18 (average 2,144 MPD), IL-32 (average 1,950 MPD), Angiogenin (average 1,901 MPD), IL-1α (average 1,895 MPD), Cystatin C (average 1,792 MPD), Resistin (average 1,699 MPD), GDF-15 (average 1,635 MPD), Angiopoietin-2 (average 1,558 MPD), TNF-α (average 1,546 MPD), PDGF-AA (average 1,536 MPD), Apolipoprotein AI (average 1,507 MPD), Osteopontin (average 1,489 MPD), Dkk-1 (average 1,447 MPD), uPAR (average 1,422 MPD), Endoglin (average 1,411 MPD), IFN-γ (average 1,394 MPD), FGF basic (average 1,381 MPD), SDF-1α (CXCL12) (average 1,378 MPD), IL-1ß (average 1,363 MPD), RAGE (average 1,327 MPD), EGF (average 1,277 MPD), IL-8 (average 1,252 MPD), IL-27 (average 1,208 MPD), BAFF (average 1,191 MPD), CD40 ligand (average 1,159 MPD), IL-34 (average 1,129 MPD), VCAM-1 (average 1,127 MPD), IL-1ra (average 1,113 MPD), MCP-1 (average 1,094 MPD), Kallikrein 3 (average 1,088 MPD), IL-12 p70 (average 1,088 MPD), IL-11 (average 1,087 MPD), ST2 (average 1,059 MPD), MMP-9 (average 1,058 MPD), IL-22 (average 1,057 MPD), C-Reactive Protein (average 1,041 MPD), BDNF (average 1,034 MPD), Vitamin D BP (average 1,032 MPD), and Lipocalin-2 (average 1,004 MPD) were identified (Fig. 22A).

[0222] Control CD56 dimWe identified 28 proteins that were quantitatively secreted at higher levels in drNK compared to pNK, and the major proteins were IL-16 (5.58-fold), BAFF (5.23-fold), CD31 (4.36-fold), ICAM-1 (3.57-fold), Emmprin (3.39-fold), VCAM-1 (3.33-fold), Flt-3 Ligand (3.32-fold), Cystatin C (3.16-fold), C-Reactive Protein (2.58-fold), IL-27 (2.44-fold), TNF-a (2.22-fold), IL-32 (2.16-fold), TIM-3 (2.01-fold), IL-12 p70 (1.94-fold), uPAR (1.53-fold), IL-18 Bpa (1.40-fold), MIF (1.39-fold), Dkk-1 (1.35-fold), IL-11 (1.33-fold), and GM-CSF. (1.30 fold), RANTES (1.29 fold), Endoglin (1.24 fold), IL-22 (1.16 fold), RAGE, Osteopontin (1.06 fold), GDF-15 (1.02 fold), Kallikrein 3 (1.02 fold), and Angiopoietin-2 (1.02 fold) were identified (Fig. 22B). In addition, three drNK-specific proteins, DDP4, M-CSF, and BDNF, were identified (Fig. 22C).

[0223]

[0224] In summary, the induced natural killer cells of the present invention have superior direct killing ability compared to the previously known control natural killer cells when treated with hepatic stellate cells (liver fibrosis cell model), alveolar epithelial cells (pulmonary fibrosis cell model), cardiac cells (cardiac fibrosis cell model), skin cells (skin fibrosis cell model), and kidney cells (kidney fibrosis cell model), which are fibrotic origin cells that induce fibrotic reactions. In addition, when the conditioned medium for the induced natural killer cells was treated to the fibrotic cell models, collagen production, activity, and viability were significantly reduced compared to the previously known control natural killer cells, indicating an excellent fibrosis inhibitory effect. This suggests that the fibrosis disease can be prevented or treated.

[0225]

[0226] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. A pharmaceutical composition for preventing or treating fibrosis, comprising directly reprogrammed natural killer (drNK) cells expressing at least one selected from the group consisting of CD56superbright, CD16superbright, and combinations thereof.

2. A composition according to claim 1, wherein the composition further comprises NKG2D or CCR5, which is a substance secreted by induced natural killer (drNK) cells.

3. A composition according to claim 1, wherein the induced natural killer (drNK) cells express at least one cytokine or chemokine gene selected from the group consisting of CCL5, IFN-γ, CXCL11, CXCL12, GDNF, VEGF, XCL1, IL16, LIF, and LTB.

4. A composition according to claim 1, wherein the induced natural killer (drNK) cells express at least one protein selected from the group consisting of DDP4, M-CSF, and BDNF.

5. In the first paragraph, the drNK cell, (a) a step of introducing a reprogramming factor into the separated cell; (b) A composition manufactured by a method including: i) culturing the cells of step (a) in a first medium containing cytokines, growth factors, and GSK3β (Glycogen synthase kinase 3β) inhibitors to directly increase the efficiency of reprogramming, and ii) culturing the cells in a second medium containing cytokines, growth factors, and AHR (Aryl hydrocarbon receptor) inhibitors to promote the production of natural killer cells.

6. A composition according to claim 5, wherein the separated cells are somatic cells other than natural killer cells.

7. A composition according to claim 1, wherein the fibrosis occurs in at least one selected from the group consisting of the lung, kidney, liver, heart, brain, blood vessel, joint, intestine, skin, soft tissue, bone marrow, penis, peritoneum, muscle, spine, testis, ovary, breast, thyroid, eardrum, pancreas, gallbladder, bladder, prostate, esophagus, stomach, uterus, spleen, lymph node, salivary gland, and nervous system.

8. A cell therapy agent for preventing or treating fibrosis, comprising drNK (Directly reprogrammed Natural killer) cells expressing at least one selected from the group consisting of CD56superbright, CD16superbright, and a combination thereof.

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