Application of NK (Natural Killer) cells in preparation of medicine for treating fibrotic diseases

Abstract

The invention relates to the field of cell therapy, in particular to application of NK cells in preparation of drugs for treating fibrosis diseases. The invention discloses application of NK (natural killer) cells in preparation of medicines for treating fibrosis diseases, and compared with nintedanib or pirfenidone, the NK cells have equivalent or even better effects in treatment of pulmonary fibrosis diseases, are higher in safety and do not cause side effects such as hepatotoxicity or renal toxicity. Also disclosed is a method of treating a subject suffering from a fibrotic disease by administering one or more NK cells to the subject.

Description

Application of NK cells in the preparation of drugs for treating fibrotic diseases Technical Field

[0001] The present invention relates to the field of cell therapy, and in particular to the application of NK cells in the preparation of medicines for treating fibrotic diseases. Background Art

[0002] When various tissues or organs are damaged or diseased, fibroblasts and pericytes within them are activated and differentiate into myofibroblasts, causing extensive tissue fibrosis. This is a key factor in the development of conditions such as heart failure with preserved ejection fraction (HFpEF), heart failure with intermediate-range ejection fraction (HFmrEF), heart failure with reduced ejection fraction (HFrEF), idiopathic pulmonary fibrosis, liver cirrhosis, and kidney disease. Fibroblast activation protein (FAP) and endosialin (CD248) are specific markers of myofibroblasts, expressed abundantly only in myofibroblasts and absent or at very low levels in other normal cells of the body.

[0003] The underlying mechanism of pulmonary fibrosis (PF) remains unclear. The primary pathological changes are diffuse alveolitis in the early stages and, in the later stages, the proliferation and transdifferentiation of fibroblasts into myofibroblasts (MFIBS), which leads to excessive deposition of extracellular matrix (ECM) and type I collagen. Inflammation and various abnormal physiological processes are associated with the development of PF.

[0004] The accumulation and secretion of the senescence-associated secretory phenotype (SASP) by senescent fibroblasts is an important factor in promoting PF. α-SMA (α-Smooth Muscle Actin, "α-SMA") is one of the surface marker proteins of myofibroblast formation.

[0005] Idiopathic pulmonary fibrosis (IPF) is a rare, chronic progressive disease of unknown etiology. Fibrosis leads to irreversible loss of lung function, manifested by symptoms such as worsening cough and dyspnea, and impaired quality of life. IPF is a large type of interstitial lung disease characterized by fibroblast proliferation, large amounts of extracellular matrix deposition, accompanied by inflammatory damage and tissue structural destruction.

[0006] Currently, FDA-approved IPF treatments such as nintedanib and pirfenidone have limited efficacy, can only delay disease progression, require high dosages, and are associated with side effects such as hepatotoxicity (nintedanib) or nephrotoxicity (pirfenidone). Patients have a poor prognosis, with a median survival of 2-3 years. Therefore, there is an urgent need for treatments that can effectively alleviate symptoms in patients with IPF and other pulmonary fibrosis, and that are safer and more effective than existing drugs to prolong patient survival.

[0007] Summary of the Invention

[0008] In view of this, in order to solve at least one of the above technical problems, one aspect of the present invention provides a use of NK cells in the preparation of a drug for treating fibrotic diseases.

[0009] In some embodiments of the present invention, the fibrotic disease includes heart failure, pulmonary fibrosis, liver cirrhosis and kidney disease; and the heart failure includes HFpEF, HFmrEF and HFrEF.

[0010] In some embodiments of the present invention, the fibrotic disease is pulmonary fibrosis.

[0011] In some embodiments of the present invention, the pulmonary fibrosis is idiopathic pulmonary fibrosis.

[0012] In some embodiments of the present invention, the NK cells include NK cells from at least one of the following sources: the subject's autologous NK cells, the subject's allogeneic NK cells, the subject's xenogeneic NK cells, and induced pluripotent stem cell-derived NK cells; the subject is an individual suffering from or suspected of suffering from the fibrotic disease.

[0013] In some embodiments of the present invention, the NK cells are autologous NK cells of the subject.

[0014] In some embodiments of the present invention, the NK cells are allogeneic NK cells of the subject.

[0015] In some embodiments of the present invention, the NK cells are isolated from allogeneic peripheral blood mononuclear cells (PBMCs) of a subject.

[0016] In some embodiments of the present invention, the NK cells include one or more NK cells derived from any of the following cells: progenitor cells, embryonic stem cells, embryonic stem cell-derived cells, embryonic germ cells, embryonic germ cell-derived cells, stem cells, stem cell-derived cells, pluripotent stem cells, induced pluripotent stem cells, hematopoietic stem cells (HSCs), and immortalized cells.

[0017] In some embodiments of the present invention, the NK cells include one or more NK cells isolated from the subject's autologous and / or allogeneic peripheral blood, umbilical cord blood, lymph nodes, ascites, pleural effusion, thymus, tumor or bone marrow.

[0018] In some embodiments of the present invention, the NK cells include one or more NK cells isolated from healthy human peripheral blood, umbilical cord blood, lymph nodes, thymus or bone marrow.

[0019] In some embodiments of the present invention, the NK cells isolated from PBMCs are expanded and cultured in vitro.

[0020] In some embodiments of the present invention, the NK cells are NK cells differentiated from induced pluripotent stem cells (iPSCs) (i-NK cells).

[0021] In some embodiments of the present invention, the i-NK cells are differentiated from human pluripotent stem cells using the method disclosed in Chinese patent publication No. CN112608895A to differentiate into NK cells.

[0022] In some embodiments of the present invention, the NK cells are CD56 + CD3 - of NK cells.

[0023] In some embodiments of the present invention, the drug comprises NK cells and at least one of the following components: pharmaceutically acceptable additives, carriers, diluents, excipients, preservation solutions and other auxiliary components.

[0024] In some embodiments of the present invention, the medicament comprises an effective amount of NK cells.

[0025] In some embodiments of the present invention, the subject is administered one or more doses of the drug.

[0026] In some embodiments of the present invention, the drug comprising an effective amount of NK cells is administered to the subject once or multiple times.

[0027] In some embodiments of the present invention, when the subject is a human, the drug comprises 2×10 6 -2×10 8 NK cells / kg (Kilogram, kg).

[0028] In some embodiments of the present invention, when the subject of administration is a human, the drug contains no more than 1×10 10 NK cells / person.

[0029] In some embodiments of the present invention, when the subject is a human, the drug comprises 2×10 6 -2×10 8 NK cells / kg, and administering the drug to the subject once or multiple times.

[0030] In some embodiments of the present invention, when the subject of administration is a human, the drug contains no more than 1×10 10 NK cells / person, and administering the drug to the subject once or multiple times.

[0031] In another aspect, the present invention provides a method of treating a subject suffering from a fibrotic disease, the method comprising administering NK cells to the subject.

[0032] In some embodiments of the present invention, NK cells are continuously administered to the subject.

[0033] In some embodiments of the present invention, the fibrotic disease includes heart failure, pulmonary fibrosis, liver cirrhosis and kidney disease; and the heart failure includes HFpEF, HFmrEF and HFrEF.

[0034] In some embodiments of the present invention, the fibrotic disease is pulmonary fibrosis.

[0035] In some embodiments of the present invention, the pulmonary fibrosis is idiopathic pulmonary fibrosis.

[0036] In some embodiments of the present invention, the NK cells include NK cells from at least one of the following sources: autologous NK cells of the subject, allogeneic NK cells of the subject, xenogeneic NK cells of the subject, and NK cells derived from induced pluripotent stem cells.

[0037] In some embodiments of the present invention, the NK cells are autologous NK cells of the subject.

[0038] In some embodiments of the present invention, the NK cells are allogeneic NK cells of the subject.

[0039] In some embodiments of the present invention, the NK cells are isolated from allogeneic PBMCs of a subject.

[0040] In some embodiments of the present invention, the NK cells include one or more NK cells derived from any of the following cells: progenitor cells, embryonic stem cells, embryonic stem cell-derived cells, embryonic germ cells, embryonic germ cell-derived cells, stem cells, stem cell-derived cells, pluripotent stem cells, induced pluripotent stem cells, hematopoietic stem cells (HSCs), and immortalized cells.

[0041] In some embodiments of the present invention, the NK cells include one or more NK cells isolated from the subject's autologous and / or allogeneic peripheral blood, umbilical cord blood, lymph nodes, ascites, pleural effusion, thymus, tumor or bone marrow.

[0042] In some embodiments of the present invention, the NK cells include one or more NK cells isolated from healthy human peripheral blood, umbilical cord blood, lymph nodes, thymus or bone marrow.

[0043] In some embodiments of the present invention, the NK cells isolated from PBMCs are expanded and cultured in vitro.

[0044] In some embodiments of the present invention, the NK cells are NK cells differentiated from iPSCs (i-NK cells).

[0045] In some embodiments of the present invention, the NK cells are i-NK cells differentiated using the method for directed differentiation of human pluripotent stem cells into NK cells disclosed in Chinese patent document CN112608895A.

[0046] In some embodiments of the present invention, the NK cells are CD56 + CD3 - of NK cells.

[0047] In some embodiments of the present invention, the NK cells and at least one of the following components are administered or continuously administered to the subject: pharmaceutically acceptable additives, carriers, diluents, excipients, preservation solutions and other auxiliary components.

[0048] In some embodiments of the present invention, the NK cells are an effective amount of NK cells.

[0049] In some embodiments of the present invention, an effective amount of NK cells is administered to the subject one or more times.

[0050] In some embodiments of the present invention, when the subject is a human, the effective amount of NK cells is 2×10 6 -2×10 8 NK cells / kg, administering the effective amount of NK cells to the subject once or multiple times.

[0051] In some embodiments of the present invention, when the subject is a human, the effective amount of NK cells is no more than 1×10 10 NK cells / person, administering the effective amount of NK cells to the subject once or multiple times.

[0052] In some embodiments of the present invention, the administration is at least one of intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection and infusion techniques.

[0053] In some embodiments of the present invention, the administration is selected from at least one of oral, nasal, intravenous, intraperitoneal, intracerebral (intracerebral parenchyma), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein, intralesional, intratumoral, subcutaneous, sustained release system and implantation device administration.

[0054] The beneficial effects of the present invention include:

[0055] NK cell therapy for pulmonary fibrosis can significantly alleviate the symptoms associated with pulmonary fibrosis, specifically by improving the lung inflammatory response in patients with pulmonary fibrosis, significantly downregulating the expression of lung fibrosis-related indicators, effectively killing myofibroblasts, and significantly downregulating the expression of inflammatory factors in the lungs of patients with pulmonary fibrosis. Moreover, compared with the use of nintedanib or pirfenidone to treat pulmonary fibrosis, the treatment effect of NK cell intervention is equivalent to or even better. More importantly, compared with the use of nintedanib or pirfenidone to treat pulmonary fibrosis, the use of NK cells will not cause side effects such as hepatotoxicity or nephrotoxicity, and is safer.

[0056] In this article:

[0057] "NK cells": Natural Killer Cells, including human NK cells, are derived from bone marrow lymphoid stem cells and are lymphocyte cytotoxic cells. NK cells can be identified by their cell surface expression of CD56 and lack of expression of CD3 (CD56 + CD3 -) recognition (Pfefferle A, et al., Frontiers In Immunology, 11: 812, 2020). NK cells sense and kill target cells that lack major histocompatibility complex (MHC) class I molecules or cells that express ligands that specifically bind to NK cell receptors. NK cell activation receptors include natural cytotoxicity receptors (NKp30, NKp44, and NKp46) and lectin-like receptors NKG2D and DNAM-1, etc. Their ligands are expressed on stressed, transformed, or infected cells, but not on normal cells, making normal cells resistant to NK cell killing (Bottino, Castriconi, et al., 2005)(Gasser, Orsulic, et al., 2005)(Lanier, 2005). NK cell activation is negatively regulated by inhibitory receptors such as killer immunoglobulin (Ig)-like receptors (KIR), NKG2A / CD94 and leukocyte Ig-like receptor-1 (LIR-1). The combination of an inhibitory receptor may be sufficient to prevent target lysis (Bryceson, Ljunggren et al., 2009). Therefore, NK cells effectively target cells expressing many stress-induced ligands and a small number of MHC-I class ligands. NK cells effectively destroy tumor cells, stress cells and virus-infected cells by various methods. The first method is to directly engage the target cell, penetrate the membrane of the target cell, and then inject proteins that cut and activate several apoptotic proteins, thereby starting the programmed cell death (apoptosis) of the target cell. The surface of the NK cell also contains a protein ligand that can bind to and activate the receptor, and the receptor is a protein ligand of the cell apoptosis-inducing ligand (TRAIL) associated with tumor necrosis factor (TNF), which opens an internal signal about apoptotic programmed cell death on the target cell. When stimulated, NK cells can also secrete cytokines such as IFN-γ and TNF-α, which not only inhibit viruses and tumors but also signal other immune cells. This broad and multimodal anti-cancer activity of NK cells has made them of great interest in the medical field.

[0058] CD56, also known as neural cell adhesion molecule, is a 200-220 kDa glycoprotein primarily expressed on human NK cells and a small number of MHC-mediated T lymphocytes. Based on the density of the CD56 differentiation antigen on the NK cell surface, human NK cells can be divided into two subpopulations: CD56 dim and CD56 bright. CD56 dim cells possess cytotoxic activity against target cells and are the primary component of NK cells, accounting for 90-95% of the total population. CD56 bright cells primarily secrete immune regulatory factors and comprise approximately 5-10% of NK cells.

[0059] CD16, also known as FcγRⅢ, is an Fc receptor on the surface of NK cells and belongs to the immunoglobulin superfamily. Binding to the Fc terminus of an antibody molecule activates NK cells and promotes their proliferation. It also mediates antibody-dependent cell-mediated cytotoxicity (ADCC), lysing and killing tumor cells.

[0060] CD94 is displayed on the surface of NK cells in either homotypic or heterotypic forms with NKG2A. Once the CD94-NKG2A complex binds to its ligand, it exerts potent inhibitory effects on NK cells. Although CD94 and its dimer form have distinct mechanisms of action, they also possess inhibitory properties. This may be related to viruses evading NK cells.

[0061] NKp30, NKp46, and NKp44: NKp30, NKp46, and NKp44 are all natural cytotoxicity receptors (NCRs). All three are members of the immunoglobulin superfamily (IgSF), but they share no homology. NCRs are expressed exclusively on the surface of NK cells and are unique to NK cells. They typically activate their killing function when KIR / KLR lose their ability to recognize "self."

[0062] "NKp30 (NCR3)": It is an important member of the NCR family, expressed on the surface of all NK cells, and plays an important role in NK cell activation and tumor killing.

[0063] "NKp44": also known as CD336, a member of IgSF, a natural cytotoxicity receptor (NCR2), which is expressed in activated NK cells. Its ligand is DAP12, and it participates in mediating the killing activity of NK cells.

[0064] "NKp46": CD335, whose extracellular region contains two Ig-like domains, while NKp30 has only one V-shaped domain. NKp46 and NKp30 have shorter cytoplasmic regions, and both contain positively charged arginine residues in their transmembrane regions.

[0065] CD107a: A functional marker that identifies NK cell activity (killing ability). CD107a, also known as LAMP-1 (lysosome-associated membrane protein-1), is a NK cell marker. It is commonly used to detect the cytotoxic activity of NK cells and other cell types, such as cytotoxic T cells.

[0066] “And / or”: should be understood to mean one or two alternatives.

[0067] "About" / "approximately": As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that ranges by ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% with respect to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0068] Unless otherwise indicated herein, any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer within the range, and, where appropriate, fractions thereof (such as tenths and hundredths of integers).

[0069] "Comprising": As used herein, unless the context requires otherwise, the word "comprising" will be understood to mean the inclusion of the specified steps, elements, or groups of steps or elements, but not the exclusion of any other steps, elements, or groups of steps or elements. In some embodiments of the present invention, the terms "including," "having," "containing," and "comprising" are used synonymously.

[0070] "Embodiments": Reference throughout this specification to "some embodiments," "some embodiments," "embodiments," "specific embodiments," "related embodiments," "an embodiment," or combinations thereof, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Therefore, various appearances of the foregoing phrases throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0071] "Treatment": As used herein, "treatment" includes any beneficial or desired effect associated with treatment. "Treatment" does not necessarily indicate complete eradication or cure of a disease or condition, or its associated symptoms.

[0072] "Subject": As used herein, "subject," "patient," or "individual" are used synonymously and include, but are not limited to, mammals, such as humans or non-human mammals, such as domestic animals, agricultural animals, or wild animals, as well as birds and aquatic animals. "Patient" includes individuals with fibrotic diseases, such as pulmonary fibrosis, particularly idiopathic pulmonary fibrosis.

[0073] "Autologous": As used herein, the term "autologous" means any material derived from the same individual that is subsequently reintroduced into that individual.

[0074] "Allogeneic": As used herein, "allogeneic" refers to a transplant derived from a different individual of the same species.

[0075] "Xenogeneic": As used herein, "xenogeneic" refers to a transplant that originates from a different individual of a different species.

[0076] "Administration" or "Administer(ed)" is synonymous with "use" or "dose". The administration disclosed in the present invention can be administered by any effective route, including intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection and infusion techniques. In some embodiments of the present invention, the administration is selected from at least one of oral, nasal, intravenous, intraperitoneal, intracerebral (intracerebral parenchyma), intraventricular, intramuscular, intraocular, intraarterial, portal vein, intralesional, sustained release system and implantation device.

[0077] "Pharmaceutically acceptable additives, carriers, diluents, excipients, preservatives, and other auxiliary ingredients" include, but are not limited to, at least one of diluents, solubilizers, emulsifiers, preservatives, preservatives, and adjuvants. Excipients are preferably non-toxic or substantially non-toxic to the recipient at the doses and concentrations employed. Such excipients include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, pharmaceutical compositions may contain substances for improving, maintaining, or preserving, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or penetration of the composition. The optimal pharmaceutical composition can be determined based on the intended route of administration, mode of delivery, and desired dosage.

[0078] "Effective amount": i.e., "Therapeutically Effective Amount", as used herein, "effective amount" is the amount of a composition or its active substance that is administered / applied to an individual to provide a beneficial effect or otherwise reduce harmful, non-beneficial events. "Effective amount" herein means a dosage that produces one or more desired or expected (e.g., beneficial) effects due to its administration / administration, which is performed once or multiple times within a specified time period. The exact dosage will depend on the purpose of the treatment and can be determined by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (Volumes 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)). In the treatment of pulmonary fibrosis, "effective amount" includes administering / applying to an individual an amount sufficient to improve the lung inflammatory response of patients with pulmonary fibrosis, down-regulate the expression of pulmonary fibrosis-related indicators, effectively kill myofibroblasts or down-regulate the expression of inflammatory factors in the lungs of patients with pulmonary fibrosis, etc., or otherwise reduce the disease burden of patients with pulmonary fibrosis.

[0079] "NK cell culture system": The NK cell culture system used in the present invention is not particularly limited in its components as long as it can culture and expand NK cells, for example, commercially available culture medium X-VIVO15 (Lonza, #04-418Q), OPTIVITRO NK Cell Expansion Kit P01 (Ecosai, #NE000-N022), ALyS505NK-EX (Zhuhai Beso Cell Science and Technology Co., Ltd., #01400P10), ALyS505NK-AC (Zhuhai Beso Cell Science and Technology Co., Ltd., #01600P02), SCGM (CELL GENIX, #20802-0500), MEMα (GICBO, Thermo Fisher, #31985070) and AIM V (GICBO, Thermo Fisher, #12055091), etc.

[0080] All publications, documents, and patents mentioned herein are hereby incorporated by reference in their entirety. In the event of a conflict, the present application (including any definitions herein) will control. However, any reference, article, publication, patent, patent publication, and patent application cited herein is not and should not be construed as an admission or any form of recommendation.

[0081] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1: Figure 1-A shows the detection results of surface markers (CD3, CD56) before and after NK cell expansion; Figure 1-B shows the detection results of surface activity markers (CD16, CD56, CD335 (NKp46)) after NK cell expansion;

[0083] Figure 2: Figure 2-A and Figure 2-B are CD56 + CD3 - The curve chart and bar chart of the detection results of NK cell expansion in vitro;

[0084] Figure 3 shows the flow cytometry results of the expression of CD56 and CD107a on the surface of amplified PB-NK cells;

[0085] FIG4 shows the test results of the killing efficiency of expanded NK cells against K-562 cells;

[0086] Figure 5 shows the results of monitoring the in vivo efficacy of NK cells in treating PF using Masson's trichrome staining;

[0087] Figure 6: Detection results of PF-related evaluation indicators after NK cell intervention;

[0088] Figure 7 shows the expression of proteins encoded by PF-related genes, COL3A1, COL1A2, and α-SMA, after NK cell intervention;

[0089] Figure 8: HE staining of the lungs of BLM-PF model mice after NK cell intervention;

[0090] Figure 9 shows the expression of inflammatory factors in the lungs of BLM-PF model mice after NK cell intervention.

[0091] Figure 10: Distribution of NK cells in the lungs of BLM-PF model mice after NK cell intervention. DETAILED DESCRIPTION

[0092] The following is a clear and complete description of the concept and technical effects of the present invention in conjunction with the embodiments, so that the purpose, features and effects of the present invention are fully understood. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments; based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without inventive effort are all within the scope of protection of the present invention.

[0093] In the following examples, the experimental methods without specific conditions are based on conventional methods and conditions known in the art, or are selected according to the product specifications. Reagents and raw materials not specified in the present invention are all commercially available.

[0094] Example 1

[0095] In vitro expansion and activity detection of PB-NK cells.

[0096] A. Day 1: Preparation of PBMCs

[0097] NK cells were expanded and cultured from healthy human PBMCs according to the instructions of the NK Cell Induction Reagent Kit (NK High-Efficiency Induction Culture Kit, Zhuhai Beso Cell Science and Technology Co., Ltd.). The specific method is as follows:

[0098] (1) Antibody coating of culture flasks

[0099] At T75cm 2 Add 9 mL of DPBS and 1 mL of ALySNK-A Reagent coating solution (Zhuhai Beso Cell Science Technology Co., Ltd., #T2020IA) to the culture flask. Gently shake the culture flask to evenly distribute the mixture on the surface of the culture flask. Incubate the flask at room temperature for 60 minutes or store it at 2-8°C overnight until use, then remove the coating solution. Rinse the culture flask once with 10 mL of DPBS (the washed culture flask should be used immediately, and be careful not to scratch the coated bottom of the flask during washing).

[0100] (2) Blood separation

[0101] Peripheral blood was collected from healthy individuals into sodium heparin blood collection tubes. 20-30 mL of blood was poured into two centrifuge tubes, each containing 15 mL of lymphocyte separation fluid. The tubes were centrifuged at 800 × g for 20 minutes at room temperature, with the volume rising and falling slowly. After centrifugation, the blood was separated into a plasma layer, a mononuclear cell layer, a separation fluid layer, and an red blood cell layer.

[0102] (3) Preparation of inactivated plasma

[0103] The upper plasma was collected with a pipette and placed in a centrifuge tube; the plasma was heated at 56°C for 30 minutes, centrifuged at 1200×g for 10 minutes at room temperature, and the supernatant was collected with a pipette into a new centrifuge tube and stored at 4°C.

[0104] (4) Preparation of PBMCs

[0105] Use a pipette to collect the second layer of mononuclear cells into a new centrifuge tube, add 35 mL of DPBS to dilute the cell suspension, and centrifuge at 500 × g for 10 minutes; remove the supernatant, repeat the dilution of the cell suspension and centrifugation steps 3 times to wash the cells, take 100 μL of the suspension after the final mixing, count the cells, and centrifuge the remaining suspension at 500 × g for 10 minutes.

[0106] B. Day 0 to Day 7: NK cell activation

[0107] NK cells in PBMCs were cultured and expanded using an NK cell culture system.

[0108] In this example, 1 mL of ALySNK-B Reagent (Zhuhai Beso Cell Science Technology Co., Ltd., #T2020IB) was added to 200 mL of ALyS505NK-AC (Zhuhai Beso Cell Science Technology Co., Ltd., #01600P02) to prepare INDM medium. 20 mL of INDM containing 10% inactivated plasma was taken to resuspend the prepared PBMCs and maintain the cell density at 1.3-1.5 × 10 6 cells / mL; add the above cell suspension to the culture flask coated with antibody; 2 Add the above cell suspension to a suspension culture flask and culture at 5% CO2 and 37°C. From Day 0 to Day 7, culture according to the culture process in Table 1 below, and add INDM medium to the culture flask.

[0109] C. Day 7 to Day 14: Expand and culture NK cells

[0110] IL-2 (Sihuan Bio, human IL-2 for injection, #SS009) was added to the serum-free culture medium of ALyS505NK-EX (Zhuhai Beso Cell Science Technology Co., Ltd., #01400P10) to prepare EXPM medium; the final concentration of IL-2 in the EXPM medium was 1000 IU / mL; when the culture was replenished on Day 7, the cell suspension in the culture flask was transferred to a culture bag, and EXPM medium was added according to the culture process shown in Table 1 below. The culture was continued in an incubation chamber at 5% CO2 and 37°C. The cultured cells were harvested on Day 14.

[0111] Table 1

[0112] D. Cell Collection

[0113] The cells were cultured for 14 days, and after successful expansion, samples were taken to test for cell phenotype, fungi, bacteria, mycoplasma, endotoxin, and other indicators; the cells were collected after 14 days of culture, and the cell suspension was transferred from the culture bag to a centrifuge bottle and centrifuged at 680×g for 10 minutes; the cell supernatant was discarded, and the cells were suspended in physiological saline containing 0.1% human serum albumin and collected into a centrifuge bottle; the centrifugation was repeated to wash the cells three times; the cells were filtered through a disposable cell sieve, collected, and injected into physiological saline containing 1% human serum albumin.

[0114] E. Flow cytometry was used to detect surface markers of NK cells before and after expansion on Day 0 and Day 14.

[0115] On Day 0 and Day 14, flow cytometry was used to detect the expression of CD56 and CD3 on the surface of NK cells before and after expansion culture. The results are shown in Figure 1-A. The expression of CD56 before and after expansion culture was calculated. + CD3 - The expansion fold of NK cells is shown in Figure 2.

[0116] As shown in Figure 1-A, on Day 14, CD56 + CD3 - The purity of NK cells increased from 14.06% on Day 0 to 97.53%;

[0117] As shown in Figure 2, Day 14, CD56 + CD3 - NK cells expanded approximately 250-fold.

[0118] On Day 14, flow cytometry was used to detect the expression of NK cell surface activation markers, CD16 and CD335 (NKp46), on the expanded NK cells. The results are shown in Figure 1-B.

[0119] As shown in Figure 1-B, after amplification, the activation markers of NK cells, CD16 or CD335, were effectively expressed. + CD56 + NK cells account for about 92.51%, CD335 + CD56 + NK cells accounted for about 91.04%, which showed that NK cells isolated from PBMCs (PB-NK cells) could be expanded in vitro, and the NK cells after large-scale expansion still had good activity.

[0120] Example 2

[0121] Detection of the killing effect of expanded PB-NK cells.

[0122] The PB-NK cells obtained by expansion and culture in Example 1 were taken, and the expression of CD56 and CD107a in the PB-NK cells was detected by flow cytometry to detect the proportion of NK cells with killing ability in the PB-NK cells. The results are shown in Figure 3;

[0123] As shown in Figure 3, in the PB-NK cells, CD56 + CD107a + NK cells (NK cells with killing ability) account for about 65.11%.

[0124] Day 0, the CD56+ CD107a + NK cells were mixed with K-562-luciferase cells (human chronic myeloid leukemia cells) at an effector-target ratio of 1:1 and 5:1, respectively. After 4 to 6 hours, the killing effect of NK cells against K-562 cells in each mixed cell group was detected and calculated using a microplate reader. The results are shown in FIG4 . The specific calculation method is:

[0125] Wells without PB-NK cells but containing only target cells K-562-luciferase cells were used as reference wells, and their fluorescence value was the total fluorescence value of the original tumor cell count. The fluorescence value of the remaining tumor cells after PB-NK cells with different effector-target ratios in each group killed K-562 cells was the residual fluorescence value. The killing efficiency was calculated as follows: Killing efficiency (%) = (total fluorescence value - residual fluorescence value) / total fluorescence value × 100%.

[0126] As shown in Figure 4, at an effector-target ratio of E:T = 5:1, the killing efficiency of NK cells is as high as 95%, indicating that NK cells are activated after mixing with K-562 cells and have the ability to effectively kill K-562 cells.

[0127] Example 3

[0128] Masson trichrome staining was used to monitor the effect of NK cell intervention in the PF model.

[0129] In this example, a bleomycin-induced PF mouse model (BLM-PF model mouse) was used, NK cells were administered to the BLM-PF model mice to treat pulmonary fibrosis, and the BLM-PF model mice treated with NK cell intervention were monitored.

[0130] On Day 0, 36 mice (approximately 20 g / mouse) were used, and 10 of them were injected with 200 μL of PBS buffer (Mock group); the remaining 26 mice were BLM-PF model mice induced with BLM (2.5 mg / mL) and divided into three groups, 10 of which were injected with PBS buffer (BLM+PBS intervention group), and 10 were injected with PBS buffer (200 μL) and PB-NK cells (1×10 7 cells / mouse) (NK cell intervention group), and 6 mice injected with PBS buffer (200 μL) and nintedanib (5 μg / mL / mouse) (nintedanib intervention group) as positive control. The specific dosing groups are shown in Table 2 below.

[0131] Table 2

[0132] On Day 14, the mice in each group were stained using Masson's trichrome staining. Lung tissues from each group of mice were prepared into tissue sections, and the staining of the lungs of each group of mice was observed under a microscope. The results are shown in Figure 5.

[0133] As shown in Figure 5, the mice in the Mock group, NK cell intervention group, and Nintedanib intervention group (upper left, lower left, and lower right) all appeared red (relatively light in the black and white picture), and the mice in the BLM+PBS intervention group (upper right) appeared blue (relatively dark in the black and white picture); NK cell intervention can significantly improve the PF symptoms of BLM-PF model mice.

[0134] Example 4

[0135] Evaluation of PF-related indicators after NK cell intervention.

[0136] The mice in each group described in Example 3 were continuously monitored. After Day 21, the mice in each group were evaluated. The results are shown in Figures 6A to 6C, respectively.

[0137] As shown in Figure 6-A, compared with the BLM+PBS intervention group, the NK cell intervention group and the nintedanib intervention group showed significant relief of pulmonary fibrosis. On Day 21, the volume of fibrosis in the NK cell intervention group accounted for approximately 20%. As shown in Figures 6-B and 6-C, the Ashcroft Assessment Scale score of the NK cell intervention group was less than 4, and the expression of collagen (a protein encoded by PF-related genes) also decreased significantly.

[0138] This proves that the therapeutic effect of the NK cell intervention group is significantly better than that of the nintedanib intervention group.

[0139] Example 5

[0140] Detection of PF-related gene and protein expression after NK cell intervention.

[0141] The mice in each group described in Example 3 were continuously monitored. After Day 21, the expression of various PF-related genes was detected by qPCR (quantitative polymerase chain reaction) and the mice in each group were evaluated. The results are shown in Figures 7A to 7C, respectively.

[0142] As shown in Figures 7-A, 7-B, and 7-C, compared with the BLM+PBS intervention group, the PF-related genes COL3A1 (type III collagen α1) and COL1A2 (type I collagen α2) in the NK cell intervention group and the nintedanib intervention group were significantly downregulated, and the expression intensity of α-SMA protein was also significantly downregulated compared with the BLM+PBS intervention group; moreover, in the NK cell intervention group, the degree of downregulation of COL3A1, COL1A2, and α-SMA proteins was better than that in the nintedanib intervention group.

[0143] This proves that the therapeutic effect of the NK cell intervention group is significantly better than that of the nintedanib intervention group.

[0144] Example 6

[0145] After NK cell intervention, the inflammatory response in the lungs of mice was monitored.

[0146] The mice in each group described in Example 3 were continuously monitored. After Day 21, the inflammatory response of the lungs of the mice in each intervention group was observed using hematoxylin-eosin staining (“HE staining”): the stained lung tissues of the mice in each group were prepared into tissue sections, and the staining of the lungs of the mice in each group was observed under a microscope. The results are shown in Figure 8; the qPCR method was used to detect the expression of inflammatory factors in the mice in each intervention group. The results are shown in Figure 9.

[0147] As shown in Figure 8 , NK cell intervention can significantly alleviate the lung inflammatory response in BLM-PF model mice;

[0148] As shown in Figure 9, after NK cell intervention, the expression of inflammatory factors IL-1β, CXCL1, CXCL2, IL-13, IL-17A and TGF-β1 in the mouse lungs were significantly downregulated, and the effect was better than that of nintedanib intervention.

[0149] Example 7

[0150] Detection of the distribution of NK cells in mouse lungs.

[0151] The mice in each group described in Example 3 were continuously monitored. After Day 21, the distribution of NK cells in the lungs of mice in each intervention group was scanned using IF technology (Immunofluorescence Technique, "IF"). The results are shown in Figure 10.

[0152] As shown in Figure 10, a large number of NK cells gathered near the lung fibroblasts (FAP) of mice in the NK cell intervention group. The lung inflammation of BLM-PF model mice treated with NK cells was alleviated, and the improvement of PF symptoms was strongly correlated with the activation and immune regulation of NK cells.

Claims

1. Application of NK cells in the preparation of drugs for the treatment of fibrotic diseases.

2. The use according to claim 1, characterized in that: The fibrotic diseases include heart failure, pulmonary fibrosis, liver cirrhosis and kidney disease; the heart failure includes HFpEF, HFmrEF and HFrEF.

3. The use according to claim 1 or 2, characterized in that: The fibrotic disease is pulmonary fibrosis.

4. The use according to claim 3, characterized in that: The pulmonary fibrosis is idiopathic pulmonary fibrosis.

5. The use according to any one of claims 1 to 4, characterized in that: The NK cells include NK cells from at least one of the following sources: autologous NK cells of the subject, allogeneic NK cells of the subject, xenogeneic NK cells of the subject, and induced pluripotent stem cell-derived NK cells; the subject is an individual suffering from or suspected of suffering from the fibrotic disease.

6. The use according to any one of claims 1 to 5, characterized in that: The medicine comprises NK cells and at least one of the following components: pharmaceutically acceptable additives, carriers, diluents, excipients, preservation solutions and other auxiliary components.

7. The use according to any one of claims 1 to 6, characterized in that: The medicament includes an effective amount of NK cells.

8. A method for treating a subject suffering from a fibrotic disease, characterized in that The method comprises administering NK cells to the subject.

9. The method according to claim 8, characterized in that The fibrotic diseases include heart failure, pulmonary fibrosis, liver cirrhosis and kidney disease; the heart failure includes HFpEF, HFmrEF and HFrEF.

10. The method according to claim 9, characterized in that The fibrotic disease is pulmonary fibrosis.

11. The method according to claim 10, characterized in that The pulmonary fibrosis is idiopathic pulmonary fibrosis.

12. The method according to any one of claims 8 to 11, characterized in that The NK cells include NK cells from at least one of the following sources: autologous NK cells of the subject, allogeneic NK cells of the subject, xenogeneic NK cells of the subject, and NK cells derived from induced pluripotent stem cells.

13. The method according to any one of claims 8 to 12, characterized in that: The NK cells are an effective amount of NK cells.

14. The method according to any one of claims 8 to 13, characterized in that: An effective amount of the NK cells is administered to the subject once or more.

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