Composition for treating inflammatory diseases induced by hyperimmune response

Abstract

The present invention relates to a composition for treating an inflammatory disease induced by a hyperimmune response, and provides a cell therapeutic agent for treating an inflammatory disease induced by a hyperimmune response. More specifically, the present invention provides a composition for treating an inflammatory disease induced by a hyperimmune response, which comprises (i) a chondrocyte or a cell capable of differentiating into a chondrocyte; and (ii) a chondrocyte into which a gene encoding TGF-β is introduced or a cell capable of differentiating into a chondrocyte into which a gene encoding TGF-β is introduced.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201680008586.3, filed on February 5, 2016, entitled "Composition for treating inflammatory diseases induced by hyperimmune response". Technical Field

[0002] This invention relates to cell therapeutic agents, and more specifically, to cell therapeutic agents for treating inflammatory diseases induced by hyperimmune responses, said cell therapeutic agents comprising (i) chondrocytes or cells capable of differentiating into chondrocytes; and (ii) chondrocytes or cells capable of differentiating into chondrocytes wherein a gene encoding TGF-β (transforming growth factor-β) is introduced therein. Background Technology

[0003] Osteoarthritis is a disease caused by the gradual loss of cartilage that protects the joints. It is caused by aging and is more likely to occur in the knee joint, which supports a large part of the body weight.

[0004] In contrast, rheumatoid arthritis, an autoimmune disease, is caused by the body's immune cells attacking the joints, leading to inflammation that persists and infiltrates the articular cartilage and bone tissue, resulting in tissue erosion. Rheumatoid arthritis frequently occurs in the fingers and wrists, and is particularly characterized by prominent intermediate knuckles in the fingers. Unlike degenerative arthritis, which mainly occurs in people aged 50 or older, rheumatoid arthritis often occurs at a younger age.

[0005] The causes, sites of onset, and ages of onset for osteoarthritis and rheumatoid arthritis are compared below (Table 1).

[0006] [Table 1]

[0007]

[0008] It has been reported that during rheumatoid arthritis, increased cytokine production in joint macrophages or fibroblasts, as well as increased IFN-γ production by Th1 cells and IL-17 production by Th17 cells, are observed. Although cytokines produced by Th1 and Th17 cells exacerbate arthritis symptoms, cytokines produced by Th2 cells, such as IL-4 and IL-10, are known to prevent or treat arthritis. It has been reported that when viral vectors containing the genes for IL-4 or IL-10, which are Th2-type cytokines, are injected into the joints of mice induced with arthritis, the therapeutic effect is applied not only to the injected leg but also to other legs (SH Kim et al., J. Immunol., 166: 3499-3505 (2001)).

[0009] Small molecules currently developed as therapeutic agents or reducing agents for rheumatoid arthritis include methotrexate, azathioprine, cyclophosphamide, and corticosteroids, as well as TNF-α antagonists such as Enbrel (etanercept), Remicade (infliximab), and Humira (adalimumab). Most of the currently developed treatments for rheumatoid arthritis have side effects such as gastrointestinal symptoms and do not significantly inhibit the progression of joint damage.

[0010] TGF-β1, one of the cell therapy components of this invention, is a multifunctional regulator of cell growth and differentiation. Neuropathic pain due to nerve injury has been reported to be significantly reduced by intrathecal injection of recombinant TGF-β1, which targets multiple cell types with multidifferentiation potency.

[0011] Recently, there have been reports of TGF-β1-expressing chondrocytes or fibroblasts being effective in treating osteoarthritis (Lee KH et al., Hum Gene Ther 2001; 12: 1805-1813, SUN U. SONG et al., Tissue Engineering 2005; 11: 1516-1526), ​​but there has been no report of their potential use in treating rheumatoid arthritis, which has different causes and pathologies than osteoarthritis.

[0012] Therefore, the inventors of this invention have made efforts to develop a cell therapy agent with anti-inflammatory effects and therapeutic efficacy for rheumatoid arthritis, and as a result, it has been confirmed that when animal models of induced rheumatoid arthritis are treated with a mixed cell composition of chondrocytes expressing TGF-β1 and chondrocytes, the symptoms of rheumatoid arthritis are improved, thereby completing this invention. Summary of the Invention

[0013] The purpose of this invention is to provide chondrocytes or cells capable of differentiating into chondrocytes and chondrocytes expressing TGF-β1 (which are not chemically synthesized drugs), or specific cells capable of differentiating into chondrocytes expressing TGF-β1, for therapeutic use in treating inflammatory diseases induced by hyperimmune responses.

[0014] To achieve the above objectives, the present invention provides, in one aspect, a composition for treating inflammatory diseases induced by hyperimmune responses, comprising (i) chondrocytes or cells capable of differentiating into chondrocytes; and (ii) chondrocytes wherein a gene encoding TGF-β is introduced, or cells capable of differentiating into chondrocytes wherein a gene encoding TGF-β is introduced.

[0015] Another aspect of the present invention provides a method for treating an inflammatory disease induced by a hyperimmune response, comprising administering a composition to a subject, the composition comprising (i) chondrocytes or cells capable of differentiating into chondrocytes; and (ii) chondrocytes wherein a gene encoding TGF-β is introduced, or cells capable of differentiating into chondrocytes wherein a gene encoding TGF-β is introduced.

[0016] Furthermore, another aspect of the present invention provides the use of a composition as a therapeutic agent for treating inflammatory diseases induced by hyperimmune responses, said composition comprising (i) chondrocytes or cells capable of differentiating into chondrocytes; and (ii) chondrocytes wherein a gene encoding TGF-β is introduced, or cells capable of differentiating into chondrocytes wherein a gene encoding TGF-β is introduced.

[0017] Another aspect of the present invention provides a method for inhibiting the expression or activity of cytokines selected from the group consisting of IL-6, IL-17A, IL-1β, and TNF-α, the method comprising administering a composition to a subject, the composition comprising (i) chondrocytes or cells capable of differentiating into chondrocytes; and (ii) chondrocytes wherein a gene encoding TGF-β is introduced, or cells capable of differentiating into chondrocytes wherein a gene encoding TGF-β is introduced. Attached Figure Description

[0018] The above and other aspects, features, and advantages of the present invention will become more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, wherein

[0019] Figure 1 This is a diagram illustrating the therapeutic mechanism of the mixed cell composition of the present invention for rheumatoid arthritis and osteoarthritis;

[0020] Figure 2 To elucidate the results of pain relief efficacy confirmed by the von Frey filament test after mixed cell therapy in animal models of osteoarthritis;

[0021] Figure 3 Elucidate the results of H&E staining performed to confirm the cartilage status of the knee joint after mixed cell therapy in an animal model with osteoarthritis;

[0022] Figure 4 To clarify the results of Masson trichrome staining performed to confirm the status of knee cartilage after mixed cell therapy in an animal model with osteoarthritis;

[0023] Figure 5 To clarify the results of immunostaining performed to confirm the content of type I and type II collagen in cartilage after mixed cell therapy in animal models with osteoarthritis;

[0024] Figures 6A-6B To clarify the results of the analysis of the expression levels of TGF-β1 and IL-10 in synovial lavage fluid in an animal model induced with osteoarthritis;

[0025] Figures 7A-7B To elucidate the results of immunochemical staining of the synovium using antibodies against the macrophage surface antigen CD68 and IL-10, in order to confirm the macrophage status in the joint cavity of cells-treated animals induced with degenerative arthritis;

[0026] Figure 8A The results of immunochemical staining of the synovium using antibodies against CD86 and arginase 1 were elucidated. Figure 8B Explaining how to convert to an inverted version using Image-Pro Plus Figure 8A The image, and Figure 8C This is a diagram illustrating the measurement of CD86 and arginase 1 positive cells;

[0027] Figure 9 The results of qRT-PCR elucidated the general pattern of gene expression in the synovium;

[0028] Figure 10 This is a graph illustrating the measurement results of reduced edema in the metatarsal region on days 19 and 26 after a mixed cell therapy-induced rheumatoid arthritis animal model.

[0029] Figure 11 To elucidate the results of cytokine alterations measured by harvesting synovial lavage fluid from the joint cavity on day 28 after a mixed cell therapy-induced rheumatoid arthritis animal model. Detailed Implementation

[0030] Hybrid cells, comprising allogeneic (non-autologous) chondrocytes and chondrocytes expressing TGF-β, are known as cell-mediated gene therapy agents for osteoarthritis. In a phase 2 clinical study in patients with osteoarthritis, the hybrid cells were shown to improve pain, activity, and cartilage structure. However, the mechanism of action of the hybrid cells remains undiscovered.

[0031] The inventors of this invention observed the effects of injecting mixed cells into an animal model of induced osteoarthritis on pain and structural transformation, and confirmed whether the mixed cells induce an anti-inflammatory environment. As a result, it was confirmed that anti-inflammatory cytokines and M2 macrophages increased in arthritic knee joints treated with mixed cells, thereby inducing an anti-inflammatory environment that relieves pain and induces joint regeneration.

[0032] Therefore, it was confirmed that mixed cells induce an environment rich in M2 macrophages with immunosuppressive properties, and under the hypothesis that mixed cells are effective against inflammatory diseases induced by hyperimmune responses in animal models of rheumatoid arthritis, chondrocytes and TGF-β-expressing chondrocytes were mixed at a predetermined ratio and used to treat the knee joint. The results showed that in the group treated with mixed cells, a reduction in metatarsal edema was confirmed, and further, a reduction in the expression of cytokines IL-6, IL-17A, IL-1β, and TNF-α, which induce and promote inflammatory responses, was confirmed in the synovial lavage fluid. The therapeutic effect of a single dose of mixed cells lasted for 26 days.

[0033] Conventionally, materials used in the treatment of rheumatoid arthritis, such as methotrexate (MTX), can cause digestive and oral ulcers and carry risks of hepatotoxicity, kidney failure, etc. Furthermore, treatments containing anti-TNF-α antibodies such as Remicade are known to impair the patient's innate immune system, leading to the expression of tuberculosis (TB) from TB carriers. In contrast, the cell therapy agent according to the present invention has the advantage of not showing the aforementioned side effects in clinical trials for degenerative arthritis. Therefore, the inventors of the present invention have confirmed the possibility that hybrid cells can be a novel therapeutic agent overcoming the disadvantages of conventional rheumatoid arthritis treatments.

[0034] Therefore, one aspect of the present invention relates to a composition for treating inflammatory diseases induced by hyperimmune responses, the composition comprising: (i) chondrocytes or cells capable of differentiating into chondrocytes; and (ii) chondrocytes wherein a gene encoding TGF-β is introduced, or cells capable of differentiating into chondrocytes wherein a gene encoding TGF-β is introduced.

[0035] Another aspect of the invention relates to a method for treating an inflammatory disease induced by a hyperimmune response, comprising administering a composition to a subject comprising (i) chondrocytes or cells capable of differentiating into chondrocytes; and (ii) chondrocytes wherein a gene encoding TGF-β is introduced or cells capable of differentiating into chondrocytes wherein a gene encoding TGF-β is introduced.

[0036] Another aspect of the invention relates to the use of a composition as a therapeutic agent for treating inflammatory diseases induced by hyperimmune responses, said composition comprising (i) chondrocytes or cells capable of differentiating into chondrocytes; and (ii) chondrocytes wherein a gene encoding TGF-β is introduced, or cells capable of differentiating into chondrocytes wherein a gene encoding TGF-β is introduced.

[0037] In this invention, inflammatory diseases induced by hyperimmune response include rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, colitis, type 1 diabetes, and encephalitis, and among these diseases, it is particularly effective in treating rheumatoid arthritis.

[0038] In this invention, the cells that can differentiate into chondrocytes are chondrocyte precursor cells or stem cells.

[0039] In this invention, (i) the chondrocytes or cells capable of differentiating into chondrocytes; and (ii) the chondrocytes in which a gene encoding TGF-β is introduced or the cells capable of differentiating into chondrocytes in which a gene encoding TGF-β is introduced can be mixed in different mixing ratios, and the ratio of (i) to (ii) is preferably from 1:1 to 100:1, and more preferably 3:1.

[0040] In one aspect of the present invention, a composition for treating inflammatory diseases induced by hyperimmune responses comprises untransduced human chondrocytes (hChonJ cells) and TGF-β1-expressing chondrocytes (hChonJb#7), more specifically, human allogeneic chondrocytes (hChonJ) and chondrocytes expressing human TGF-β1. The two cell types can be mixed in various mixing ratios. For example, the two cell types can be mixed in a 3:1 ratio, i.e., a ratio of 3 hChonJ cells to 1 hChonJb#7. Cells mixed in a 3:1 ratio and methods for producing them are disclosed in the paper [Cytotherapy, 2012 Feb; 14(2): 247-256] and U.S. Patent Registration Nos. 7,005,127 and 7,282,200.

[0041] In one aspect of this invention, in a rat model of arthritis treated with hybrid cells, pain began to decrease 15 days after treatment and the pain relief effect lasted until 56 days after treatment (the end date of the experiment). Pain relief has been confirmed to be associated with structural improvement of the arthritic knee joint, and the treated hybrid cells were found to be effective for both pain relief and chondrocyte regeneration. That is, the hybrid cells used in this invention are a cell-mediated gene therapy targeting pain relief and cartilage tissue regeneration.

[0042] In another aspect of the invention, the cartilage tissue in a rat model of arthritis treated with mixed cells was stained with type I collagen and type II collagen and subjected to histochemical analysis. The results confirmed that the mixed cells regenerated hyaline cartilage tissue positive for type II collagen.

[0043] Based on their activation phenotype, macrophages, which are innate immune cells, are classified into activated "M1 macrophages" with pro-inflammatory properties and activated "M2 macrophages" with immunosuppressive properties. IFN-γ is a key cytokine involved in the activation of M1 macrophages, and TLR signaling induced by pathological recognition contributes to the differentiation of M1 macrophages. M1 macrophages are known to increase the expression of TNFα, IL-12, and IL-23 and decrease the expression of IL-10 to induce Th1 and Th17 immune responses that cause tissue damage. Conversely, M2 macrophages increase the expression level of IL-10 and decrease the expression of IL-12, thereby reducing inflammation and promoting tissue regeneration.

[0044] In one aspect of the invention, TGF-β expressed in human chondrocytes hChonJb#7 (induced by human chondrocyte hChonJ) expressing TGF-β1 and IL-10 induces the generation of M2 macrophages, and the generated M2 macrophages secrete IL-10 to create an anti-inflammatory environment in joints treated with hybrid cell therapy. Histochemical analysis of the synovium in an animal model treated with hybrid cell therapy confirmed an increase in M2 macrophages (…). Figures 8A-8C ).

[0045] Furthermore, it was determined that the immunosuppressive effect of the compounds of the present invention was induced by increased expression of TGF-β1 and arginase 1, and in one aspect of the present invention, arginase 1 expression was increased in the synovium of the knee joint in an animal model using mixed cell therapy.

[0046] Increased arginase 1 expression and decreased L-arginine levels are known to induce strong immunosuppressive effects in the immune system, and TGF-β has been reported to induce arginase 1 transcription and arginase activity (Durante et al., Circulation, 103(8), 1121-7, 2001). Therefore, it has been determined that M2 macrophages can more effectively regulate immunity in the presence of TGF-β1.

[0047] Furthermore, in the anti-inflammatory effects of the compositions of the present invention, it is shown that IL-10 expressed in M2 macrophages induced by mixed cell therapy activates STAT3 and PI3K, thereby demonstrating an anti-inflammatory effect regulated by Hmox-1.

[0048] Meanwhile, recently, cytokines and growth factors such as interleukin-1 (IL-1), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) have been detected in the synovium and synovial fluid of patients with chronic rheumatoid arthritis (Nouri et al., Clin. Exp. Immunol. 55: 275-372, 1984; Thornton et al., Clin. Exp. Immunol. 86:79-86, 1991; Saxne et al., Arthritis Rheum. 31:1041-1045, 1988; Seitz et al., J. Clin. Invest. 87: 463-469, 1991; Lafyatis et al., J. Immunol. 143: 1142-1148). 1989;Melnyk et al., Arthrtis Rheum. 33: 493-500, 1990).

[0049] IL-6 is a cytokine known as B cell stimulating factor 2 or interferon β2. IL-6 is a differentiation factor involved in B lymphocyte activation (Hirano T. et al., Nature 324, 73-76, 1986) and is known to be a multifunctional cytokine affecting multiple cell types (Akira, S. et al., Adv. In Immunology 54, 1-78, 1993).

[0050] High levels of interleukin-6 and soluble IL-6 receptors have been reported in the serum or synovial fluid of patients with chronic rheumatoid arthritis (Houssiau et al., Arthritis Rheum. 31: 784-788, 1988; Hirano et al., Eur. J. Immunol. 18: 1797-1801, 1988; Yoshioka et al., Japn. J. Rheumatol. in print). Similar results have been obtained even in animal models of rheumatoid arthritis (Takai et al., Arthritis Rheum. 32: 594-600, 1989; Leisten et al., Clin. Immunol. Immunopathol. 56: 108-115, 1990), and these results indicate that IL-6 is known to be a factor associated with chronic rheumatoid arthritis.

[0051] Meanwhile, in the clinical progression of rheumatoid arthritis (RA), secukinumab, as a biologic agent, is a high-affinity human monoclonal antibody that inhibits IL-17A activity. In a proof-of-concept (PoC) study of RA, patients with active RA treated with a stable dose of methotrexate (MTX) received co-treatment with secukinumab. The results confirmed that the secukinumab treatment group rapidly improved clinical symptoms of RA compared to placebo (Hueber et al. (2010) Sci. Transl. Med. 2(52):52-72). This data suggests that IL-17A neutralization may be effective in patients with RA.

[0052] In another aspect of the invention, it was confirmed that treating rat paws with the mixed cells, in which rheumatoid arthritis was induced by full Freund's adjuvant (CFA) treatment and metatarsal edema was confirmed for 28 days, was effective. As a result, in the group treated with the mixed cells, a reduction in metatarsal edema was confirmed. Figure 10 The analysis of pro-inflammatory cytokines IFN-γ, IL-6, IL-17A, IL-1β and TNF-α that induce and promote inflammatory responses confirmed that metatarsal edema was statistically significantly reduced in both the mixed cell therapy group and the MTX treatment group compared to the control group.

[0053] Therefore, the composition of the present invention has the effect of inhibiting the expression of inflammatory cytokines IL-6, IL-17A, IL-1β and TNF-α to reduce edema, said inflammatory cytokines being excessively secreted from the synovial lavage fluid of patients with rheumatoid arthritis.

[0054] The invention will now be described in more detail by way of examples. These examples are merely illustrative, and it will be apparent to those skilled in the art that the scope of the invention is not limited to these examples.

[0055] Example 1: Induction of pain relief and Col II-positive cartilage regeneration through treatment with mixed cells in an animal model of arthritis

[0056] In a phase 2 clinical trial of mixed cell therapy in patients with arthritis, reduced pain and structural improvements in the joint environment were observed. To confirm the mechanism, mixed cell therapy, joint histochemistry, and von Frey filament assays were performed in an animal model of MIA-induced osteoarthritis.

[0057] 1-1: Preparation of MIA-induced arthritis animal model

[0058] Six-week-old male rats (Spargue-Dawley, 200–225 g, Nara Biotech, Korea) were used in the preparation of the animal model. Animal experiments were conducted under veterinary control with approval from the Kolon LifeScience (Korea) Institutional Animal Care and Use Committee (IACUC No. KLS IACUC 2013-04).

[0059] To induce arthritis, the left knee joint cavity of rats was treated with 50 μl of MIA (sodium iodoacetate monosodium, Sigma, USA) solution at a concentration of 60 mg / mL using a 31G syringe.

[0060] 1-2: Preparation of cells for cell therapy

[0061] The hChonJ cells used in this invention are human-derived chondrocytes, and hChonJb#7 are chondrocytes transformed to secrete TGF-β. Methods for preparing the chondrocytes and the mixing ratios are disclosed in the paper [Cytotherapy, 2012 Feb; 14(2): 247-256] and U.S. Patent Registration Nos. 7,005,127 and 7,282,200.

[0062] hChonJb#7 can be prepared by injecting TGF-β cDNA into cells using known methods. For example, a vector containing TGF-β cDNA can be prepared by including promoters such as metallothionein and TGF-β in a vector known to contain resistance genes such as ampicillin and neomycin [e.g., pUC19 (containing the ampicillin resistance gene), Gibco-BRL]. Subsequently, the vector containing TGF-β cDNA is injected into chondrocytes using known methods such as the calcium phosphate method or the liposome method to prepare hChonJb#7.

[0063] 1-3: Confirm the pain relief and cartilage regeneration effects of the mixed cell therapy.

[0064] Two weeks after MIA injection, mixed cells {(1.2 × 10 6 Cells: hChonJ (9 × 10⁻⁶) 5 Cells) +hChonJb# 7 (3.0 × 10 5 Cells)}, hChonJ (9 × 10 5 Cells), hChonJb#7 (3.0 × 10⁻⁶) 5The joint cavity of the left knee was treated with either cells or the control {CryoStor-10 (CS-10)}. General symptoms were observed, and weight was measured weekly.

[0065] Pain relief was observed through the implementation of the von Frey fiber test, and the results were as follows: Figure 2 As shown, pain levels decreased 15 days after cell therapy, and this effect persisted until the end of the observation period (day 56 post-treatment). This was observed when using hChonJb#7 cells expressing TGF-β (3.0 × 10⁻⁶). 5 In the group treated with hChonJ cells (9 × 10⁻⁶ cells), pain relief was observed from day 15, but the effect disappeared after 6 weeks. Conversely, in the group treated with normal human chondrocytes (hChonJ cells), pain relief was observed from day 15, but the effect disappeared after 6 weeks. 5 No pain relief was observed in the cell therapy group.

[0066] To confirm whether the pain-relieving effect of mixed cells is related to cartilage regeneration, left knee joints from five experimental groups were collected and histochemically analyzed using H&E staining and Masson's trichrome staining (see [link to study]). Figure 3 and Figure 4 ).

[0067] No cartilage regeneration was observed in the control (CS-10) and hChonJ treatment groups. In contrast, more cartilage was observed in the hChonJb#7 treatment group and the mixed cell therapy group compared to the other control group.

[0068] In addition, the results of immunohistochemical staining are as follows: Figure 5 As shown, it was confirmed that the content of type II collagen in the cartilage of the mixed cell therapy group was higher than that of the hChonJb#7 treatment group, and from this result, it can be seen that the cartilage in the mixed cell therapy group is close to hyaline cartilage.

[0069] To assess the degree of improvement in joint condition, the histopathological results and pathological scores of H&E-stained joints were evaluated using the methods disclosed in the paper [Kobayasi, K., J. et al., J. Vet. Med. Sci., 65:1195-1199, 2003], and the results are summarized in Table 2 below. Among the following experimental results, the mixed cell therapy group showed the greatest improvement in cartilage.

[0070] [Table 2]

[0071]

[0072]

[0073] Example 2: Analysis of the effect of mixed cell therapy on the secretion of anti-inflammatory cytokines

[0074] The effects of mixed cell therapy on the profile of cytokine expression in synovial fluid within the joint cavity were confirmed in an animal model.

[0075] To confirm whether macrophages were induced at the cellular site, CD68-positive cells were confirmed to be present in the synovium.

[0076] Tissue sections were prepared by microdissecting paraffin-embedded synovium with a thickness of 4 μm, and then immunohistochemical staining was performed using CD68 antibody (Novus, Co, USA).

[0077] The result is, as Figure 7A As shown, CD68-positive cells were significantly increased in the mixed cell therapy group.

[0078] IL-10 is known to have a pain-relieving effect. Based on the above reports, an arthritis model was generated and treated with mixed cells, and the expression of IL-10 was subsequently confirmed. The expression of IL-10 was measured in the synovial lavage fluid of rats with induced arthritis using a 96-well plate assay with magnetic beads (#RECYTMAG-65K, Milpore, USA).

[0079] The result is, as Figure 6B As shown, increased IL-10 expression was confirmed in both the mixed cell therapy group and the hChonJ treatment group. Immunological analysis confirmed an increase in IL-10-positive cells in the mixed cell therapy group. These results indicate that mixed cell therapy in arthritic knee joints induces the anti-inflammatory cytokine IL-10.

[0080] Example 3: Confirmation of the M2 macrophage enrichment environment induced by mixed cells

[0081] There are two types of macrophages. M1 macrophages are responsible for acute inflammation by secreting high levels of inflammatory cytokines such as IL-12 and TNF-α, while M2 macrophages play an important role in wound healing and anti-inflammatory regulation.

[0082] In Example 2, high levels of IL-10 expression were confirmed in the mixed cell therapy group, and as a result, the expression of CD86, a marker of M1 macrophages, and arginase 1, a marker of M2 macrophages, were confirmed to confirm whether the mixed cells induced macrophages in the knees of animals with induced arthritis and in the synovium of animal models.

[0083] Therefore, paraffin-embedded synovium was immunostained using CD86 antibody (AbD serotec, NC, USA) and mouse arginase 1 antibody (BDBioscience, USA).

[0084] The result is, as Figures 8A-8C As shown, CD86 expression was not different between the control and mixed cell therapy groups, but arginase 1 was significantly higher in the mixed cell therapy group. This indicates that mixed cells induce M2 macrophages.

[0085] Example 4: Confirmation of M2 macrophage enrichment environment induced by mixed cells:RNA

[0086] To confirm the gene expression levels induced by M2 macrophages as identified in Example 3, total RNA was isolated from animal synovium and the expression of genes associated with M1 and M2 macrophages was confirmed.

[0087] RNA was isolated from the synovium using the RNeasy Lipid Tissue Mini Kit (QIAGEN, USA) and then isolated using SuperScript. TM The III First-Strand Synthesis System (Invitrogen, USA) synthesized cDNA from isolated RNA.

[0088] Real-time PCR was then performed using an ABI 7900 instrument. A total of 50 μL of PCR reaction mixture was used, comprising 1 μL of cDNA, 0.2 μM of each primer, and 10 μL of SYBR Premix Ex Taq (TAKARA, Japan), with 40 cycles performed at 95°C for 10 seconds and 60°C for 30 seconds. Gene expression levels were confirmed using the 2-ΔΔCt method, and the qRT-PCR primers used are listed in Table 3.

[0089] [Table 3] Primer sequences used for qRT-PCR

[0090]

[0091] An overview of macrophage-related gene expression is shown in Figure 9 CD69 expression was high in the mixed cell therapy group. TNF-α showed no difference between the control and mixed cell therapy groups. IL-10, an anti-inflammatory cytokine, was highly expressed in the mixed cell therapy group.

[0092] To confirm whether increased IL-10 mRNA expression induced protein expression, a multiplex cytokine assay was performed in the synovium. The results were as follows: Figure 10As shown, a rapid increase in IL-10 protein levels was confirmed in the mixed cell therapy group. Significant increases in mRNA levels of additional macrophage markers, IL-10RA (interleukin-10 receptor α subunit) and Hmox1 (heme oxygenase 1), were also confirmed.

[0093] When summarizing the results, an increased induction of M2 macrophages was observed in the case of administering mixed cells in the arthritis model.

[0094] Example 5: Confirmation of the efficacy of mixed cells in an animal model of rheumatoid arthritis

[0095] 5-1: Preparation of animal models of rheumatoid arthritis

[0096] In the preparation of an animal model of induced rheumatoid arthritis, 7-week-old female rats (160 to 180 g Lewis, Central laboratory animals, Japan) were used. Animal experiments were conducted with the approval of the Animal Care and Use Committee of KOL Life Sciences (Korea) (IACUC No. KLS IACUC 2015-07).

[0097] To induce rheumatoid arthritis, 50 μl of a 5 mg / mL CFA (Complete Freund's Adjuvant, manufacturer: Chondrex, Inc.) solution was administered subcutaneously to the subcutaneous sole of rats using a 31G syringe. After 3 weeks, the induction of arthritis was confirmed by measuring the size of metatarsal edema using calipers. Subsequently, when edema induction was most severe, the experimental material was administered into the left knee joint cavity of Lewis rats.

[0098] 5-2: Testing the therapeutic effect of the test material on animal models

[0099] Rheumatoid arthritis was induced in rats in groups of 6, and after 3 weeks of CFA treatment, mixed cells {(1.2 × 10⁻⁶)} were collected. 6 Cells: hChonJ (9 × 10⁻⁶) 5 Cells) + hChonJb#7 (3.0 ×10 5 Cells)}, hChonJ (9 ×10 5 Cells), hChonJb#7 (3.0 ×10⁻⁶) 5 The cells and negative control (CS-10) were treated into the left knee joint cavity. In the positive control case, 0.4 mg / kg of MTX (methotrexate) was administered orally once a week. Metatarsal diameter and body weight were measured every three days.

[0100] Table 4 illustrates the treatment conditions for each treatment group.

[0101] [Table 4] Treatment conditions for each experimental group

[0102]

[0103] Treatment conditions for each experimental group

[0104] 5-3: Effects of mixed cells in animal models of rheumatoid arthritis

[0105] The test material was processed onto an arthritis model, and the size of metatarsal edema was measured using calipers. The results are shown in... Figure 10 In the control (CS-10), hChonJ, hChonJb#7, mixed cell therapy, and MTX (methotrexate) treatment groups, reductions in edema size were measured on days 19 and 26, and the results showed statistically significant reductions compared to the control in the mixed cell therapy and MTX treatment groups.

[0106] 5-4: Analysis of the overall profile of cytokine secretion through mixed cell therapy

[0107] Samples were processed into an arthritis model. On day 28, synovial lavage fluid was collected from the left knee joint cavity of the experimental rats and cytokines were analyzed. The results are shown in... Figure 11 Synovial lavage fluid was collected from the joint cavity of rats by administering 50 μL of saline solution via a 31G insulin syringe and collecting the solution immediately upon syringe puncture. IFN-γ, IL-6, IL-17, IL-1β, and TNF-α as cytokines were analyzed using a cytokine / chemokine multicomplex (RECYTMAG-65K, Millipore). Analysis of pro-inflammatory cytokines IFN-γ, IL-6, IL-17, IL-1β, and TNF-α, which induce and promote inflammatory responses, was conducted, and the results showed a statistically significant reduction in cytokines compared to the control group in both the mixed cell therapy group and the MTX (methotrexate) treatment group.

[0108] The hybrid cell composition of the present invention can improve symptoms of inflammatory diseases, including rheumatoid arthritis, and suppress hyperimmune responses through cell therapy without the side effects of other types of conventional drug therapies.

[0109] Although specific parts of the invention have been described in detail, it will be apparent to those skilled in the art that such detailed description is merely a preferred embodiment and that the scope of the invention is not limited thereto. Therefore, the essential scope of the invention will be defined by the appended claims and their equivalents.

[0110] This disclosure relates to the following implementation plan.

[0111] 1. A composition for treating inflammatory diseases induced by hyperimmune responses, the composition comprising: (i) chondrocytes or cells capable of differentiating into chondrocytes; and (ii) chondrocytes or cells capable of differentiating into chondrocytes wherein a gene encoding TGF-β (transforming growth factor-β) is introduced therein.

[0112] 2. The composition according to embodiment 1, wherein the inflammatory disease induced by the hyperimmune response is selected from the group consisting of: rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, colitis, type 1 diabetes, and encephalitis.

[0113] 3. The composition according to embodiment 2, wherein the inflammatory disease induced by hyperimmune response is rheumatoid arthritis.

[0114] 4. The composition according to embodiment 1, wherein the cells capable of differentiating into chondrocytes are chondrocyte precursor cells or stem cells.

[0115] 5. The composition according to embodiment 1, wherein (i) chondrocytes or cells capable of differentiating into chondrocytes; and (ii) chondrocytes or cells capable of differentiating into chondrocytes wherein a gene encoding TGF-β (transforming growth factor-β) is introduced therein, in a ratio ranging from 1:1 to 100:1.

[0116] 6. The composition according to embodiment 5, wherein (i) chondrocytes or cells capable of differentiating into chondrocytes; and (ii) chondrocytes or cells capable of differentiating into chondrocytes wherein a gene encoding TGF-β (transforming growth factor-β) is introduced therein, in a ratio ranging from 1:1 to 100:1.

[0117] 7. A method of treating an inflammatory disease induced by a hyperimmune response, comprising administering a composition to a subject, said composition comprising (i) chondrocytes or cells capable of differentiating into chondrocytes; and (ii) chondrocytes wherein a gene encoding TGF-β is introduced, or cells capable of differentiating into chondrocytes wherein a gene encoding TGF-β is introduced.

[0118] 8. Use of the composition as a therapeutic agent for treating inflammatory diseases induced by hyperimmune response, said composition comprising (i) chondrocytes or cells capable of differentiating into chondrocytes; and (ii) chondrocytes wherein a gene encoding TGF-β is introduced, or cells capable of differentiating into chondrocytes wherein a gene encoding TGF-β is introduced.

[0119] 9. A method for inhibiting the expression or activity of cytokines selected from the group consisting of IL-6, IL-17A, IL-1β, and TNF-α1, the method comprising administering a composition to a subject, the composition comprising (i) chondrocytes or cells capable of differentiating into chondrocytes; and (ii) chondrocytes wherein a gene encoding TGF-β is introduced, or cells capable of differentiating into chondrocytes wherein a gene encoding TGF-β is introduced.

Claims

1. A composition for treating rheumatoid arthritis, the composition comprising: (i) chondrocytes; and (ii) hChonJb#7 wherein a gene encoding TGF-β (transforming growth factor-β) is introduced, wherein the composition has reduced expression of IL6, IL-17A, IL-1β and TNF-α, and the composition is administered into the joint cavity.

2. The composition according to claim 1, wherein (i) chondrocytes; and (ii) hChonJb#7 wherein the gene encoding TGF-β (transforming growth factor-β) is introduced in a ratio ranging from 1:1 to 100:

1.

3. The composition according to claim 2, wherein (i) chondrocytes; and (ii) hChonJb#7 wherein the gene encoding TGF-β (transforming growth factor-β) is introduced in a ratio ranging from 1:1 to 3:

1.

4. Use of the composition in the preparation of a medicament for treating rheumatoid arthritis, wherein the composition comprises (i) chondrocytes; and (ii) hChonJb#7 wherein a gene encoding TGF-β is introduced, wherein the composition has reduced expression of IL6, IL-17A, IL-1β and TNF-α, and wherein the composition is administered into the joint cavity.

5. The use according to claim 4, wherein (i) chondrocytes; and (ii) hChonJb#7 wherein the gene encoding TGF-β (transforming growth factor-β) is introduced, the ratio of which is in the range of 1:1 to 100:

1.

6. The use according to claim 5, wherein (i) chondrocytes; and (ii) hChonJb#7 wherein the gene encoding TGF-β (transforming growth factor-β) is introduced, in a ratio ranging from 1:1 to 3:1.

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