WO2012099441A2 - Immune response inhibiting composition containing stem cells expressing tryptophan metabolizing enzyme genes - Google Patents

Abstract

The present invention relates to mesenchymal stem cells expressing tryptophan metabolizing enzyme genes and to an immune inhibiting composition comprising the same. More particularly, kynurenine-3-hydroxylase and kynureninase which are tryptophan metabolizing enzymes which are not naturally expressed from mesenchymal stem cells are expressed in mesenchymal stem cells, to thereby effectively inhibit alloantigen-reactive T cells of mesenchymal stem cells and thus increase immunosuppressive effects. The mesenchymal stem cells in which kynurenine-3-hydroxylase and kynureninase are expressed may provide more enhanced immunosuppressive effects than mesenchymal stem cells in which those enzymes are not expressed, and therefore, may effectively prevent and treat diseases caused by T-cell overactivation, including graft-versus-host diseases.

Description

Composition for inhibiting immune response containing stem cells expressing tryptophan metabolic enzyme gene

The present invention relates to a stem cell expressing a tryptophan metabolase gene and a composition for inhibiting an immune response comprising the same.

Immunosuppressants refer to a variety of substances that are used to reduce or block the host's ability to produce antibodies (a humoral immune response) or a cellular immune response to antigenic stimulation. Mainly used cases can be classified into three categories. Firstly, they are used to prevent rejection after organ transplant surgery. Immunosuppressants are used for the purpose of treatment and prevention to maintain transplanted tissues or organs. The use of immunosuppressive agents to suppress acute transplant rejection after surgery and slow rejection after a certain period of time has increased the success rate of tissue and organ transplantation. However, despite numerous immunosuppressive agents for inhibiting organ transplant rejection, immunosuppressant safety is still insufficient.

Second, immunosuppressants are used to treat autoimmune diseases. Autoimmune disease occurs when an individual's immune system recognizes endogenous proteins as foreign antigens. As a result, the formed antibody or immune T cells respond to this antigen present in the tissue, destroying the tissue with the antigen. Immunosuppression has been shown to be effective in inhibiting this autoimmune response. Most autoimmune diseases have yet to be elucidated on the path or mechanism of their development, so they rely on immunosuppressive agents such as steroids without fundamental treatment to see only temporary symptoms. These diseases include rheumatoid arthritis, nephritis, osteoarthritis, systemic lupus erythematosus, atopic dermatitis, psoriasis, aphthosis, asthma, and pediatric diabetes. .

Third is the use of antibodies to specific antigens for selective immunosuppression. One of the most effective and selective immunosuppressive regimens currently used is the prevention of Rh hemolytic disease of the newborn. Rh-negative mothers whose mothers are pregnant with Rh-positive fetuses are exposed to maternal circulating blood flow to fetal erythrocytes due to childbirth, miscarriage, or ectopic pregnancy. The removal of can block the antibody formation against Rh-positive erythrocytes of the parent. This treatment prevents the Rh-negative mother from causing multiple Rh-positive babies to cause fetal bloat caused by the mother's red blood cell mismatch.

Looking at the classification of immunosuppressive agents according to each action mechanism and the development status of immunosuppressive agents according to each mechanism, immunosuppressive agents are classified into five types according to the action mechanism.

First, it is an immunosuppressive agent that inhibits the regulators of gene expression of proteins involved in inflammation and immunity. Representative drugs include glucocorticoids. The drug regulates the expression of many cytokine (IL-1, IL-2, IL-6, etc.) genes directly and indirectly by entering cells and binding to specific positions in the nucleus. It is known that the proliferation of T cells and B cells is reduced, and cytokine production and activity are suppressed.

Second, alkylating agents are drugs that alkylate DNA and interfere with DNA replication in T and B cells, thereby inhibiting and killing their function. In this case, B cells are slower in recovery than T cells, and thus the drug effect on B cells is stronger, resulting in more suppression of humoral immunity. However, when a large dose of the drug is administered, immunotolerance to new antigens exposed at the same time may occur, and there is a high risk of metastasis and metastasis to cancer. Representative drugs include cyclophosphamide.

Third, kinase and phosphatase inhibitors (inhibitors of kinase and phosphatases). They inhibit the intracellular signal transduction system, which is a sequential reaction of phosphorylation and dephosphorylation, resulting in immunosuppressive effects. Representative drugs include Cyclosporine and FK506.

Fourth, new inhibitors of de novo purine synthesis act as potent antagonists of inosine monophosphate dehydrogenase (IMPDH), which is essential for purine base neosynthesis. Therefore, it inhibits lymphocyte proliferation and antibody production by B cells, depletes guanine nucleotides of leukocytes, inhibits leukocyte proliferation and migration to inflammatory sites, and glycosylation of glycoproteins on the surface of lymphocyte cells. It interferes with glycosylation.

Finally, new inhibitors of pyrimidine synthesis inhibit the proliferation of activated T cells by inhibiting the dihydroorotate dehydrogenase (DHODH), an enzyme essential for the biosynthesis of pyrimidine among the bases required for DNA synthesis. Suppress This inhibition of ribonucleotide synthetase can stop cell division in the G1 phase and thus inhibit the proliferation of stimulated lymphocytes. Drugs of this mechanism include leflunomide.

However, the commonly used immunosuppressants, despite their popularity, some of them are not sufficient in effect or have serious side effects such as anemia, hair loss, hypertension, hyperlipidemia, hyperglycemia, stomach ulcer, liver and kidney dysfunction.

Thus, candidates for new immunosuppressants have recently emerged to alleviate the side effects of existing drugs and to produce sufficient immunosuppressive effects, and new concept pharmaceuticals such as biopharmaceuticals and cell therapies for suppressing excessive immune responses are emerging.

In related prior art, Korean Patent No. 0963030 and "Mesenchymal Stem Cell-mediated Autologous Dendritic Cells with Increased Immune Suppression Capacity," such as "CD70 expressing neural stem cells capable of inhibiting a transplant immune response and its use", Techniques such as Korean Laid-Open Publication No. 2008-0109705 can be given.

Thus, the present inventors have been studying the development of a new cell therapy that can effectively suppress the excessive immune response, and found that stem cells expressing tryptophan metabolizing enzymes effectively inhibit T cell overactivation to enhance immune suppression effect. The present invention has been completed.

Accordingly, it is an object of the present invention to provide a composition for inhibiting an immune response comprising stem cells expressing tryptophan metabolase gene.

It is also an object of the present invention to provide a composition for preventing and treating graft-versus-host disease comprising stem cells expressing tryptophan metabolic enzyme gene.

In addition, an object of the present invention is to provide a stem cell expressing tryptophan metabolase gene and a method for producing the same.

In order to achieve the above object, the present invention provides a composition for inhibiting immune response comprising stem cells expressing tryptophan metabolase gene.

In one embodiment of the present invention, the tryptophan metabolase may be kynurenine-3-hydroxylase (KMO) or kynureninase (KYNU).

In one embodiment of the present invention, the kynurenine 3-hydroxylase (KMO) may have an amino acid sequence of SEQ ID NO: 9, kynureninase (KYNU) is a sequence It may have an amino acid sequence of the list 10.

In one embodiment of the present invention, the stem cell may be a stem cell introduced with a recombinant expression vector comprising a nucleic acid encoding a tryptophan metabolase.

In one embodiment of the present invention, the recombinant expression vector may be pBI-CMV1-KMO-KYNU having a cleavage map of FIG.

In one embodiment of the present invention, the stem cells may be adult stem cells.

In one embodiment of the present invention, the adult stem cells are mesenchymal stem cells, knapsack, placenta, umbilical cord, cord blood, skin, peripheral blood, bone marrow, fat, muscle, liver, nerve tissue, periosteum, fetal membrane, synovial membrane , Synovial fluid, amniotic membrane, meniscus, meniscus ligament, articular chondrocytes, indwelling, perivascular cells, striatal bone, subpatellar mass, spleen and thymus.

In one embodiment of the present invention, the immune response may be caused by T-cell overactivation.

In one embodiment of the invention, the immune response may be an immune response against transplanted organs, tissues or cells.

In one embodiment of the invention, the transplanted organ, tissue or cell may be a homologous graft.

In one embodiment of the invention, the transplanted organ, tissue or cell may be a xenograft.

In one embodiment of the present invention, the composition can be used for the prevention and treatment of organ transplant rejection, graft versus host reaction, autoimmune disease or chronic inflammatory disease.

In one embodiment of the present invention, the autoimmune disease is rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, Addison's disease, vitiligo, scleroderma, Goodpasture syndrome, Bezette's disease, Crohn's disease, ankylosing Spondylitis, uveitis, thrombocytopenic purpura, vulgaris, pediatric diabetes mellitus, autoimmune hemolytic anemia, cryoglobulinosis, adrenal proteinuria and systemic lupus erythematosus.

The present invention also provides a composition for preventing or treating graft-versus-host disease comprising stem cells expressing the tryptophan metabolase gene.

The present invention effectively inhibits allogene-antigen-responsive T cells of stem cells by introducing and expressing kynurenine-3-hydroxylase and kynureninase, which are tryptophan metabolizing enzymes not naturally expressed in stem cells, into stem cells. Since the inhibitory effect can be enhanced, stem cells expressing kynurenine-3-hydroxylase and kynureninase have an improved immunosuppressive effect compared to stem cells that do not express these enzymes. The disease caused by cell overactivation can be effectively prevented and treated.

1a and 1b show the results of morphological changes and immunophenotype analysis of mesenchymal stem cells, respectively.

Figures 2a and 2b shows the weak immunosuppressive effect of IDO, Figure 2a shows the gene and protein expression pattern of IDO, Figure 2b shows the effect of homologous antigen-responsive T cells.

Figure 3 examines the enzyme expression of mesenchymal stem cells themselves.

Figure 4 shows the recombinant vector pBI-CMV1-KMO-KYNU according to an embodiment of the present invention,

Figure 5 examines the enzyme expression of mesenchymal stem cells according to the present invention expressing KMO and KYNU.

6a and 6b examine the effect of homologous antigen-responsive T cells in the metabolism of tryptophan premetabolism of mesenchymal stem cells according to the present invention expressing KMO and KYNU.

The present invention provides a pharmaceutical composition for immunosuppression which can suppress an excessive immune response caused by T cell overactivation, including stem cells expressing tryptophan metabolase gene.

The stem cells expressing the tryptophan metabolase gene according to the present invention, tryptophan metabolase gene that is not expressed in natural stem cells, that is, kynurenine 3-hydroxylase (KMO) or kynu Refers to stem cells expressing the enzyme gene by introducing Reninase (kynureninase (KYNU) from the outside.

The tryptophan metabolase refers to an enzyme that acts on tryptophan metabolism pathway leading from tryptophan to tryurtophan metakinism through kynurenine to 3-hydroxykynurenine and decomposing tryptophan into kynurenine. Indoleamine 2,3-dioxygenase (IDO), which plays a role, is representative, and kynurenine 3-hydroxylase (KMO), kynureninase; KYNU), kynurenine amino-transferase, and the like.

The present inventors found that indoleamine 2,3-dioxygenase (IDO) gene is naturally expressed in INF-ν treatment of stem cells, but it is an enzyme after decomposing kynurenine, that is, kynurenine-. It was found that 3-hydroxylase (KMO) and kynureninase (KYNU) were not expressed in stem cells, and the immune response was suppressed through the external introduction of the two enzymes into stem cells. It was confirmed that can be maximized.

According to one embodiment of the present invention, after introducing kynurenine-3-hydroxylase (KMO) and kynureninase (KYNU) genes into stem cells using a recombinant vector (execution) As a result of confirming that the proliferation of lymphocytes in response to homologous antigens is suppressed, the proliferation of T cells is markedly reduced, indicating that stem cells according to the present invention suppress excessive T cell immune responses ( See Example 6.

Looking at the method for producing the stem cells according to the present invention in detail, first it is necessary to separate the stem cells from the tissue and culture.

Here, "stem cells" include both embryonic stem cells, which are pluripotent stem cells, and adult stem cells, which are multipotent stem cells.

The “adult stem cells” refer to undifferentiated cells that are capable of self-reproduction as stem cells occurring in differentiated tissues and can be differentiated into all cell types of derived tissues. Specifically, the adult stem cells are umbilical cord blood (umbilical cord blood), bone marrow, spleen, ovary, testis, peripheral blood, amniotic fluid, brain, blood vessels, skeletal muscle, skin or gastrointestinal epithelium, cornea, tooth pulp, retina, It can be extracted from the liver or pancreas, and means primitive cells just before differentiation into cells of specific organs such as bone, liver, and blood. For example, the spinal cord has been reported to have hematopoietic stem cells (HSCs) and mesenchymal stem cells (Mesenchymal Stem Cells), and neural stem cells, stem cells derived from the brain, have subventricular zones ( It is known to be isolated from the subventricular zone, ventricular zone and hippocampus of the central nervous system (CNS). In general, adult stem cells are known to be difficult to proliferate, but have a strong tendency to differentiate. As a result, adult stem cells can be used not only to regenerate organs required by actual medicine but also to characterize each organ after transplantation. It has characteristics that can be differentiated accordingly.

Therefore, the stem cells used in the present invention include both embryonic stem cells and adult stem cells, preferably adult stem cells, most preferably mesenchymal stem cells of adult stem cells.

The “mesenchymal stem cell” is a primordial cell of mesoderm origin, which can proliferate in an undifferentiated state and is separated from various tissues such as bone marrow, adipose tissue, liver, tendon, synovial cord and cord blood. However, there is no single marker that can be clearly defined as mesenchymal stem cells. However, CD14, CD34 and CD45 are well known as hematopoietic stem markers, and SH-2 (CD105), SH-3 (CD73), SH-4 and Thy-1 are known as mesenchymal markers. Mesenchymal stem cells express major histocompatibility complex (MHC) class I, and MHC class II can be induced by interferon gamma (IFN-γ) and can be expressed by Fas or Fas ligand ( Since it does not express FAS L, CD40) and costimulatory molecules (B7), it does not induce an immune response and is free from cytolysis by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. It is.

The mesenchymal stem cells according to the present invention can be used anywhere in the tissue where mesenchymal stem cells are present, ie, backpack, placenta, umbilical cord, cord blood, skin, peripheral blood, bone marrow, fat, muscle, liver, nerve tissue, periosteum, fetus. Membrane, synovial membrane, synovial fluid, amniotic membrane, meniscus, trans cruciate ligament, articular chondrocyte, indwelling, perivascular cells, striatum, subpatellar mass, spleen or thymus can be separated. In one embodiment of the present invention used mesenchymal stem cells isolated from the bone marrow.

After the separation of the stem cells is preferably added to passaging step. In one embodiment of the present invention, the stem cells were subcultured for 7 to 8 generations.

Thereafter, a step of transforming stem cells by introducing tryptophan metabolase gene into the cultured stem cells is required. That is, in this step, the nucleic acid molecule encoding the tryptophan metabolizing enzyme is introduced into the stem cells. The tryptophan metabolizing enzymes correspond in particular to kynurenine 3-hydroxylase (KMO) and kynureninase (KYNU).

Tryptophan metabolases of the present invention include not only proteins having their wild type amino acid sequence, but also variants thereof. The variant refers to a protein in which the natural amino acid sequence of tryptophan metabolase and one or more amino acid residues have different sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. The variant may be a functional equivalent exhibiting the same biological activity as the native protein or a variant in which the physicochemical properties of the protein are modified as necessary. It is a variant with increased structural stability to physicochemical environment or enhanced physiological activity.

Preferably, the enzyme encodes a kynurenine 3-hydroxylase (KMO) having an amino acid sequence of SEQ ID NO: 9 and a kynureninase (KYNU) enzyme having an amino acid sequence of SEQ ID NO: 10. It is provided in the form of a nucleic acid having a nucleotide sequence. The nucleotide sequence encoding the tryptophan metabolase is a nucleotide sequence encoding an enzyme in wild-type or variant form as described above, wherein one or more bases can be mutated by substitution, deletion, insertion, or a combination thereof. Or by chemical synthesis.

Nucleic acid having a nucleotide sequence encoding the above tryptophan metabolase may be single or double chain, DNA molecules (genome, cDNA) or RNA molecules.

The nucleic acid encoding the tryptophan metabolase is introduced into the cell by known methods in the art, for example, naked DNA in the form of a vector (Wolff et al. Science, 1990: Wolffet al. J Cell Sci. 103: 1249-59). , 1992), liposomes, cationic polymers (Cationic polymer) and the like can be introduced into the cell. Liposomes are phospholipid membranes prepared by mixing cationic phospholipids such as DOTMA or DOTAP for gene introduction. When liposomes and anionic nucleic acids are mixed in a proportion, a nucleic acid-liposomal complex is formed.

Preferably, in introducing a gene of tryptophan metabolizing enzyme into stem cells, a recombinant vector may be used as a carrier of the enzyme gene.

As used herein, "recombinant" refers to a cell expressing a heterologous nucleic acid, expressing the nucleic acid, or expressing a protein encoded by a peptide, heterologous peptide, or heterologous nucleic acid. The recombinant cell transformed with the vector may express a gene or a gene fragment which is not expressed in the natural form of the cell in one of the sense and antisense forms. In addition, recombinant cells may express genes expressed in cells in a natural state, but the genes are modified and reintroduced into cells by artificial means.

"Vector" as used herein refers to DNA fragment (s), nucleic acid molecules that are delivered into a cell. Vectors can replicate DNA and be reproduced independently in host cells. "Delivers" can often be used interchangeably with "vectors." An "expression vector" refers to a recombinant DNA molecule comprising a coding sequence of interest and an appropriate nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

As used herein, "operably linked" refers to the functional linkage of a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein of interest to perform a general function. Operational linkage with recombinant vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation employs enzymes commonly known in the art.

Vectors of the present invention include signal or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals, enhancers, and can be prepared in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. In addition, the expression vector includes a selectable marker for selecting a host cell containing the vector and, in the case of a replicable expression vector, a replication origin. Vectors can self replicate or integrate into host DNA.

Vectors include plasmid vectors, cosmid vectors, viral vectors, and the like. Preferably, it is a viral vector. Viral vectors are retroviruses such as Human immunodeficiency virus HIV (Murineleukemia virus) Avian sarcoma / leukosis (ASLV), Spleen necrosis virus (SNV), Rous sarcoma virus (RSV), and Mouse mammary tumor viruses, such as, but not limited to, vectors derived from Adenovirus, Adeno-associated virus, Herpes simplex virus, CMV (cytomegalovirus), and the like.

The recombinant expression vector according to the present invention may use any replicable expression vector capable of expressing kynurenine 3-hydroxylase (KMO) and kynureninase (KYNU). In an embodiment of the present invention, pBI-CMV1-KMO-KYNU having a cleavage map of FIG. 4 was used as the recombinant expression vector.

Through the above processes, after introducing tryptophan metabolase into stem cells, a step of measuring the expression and activity of the tryptophan metabolase gene in the transformed stem cells is required. Although not limited thereto, the transformed stem cells may measure the expression and activity of tryptophan metabolase gene using mRNA expression or high performance liquid chromatography (HPLC).

Accordingly, the present invention provides a stem cell expressing tryptophan metabolizing enzyme prepared by the above steps.

Such stem cells may be used as pharmaceutical compositions for use in suppressing immune responses. In other words, the composition can inhibit the immune response caused by T-cell overactivation. In addition, the immune response may be an immune response against the transplanted organ, tissue or cell, which may be an allograft or xenograft.

Thus, the composition according to the present invention can be used for the prevention and treatment of organ transplant rejection, graft-versus-host reaction, autoimmune disease or chronic inflammatory disease.

The "autoimmune disease" is rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, Addison's disease, vitiligo, scleroderma, Goodpasture syndrome, Bezette's disease, Crohn's disease, ankylosing spondylitis, uveitis, thrombocytopenic Purpura, vulgaris, pediatric diabetes mellitus, autoimmune hemolytic anemia, cryoglobulinosis, adrenal protein dystrophy or systemic lupus erythematosus.

In addition, the stem cells according to the present invention can be used as a composition for preventing or treating graft-versus-host disease, inhibiting the activity of allogeneic antigen-responsive t cells.

Allogeneic haematopoietic stem cell transplantation (HSCT) is used to treat malignant blood diseases such as acute myeloid leukemia, acute lymphoma, multiple myeloma, aplastic anemia and immunodeficiency. It is one of the most effective and permanent treatments for the world, and is used to treat more than 20,000 patients worldwide annually (reported by the Center for International Blood and Marrow Transplant Research). A major obstacle to the success of allogeneic hematopoietic stem cell transplantation is graft-versus-host disease (GVHD), an immune complication caused by the donor's immune response to the recipient's allogeneic antigen (HLA).

Graft-versus-host disease (GVHD) is divided into acute and chronic. Usually occurs 100 days after transplantation, acute graft-versus-host disease (aGVHD), 100 days after Is called chronic graft-versus-host disease (cGVHD), but it is now classified according to clinical features and does not necessarily include a 100-day period. If the chronic disease pattern is a complex of allogeneic and autoimmune reactions, acute disease is allogeneic immune alone. That is, aGVHD is a disease that causes an inflammatory response in response to homologous antigens present in host cells of T cells in the graft, and mainly causes damage to epithelial cells of skin, liver and gastrointestinal tract.

The target organs of aGVHD are skin, liver, and gastrointestinal tract, but the immune organs are also important target organs, resulting in significant immunity. Moreover, because immunosuppressive agents used to treat aGVHD exacerbate these immunocompromises, aGVHD is often accompanied by unexpected infections, and pneumonia, hepatitis and gastroenteritis caused by cytomegalovirus (CMV) are typical diseases. Therefore, if aGVHD occurs, the CMV antigen should be quantified. If there is any signs of increase in antigen, antiviral agents should be administered immediately before the disease caused by CMV. You must be nervous. This is not because aGVHD disease itself, but the accompanying infection is the leading cause of death.

The basis of aGVHD treatment is a steroid preparation, which is usually used as a treatment if cyclosporin is being used to prevent aGVHD, and steroids are added to it. However, if the steroid drug does not work, tacrolimus, siromirus, antilymphocyte serum, budesonide (for gastrointestinal disease), beclomethazole (for gastrointestinal disease), mycofinolate, warmth, pentostatin, Many treatments are available, including diclizumab, infliximab, and alemtuzumab, but there are no standard treatments. Since it is very difficult to balance the immunosuppression caused by treatment with the opportunistic infection caused by immunosuppression, there is an urgent need to develop a new effective treatment method with fewer side effects.

Meanwhile, mesenchymal stem cells among stem cells are known to inhibit T-cell proliferation in a concentration-dependent manner during mixed lymphocyte reaction (MLR), and inhibit proliferation of B-cells and production of immunoglobulins. Although it was attracting attention as a candidate drug for cell therapy for suppressing immune response, the effect is so small that it is only used in pediatric cancer despite the advantages of high stability and fewer side effects in cell therapy. Recently, the immunosuppressive activity of mesenchymal stem cells can be changed by regulation of indoleamine 2,3-dioxygenase (IDO) activity, one of tryptophan metabolase enzymes, and is independent of IDO. Conflicting reports have coexisted.

Thus, the present inventors, as described above, in the stem cells tryptophan metabolizing enzyme IDO is expressed, but its downstream enzymes kynurenine-3-hydroxylase (KMO) and kynureninase (kynureninase) The present invention was completed by discovering that KYNU) is not expressed and effectively inhibiting T cell overactivation of stem cells when KMO and KYNU are expressed.

Accordingly, the present invention provides a stem cell expressing a tryptophan metabolase gene, such as kynurenine 3-hydroxylase (KMO) and kynureninase (KYNU), and the immunity comprising the same. Provided are compositions for inhibition and compositions for preventing or treating graft-versus-host disease.

The composition according to the present invention may include a pharmaceutically effective amount of stem cells alone or may include one or more pharmaceutically acceptable carriers, excipients or diluents. The pharmaceutically effective amount herein refers to an amount sufficient to prevent, ameliorate and treat immunosuppressive or graft-versus-host disease. The therapeutically effective amount of the pharmaceutical composition is not particularly limited, but is preferably 1 × 10 ⁴ cell / kg to 1 × 10 ⁸ cell / kg, and preferably 1 × 10 ⁵ cell / kg to 1 × 10 ⁷ cell / kg. More preferably, it is most preferable that it is 5 * 10 <^5> cell / kg-5 * 10 <^6> cell / kg. However, the pharmaceutically effective amount may be appropriately changed depending on the degree of symptoms of the disease, the age, weight, health condition, sex, route of administration and duration of treatment of the patient.

In addition, "pharmaceutically acceptable" as used herein refers to a composition which is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorders, dizziness or the like when administered to a human. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.

In addition, the compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.

The sterile composition for injection can be prescribed according to a conventional formulation practice using an excipient such as distilled water for injection. As the aqueous solution for injection, for example, isotonic solutions containing physiological saline, glucose or other auxiliary agents, for example D-sorbitol, D-mannose, D-mannitol, sodium chloride, and suitable dissolution aids, for example Alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate 80 (TM), HCO-50. Examples of the oily liquid include sesame oil and soybean oil, and it can be used in combination with benzyl benzoate and benzyl alcohol as a dissolution aid. It can also be combined with buffers such as phosphate buffers, sodium acetate buffers, analgesics such as procaine hydrochloride, stabilizers such as benzyl alcohol, phenols, antioxidants. The prepared injection solution is usually filled in a suitable ampoule.

Administration of the patient into the body is preferably parenteral, and can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular, the dosage of active ingredient being determined by the route of administration, age, sex, It may be appropriately selected depending on various factors such as the weight and the severity of the patient. In addition, the administration time may be a short time or a long time continuous administration. More specifically, injection type, transdermal administration type and the like can be given. As an example of an injection formulation, it can administer by intravenous injection, intraarterial injection, selective intraarterial injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, intraventricular injection, intracranial injection, intramedullary injection, etc., for example.

In particular, the composition according to the present invention is a stem cell as an active ingredient, in order to prevent graft-versus-host disease, it is effective to transplant the patient with hematopoietic stem cells during the hematopoietic stem cell transplantation.

In addition, the composition of the present invention can be administered in parallel with known components that have an existing immunosuppressant or graft-versus-host disease treatment effect.

Furthermore, the composition according to the present invention not only provides an effect of suppressing excessive immune response and alleviating graft-versus-host disease through the excellent T-cell hyperactivation inhibitory effect, but also has no toxicity and side effects in the human body as a cell therapy. Can be used.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

<Example 1>

Stem Cell Culture and Immunophenotype Analysis

For isolation of stem cells to be used in the present invention, subjects were selected as mesenchymal stem cells of adult stem cells, and after 7-8 weeks of age, C57BL / 6 mice were dislocated from the cervical spine, and then the femur, tibia, and humerus were removed and cultured with a syringe ( The bone marrow cells were extracted by passing through RPMI media). The suspension was passed through a 100 μm nylon mesh, centrifuged and erythrocytes were hemolyzed by the addition of erythrocyte hemolysis solution, followed by centrifugation to separate only nucleated cells. The nucleated cells were suspended in MesenCult MSC base medium containing 1 × penicillin / streptomycin, Mesenchymal stem cell (MSC) stimulatory supplements, and cultured in a 37 ° C., 5% CO ₂ incubator. After 3 days, half of the culture medium was removed, and a new culture solution was added, and the culture solution was changed every 7 days while culturing under the same conditions. When the cells were grown to a density of about 80-90%, the cells attached with trypsin-EDTA were detached, washed with phosphate buffer solution, and subcultured under the same conditions.

To analyze surface antigens of MSCs, they were stained with monoclonal antibodies of CD11b, CD44, CD45 and Sca-1 fluorescently labeled with fluoresein isothiocyanate (FITC) or phycoerythrin (PE). The corresponding isotype control antibody was used as a negative control, and analyzed using a flow cytometer (FACS).

As a result, as shown in Figure 1a after the cultivation of the radial projections of the cells attached to the growing on the bottom was observed, when the cultured cells were more than 90% density was passaged. Morphological changes were not observed during subculture.

In addition, when the surface antigens of the cultured cells were analyzed by flow cytometry, hematopoietic markers CD44 and Sca-1 surface antigens were positive, and monocyte markers CD11b and CD45 were not expressed as shown in FIG. 1B. From this, it was confirmed that the cultured cells maintained the characteristics of the MSC well.

<Example 2>

Review of immunosuppressive effects of IDO (indoleamine 2,3-dioxygenase)

<2-1> Analysis of gene and protein expression patterns of IDO

MSC cells isolated from normal mice and IDO KO (knock-out) mice were cultured with 100 units / ml of INF-ν-treated and untreated groups to extract total RNA, quantified and synthesized cDNA. The IDO gene expression was observed by PCR. At this time, PCR conditions are shown in Table 1 below.

Table 1

Target	Sequence [F (forward), R (reverse)]	Size (bp)	Annealing Tem (℃)	Cycle
IDO	F 5'-GAACCGAGGGGATGACGATGT-3 '(SEQ ID NO: 1)	300	57	27
	R 5'-TCGTGCAGTGCCTTTTCCAA-3 '(SEQ ID NO: 2)
KMO	F 5'-GCCTTGAAAGCCATTGGTC-3 '(SEQ ID NO: 3)	230	57	28
	R 5'-GCACTGTGAGTACCCCTTCC-3 '(SEQ ID NO: 4)
KYNU	F 5'-AAATTCCCTTGGCCTTCAAC-3 '(SEQ ID NO: 5)	103	64	30
	R 5'-GCGTTTGCCTACATCATGG-3 '(SEQ ID NO: 6)
GAPDH	F 5'-TTCACCACCATGGAGAAGGC-3 '(SEQ ID NO: 7)	230	57	23
	R 5'-GGCATGGACTGTGGTCATGA-3 '(SEQ ID NO: 8)

Groups treated with INF-ν at 100 units / ml and untreated groups were eluted with lysis solution, and quantified by electrophoresis on 12% SDS polyacrylamide gels. Was implemented. The protein on the gel was transferred to the membrane and then blocked with Tris buffered saline-tween (TBST) solution containing 5% skim milk powder for 1 hour at room temperature. The membrane was treated with a primary antibody, shaken slowly at 4 ° C., and reacted overnight. The primary antibody was removed, washed three times with TBST solution, and then reacted with a horse peroxidase-conjugated anti-rabbit secondary antibody for two hours. I was. The membrane was developed by using LAS-3000 after emitting light by chemical fluorescence.

As a result of observing the gene and protein expression patterns of IDO, as shown in FIG. 2A, neither IDO was expressed in MSC of normal mice not treated with IFN-ν and MSC of IDO KO mice, and IFN-ν was expressed in MSCs of normal mice. Expression of IDO was observed only after treatment.

<2-2> Examination of alloantigen-responsive T cell inhibitory effect

Mixed lymphocyte responses were performed to investigate the inhibitory effect of mesenchymal stem cells (MSC) on allogeneic antigens. C57BL / 6 (H-2 ^b ) T cells labeled with CFSE were used as the response cells, and B6D2F1 (H-2 ^{b / d} ) splenocytes were used as stimulatory cells. Reaction cells 2X10 ⁵ and stimulation cells 4X10 ⁵ (final volume 0.2ml) per 96 well plate were incubated in 37 ° C., 5% CO ₂ animal cell incubator for 4 days. The degree of cell proliferation by mixed lymphocytes was analyzed by flow cytometry for the decrease of CFSE fluorescence sensitivity in CD3 positive cells.

As a result, 64.2% T cell proliferation was observed in the absence of MSC as shown in FIG. 2B, while T cell proliferation was reduced to 46.1% when the MSCs were treated together. In case of T cell proliferation rate of 50.1%, the proliferation rate was slightly increased than that of normal MSC treatment. That is, the present inventors were able to confirm the T cell proliferation inhibitory activity of the mesenchymal stem cells themselves, and it was found that the IDO enzyme was weakly involved in the immunosuppressive ability of MSC.

<Example 3>

Tryptophan Metabolizing Enzyme Gene Expression Analysis

After treatment with normal mouse MSC IFN-ν, tryptophan metabolase gene expression was observed by PCR. In this case, PCR conditions are shown in Table 1 above.

As a result, as shown in FIG. 3, all of the tryptophan metabolase genes, ie, IDO, KMO, KYNU and GAPDH genes, were not expressed at all in MSCs of normal mice, and only IDO was expressed upon IFN-ν treatment.

<Example 4>

Mesenchymal stem cell production according to the present invention

<4-1> Recombinant Vector Preparation

Recombinant expression vector pBI-CMV1-KMO-KYNU (about 6KDa) containing tryptophan metabolase gene was inserted into the pBI-CMV1 vector by cloning and expressing the mouse tryptophan metabolase KMO and KYNU genes. ) Was prepared.

<4-2> Mesenchymal Stem Cell Preparation

MSCs of normal mice of ⁵ × 10 ⁵ cells were transformed with pBI-CMV1 vector and recombinant expression vector pBI-CMV1-KMO-KYNU using Amaxa Nucleofactor reagent (V solution).

Recombinant expression vector pBI-CMV1-KMO-KYNU was transformed into normal mouse MSC to produce transgenic mesenchymal stem cells expressing not only IDO but also downstream enzymes KMO and KYNU. Mesenchymal stem cells transformed only with the pBI-CMV1 vector were prepared as controls.

Example 5

Identification of Transformed Mesenchymal Stem Cells According to the Invention

After treatment with IFN-v in mouse MSC transformed with a control vector and a vector expressing KMO-KYNU simultaneously, tryptophan metabolase gene expression was observed by PCR. In this case, PCR conditions are shown in Table 1 above.

As a result, in the MSC transformed with the control vector, only IDO was expressed in the tryptophan metabolase gene as shown in FIG. 5, and in the MSC transformed with the vector expressing KMO-KYNU simultaneously, not only IDO, but also KMO and its downstream enzymes. It was confirmed that KYNU is also expressed.

<Example 6>

Examination of alloantigen-responsive T cell inhibitory effect of mesenchymal stem cells according to the present invention

Mixed lymphocyte response was performed to investigate the effect of transforming MSCs on inhibitory lymphocyte proliferation against homologous antigens. C57BL / 6 (H-2 ^b ) T cells labeled with CFSE were used as the response cells, and B6D2F1 (H-2 ^{b / d} ) splenocytes were used as stimulatory cells. Reaction cells 2X10 ⁵ and stimulation cells 4X10 ⁵ (final volume 0.2ml) per 96 well plate were incubated in 37 ° C., 5% CO ₂ incubator for 4 days.

The extent of cell proliferation by mixed lymphocytes was first analyzed by flow cytometry for the decrease of CFSE fluorescence sensitivity in CD3 positive cells.

As a result, 72.5% T cell proliferation was observed in the absence of MSC as shown in FIG. 6A, while T cell proliferation was slightly reduced to 67.8% when the MSC transformed with the control vector was treated together. In addition, when the MSC transformed with the recombinant vector including the tryptophan metabolase according to the present invention, it was observed that T cell proliferation was significantly reduced to 54.2%.

In addition, T cell proliferation rate was observed by measuring the radioactivity of harvested cells after adding [3H] cymidine (1.0 / well) 18 hours before cell harvest while incubating in 96 well plates for 96 hours.

As a result, as shown in FIG. 6B, the T cell proliferation was significantly increased when MSC was not present, whereas T cell proliferation was slightly decreased when the MSC transformed with the control vector was treated. The treatment of MSC transformed with a recombinant vector containing tryptophan metabolase showed a significant decrease in T cell proliferation. Therefore, the present inventors were able to confirm that the transformed MSC according to the present invention has a very excellent ability to inhibit T cell proliferation.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (13)

1. Composition for inhibiting immune response comprising stem cells expressing tryptophan metabolase gene.
2. The method of claim 1,

   The tryptophan metabolase is a kynurenine-3-hydroxylase (KMO) or kynureninase (KYNU) composition for inhibiting immune response, characterized in that.
3. The method of claim 1,

   The stem cell is an immune response inhibiting composition, characterized in that the stem cell introduced recombinant expression vector containing a nucleic acid encoding a tryptophan metabolase.
4. The method of claim 3,

   The recombinant expression vector is a pBI-CMV1-KMO-KYNU having a cleavage map of Figure 4 composition for inhibiting immune response.
5. The method of claim 1,

   The stem cells are immune stem composition, characterized in that the adult stem cells.
6. The method of claim 5,

   The adult stem cells are mesenchymal stem cells, knapsack, placenta, umbilical cord, cord blood, skin, peripheral blood, bone marrow, fat, muscle, liver, nerve tissue, periosteum, fetal membrane, synovial membrane, synovial fluid, amnion, meniscus, A composition for inhibiting immune response, characterized in that isolated from tissues selected from the group consisting of transverse ligaments, articular chondrocytes, teeth, perivascular cells, striatum, subpatellar mass, spleen and thymus.
7. The method of claim 1,

   The immune response is a composition for inhibiting immune response, characterized in that generated by T-cell overactivation.
8. The method of claim 1,

   The immune response is an immune response inhibiting composition, characterized in that the immune response to the transplanted organ, tissue or cell.
9. The method of claim 8,

   The transplanted organ, tissue or cell composition for inhibiting immune response, characterized in that the allograft.
10. The method of claim 8,

    The transplanted organ, tissue, or cell is a composition for inhibiting immune response, characterized in that the xenograft.
11. The method of claim 1,

    The composition is an organ transplant rejection reaction, graft-versus-host reaction, autoimmune disease or chronic inflammatory disease, characterized in that used for the prevention and treatment of chronic inflammatory diseases.
12.  The method of claim 11,

    The autoimmune diseases include rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, Addison's disease, vitiligo, scleroderma, Goodpasture syndrome, Bezette's disease, Crohn's disease, ankylosing spondylitis, uveitis, thrombocytopenic purpura, A composition for suppressing an immune response, characterized in that selected from the group consisting of vulgaris, pediatric diabetes mellitus, autoimmune hemolytic anemia, cryoglobulinosis, adrenal protein dystrophy and systemic lupus erythematosus.
13. A composition for preventing or treating graft-versus-host disease comprising stem cells expressing tryptophan metabolizing enzyme gene.

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