Composition for treating graft-versus-host disease

Abstract

To provide a novel cell therapeutic agent that can improve GVHD treatment efficacy by strengthening immunomodulatory activity of MSC through use of a TLR5 agonist.SOLUTION: The present invention relates to a pharmaceutical composition for prevention or treatment of graft-versus-host disease, the composition containing, as an active ingredient, mesenchymal stem cells treated with a TLR5 (Toll-like receptor 5) agonist. The composition of the present invention is excellent in ability to reduce variability under a low-grade inflammatory environment and to exert an immunomodulatory effect, and can be utilized as an MSC-based therapeutic agent for steroid-resistant graft-versus-host disease (GVHD).SELECTED DRAWING: Figure 5b

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition for preventing or treating graft-versus-host disease, which comprises mesenchymal stem cells treated with a TLR5 agonist as an active ingredient. [Background technology]

[0002] Graft-versus-host disease (GVHD) is a severe inflammatory complication that primarily occurs after allogeneic hematopoietic stem cell transplantation (HSCT). It is induced by immune attack of donor T cells against recipient tissues. GVHD is a major cause of increased mortality after HSCT, and complications such as infection and organ toxicity also significantly affect patient survival. Accordingly, various cell-based therapeutic strategies have been developed to prevent or treat GVHD.

[0003] Mesenchymal stem cells (MSCs) are immune-privileged adult stem cells that do not induce immune rejection due to their low expression of MHC class I and absence of MHC class II, and they exhibit anti-inflammatory effects by secreting various immunoregulatory factors. Based on these properties, MSCs have been used to prevent and treat acute GVHD (aGVHD), and their clinical effectiveness has been reported through their suppression of lymphocyte activity and induction of macrophages into an anti-inflammatory M2 phenotype.

[0004] However, MSC-based therapies have limitations, such as variable immunostimulatory or immunosuppressive properties depending on the inflammatory stimulus, only inducing immunosuppressive functions in a high-grade inflammatory environment, while showing minimal therapeutic effects in low-grade inflammation. In clinical practice, calcineurin inhibitors and high-dose steroids are used in combination after HSCT, which reduces the inflammatory environment and limits the effectiveness of MSCs. Due to these issues, the clinical efficacy of MSCs in GVHD treatment has been inconsistent, and the therapeutic mechanism has not been clearly elucidated.

[0005] On the other hand, MSCs are known to express various Toll-like receptors (TLRs), which allow them to respond to pathogen-derived stimuli and regulate immune responses via the NF-κB signaling pathway. TLR5, in particular, is a receptor involved in innate immunity and has been reported to induce cytokines such as granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6) upon stimulation, and to be involved in the recovery of tissue injury and the suppression of inflammation.

[0006] KMRC011 is a TLR5 stimulator that can induce the immunomodulatory activity of MSCs even in low-grade inflammatory environments. NF-κB signaling is induced in MSCs through TLR5 agonists, which has been shown to increase the secretion of immunosuppressive factors such as indoleamine 2,3-dioxidase (IDO), cyclooxygenase-2 (COX-2), interleukin-10 (IL-10), and macrophage colony-stimulating factor (M-CSF), even without the overexpression of inflammatory cytokines.

[0007] These findings suggest that KMRC011-pretreated MSCs effectively suppress lymphocyte proliferation and induce macrophage differentiation toward an anti-inflammatory M2 phenotype, thereby alleviating disease severity in a real aGVHD mouse model.

[0008] Therefore, there is a pressing need for the development of new cell therapy agents that can improve the therapeutic efficacy of GVHD by enhancing the immunoregulatory activity of MSCs using TLR5 agonists. Summary of the Invention [Problem to be solved by the invention]

[0009] Against this background, the present inventors have completed the present invention by developing a pharmaceutical composition that can improve the therapeutic efficacy of GVHD by enhancing the immunoregulatory activity of MSCs using a TLR5 agonist.

[0010] An object of the present invention is to provide a pharmaceutical composition for preventing or treating graft-versus-host disease, which comprises mesenchymal stem cells treated with a TLR5 (Toll-like receptor 5) agonist as an active ingredient.

[0011] Another object of the present invention is to provide a method for producing mesenchymal stem cells with improved anti-inflammatory function, which comprises treating mesenchymal stem cells with a TLR5 agonist. [Means for solving the problem]

[0012] The present inventors have made intensive research efforts to develop a new cell therapy agent that can improve the therapeutic efficacy of GVHD by enhancing the immunoregulatory activity of MSCs using a TLR5 agonist.

[0013] As a result, the present inventors have developed a method for treating graft-versus-host disease by treating mesenchymal stem cells with a TLR5 agonist, thereby completing the present invention.

[0014] The configuration of the present invention will be described in detail below.

[0015] One aspect of the present invention is a pharmaceutical composition for preventing or treating graft-versus-host disease, comprising mesenchymal stem cells treated with a TLR5 (Toll-like receptor 5) agonist as an active ingredient.

[0016] The term "TLR5 agonist" as used herein refers to a substance that selectively stimulates the innate immune receptor TLR5, and is primarily composed of flagellin, a bacterial flagellar protein, or its derivatives. It binds to TLR5 in immune cells (particularly dendritic cells and macrophages) to activate the NF-κB pathway and induce anti-inflammatory or immunostimulatory cytokines, thereby exerting immunomodulatory effects. This agonist acts primarily at sites where TLR5 is expressed, such as the gastrointestinal mucosa, respiratory epithelial cells, and immune cells in tissues.

[0017] Mesenchymal stem cells (MSCs), as used herein, are adult stem cells that can be derived from autologous or allogeneic tissues. They are isolated from bone marrow, adipose tissue, umbilical cord blood, etc. and possess not only the ability to differentiate into various tissues but also strong immunoregulatory functions, making them useful for the development of cell therapy. In particular, in graft-versus-host disease (GVHD), donor T cells attack the recipient's tissues, triggering a severe inflammatory response. MSCs suppress T cell proliferation and activation, and regulate the immune response in GVHD by secreting anti-inflammatory cytokines and inducing M2 macrophages. Thanks to these mechanisms, MSCs are one of the stem cell types most actively used clinically as a treatment for GVHD, and their potential for treating steroid-refractory GVHD patients, who do not respond to conventional immunosuppressants, is attracting particular attention.

[0018] In the present invention, the TLR5 agonist may be one or more selected from the group consisting of KMRC011, flagellin, Entolimod (CBLB502), VAX102 / VAX124, and recombinant flagellin C, for example, but is not limited to, KMRC011.

[0019] Flagellin is a protein that makes up bacterial flagella and is a naturally occurring TLR5 agonist, while entrimod is a recombinant protein consisting of only the D0 and D1 domains of flagellin. Entrimod contains a hexa-histidine tag, which can induce unwanted immune responses, induce toxicity, and exhibit low specificity.

[0020] KMRC011 is a biologic developed as a Toll-like receptor 5 (TLR5) agonist, consisting of a recombinant protein derived from Salmonella flagellin. The hexa-histidine tag of KMRC011 has been removed to enhance TLR5 binding specificity and eliminate toxicity.

[0021] According to one embodiment of the present invention, MSCs treated with KMRC011 have enhanced immunomodulatory effects by increasing NFκB expression and secreting cytokines CCL2, CCl5, and CXCL2, which promote the differentiation and migration of M2 macrophages, and CD3 + It suppressed T cell proliferation.

[0022] Furthermore, in an animal model of induced GVHD, the group receiving KMRC011-treated MSCs showed greater protection from GVHD, with reduced inflammatory infiltration, maintained long small intestinal villi, a significant decrease in the level of Th1 cells expressing IFN-γ, a significant increase in the level of Treg cells expressing FoxP3, and an increase in the proportion of M2 macrophages.

[0023] In the present invention, mesenchymal stem cells may express one or more positive markers selected from the group consisting of COX2, IDO, CCL2, CCL5 and CXCL2, but are not limited thereto.

[0024] In the present invention, mesenchymal stem cells may express one or more negative CD markers selected from the group consisting of c-tyrosine protein kinase (Kit), CD11b, and CD45, but are not limited thereto.

[0025] The term "agent" as used herein refers to pharmaceutical products (as defined by the U.S. FDA) that are made from cells and tissues isolated, cultured, and specially manipulated from humans and used for therapeutic, diagnostic, and preventive purposes. This refers to pharmaceutical products in which live autologous, allogeneic, or xenogeneic cells are expanded and selected in vitro to restore the function of the cells or tissue, or the biological properties of the cells are altered in other ways, and in which such cells are used for the treatment, diagnosis, and prevention of disease.

[0026] The preferred dose of the composition of the present invention varies depending on the condition and weight of the individual, the severity of the disease, the drug form, the route and duration of administration, and can be appropriately selected by those skilled in the art. The administration may be once a day or in several divided doses, and the above dose does not limit the scope of the present invention in any aspect.

[0027] In the present invention, the pharmaceutical composition for treatment may further include a support, preferably a biodegradable support, for receiving the mesenchymal stem cells treated with a TLR5 agonist, which may be a hydrogel such as, but not limited to, fibrin glue, hyaluronic acid, gelatin, collagen, alginic acid, cellulose, pectin, chitin, polyglycolic acid, or polylactic acid.

[0028] In the present invention, the pharmaceutical composition for treatment may further comprise a pharmaceutically acceptable carrier, which may be saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of one or more of these components, and may contain other common additives such as antioxidants, buffers, and bacteriostatic agents, if necessary.

[0029] In the present invention, the therapeutic pharmaceutical composition may be formulated into an injectable dosage form such as, but not limited to, an aqueous solution, a suspension, an emulsion, etc. by additionally adding a diluent, a dispersant, a surfactant, a binder, and a lubricant.

[0030] In the present invention, "prevention" means any action of suppressing or delaying the progression of critical limb ischemia by administering the composition of the present invention.

[0031] In the present invention, "treatment" and "amelioration" refer to any action in which the symptoms of graft-versus-host disease are improved or beneficially altered by administering a composition of the present invention.

[0032] In the present invention, a therapeutically effective amount of a pharmaceutical composition refers to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal, or human that is anticipated by a researcher, veterinarian, physician, or other clinical practitioner, including an amount that induces alleviation of symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the content (number) of cells contained in the cellular therapy composition of the present invention will vary depending on the desired effect. Therefore, the optimal content of the cellular therapy composition can be easily determined by those skilled in the art and can be adjusted depending on various factors, including, but not limited to, the type of disease, the severity of the disease, the content of other components contained in the composition, the type of dosage form, the patient's age, weight, general health, sex, and diet, the time of administration, the route of administration, the excretion rate of the composition, the duration of treatment, and concomitant drugs.

[0033] In the present invention, the composition contains mesenchymal stem cells treated with a TLR5 agonist at a concentration of 1 × 10 per kg of body weight of the individual to be administered. 6 ~1 X 10 7 It can be administered by cell number.

[0034] Another aspect of the present invention is a method for producing mesenchymal stem cells with improved anti-inflammatory function, comprising the step of treating mesenchymal stem cells with a TLR5 agonist.

[0035] In the present invention, the TLR5 agonist may be one or more selected from the group consisting of KMRC011, flagellin, Entolimod (CBLB502), VAX102 / VAX124, and recombinant flagellin C, but is not limited to these.

[0036] In the present invention, the anti-inflammatory function may be, but is not limited to, acting in a low-inflammatory environment.

[0037] According to yet another aspect of the present invention, a customized kit for preventing or treating graft-versus-host disease may be provided, comprising the pharmaceutical composition according to the present invention. The kit may be prepared according to a kit preparation method used in the art, with the proviso that the kit may contain, as an active ingredient capable of inducing a therapeutic effect, mesenchymal stem cells treated with the TLR5 agonist of the present invention. [Effects of the Invention]

[0038] The present invention relates to a pharmaceutical composition for preventing or treating graft-versus-host disease (GVHD), which contains mesenchymal stem cells treated with a TLR5 (Toll-like receptor 5) agonist as an active ingredient. The composition of the present invention has excellent abilities to reduce variability and exert immunomodulatory effects in a low-grade inflammatory environment, and can be used as an MSC-based therapeutic agent for steroid-resistant GVHD. [Brief explanation of the drawings]

[0039] [Figure 1] FIG. 1 shows the results of a comparative evaluation of the activation reactions of flagellin and KMRC011. [Figure 2] FIG. 1 shows the results of evaluating the increased expression of NFκB and p65 phosphorylation levels in MSCs treated with KMRC011. [Figure 3] This figure shows the results of evaluating the increase in COX2 and IDO expression in MSCs treated with KMRC011, the increase in secretion of M2 macrophage-induced cytokines such as G-CSF and M-CSF, and the increase in cytokine gene expression such as CCL2, CCL5, and CXCL2, which are related to macrophage influx. [Figure 4] FIG. 1 shows the results of evaluating the reduction in CD3+ T cell proliferation in MSCs treated with KMRC011. [Figure 5] FIG. 1 shows the results of evaluating survival rates and clinical indicators after injecting KMRC011-treated MSCs into a GVHD animal model. [Figure 6] FIG. 1 shows the results of histopathological and immunohistochemical analyses observed in the major target organs of GVHD (skin, liver, small intestine, and large intestine) after injection of KMRC011-treated MSCs. [Figure 7] FIG. 1 shows the results of evaluating the splenic M2 ratio and macrophage migration in a GVHD model injected with KMRC011-treated MSCs. DETAILED DESCRIPTION OF THE INVENTION

[0040] The advantages and features of the present invention, as well as methods for achieving them, will become apparent from the following detailed description of the embodiments. However, the present invention is not limited to the embodiments disclosed below, and may be realized in various different forms. The embodiments are provided solely for the purpose of complete disclosure of the present invention and to fully convey the scope of the invention to those skilled in the art. The present invention is defined only by the scope of the claims.

[0041] Preparation Example 1. Preparation of mesenchymal stem cells for TLR5 agonist treatment Mesenchymal stem cells (MSCs) were isolated from bone marrow from the femurs and tibias of C57BL / 6 mice and cultured in complete Dulbecco's modified Eagle's medium (Gibco, Carlsbad, CA, USA). The medium contained 10% fetal bovine serum (FBS, Gibco), 2 mM L-glutamine (Gibco), and 1% antibiotics (penicillin [10 U / mL]-streptomycin [10 g / mL]). The FBS was heat-inactivated, and the endotoxin content was maintained below 5 EU / mL, and the hemoglobin concentration was maintained below 10 mg / dL. The immunophenotype of the cells was consistently positive for spinocerebellar ataxia type 1 (SCA1), CD44, and CD29, and negative for c-tyrosine protein kinase (Kit), CD11b, and CD45, and this was maintained after more than 15 passages. The cells used in this study were isolated at passages 15–20.

[0042] Example 1. Screening of TLR5 agonists The TLR5 activation potency of KMRC011 and flagellin was compared in HEK-Dual cells (human kidney cells dually expressing core gene reporters) transfected with the TLR5 gene. IL-8 responses were monitored using a luminometer using HEK-293 reporter cells.

[0043] The experimental results showed that at low concentrations (0.001-0.01 ng / ml), KMRC011 activated TLR5 much more effectively than flagellin (Figure 1A). In contrast, no biological activity of TLR5 was observed when LPS, a negative control, was used (Figure 1A).

[0044] In addition, NF-κB response was confirmed by alkaline phosphatase activity (AP) reaction measurement method under the same conditions.

[0045] As a result, we confirmed that KMRC011 showed higher reactivity than flagellin at a concentration of 0.01 ng / ml (Figure 1B). The significance level for validation was *P<0.05, **P<0.01. Each result is a representative result of two repeated experiments.

[0046] Manufacturing Example 2: Treatment of mesenchymal stem cells with TLR5 agonist 2-1. Establishment of animal models An animal model was established for the experiments. Baguio albino (BALB / c (H-2d)) mice (8–10 weeks old) and C57 black (BL) / 6 (H-2b) mice were purchased from OrientBio (Seongnam, Korea). Mice were maintained in a pathogen-free (SPF) environment in an animal facility with controlled humidity (55 ± 5%), photoperiod (12 h light / 12 h dark), and temperature (22 ± 1°C). The air in the facility was filtered through a high-efficiency particulate air (HEPA) filter system to block bacteria and viruses. Animals were provided with mouse chow and tap water ad libitum. The animal care and euthanasia procedures used in this animal model were approved by the Animal Experiment Ethics Committee of Korea University and The Catholic University of Japan College of Medicine (approval number: 2023-0220-01).

[0047] 2-2. Transplantation of MSCs pretreated with TLR5 agonists into animal models The TLR5 agonist used in this experiment was KMRC011, which was provided in lyophilized powder form by Connext (Daegu, Korea) and contained a dose of 150 μg per vial. KMRC011 was prepared according to the manufacturer's guidelines and prepared as a 150 μg / mL solution by adding 1 mL of distilled water to the vial. All drugs were dissolved immediately before use, and the remaining solution was discarded. MSCs were treated with the TLR5 agonist (100 ng / mL) 24 hours before use, and 2.8 × 10 4 / cm 2For in vivo experiments, MSCs pretreated with TLR5 agonists were seeded at a concentration of 1 × 10 on the day of bone marrow transplantation (BMT) and 4 days later. 6 Two cell doses were administered to mice.

[0048] Example 2. Evaluation of signal transduction via the NFκB kinase pathway After pretreatment of MSCs with KMRC011 for 24 hours, the increased expression and activation signals of NFκB were analyzed using quantitative reverse transcription PCR (qRT-PCR) and Western blot.

[0049] Total ribonucleic acid (RNA) was extracted using the RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Total RNA (2 μg) was reverse transcribed at 50°C for 2 min and then at 60°C for 30 min. Quantitative PCR was performed using an iQ PCR kit. TM The PCR was performed using SYBR® Green Supermix and a real-time PCR instrument CFX96 Touch (Bio-Rad, CA, USA) according to the manufacturer's instructions. The crossover point was defined as the maximum value of the second derivative of the fluorescence curve. The negative control group contained all components of the reaction mixture except template DNA. For quantitative analysis, the relative mRNA expression level of a specific gene was calculated using the ΔΔCt method relative to β-actin.

[0050] qRT-PCR results showed that the KMRC011-treated group had increased NFκB expression compared to non-pretreated MSCs, and when treated with IFN-γ and TNF-α (5 ng / mL each) to create a low-grade inflammatory environment, NFκB expression was increased compared to when no inflammatory environment was created (Figure 2A).

[0051] For Western blot experiments, MSCs were stimulated with or without IFN-γ and TNF-α (5 ng / mL each) to induce an inflammatory environment, and then pretreated with a TLR5 agonist for 24 hours. The medium was discarded, and the cells were washed twice with cold phosphate-buffered saline (PBS). Radioimmunoprecipitation assay lysis solution was then added to each well to lyse the cells, followed by centrifugation to obtain total cellular protein. Total protein concentration was measured using a bicinchoninic acid kit (Thermo Scientific, USA). Proteins were separated by 10% SDS-PAGE and electrotransferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% nonfat dry milk at room temperature for 1 hour, followed by overnight incubation with primary antibodies at 4°C. The membrane was washed with Tris-buffered saline and 0.1% Tween-20 (TBST) and then incubated with HRP-conjugated anti-rabbit IgG antibody (1:10,000, USA) at room temperature for 1 hour. After three washes, proteins were detected using an ECL detection kit and Hyperfilm-ECL reagent (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Relative protein expression levels were analyzed using ImageJ software. Antibodies used were: p-p65 (phospho Ser536) (Cell Signaling Technology, Danvers, MA, USA), β-actin.

[0052] Western blot analysis of p-p65 revealed that KMRC011-pretreated MSCs had higher p65 phosphorylation levels than non-pretreated MSCs, even in low-grade inflammatory conditions (Figure 2B). Because TLRs typically activate the NFκB pathway, p-p65 was analyzed to confirm this.

[0053] This confirmed that MSCs treated with KMRC011 increased signaling through the NFκB kinase pathway.

[0054] Example 3. Evaluation of cytokine secretion and expression in MSCs due to increased NFκB signaling To evaluate the immunomodulatory effects of KMRC011, we pretreated mesenchymal stem cells (MSCs) with the TLR5 agonist KMRC011 for 24 hours and then analyzed the gene and protein expression levels in a low-grade inflammatory environment (treated with low-dose TNF-α or LPS). First, RNA was extracted from KMRC011-pretreated MSCs and qRT-PCR was performed to confirm the increased expression of the immunosuppressive factors COX2 and IDO, confirming the enhanced immunosuppressive function of MSCs (Figure 3A). Next, ELISA analysis of conditioned medium collected under the same conditions confirmed the increased secretion of M2 macrophage-inducing cytokines, such as G-CSF and M-CSF (Figure 3B). Next, qRT-PCR was performed to target cytokine genes, such as CCL2, CCL5, and CXCL2, which are associated with macrophage influx. The expression of these genes was highest in KMRC011-pretreated MSCs under a low-grade inflammatory environment (Figure 3C).

[0055] This confirmed that KMRC011 pretreatment not only enhanced the immunosuppressive function of MSCs, but also enhanced their immunomodulatory effects by increasing cytokine secretion that promoted the differentiation and migration of M2 macrophages.

[0056] Example 4. MSC / peripheral blood mononuclear cell co-culture MSC (1.25 × 10 4) were cultured in Roswell Park Memorial Institute 1640 medium (Gibco) containing 10% FBS, penicillin (100 U / mL), and streptomycin (100 mg / mL). The medium was supplemented with 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Gibco), 2 mM L-glutamine, 10% heat-inactivated FBS, 100 mM sodium pyruvate, and a 1% antibiotic mixture containing penicillin (10 U / mL) and streptomycin (10 mg / mL). For TLR5 agonist pretreatment, MSCs were incubated with KMRC011 (100 ng / mL) for 24 hours to inhibit cell proliferation. 137 The mice were irradiated with 2,000 rads of radiation using a Cs source (GammaCell 3000 Elan, Nordion, Ontario, Canada). Human PBMCs were isolated from peripheral blood of healthy donors using Lymphoprep (STEMCELL Technologies, Vancouver, Canada) and stimulated with anti-CD3 and anti-CD28 antibodies. Then, PBMCs (1 × 10 5 ) were seeded into 96-well plates in a total volume of 200 μL at a ratio of 1:8 with or without irradiated MSCs.

[0057] On day 0, growth-inhibited MSCs were co-cultured with anti-CD3 / anti-CD28-stimulated PBMCs at a ratio of 1:8. On day 4, cells were harvested and analyzed by flow cytometry (FACS). Cells were collected into flow cytometry tubes and centrifuged at 1,800 rpm for 3 minutes to separate the cells. The cells were then washed twice with FACS staining buffer. Each sample was diluted and resuspended according to the manufacturer's instructions and incubated for 30 minutes at 4°C in the dark. Intracellular staining was performed using an intracellular staining kit (eBioscience) according to the manufacturer's instructions. Flow cytometry was performed using an LSRFortessa flow cytometer (Becton Dickinson), and data were analyzed using Flowjo software v10.7.1 (Tristar, Ashland, OR). Mononuclear cells were immunostained with the following combinations of fluorescently labeled antibodies: Ly-6A / E (Sca-1)-PE, CD285 (TLR5)-PE, CD29-FITC, CD117 (c-Kit)-FITC, IL-10-Pacific Blue, CD206-PE, F4 / 80-Alexa Fluor 700 (BioLegend), CD44-PE, and mouse IDO (mIDO)-eFluor. TM The following antibodies were used: CD11b-FITC, CD45-FITC, IL-4-PE, mouse TGF-β1 (mTGF-β1)-PE, CD86-APC, CD11b-PerCP-Cyanine 5.5 (BD Biosciences, San Diego, CA, USA), and mouse COX2 (mCOX2)-FITC (Bioss). Before intracellular cytokine staining, cells were stimulated for 13 hours in a 5% CO2, 37°C incubator with medium containing monensin (GolgiStop, 1 μL / mL; BD PharMingen). T cells immunostained for CD3 were gated and analyzed for expression of the proliferation marker Ki-67.

[0058] Experimental results showed that in the absence of non-pretreated MSCs or KMRC011-pretreated MSCs, CD3 + More than 70% of T cells were Ki-67 positive, indicating active proliferation, but we confirmed that T cell proliferation was significantly reduced in the presence of KMRC011-pretreated MSCs (Figure 4).

[0059] This confirmed that the inhibitory effect of MSCs on T cell proliferation was enhanced through KMRC011 pretreatment.

[0060] Example 5. Assessment of survival rate in GVHD animal models All recipient BALB / c H-2d mice were irradiated with a total of 800 cGy at a rate of 70 cGy / min using a Mevatron MXE-2 machine (Siemens, New York, USA). Subsequently, the disease induction group (GVHD group, n = 14) was treated with 5 × 10 bone marrow cells isolated from donor mice (C57BL / 6, H-2b). 6 and 5 x 10 spleen cells 6 The GVHD mice were then injected intravenously into the tail vein with 1 × 10 mice (Fig. 5a). 6 MSCs, 1 × 10 6 Mice were randomly assigned to one of three groups: one with or without KMRC011-pretreated MSCs. Control mice (n = 14) were irradiated in the same manner, but donor bone marrow and spleen cells were derived from the same BALB / c H-2d mice to prevent GVHD. Post-BMT survival was monitored daily, and the severity of clinical aGVHD was assessed three times weekly by summing scores for five clinical indicators (weight loss, posture, activity, coat condition, and skin condition). Mice with a total score of 7 or more were considered comatose and were euthanized 35 days after transplantation.

[0061] Experimental results showed that mice systemically injected with MSCs had a lower mortality rate and increased survival rate compared to the control group, and KMRC011-pretreated MSCs demonstrated superior therapeutic effects compared to non-pretreated MSCs (Figure 5b). Mice transplanted with allogeneic BMCs and SCs had lower survival rates and higher GVHD scores within 27 days (Figures 5b-5c). The KMRC011-pretreated MSC group demonstrated greater protection from GVHD, with the lowest clinical scores and alleviation of GVHD symptoms, including weight loss (Figures 5c-5d).

[0062] Example 6. Histopathological evaluation of GVHD major target organs After sacrificing the mice, small intestinal and liver tissues were fixed in 4% formaldehyde solution and embedded in paraffin. Five-μm-thick sections were prepared from each organ and subjected to H&E staining or immunohistochemistry (IHC) staining for histological analysis. A single pathologist read the slides in a blinded fashion to assess the severity of GVHD.

[0063] For the small intestine, scores were assigned on a scale of 0 to 5 based on the criteria of Cooke et al. for the following six items: (1) superficial colonic epithelial cell lesions or flattening of villi, (2) glands and regeneration, (3) glands and epithelial cell death, (4) glands and loss, (5) lamina propria inflammation, and (6) mucosal ulcers.

[0064] For the liver, scores were assigned on a scale of 0 to 3 for the following seven items: (1) portal inflammation, (2) bile duct lesions, (3) periportal necrosis, (4) vascular endotheliitis, (5) lobular necrosis-inflammatory reaction, (6) segmental necrosis, and (7) vascular arterial lymphocytic infiltration.

[0065] The scores for each item were summed to calculate a total score for each organ, and the scores for all target organs for each mouse were summed to calculate an overall histological score. Immunohistochemistry (IHC) staining results were evaluated according to a semiquantitative scoring system previously published. A score of 1 to 4 was assigned based on the percentage of epithelial cells showing strong nuclear staining, with the following criteria: 1 point for 1%-10%, 2 points for 11%-50%, 3 points for 51%-80%, and 4 points for 81%-100%.

[0066] Histopathological analysis of the major target organs of GVHD (skin, liver, small intestine, and large intestine) on day 21 posttransplant clearly demonstrated that the KMRC011-pretreated MSC group exhibited a more ameliorated GVHD pathology than the non-pretreated MSC group (Figure 6A). Inflammatory infiltration was observed in the liver tissue of the disease-induced group, but the degree of infiltration was reduced in the non-pretreated MSC group and was lowest in the KMRC011-pretreated MSC group. Small intestinal villi were severely disrupted and proliferative, exhibiting hyperchromatic nuclei and many cells showing signs of cell death. The KMRC011-pretreated MSC group maintained a longer length of small intestinal villi than the MSC group. Furthermore, immunohistochemical evaluation of T cell expression levels in the small intestine on day 21 after transplantation revealed that the KMRC011-pretreated MSC group had a significantly decreased level of Th1 cells expressing IFN-γ, and a significantly increased level of Treg cells expressing FoxP3 compared with the non-pretreated MSC group (Figure 6B).

[0067] Example 7. Evaluation of splenic M2 ratio and macrophage migration in a GVHD model Mesenchymal stem cells (MSCs) pretreated with KMRC011 (100 ng / mL) for 24 hours were intravenously administered to GVHD-induced mice, and the spleens were harvested on the 21st and 28th days to prepare single cell suspensions. + F4 / 80 + Flow cytometry was performed on macrophages for M1 markers (iNOS, CD86) and M2 markers (Arg1, CD206), and the differentiation pattern of macrophages and the M1 / M2 ratio in each group were compared.

[0068] The experimental results showed that at 21 days, the non-pretreated MSC group had a high proportion of M1 macrophages, while the KMRC011-pretreated MSC group had an increased proportion of M2 macrophages (Figures 7a-7d). The ratio of M1 to M2 macrophages was lowest in the KMRC011-pretreated MSC group (Figure 7b). At 28 days of treatment, the proportion of M1 macrophages significantly decreased in the KMRC011-pretreated MSC group compared to the non-pretreated MSC group (Figures 7e-7g), while the proportion of M2 macrophages significantly increased (Figures 7e-7f, 7h).

[0069] This confirmed that KMRC011-pretreated MSCs can alleviate GVHD by increasing the ratio of M2 macrophages, and that the M1 / M2 ratio continuously decreased as treatment progressed.

Claims

1. A pharmaceutical composition for preventing or treating graft-versus-host disease, comprising mesenchymal stem cells treated with a TLR5 (Toll-like receptor 5) agonist as an active ingredient.

2. 2. The pharmaceutical composition for preventing or treating graft-versus-host disease according to claim 1, wherein the TLR5 agonist is one or more selected from the group consisting of KMRC011, flagellin, Entolimod (CBLB502), VAX102 / VAX124, and recombinant flagellin C.

3. The pharmaceutical composition for preventing or treating graft-versus-host disease according to claim 1, wherein the mesenchymal stem cells express one or more positive markers selected from the group consisting of COx2, IDO, CCL2, CCL5 and CXCL2.

4. The pharmaceutical composition for preventing or treating graft-versus-host disease according to claim 1, wherein the mesenchymal stem cells express one or more negative CD markers selected from the group consisting of c-tyrosine protein kinase (Kit), CD11b, and CD45.

5. A method for producing mesenchymal stem cells with improved anti-inflammatory function, comprising treating mesenchymal stem cells with a TLR5 agonist.

6. 6. The method for producing mesenchymal stem cells with improved anti-inflammatory function according to claim 5, wherein the TLR5 agonist is one or more selected from the group consisting of KMRC011, flagellin, Entolimod (CBLB502), VAX102 / VAX124, and recombinant flagellin C.

7. The method for producing mesenchymal stem cells according to claim 5, wherein the anti-inflammatory function acts in a low-inflammatory environment.

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