Mass production of immunosuppressive T cells using human serum albumin

Abstract

The present invention relates to a method for mass-producing immunosuppressive T cells using human serum albumin. According to the present invention, when peripheral blood stem cells (PBSCs) obtained from individuals administered with granulocyte colony-stimulating factor (G-CSF) are cultured in a medium containing human serum albumin (HSA) or HSA and mercaptoethanol, immunosuppressive T cells having immunosuppressive effects can be mass-produced. It has been confirmed that the immunosuppressive T cells exhibit excellent therapeutic effects against acute graft-versus-host disease. Therefore, the present invention is expected to be useful in the treatment of immune diseases such as acute graft-versus-host disease.

Description

[Technical Field]

[0001] The present invention relates to a method for mass production of immunosuppressive T cells using human serum albumin. [Background technology]

[0002] Acute graft-versus-host disease (aGVHD) is an immune response caused by a major histocompatibility complex (MHC) mismatch between transplanted immune cells and the patient's own cells after allogeneic hematopoietic stem cell transplantation. Approximately 20% of patients with aGVHD do not respond to commonly used steroid treatment and end up on long-term use of various immunosuppressants. This not only reduces their quality of life due to side effects, but ultimately leads to death from infection. To prevent organ damage caused by aGVHD and improve survival rates, cell therapy that corrects the imbalance between immune cells is ideal, rather than the combination of various immunosuppressants. However, mesenchymal stem cells (MSCs), currently used as a cell therapy, have only temporary and weak therapeutic effects, and are therefore not widely used in clinical practice.

[0003] On the other hand, recent research reports have shown that T lymphocytes expressing surface antigens such as PD1, TIM3, TIGIT, and LAG3 on their cell surface are known to have the effect of suppressing immune responses, unlike other T lymphocytes. However, CD3, which has an immunosuppressive effect, + PD1 + TIM3 + CD3 cells are present in very small amounts in the blood. + The lymphocyte concentration is 3.3±2.9%, which makes it difficult to use as a cell therapy in clinical settings.

[0004] Therefore, we investigated the mass production of T cells with excellent immunosuppressive effects and found that CD3 T cells could be produced in a medium containing human serum albumin (HSA) and mercaptoethanol. + When lymphocytes are cultured, the surface antigen is CD3 + PD1 + TIM3 + Lymphocytes and CD3 + PD1 + TIM3 - The present invention was completed based on the finding that immunosuppressive T lymphocytes, such as lymphocytes, are significantly increased. Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a method for mass production of immunosuppressive T cells.

[0006] Another object of the present invention is to provide a method for the in vitro differentiation of T cells into CD3 + PD1 + TIM3 + , CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - The present invention provides a method for inducing T cells having one or more phenotypes selected from the group consisting of:

[0007] Another object of the present invention is to differentiate T cells into CD3 + PD1 + TIM3 + The present invention provides a method for inducing T cells having a phenotype of

[0008] Another object of the present invention is to provide a medium composition for mass production of immunosuppressive T cells.

[0009] Another object of the present invention is to provide a kit for mass production of immunosuppressive T cells.

[0010] Another object of the present invention is to + PD1 + TIM3 + , CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - The present invention provides a medium composition for expanding immunosuppressive T cells having one or more phenotypes selected from the group consisting of:

[0011] Another object of the present invention is to + PD1 + TIM3 + The present invention aims to provide a medium composition for expanding immunosuppressive T cells having the phenotype of

[0012] Another object of the present invention is to + PD1 + TIM3 + , CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - The present invention provides a kit for expanding immunosuppressive T cells having one or more phenotypes selected from the group consisting of:

[0013] Another object of the present invention is to + PD1 + TIM3 + The present invention provides a kit for expanding immunosuppressive T cells having the phenotype of

[0014] It is yet another object of the present invention to provide a pharmaceutical composition for preventing or treating autoimmune diseases.

[0015] It is still another object of the present invention to provide a cell therapeutic composition for preventing or treating autoimmune diseases.

[0016] It is yet another object of the present invention to provide a method for preventing or treating autoimmune diseases. [Means for solving the problem]

[0017] To achieve the above object, the present invention provides a method for mass-producing immunosuppressive T cells, which comprises the steps of: extracting CD3 + sorting the cells; and + Culturing the cells in a medium containing human serum albumin (HSA).

[0018] In order to achieve the above-mentioned other objects, the present invention provides a method for mass-producing immunosuppressive T cells, which comprises the following steps: extracting CD3 T cells from a sample isolated from an individual administered granulocyte colony stimulating factor (G-CSF); + a step of sorting the cells; and + The cells are cultured in a medium containing human serum albumin (HSA) and mercaptoethanol for 1 to 7 days.

[0019] In order to achieve the above and other objects, the present invention provides a method for in vitro differentiation of T cells into CD3 T cells, which comprises the following steps: + PD1 + TIM3 + , CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - The present invention provides a method for inducing T cells having one or more phenotypes selected from the group consisting of: CD3 T cells isolated from a sample from an individual administered granulocyte colony stimulating factor (G-CSF). + a step of sorting the cells; and+ The cells are cultured in a medium containing human serum albumin (HSA) and mercaptoethanol for 1 to 3 days.

[0020] In order to achieve the above and other objects, the present invention provides a method for in vitro differentiation of T cells into CD3 T cells, which comprises the following steps: + PD1 + TIM3 + The present invention provides a method for inducing T cells having a phenotype of CD3 from a sample isolated from an individual administered granulocyte colony stimulating factor (G-CSF). + sorting the cells; and + The cells are cultured in a medium containing human serum albumin (HSA) and mercaptoethanol for 4 to 7 days.

[0021] In order to achieve the above and other objects, the present invention provides a medium composition for mass production of immunosuppressive T cells, which comprises human serum albumin (HSA).

[0022] In order to achieve the above and other objects, the present invention also provides a kit for mass production of immunosuppressive T cells, which kit includes granulocyte colony stimulating factor (G-CSF) and human serum albumin (HSA).

[0023] To achieve the above and other objects, the present invention provides a medium composition for expanding immunosuppressive T cells having one or more phenotypes selected from the group consisting of CD3+PD1+TIM3+, CD3+PD1+TIM3-, and CD3+PD1-TIM3-, which comprises human serum albumin (HSA) and peripheral blood stem cells (PBSCs) cultured for 1 to 3 days.To achieve the above and other objects, the present invention provides a medium composition for expanding immunosuppressive T cells having one or more phenotypes selected from the group consisting of CD3+PD1+TIM3+, CD3+PD1+TIM3-, and CD3+PD1-TIM3-, which comprises human serum albumin (HSA) and peripheral blood stem cells (PBSCs) cultured for 4 to 7 days. + PD1 + TIM3 + The present invention provides a medium composition for expanding immunosuppressive T cells having the phenotype of

[0024] In order to achieve the above and other objects, the present invention provides a method for producing a CD3 induced inflammatory cytokine, comprising culturing peripheral blood stem cells (PBSCs) for 1 to 3 days using a granulocyte colony stimulating factor (G-CSF) and human serum albumin (HSA). + PD1 + TIM3 + , CD3 + PD1+TIM3 - and CD3 + PD1 - TIM3 - The present invention provides a kit for expanding immunosuppressive T cells having one or more phenotypes selected from the group consisting of:

[0025] In order to achieve the above and other objects, the present invention provides a method for producing a CD3 induced inflammatory cytokine, comprising culturing peripheral blood stem cells (PBSCs) for 4 to 7 days using a granulocyte colony stimulating factor (G-CSF) and human serum albumin (HSA). + PD1 + TIM3 + The present invention provides a kit for expanding immunosuppressive T cells having the phenotype of

[0026] In order to achieve the above and other objects, the present invention also provides a pharmaceutical composition for preventing or treating autoimmune diseases, comprising immunosuppressive T cells produced by the method for mass-producing immunosuppressive T cells according to the present invention.

[0027] In order to achieve the above and other objects, the present invention provides a method for mass-producing immunosuppressive T cells according to the present invention. + PD1 + TIM3 + The present invention provides a cell therapy composition for preventing or treating autoimmune diseases, which comprises immunosuppressive T cells having the phenotype of

[0028] In order to achieve the above and other objects, the present invention also provides a method for preventing or treating an autoimmune disease, comprising administering to an individual immunosuppressive T cells produced by the method for mass-producing immunosuppressive T cells according to the present invention. [Effects of the Invention]

[0029] The present inventors have confirmed that when peripheral blood stem cells (PBSCs) obtained from individuals administered granulocyte colony-stimulating factor (G-CSF) are cultured in a medium containing human serum albumin (HSA) or HSA and mercaptoethanol, immunosuppressive T cells with immunosuppressive effects can be produced in large quantities, and that the immunosuppressive T cells exhibit excellent therapeutic effects against acute graft-versus-host disease. [Brief explanation of the drawings]

[0030] [Figure 1] These are the results of examining the cell count when CD3+ lymphocytes isolated from G-CSF mobilized peripheral blood stem cells (G-PBSCs) were cultured in a medium containing HSA (human serum albumin) or a medium containing HSA and mercaptoethanol. [Figure 2] The figures show the results of counting the numbers of CD3+PD1+, CD3+PD1+TIM3+, CD3+PD1+LAG3+, and CD3+PD1+TIGIT+ lymphocytes in CD3+ lymphocytes (CD3+PBSCs) isolated from the blood of healthy individuals, CD3+G-PBSCs before culture (D0 CD3+G-PBSCs), and CD3+G-PBSCs cultured for 4 days by the method of the present invention (D4 CD3+G-PBSCs). [Figure 3] 1 shows the results of a mixed lymphocyte reaction measured in CD3+PD1+TIM3+ and CD3+PD1-TIM3- lymphocytes isolated from CD3+G-PBSCs cultured for 4 days by the method of the present invention. [Figure 4] FIG. 10 is a diagram confirming the expression of TIGIT and LAG3 surface antigens in CD3+PD1+TIM3+ lymphocytes cultured for 4 days by the method according to the present invention. [Figure 5] FIG. 1 shows mixed lymphocyte reaction measured in CD3+ G-PBSCs before culture (control group) and CD3+ G-PBSCs cultured for 2 or 4 days by the method according to the present invention. [Figure 6] FIG. 1 shows the numbers of CD3+PD1+TIM3+, CD3+PD1+TIM3-, and CD3+PD1-TIM3- lymphocytes in CD3+G-PBSCs before culture (control group) and CD3+G-PBSCs cultured for 2 and 4 days by the method of the present invention. [Figure 7] CD3+PD1+TIM3+, CD3+PD1-TIM3-, and CD3+PD1+TIM3- lymphocytes were isolated from CD3+G-PBSCs cultured for 2 or 4 days by the method of the present invention, and the results of the mixed lymphocyte reaction were measured for each lymphocyte type. The results were also measured after treating the isolated lymphocytes with anti-PD1 mAb or anti-Tim3 mAb depending on the cell surface antigen expression. [Figure 8] Only CD3+PD1-TIM3- and CD3+PD1+TIM3- lymphocytes were isolated from CD3+G-PBSCs before culture, and then cultured for 4 days using the method of the present invention. The results show that the amount of CD3+PD1+TIM3+ lymphocytes differentiated from the CD3+PD1-TIM3- and CD3+PD1+TIM3- lymphocytes was measured. [Figure 9] FIG. 1 shows the results of evaluating the therapeutic effect of CD3+ G-PBSC cells cultured for 4 days by the method of the present invention using a xenograft aGVHD mouse model (NOD-scid IL-2Rγ null). [Figure 10] FIG. 10 is a diagram showing the evaluation of the therapeutic effect of CD3+ G-PBSC cells cultured for two days by the method of the present invention using a xenograft aGVHD mouse model (NOD-scid IL-2Rγ null). DETAILED DESCRIPTION OF THE INVENTION

[0031] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, detailed descriptions of well-known technologies known to those skilled in the art may be omitted. Furthermore, when describing the present invention, if it is determined that a detailed description of related well-known functions or configurations may unnecessarily obscure the gist of the present invention, such detailed description may be omitted. Furthermore, the terminology used in this specification is used to appropriately describe preferred embodiments of the present invention, and may vary depending on the intentions of users or operators, or the practices in the field to which the present invention pertains.

[0032] Therefore, the definition of this term should be based on the contents of the entire specification. Throughout the specification, when a part "includes" a certain component, this does not mean that other components are excluded, but that other components may also be included, unless otherwise specified.

[0033] The present invention provides a method for mass production of immunosuppressive T cells, which comprises the steps of: extracting CD3 + sorting the cells; and + Culturing the cells in a medium containing human serum albumin (HSA).

[0034] In the present invention, the term "CD3" refers to a molecule that is a T cell co-receptor and is involved in the activation of cytotoxic T cells and helper T cells.

[0035] In the present invention, the term "human serum albumin" refers to a receptor protein that constitutes most of the human serum proteins and is capable of regulating the osmotic pressure of blood and transporting substances through the blood.

[0036] In the present invention, the sample may be any one or more selected from the group consisting of blood, tissue, cells, plasma, feces, urine, and semen, but is not limited thereto. Preferably, the sample may be any one or more selected from the group consisting of blood and peripheral blood stem cells (PBSCs), more preferably PBSCs.

[0037] In the present invention, the term "peripheral blood stem cells (PBSCs)" refers to hematopoietic stem cells present in peripheral blood vessels. Hematopoietic stem cells are stem cells that produce blood, including all cells in the blood, such as red blood cells, white blood cells, platelets, and T cells.

[0038] Furthermore, the individual may be a healthy allogeneic individual. Preferably, the individual may be a healthy allogeneic individual to which the immunosuppressive T cells of the present invention are administered.

[0039] In addition, the individual has CD3 + The cells are characterized by being mobilized into the peripheral blood. + It can mean that the cells have migrated into the peripheral blood. + Cells can be mobilized into peripheral blood by any method known in the art, without limitation. In one embodiment of the present invention, leucocyte growth factor was administered to an individual for the mobilization.

[0040] Thus, the individual may be an individual to whom a leucocyte growth factor has been administered, and the leucocyte growth factor may be a granulocyte colony stimulating factor (G-CSF).

[0041] In the present invention, the term "granulocyte colony stimulating factor (G-CSF)" refers to a glycoprotein that promotes the proliferation and differentiation of hematopoietic stem cells and enhances the viability of neutrophils, and can be used to prevent and treat neutropenia.

[0042] According to one embodiment of the present invention, in the method for mass production of immunosuppressive T cells of the present invention, CD3 + When lymphocytes were cultured in a medium containing HSA, the cell number increased by 0.4±0.1 times compared to before culture. Furthermore, when cultured in a medium containing HSA and mercaptoethanol, the cell number increased by 1.0±0.6 times compared to before culture, and CD3 + A significant increase in lymphocyte yield was observed.

[0043] Therefore, in the method for mass production of immunosuppressive T cells of the present invention, the medium may further contain mercaptoethanol.

[0044] In the present invention, the term "mercaptoethanol" refers to a compound that can reduce disulfide bonds and remove hydroxyl radicals, and is therefore used as a biological reducing agent and antioxidant.

[0045] The concentration of mercaptoethanol may be, but is not limited to, 1 μM to 200 μM. Preferably, the concentration of mercaptoethanol may be, but is not limited to, 20 μM to 100 μM, and more preferably, the concentration of mercaptoethanol may be, but is not limited to, 30 μM to 70 μM. Even more preferably, the concentration of mercaptoethanol may be, but is not limited to, 30 μM to 60 μM, 30 μM to 50 μM, 40 μM to 70 μM, 40 μM to 60 μM, 40 μM to 50 μM, 50 μM to 70 μM, or 50 μM to 60 μM.

[0046] The concentration of the human serum albumin (HSA) may be, but is not limited to, 1% to 10% by weight. Preferably, the concentration of the human serum albumin may be, but is not limited to, 1% to 8% by weight. More preferably, the concentration of the human serum albumin may be, but is not limited to, 2% to 8% by weight. Even more preferably, the concentration of the human serum albumin may be, but is not limited to, 2% to 7% by weight, 2% to 6% by weight, 2% to 5% by weight, 3% to 8% by weight, 3% to 7% by weight, 3% to 6% by weight, 3% to 5% by weight, 4% to 8% by weight, 4% to 7% by weight, 4% to 6% by weight, 4% to 5% by weight, 5% to 8% by weight, 5% to 7% by weight, or 5% to 6% by weight.

[0047] Human serum albumin is a homologous protein, not a xenoprotein like fetal bovine serum (FBS), making it safer for clinical use as a cell therapy agent. Furthermore, when the concentration of human serum albumin is 1% to 10% by weight, it can significantly increase the production yield of immunosuppressive T cells, making it useful for mass production of immunosuppressive T cells.

[0048] According to another embodiment of the present invention, the peripheral blood of a healthy person has a concentration of 1×10 3 CD3 + G-PBSC CD3 per cell + PD1 + TIM3 + However, after subcutaneous injection of G-CSF for 5 days in healthy individuals, the CD3 + When G-PBSCs were collected and cultured for 4 days in a medium containing 5% HSA and mercaptoethanol, 1 × 10 3 CD3 + CD3 per cell + PD1 + TIM3 +The number of lymphocytes was measured to be 865±88. That is, the method of the present invention + PD1 + TIM3 + It was confirmed that the number of lymphocytes could be increased 433 times compared to peripheral blood and approximately 13 times more than before culture.

[0049] Furthermore, according to another embodiment of the present invention, CD3 + When G-PBSC cells were cultured, CD3 + PD1 + TIM3 + The cell number increased as the culture time progressed, but before culture, it was 1 × 10 3 CD3 + The number of CD3 cells per G-PBSC was 68±8, but after 2 days of culture it increased to 212±44, and after 4 days of culture it increased to 865±88. + PD1 + TIM3 - Lymphocytes were cultured at 1 × 10 3 CD3 + The number of cells per G-PBSC was 197±52, but after 2 days of culture it was 172±64, and after 4 days of culture, almost all cells had disappeared. + PD1 - TIM3 - The lymphocyte count was 1 × 10 before culture. 3 CD3 + The number of cells per G-PBSC was 735±105, but after 2 days of culture it was 616±192, and after 4 days of culture it decreased to 67±38.

[0050] In particular, according to another embodiment, CD3 + From G-PBSCs, CD3 + PD1 - TIM3 - and CD3 + PD1 + TIM3 - Lymphocytes were isolated and cultured for 4 days using the method of the present invention. + PD1 - TIM3- Among lymphocytes, 70.8±3.1% of cells were CD3 + PD1 + TIM3 - Among lymphocytes, 80.7±5.2% of cells were CD3 + PD1 + TIM3 + It was confirmed that the cells differentiated into lymphocytes. + PD1 - TIM3 - Lymphocytes and CD3 + PD1 + TIM3 - Lymphocytes gradually increased in number over time in culture. + PD1 + TIM3 + They are presumed to differentiate into lymphocytes.

[0051] Therefore, in the present invention, the term "mass production" means securing cells in a therapeutic dose. + CD3 cell fraction with excellent immunosuppressive effects + PD1 + TIM3 + This means that cells containing approximately 87% (86.5±8.8%) lymphocytes are produced.

[0052] The terms "PD1" and "Tim3" refer to co-inhibitory molecules, and T cells expressing these factors can block excessive immune responses and maintain immune tolerance.

[0053] In the present invention, the term "immune tolerance" refers to a state in which an immune response to a specific antigen is not exhibited. Therefore, the immunosuppressive T cells according to the present invention refer to T cells that induce tolerance to self-antigens and suppress T cell proliferation, and for the purposes of the present invention, the immunosuppressive T cells refer to T cells that induce immune tolerance to self-antigens that cause autoimmune diseases, preferably graft-versus-host disease.

[0054] In the present invention, the culture can be performed for 1 to 7 days, but is not limited thereto. The immunosuppressive T cells cultured for 1 to 7 days by the method of the present invention are CD3 + PD1 + TIM3 + It is characterized by having a phenotype of

[0055] When the culture is carried out for 1 to 3 days, the immunosuppressive T cells are CD3 + PD1 + TIM3 + , CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - The phenotype may be one or more selected from the group consisting of:

[0056] Furthermore, the present invention provides a method for mass production of immunosuppressive T cells, which comprises the steps of: extracting CD3 T cells from a sample isolated from an individual administered granulocyte colony stimulating factor (G-CSF); + sorting the cells; and + The cells are cultured in a medium containing human serum albumin (HSA) and mercaptoethanol for 1 to 7 days.

[0057] The present invention also provides a method for in vitro differentiation of T cells into CD3 + PD1 + TIM3 + , CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 -The present invention provides a method for inducing T cells having one or more phenotypes selected from the group consisting of: CD3 T cells isolated from a sample from an individual administered granulocyte colony stimulating factor (G-CSF). + sorting the cells; and + The cells are cultured in a medium containing human serum albumin (HSA) and mercaptoethanol for 1 to 3 days.

[0058] The present invention also provides a method for in vitro differentiation of T cells into CD3 + PD1 + TIM3 + The present invention provides a method for inducing T cells having a phenotype of CD3 from a sample isolated from an individual administered granulocyte colony stimulating factor (G-CSF). + sorting the cells; and + The cells are cultured in a medium containing human serum albumin (HSA) and mercaptoethanol for 4 to 7 days.

[0059] The granulocyte colony-stimulating factor (G-CSF) may be administered at a concentration of 1 to 50 μg / kg, but is not limited thereto. Preferably, it may be administered at a concentration of 1 to 30 μg / kg, and more preferably, at a concentration of 5 to 20 μg / kg, but is not limited thereto. Even more preferably, it may be administered at a concentration of 5 to 15 μg / kg, 5 to 10 μg / kg, 6 to 20 μg / kg, 6 to 15 μg / kg, 6 to 10 μg / kg, 7 to 20 μg / kg, 7 to 15 μg / kg, 7 to 10 μg / kg, 8 to 20 μg / kg, 8 to 15 μg / kg, 8 to 10 μg / kg, 9 to 20 μg / kg, 9 to 15 μg / kg, 9 to 10 μg / kg, 10 to 20 μg / kg, or 10 to 15 μg / kg, but is not limited thereto.

[0060] The present invention also provides a medium composition for mass production of immunosuppressive T cells, which comprises human serum albumin (HSA).

[0061] The medium composition may further comprise mercaptoethanol.

[0062] The concentration of mercaptoethanol may be, but is not limited to, 1 μM to 200 μM, preferably 20 μM to 100 μM, and more preferably 30 μM to 70 μM, but is not limited to these.

[0063] The concentration of the human serum albumin (HSA) may be, but is not limited to, 1% to 10% by weight, preferably 1% to 8% by weight, and more preferably 2% to 8% by weight, but is not limited to these.

[0064] The medium composition according to the present invention can be used for culturing blood or peripheral blood stem cells (PBSCs), and more preferably for culturing PBSCs.

[0065] The immunosuppressive T cells cultured using the medium composition of the present invention are CD3 + PD1 + Tim3 + It is characterized by having an immunophenotype of

[0066] In the present invention, the term "media" refers to a medium capable of supporting cell growth and survival in vitro and includes all commonly used media suitable for cell culture. The medium and culture conditions can be selected depending on the type of cell. The medium used for culture is preferably cell culture minimum medium (CCMM), which generally contains a carbon source, a nitrogen source, and trace element components. Any medium commonly used for culturing animal cells can be used. Preferably, a medium containing serum (e.g., fetal bovine serum, horse serum, or human serum) can be used. Media that can be used in the present invention include, but are not limited to, the RPMI series (e.g., RPMI 1640), Eagle's MEM (Eagle's minimum essential medium, Eagle), α-MEM, Iscove's MEM, 199 medium, CMRL 1066, F12, F10, DMEM (Dulbecco's modification of Eagle's medium), a mixture of DMEM and F12, Waymouth's MB752 / 1, McCoy's 5A, and MCDB series. The media may contain other components, such as antioxidants or antibiotics such as penicillin, streptomycin, and gentamicin.

[0067] Furthermore, the present invention provides a kit for mass production of immunosuppressive T cells, which comprises granulocyte colony-stimulating factor (G-CSF) and human serum albumin (HSA). The kit may further comprise mercaptoethanol.

[0068] The concentrations of G-CSF, HSA, and mercaptoethanol contained in the kit are as described above.

[0069] The kit is not limited to the above and may include other reagents and tools, such as a culture plate for culturing target cells and reagents necessary for assaying immunosuppressive activity (e.g., mixed lymphocyte reaction (MLR)). The kit may also include PBSCs to be cultured.

[0070] The kit according to the present invention can be provided in a single container or separate containers containing a medium containing G-CSF and HSA or G-CSF, HSA and mercaptoethanol, and all other components and reagents necessary for culture in appropriate volumes and / or forms.

[0071] The kit according to the present invention may include instructions describing the order in which to carry out the above-described method according to the present invention.

[0072] The present invention also provides a method for producing a CD3 immunization medium comprising culturing peripheral blood stem cells (PBSCs) for 1 to 3 days, the method comprising the steps of: + PD1 + TIM3 + , CD3 + PD1 + TIM3 - and CD3 + PD1- TIM3 - The present invention provides a medium composition for expanding immunosuppressive T cells having one or more phenotypes selected from the group consisting of:

[0073] In addition, the present invention provides a method for producing a CD3 immunization medium containing human serum albumin (HSA) and peripheral blood stem cells (PBSCs) cultured for 4 to 7 days. + PD1 + TIM3 + The present invention provides a medium composition for expanding immunosuppressive T cells having the phenotype of

[0074] The medium composition for expanding immunosuppressive T cells according to the present invention may further contain mercaptoethanol.

[0075] The specific details of the medium composition, including the concentrations of HSA and mercaptoethanol, are as described above.

[0076] The present invention also provides a method for producing a CD3 immunization medium comprising culturing peripheral blood stem cells (PBSCs) for 1 to 3 days, the method comprising culturing a CD3 immunization medium containing granulocyte colony stimulating factor (G-CSF) and human serum albumin (HSA). + PD1 + TIM3 + , CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - The present invention provides a kit for expanding immunosuppressive T cells having one or more phenotypes selected from the group consisting of:

[0077] In addition, the present invention provides a method for producing a CD3 immunization medium containing granulocyte colony stimulating factor (G-CSF) and human serum albumin (HSA), which is characterized by culturing peripheral blood stem cells (PBSCs) for 4 to 7 days. + PD1 + TIM3 + The present invention provides a kit for expanding immunosuppressive T cells having the phenotype of

[0078] The kit for expanding immunosuppressive T cells according to the present invention may further contain mercaptoethanol.

[0079] In the kit of the present invention, the specific details including the concentrations of the G-CSF, HSA and mercaptoethanol are as described above.

[0080] According to another embodiment of the present invention, total CD3 + As a result of mixed lymphocyte reaction performed on G-PBSCs, CD3 + In the case of G-PBSC (control group), 0.5±0.1% of T cells were not detected to proliferate against allogeneic mononuclear cells, and CD3 + For G-PBSCs, 13.9±6.4% were CD3 + In the case of G-PBSCs, 43.2±10.1% of T cells were confirmed not to proliferate against allogeneic mononuclear cells. + G-PBSCs have the best immune response suppression effect, and the immune response suppression effect of these cells was significantly higher than that of CD3 cells isolated after 4 days of culture. + PD1 + TIM3 + That is, it was confirmed that the T cells cultured by the method of the present invention exhibited a superior immune response suppression effect, and that this effect was far superior in the T cells cultured for 2 days.

[0081] According to another embodiment of the present invention, CD3 + Injection of G-PBSC cells into a xenograft aGVHD mouse model significantly increased mouse survival. In particular, the CD3 + 1 × 10 G-PBSCs 7 In the experimental group infused with 100 cells / kg, 100% of the control mice survived 80 days after cell infusion, while 4-day cultured CD3 + The T cells cultured for 2 days showed a much better therapeutic effect than G-PBSCs. That is, it was confirmed that the T cells cultured by the method of the present invention showed a superior therapeutic effect against graft-versus-host disease, and that the effect was far superior for the T cells cultured for 2 days.

[0082] Therefore, the present invention provides a pharmaceutical composition for preventing or treating autoimmune diseases, which comprises immunosuppressive T cells produced by the method for mass-producing immunosuppressive T cells according to the present invention.

[0083] In the pharmaceutical composition of the present invention, the immunosuppressive T cells are CD3 + PD1 + TIM3 + Furthermore, the immunosuppressive T cells are characterized by having a phenotype of CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - The present invention may further have one or more phenotypes selected from the group consisting of:

[0084] In the pharmaceutical composition according to the present invention, the autoimmune disease is selected from the group consisting of graft-versus-host disease, rheumatoid arthritis, asthma, dermatitis, psoriasis, cystic fibrosis, post-transplantation late and chronic solid organ rejection, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, Hashimoto thyroiditis, polymyositis, scleroderma, Addison's disease, vitiligo, pernicious anemia, glomerulonephritis, and pulmonary fibrosis. fibrosis), Inflammatory Bowel Diseases, Autoimmune Diabetes, Diabetic retinopathy, Rhinitis, Ischemia-reperfusion injury, Post-angioplasty restenosis, Chronic obstructive pulmonary diseases;The disease may be one or more diseases selected from the group consisting of COPD, Graves' disease, gastrointestinal allergies, conjunctivitis, atherosclerosis, coronary artery disease, angina, cancer metastasis, and arteriolar disease, and preferably may be graft-versus-host disease, but is not limited to these;

[0085] In the present invention, the term "graft-versus-host disease" refers to a disease in which lymphocytes transfused during hematopoietic stem cell transplantation attack a host (the body of the transfused person) with a weakened immune system, causing symptoms such as fever, rash, abnormal liver function, diarrhea, and pancytopenia (a condition in which white blood cells, red blood cells, and platelets are all reduced). There are two types of graft-versus-host disease that occur after transplantation: acute and chronic. In addition to cases that occur during hematopoietic stem cell transplantation, graft-versus-host disease can also occur during blood transfusions, although this is extremely rare. Once a graft-versus-host reaction occurs after a transfusion, it can be extremely fatal.

[0086] The pharmaceutical composition of the present invention can be administered to an "individual" who has or may have an autoimmune disease, and the "individual" can mean any animal, including humans, and may be an individual of the same species as the individual from which the immunosuppressive T cells of the present invention are derived.

[0087] In the present invention, the term "prevention" means any action of suppressing the symptoms of a specific disease or delaying its progression by administering the composition of the present invention.

[0088] In the present invention, the term "treatment" means any action that improves or beneficially changes the symptoms of a specific disease by administering the composition of the present invention.

[0089] The pharmaceutical composition of the present invention may further contain an adjuvant in addition to the active ingredient. Any adjuvant known in the art may be used without limitation, and for example, Freund's complete or incomplete adjuvant may be further contained to enhance its effect.

[0090] The pharmaceutical composition of the present invention can be prepared by mixing the active ingredient with a pharmaceutically acceptable carrier. Here, pharmaceutically acceptable carriers include carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0091] The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as acids, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions, according to the usual methods.

[0092] When formulated, the formulation can be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like. Such solid formulations can be prepared by mixing the active ingredient with at least one or more excipients, such as starch, calcium carbonate, sucrose, lactose, and gelatin. Lubricants such as magnesium stearate and talc can also be used in addition to simple excipients. Liquid formulations for oral administration include suspensions, oral solutions, emulsions, syrups, and the like. In addition to commonly used diluents such as water and liquid paraffin, various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, can be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Examples of non-aqueous solvents and suspending agents that can be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Examples of suppository bases that can be used include witepsol, tween 61, cacao butter, laurin butter, and glycerogelatin.

[0093] Pharmaceutical compositions according to the present invention can be administered to an individual by a variety of routes, including, for example, oral, intravenous, intramuscular, subcutaneous, or intraperitoneal injection, although any mode of administration is contemplated.

[0094] The dosage of the pharmaceutical composition according to the present invention is selected taking into consideration the age, weight, sex, physical condition, etc. of the individual. It is obvious that the concentration of the active ingredient contained in the pharmaceutical composition can be selected in various ways depending on the subject, and preferably, the active ingredient is contained in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg / ml. If the concentration is less than 0.01 μg / ml, the active ingredient may not exhibit pharmacological activity, and if it exceeds 5,000 μg / ml, it may be toxic to the human body.

[0095] In the present invention, the pharmaceutical composition for preventing or treating autoimmune diseases may further contain, in addition to the immunosuppressive T cells as the active ingredient, any compound or natural extract whose safety has already been verified and whose therapeutic effect is known to enhance the therapeutic effect of an autoimmune disease, preferably graft-versus-host disease. Furthermore, the pharmaceutical composition for preventing or treating autoimmune diseases of the present invention may be used in conjunction with surgical treatment of an autoimmune disease, preferably graft-versus-host disease.

[0096] In addition, the present invention provides a method for mass-producing immunosuppressive T cells according to the present invention. + PD1 + TIM3 + The present invention provides a cell therapy composition for preventing or treating autoimmune diseases, which comprises immunosuppressive T cells having a phenotype of CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - The present invention may further have one or more phenotypes selected from the group consisting of:

[0097] In the present invention, the term "cellular therapeutic agent" refers to a pharmaceutical product (as defined by the U.S. FDA) that is made from cells and tissues isolated, cultured, and specially processed from humans and used for the purposes of treatment, diagnosis, and prevention of diseases. This pharmaceutical product is made by selecting and growing living autologous, allogeneic, or xenogeneic cells in vitro, or by changing the biological properties of the cells in other ways, in order to restore the function of the cells or tissues.

[0098] The cell therapy composition of the present invention can be administered directly to the lesion site, and in this case, it can be administered in the form of an injection. In particular, the cell therapy composition of the present invention is a cell therapy composition containing CD3 + PD1 + TIM3 +For direct administration of immunosuppressive T cells having the phenotype, the immunosuppressive T cells can be formulated into an injectable form, in which case a hydrogel can be further included to make the immunosuppressive T cells suitable for injection.

[0099] Hydrogels that can be used in the cell therapy agent of the present invention may include, without limitation, hydrogels known in the art that are suitable for injection, and may be one or more selected from the group consisting of small intestinal submucosa, hyaluronic acid, carboxymethylcellulose (CMC), alginate, chitosan, polyacrylamide, poly(N-isopropylacrylamide), β-glycerophosphate, Pluronic (Poly(ethylene oxide) poly(propylene oxide) poly(ethylene oxide)), fibrin, polyethylene oxide (PEO), and a mixture of carboxymethylcellulose (CMC) and polyethyleneimine (PEI), but are not limited to these.

[0100] The preferred dosage of the cell therapy 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. Administration can be once a day or in several divided doses, and the above dosage does not limit the scope of the present invention in any aspect.

[0101] The present invention also provides a method for preventing or treating an autoimmune disease, comprising the step of administering to an individual immunosuppressive T cells produced by the method for mass-producing immunosuppressive T cells according to the present invention.

[0102] The term "individual" refers to a subject that has or may have an autoimmune disease, and may refer to any animal including a human.

[0103] The autoimmune diseases include graft-versus-host disease, rheumatoid arthritis, asthma, dermatitis, psoriasis, cystic fibrosis, post-transplantation late and chronic solid organ rejection, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, Hashimoto thyroiditis, polymyositis, scleroderma, Addison's disease, vitiligo, pernicious anemia, glomerulonephritis, and pulmonary fibrosis. The disease may be one or more diseases selected from the group consisting of inflammatory bowel diseases, inflammatory bowel diseases, autoimmune diabetes, diabetic retinopathy, rhinitis, ischemia-reperfusion injury, post-angioplasty restenosis, chronic obstructive pulmonary diseases (COPD), Graves' disease, gastrointestinal allergies, conjunctivitis, atherosclerosis, coronary artery disease, angina, cancer metastasis, and arteriolar disease, and is preferably, but not limited to, graft-versus-host disease. [Example]

[0104] The present invention will be described in more detail below with reference to examples. These examples are merely for the purpose of more specifically illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not limited to these examples.

[0105] Example 1: Cell collection and proliferation method After subcutaneous injection of G-CSF (neutrogin, 10 μg / kg) for 5 days to normal peripheral blood stem cell (PBSC) donors, G-CSF-mobilized peripheral blood stem cells (G-PBSC) were collected using a COBE Spectra cell sorter. CD3 Dynabeads (trademark pending) were used to separate the collected G-PBSCs using magnetic-activated cell sorting (MACS). + Lymphocytes were isolated. CD3 + Lymphocytes were cultured at 5 × 10 in 5% HSA (human serum albumin). 5 The cells were cultured at a concentration of 100 cells / mL for 2 or 4 days in RPMI 1640 medium containing 2 mM L-glutamine, 25 mM HEPES (hydroxyethyl piperazine ethane sulfonic acid), 1 mM sodium pyruvate, and 50 μM mercaptoethanol.

[0106] CD3 + After culturing G-PBSC lymphocytes in a medium containing HSA and mercaptoethanol for 4 days, the total cell number increased by 1.0±0.6 times compared to before culturing, while the cell number increased by 0.4±0.1 times when cultured with HSA alone (see Figure 1). + By culturing lymphocytes with HSA and mercaptoethanol, CD3 +It was confirmed that the yield of lymphocytes can be increased.

[0107] <Example 2> Confirmation of the phenotype of proliferated cells Flow cytometry using a fluorescence-activated cell sorter (FACS) was performed to confirm the expression of the co-inhibitory molecules PD1, TIM3, LAG3, and TIGIT in the cells cultured in Example 1. At this time, the expression of PD1, TIM3, LAG3, and TIGIT was also confirmed in the peripheral blood of healthy individuals (control group).

[0108] As a result, in the peripheral blood of healthy individuals, the concentration was 1 × 10 3 CD3 + G-PBSC CD3 per cell + PD1 + TIM3 + , CD3 + PD1 + LAG3 + and CD3 + PD1 + TIGIT + The lymphocyte counts were extremely low at 2±1, 3±1, and 3±1, respectively. On the other hand, after subcutaneous injection of G-CSF for 5 days, G-PBSCs were collected and CD3 + Lymphocyte isolation revealed 1 × 10 3 CD3 + CD3 per G-PBSC + PD1 + TIM3 + , CD3 + PD1 + LAG3 + and CD3 + PD1 + TIGIT + The lymphocyte counts were confirmed to increase to 68±8, 105±18, and 38±6, respectively. + G-PBSCs were cultured in 5% HAS for 4 days, resulting in 1 × 10 3 CD3 +CD3 per G-PBSC + PD1 + LAG3 + and CD3 + PD1 + TIGIT + The lymphocyte counts were measured as 865±89, 295±36, and 5±4, respectively. In this result, the CD3 + PD1 + TIM3 + The number of lymphocytes was confirmed to be 433 times higher than that in peripheral blood and approximately 13 times higher than before culture (see Figure 2). + PD1 + TIM3 + In lymphocytes, the LAG3 surface antigen was expressed in 48.9±5.2% of all cells, and TIGIT was hardly expressed (see FIG. 4).

[0109] Specifically, CD3 cells cultured for 4 days in a medium containing 5% HSA and 50 μM mercaptoethanol were + G-PBSCs are CD3 + PD1 + TIM3 + and CD3 + PD1 - TIM3 - The CD3 lymphocytes differentiated into two groups, each expressing a surface antigen (see Figures 3 and 6). In contrast, the CD3 lymphocytes cultured for 2 days in a medium containing 5% HSA and 50 μM mercaptoethanol + G-PBSCs are CD3 + PD1 + TIM3 + , CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - The lymphocytes differentiated into three groups on which surface antigens were expressed (see Figure 6).

[0110] CD3 cells cultured in medium containing 5% HSA and 50 μM mercaptoethanol + G-PBSC medium, CD3 + PD1 + TIM3 + The cell number increased with the passage of culture time, but before culture, it was 1 × 10 3 CD3 + The number of CD3 cells per G-PBSC was 68±8, but increased to 212±44 after 2 days of culture and to 865±88 after 4 days of culture (see Figure 6). + PD1 + TIM3 - Lymphocytes were cultured at 1 × 10 3 CD3 + The number of cells per G-PBSC was 197±52, but after 2 days of culture it was 172±64, and after 4 days of culture almost all cells had disappeared (see Figure 6). + PD1 - TIM3 - The number of lymphocytes also decreased with the passage of culture time, but before culture, the number was 1 × 10 3 CD3 + The number of CD3+ / -105 cells per G-PBSC was 735±105 cells per G-PBSC, but after 2 days of culture, it was 616±192 cells, and after 4 days of culture, it decreased to 67±38 cells (see Figure 6). + PD1 - TIM3 - Lymphocytes and CD3 + PD1 + TIM3 - Lymphocytes gradually increased in number over time in culture. + PD1 + TIM3 + It is assumed that they will differentiate into lymphocytes. + G-PBSC to CD3 + PD1 - TIM3 - and CD3 + PD1 + TIM3 - Lymphocytes were isolated and cultured for 4 days. As a result, after 4 days of culture, CD3 + PD1 - TIM3- Among lymphocytes, 70.8±3.1% of cells were CD3 + PD1 + TIM3 - Among lymphocytes, 80.7±5.2% of cells were CD3 + PD1 + TIM3 + It was confirmed that the cells were differentiated into lymphocytes (see Figure 8).

[0111] Example 3: Confirmation of the immune response suppression effect of cultured lymphocytes using a mixed lymphocyte reaction The immune response suppression effect of the cells cultured by the method of the present invention was confirmed using a mixed lymphocyte reaction (MLR). The mixed lymphocyte reaction is an in vitro experimental method for measuring the immune response of T lymphocytes to alloantigens. Specifically, T lymphocytes were cultured with other human monocytes (CD14 + When T lymphocytes are mixed with human monocytes (human T lymphocytes) and cultured, the MHC mismatch between the two cells causes the cultured T lymphocytes to recognize the other human monocytes as alloantigens and proliferate. The immune response of T lymphocytes to alloantigens is measured by measuring the level of T lymphocyte proliferation. To prevent allogeneic monocytes from proliferating outside of the T lymphocytes, the allogeneic monocytes are irradiated. The T lymphocytes are then stained with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to measure their proliferation. The stained CFSE penetrates the cell membrane and remains by nonspecifically binding to intracellular proteins. The fluorescence intensity of CFSE decreases by half with each cell division. After culturing using this, the intracellular CFSE fluorescence can be measured to confirm the level of lymphocyte proliferation. Based on this, T lymphocytes cultured according to the method of the present invention are used for mixed lymphocyte reaction (MLL). + PD1 + TIM3 + , CD3 + PD1 - TIM3 - and CD3 + PD1 + TIM3- Lymphocytes were separated and the separated lymphocytes were then cultured with allogeneic mononuclear cells (CD14 + The cells were mixed with PD-1 mAb (cells) at a cell number ratio of 1:2 and cultured for 3 days. Afterwards, all cultured cells were harvested, and the CSFE fluorescence level was measured to determine the percentage of lymphocytes that did not proliferate in response to the alloantigen. Anti-PD1 mAb and anti-Tim3 mAb were used at concentrations of 20 μg / mL and 40 μg / mL, respectively, in 5% HSA cell culture medium.

[0112] CD3 cells cultured for 4 days in medium containing 5% HSA and 50 μM mercaptoethanol + G-PBSCs are CD3 + PD1 + TIM3 + and CD3 + PD1 - TIM3 - The cells differentiated into two groups of lymphocytes: CD3 + PD1 + TIM3 + and CD3 + PD1 - TIM3 - A mixed lymphocyte reaction was performed on lymphocytes, and CD3 + PD1 + TIM3 + In lymphocytes, 34.7±1.8% of T cells did not proliferate against allogeneic mononuclear cells, and CD3 + PD1 - TIM3 - In the case of lymphocytes, T cells did not proliferate in 18.7±0.6% of cases (see Figure 3). + Among the lymphocytes differentiated in G-PBSCs, CD3 + PD1 + TIM3 + It was confirmed that it has a high effect of suppressing the immune response of lymphocytes.

[0113] In addition, CD3 + To assess the immune response suppression effect of G-PBSC cells, CD3 cells were cultured for 2 or 4 days in a medium containing 5% HSA and 50 μM mercaptoethanol. +Mixed lymphocyte reaction (MLL) was performed on G-PBSC cells. + In the case of G-PBSCs, 0.5±0.1% of T cells were found not to proliferate against allogeneic mononuclear cells, but CD3 T cells were found to proliferate against allogeneic mononuclear cells when cultured for 2 days in a medium containing 5% HSA and 50 μM mercaptoethanol. + In the case of G-PBSCs, 43.2±10.1% of T cells did not proliferate against allogeneic mononuclear cells, and CD3 T cells were not proliferated against allogeneic mononuclear cells after 4 days of culture in a medium containing 5% HSA and 50 μM mercaptoethanol. + In the case of G-PBSCs, 13.9±6.4% of T cells did not proliferate against allogeneic mononuclear cells (see Figure 5). + We confirmed that G-PBSCs have the best immunosuppressive effect (see Figure 5). The immunosuppressive effect of these cells was significantly higher than that of isolated CD3 + PD1 + TIM3 + It was shown to be higher than lymphocytes (see Figure 3).

[0114] In addition, CD3 + G-PBSCs are CD3 + PD1 + TIM3 + , CD3 + PD1 - TIM3 - and CD3 + PD1 + TIM3 - Lymphocytes were differentiated into three groups (see Figure 6). After separating the lymphocytes from the three groups, a mixed lymphocyte reaction was performed on each group. The results showed that CD3 + PD1 + TIM3 + In lymphocytes, 19.5±3.0% of T cells did not proliferate against allogeneic mononuclear cells, and CD3 + PD1 + TIM3 - In lymphocytes, 29.1±12.6% of T lymphocytes were CD3 + PD1 - TIM3 -In the case of lymphocytes, 20.3±7.9% of T lymphocytes were confirmed to not proliferate against allogeneic mononuclear cells (see Figure 7). + Among G-PBSCs, 43.2±10.1% of T lymphocytes did not proliferate against allogeneic mononuclear cells (see Figure 5). + PD1 + TIM3 + , CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - The immunosuppressive ability of lymphocytes was significantly reduced (see Figure 7). Taking the above results together, the CD3 lymphocytes cultured for 2 days by the method of the present invention were required for suppressing immune responses. + We concluded that the most effective method was to use whole G-PBSCs.

[0115] In addition, CD3 + Within G-PBSCs, CD3 + PD1 + TIM3 + The number of lymphocytes was confirmed to be 86.5±8.8%, which was an increase from 21.2±4.4% after 2 days of culture. However, the number of CD3 + The immunosuppressive effect of G-PBSCs was significantly reduced (see Figures 5 and 6). This was confirmed by the CD3 + PD1 + TIM3 + This suggests that lymphocytes may have different immunosuppressive abilities even if they express the same surface antigen. + PD1 + TIM3 +The cells were isolated and treated with a monoclonal antibody against PD1 (anti-PD1 mAb) and a monoclonal antibody against Tim3 (anti-Tim3 mAb), respectively, followed by a mixed lymphocyte reaction. Generally, treatment with anti-PD1 mAb or anti-Tim3 mAb is expected to counteract the suppressive effect of immunosuppressive T lymphocytes on the allogeneic immune response, resulting in a decrease in T lymphocytes that do not respond to allogeneic mononuclear cells. Thus, CD3 cells isolated after 4 days of culture were + PD1 + TIM3 + When lymphocytes were treated with anti-PD1 mAb and anti-Tim-3 mAb, the percentage of T lymphocytes that did not proliferate against allogeneic monocytes decreased from 34.7±1.8% to 17.7±7.0% and from 34.7±1.8% to 15.3±4.4%, respectively, demonstrating that the suppressive effects of anti-PD1 mAb and anti-Tim-3 mAb on allogeneic immune responses were counteracted. On the other hand, CD3s isolated after 2 days of culture were significantly reduced. + PD1 + TIM3 + When treated with anti-PD1 mAb and anti-Tim-3 mAb, the percentage of T lymphocytes not proliferating against allogeneic monocytes decreased from 19.5±3.0% to 17.6±7.8%, from 19.5±3.0% to 19.6±3.3%, and to 19.7±3.6%, respectively. The expected immune response enhancement effect of anti-PD1 mAb and anti-Tim-3 mAb was not observed. In addition, the CD3 + CD3 isolated from G-PBSCs + PD1 + TIM3 - When cells were treated with anti-PD1 mAb, the percentage of T lymphocytes not proliferating against allogeneic monocytes was reduced from 29.1±12.6% to 23.0±11.1%, and the percentage of CD3 + PD1 + TIM3 - We were able to confirm the immune response enhancing effect of anti-PD1 mAb on CD3 cells (see Figure 7). + PD1 + TIM3+ Unlike lymphocytes, CD3 + PD1 + TIM3 + Lymphocytes are cells whose immune response suppression effects are not counterbalanced by anti-PD1 mAb and anti-Tim3 mAb, and it was confirmed that although the two cells express the same surface antigen, they are functionally different types of cells.

[0116] Example 4: Confirmation of the immune response suppression effect of cultured lymphocytes using a xenograft GVHD mouse model Cultured CD3 + To confirm the therapeutic effect of G-PBSC cells, we conducted experiments using a xenograft aGVHD mouse model lacking immune function (NOD-scid IL-2Rγ null). NOD-scid IL-2Rγ null mice lack T and B lymphocyte function. After irradiating the NOD-scid IL-2Rγ null mice with 3 Gy of radiation, 3.5 ± 1.8 × 10 cells isolated from normal allogeneic individuals were transplanted. 7 PBMCs were intraperitoneally injected into mice at a dose of 1000 / kg to induce aGVHD. + G-PBSC lymphocytes were gradually increased (control group vs. 1 × 10 6 cells / kg vs. 1×10 7 cells / kg vs. 5×10 7 cells / kg vs. 10×10 7 cells / kg) into the tail vein of mice, and the survival of the mice was confirmed. + The therapeutic effect of G-PBSC lymphocytes was evaluated.

[0117] As a result, in the control group (n=7), only 14% of mice survived 100 days after peripheral mononuclear cell injection, whereas the CD3 + G-PBSC lymphocytes 1 × 10 6 In the group injected with 1 × 10 cells / kg (n = 5), 20% of the control mice survived.7 In the group injected with 100 cells / kg (n=7), 71% of the mice survived and developed CD3 + G-PBSCs: 1 x 10 7 It was confirmed that the dose of CD3 cells / kg was appropriate for treatment (see Figure 9). + The volume of G-PBSC lymphocytes was increased by five times, to 5 × 10 7 cells / kg (n=3), or 10 times the volume, 10 x 10 7 When cells / kg (n=3) were injected, most of the control mice died within 20 days (see FIG. 9).

[0118] CD cultured for 2 days in the same manner 3 +G-PBSCs were infused into the mice, and the therapeutic effect on aGVHD was evaluated (see Figure 10). 7 In the group injected with 100 cells / kg (n=4), 100% of the control mice were confirmed to survive 80 days after cell injection (see Figure 10). + G-PBSC lymphocytes 1 × 10 7 5x10 cells / kg 7 When 10 × 10 cells / kg (n = 2) were injected, 50% of the target mice survived, and 10 × 10 cells / kg (n = 2) were injected. 7 It was confirmed that all of the mice in the group injected with 1000 cells / kg (n=2) survived without death (see Figure 10). + G-PBSC lymphocytes were also confirmed to effectively suppress immune responses in vivo, and CD3 + G-PBSC lymphocytes 1 × 10 7 It was confirmed that administration at a dose of cells / kg was the most effective way to suppress graft-versus-host reaction.

[0119] From the results of the above example, it was found that CD3 + When G-PBSCs were cultured for 4 days, most of the cells (86.5±8.8%) were CD3 + PD1+ TIM3 + Differentiated CD3 cells differentiated into lymphocytes + PD1 + TIM3 + It was confirmed that lymphocytes have an excellent immune response suppression effect. + G-PBSCs express a large amount of CD3 + PD1 + TIM3 - Lymphocytes and CD3 + PD1 + TIM3 + Lymphocytes were included, and CD3 cells were isolated after 4 days of culture. + PD1 + TIM3 + Therefore, the CD3 cells cultured for 2 days according to the method of the present invention showed a superior immunosuppressive effect to the lymphocytes. + G-PBSCs are the most ideal cell therapy agent, and it is expected that they will be useful in the future for treating immune diseases such as graft-versus-host disease.

Claims

1. A method for mass production of immunosuppressive T cells, comprising the steps of: CD3 from samples isolated from individuals ＋ Sorting the cells; and The CD3 ＋ Culturing the cells in a medium containing human serum albumin (HSA).

2. The method for mass-producing immunosuppressive T cells according to claim 1, wherein the sample is at least one selected from the group consisting of blood, tissue, cells, plasma, feces, urine, and semen.

3. 2. The method of claim 1, wherein the sample is at least one selected from the group consisting of blood and peripheral blood stem cells (PBSCs).

4. The method for mass production of immunosuppressive T cells according to claim 1 , wherein the individual is a healthy allogeneic individual.

5. The individual has a CD3 ＋ The method for mass production of immunosuppressive T cells according to claim 1, wherein the cells are mobilized into peripheral blood.

6. 2. The method for mass-producing immunosuppressive T cells according to claim 1, wherein the individual is an individual to whom a leukocyte growth factor has been administered.

7. 7. The method for mass-producing immunosuppressive T cells according to claim 6, wherein the leukocyte growth factor is granulocyte colony stimulating factor (G-CSF).

8. The method for mass-producing immunosuppressive T cells according to claim 1, wherein the medium further contains mercaptoethanol.

9. The method for mass-producing immunosuppressive T cells according to claim 8, wherein the concentration of mercaptoethanol is 1 μM to 200 μM.

10. 2. The method for mass-producing immunosuppressive T cells according to claim 1, wherein the concentration of human serum albumin (HSA) is 1% to 10% by weight.

11. The method for mass-producing immunosuppressive T cells according to claim 1, wherein the culturing is carried out for 1 to 7 days.

12. The immunosuppressive T cells are CD3 ＋ PD1 ＋ TIM3 ＋ 2. The method for mass production of immunosuppressive T cells according to claim 1, wherein the immunosuppressive T cells have a phenotype of:

13. When the culture is carried out for 1 to 3 days, the immunosuppressive T cells are CD3 ＋ PD1 ＋ TIM3 ＋ , CD3 ＋ PD1 ＋ TIM3 － and CD3 ＋ PD1 － TIM3 － The method for mass-producing immunosuppressive T cells according to claim 1, characterized in that the immunosuppressive T cells have one or more phenotypes selected from the group consisting of:

14. A method for mass production of immunosuppressive T cells, comprising the steps of: CD3 from samples isolated from individuals administered granulocyte colony stimulating factor (G-CSF) ＋ Sorting the cells; and The CD3 ＋ Culturing the cells in a medium containing human serum albumin (HSA) and mercaptoethanol for 1 to 7 days.

15. A method for in vitro transfection of T cells with CD3 ＋ PD1 ＋ TIM3 ＋ , CD3 ＋ PD1 ＋ TIM3 － and CD3 ＋ PD1 － TIM3 － A method for inducing T cells having one or more phenotypes selected from the group consisting of: CD3 from samples isolated from individuals administered granulocyte colony stimulating factor (G-CSF) ＋ Sorting the cells; and The CD3 ＋ Culturing the cells in a medium containing human serum albumin (HSA) and mercaptoethanol for 1 to 3 days.

16. A method for in vitro transfection of T cells with CD3 ＋ PD1 ＋ TIM3 ＋ A method for inducing T cells having a phenotype of: CD3 from samples isolated from individuals administered granulocyte colony stimulating factor (G-CSF) ＋ Sorting the cells; and The CD3 ＋ Culturing the cells in a medium containing human serum albumin (HSA) and mercaptoethanol for 4 to 7 days.

17. 15. The method for mass-producing immunosuppressive T cells according to claim 14, wherein the granulocyte colony stimulating factor (G-CSF) is administered at a concentration of 1 to 50 μg / kg.

18. A medium composition for mass production of immunosuppressive T cells, comprising human serum albumin (HSA).

19. The medium composition for mass production of immunosuppressive T cells according to claim 18, further comprising mercaptoethanol.

20. The medium composition for mass production of immunosuppressive T cells according to claim 19, wherein the concentration of mercaptoethanol is 1 μM to 200 μM.

21. The medium composition for mass production of immunosuppressive T cells according to claim 18, wherein the concentration of the human serum albumin (HSA) is 1% to 10% by weight.

22. The medium composition for mass production of immunosuppressive T cells according to claim 18, wherein the medium composition is used for culturing blood or peripheral blood stem cells (PBSCs).

23. The immunosuppressive T cells are CD3 ＋ PD1 ＋ Time 3 ＋ The medium composition for mass production of immunosuppressive T cells according to claim 18, characterized in that the immunosuppressive T cells have an immunophenotype of:

24. A kit for mass production of immunosuppressive T cells containing granulocyte colony stimulating factor (G-CSF) and human serum albumin (HSA).

25. The kit for mass production of immunosuppressive T cells according to claim 24, further comprising mercaptoethanol.

26. containing human serum albumin (HSA), A method for producing CD3 cells, characterized by culturing peripheral blood stem cells (PBSCs) for 1 to 3 days. ＋ PD1 ＋ TIM3 ＋ , CD3 ＋ PD1 ＋ TIM3 － and CD3 ＋ PD1 － TIM3 － A medium composition for expanding immunosuppressive T cells having one or more phenotypes selected from the group consisting of:

27. The CD3 according to claim 26, characterized in that the medium composition further contains mercaptoethanol. ＋ PD1 ＋ TIM3 ＋ , CD3 ＋ PD1 ＋ TIM3 － and CD3 ＋ PD1 － TIM3 － A medium composition for expanding immunosuppressive T cells having one or more phenotypes selected from the group consisting of:

28. containing human serum albumin (HSA), A method for producing CD3 cells, characterized by culturing peripheral blood stem cells (PBSCs) for 4 to 7 days. ＋ PD1 ＋ TIM3 ＋ A medium composition for expanding immunosuppressive T cells having the phenotype of

29. The CD3 according to claim 28, characterized in that the medium composition further contains mercaptoethanol. ＋ PD1 ＋ TIM3 ＋ A medium composition for expanding immunosuppressive T cells having the phenotype of

30. granulocyte colony stimulating factor (G-CSF) and human serum albumin (HSA), A method for producing CD3 cells, characterized by culturing peripheral blood stem cells (PBSCs) for 1 to 3 days. ＋ PD1 ＋ TIM3 ＋ , CD3 ＋ PD1 ＋ TIM3 － and CD3 ＋ PD1 － TIM3 － A kit for expanding immunosuppressive T cells having one or more phenotypes selected from the group consisting of:

31. The CD3 of claim 30, wherein the kit further comprises mercaptoethanol. ＋ PD1 ＋ TIM3 ＋ , CD3 ＋ PD1 ＋ TIM3 － and CD3 ＋ PD1 － TIM3 － A kit for expanding immunosuppressive T cells having one or more phenotypes selected from the group consisting of:

32. granulocyte colony stimulating factor (G-CSF) and human serum albumin (HSA), A method for producing CD3 cells, characterized by culturing peripheral blood stem cells (PBSCs) for 4 to 7 days. ＋ PD1 ＋ TIM3 ＋ A kit for expanding immunosuppressive T cells having the phenotype of

33. The CD3 of claim 32, wherein the kit further comprises mercaptoethanol. ＋ PD1 ＋ TIM3 ＋ A kit for expanding immunosuppressive T cells having the phenotype of

34. A pharmaceutical composition for preventing or treating an autoimmune disease, comprising immunosuppressive T cells produced by the method for mass-producing immunosuppressive T cells according to claim 1.

35. The autoimmune diseases include graft-versus-host disease, rheumatoid arthritis, asthma, dermatitis, psoriasis, cystic fibrosis, post-transplantation late and chronic solid organ rejection, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, and the like. syndrome, chronic thyroiditis (Hashimoto thyroiditis), polymyositis, scleroderma, Addison's disease, vitiligo, pernicious anemia, glomerulonephritis and pulmonary fibrosis, inflammatory bowel diseases, autoimmune diabetes, diabetic retinopathy retinopathy, rhinitis, ischemia-reperfusion injury, post-angioplasty restenosis, chronic obstructive pulmonary disease (COPD), Graves' disease, gastrointestinal allergies, conjunctivitis, atherosclerosis, and coronary artery disease.

35. The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 34, wherein the composition is for one or more diseases selected from the group consisting of angina, cancer metastasis, and arteriolar disease.

36. The immunosuppressive T cells are CD3 ＋ PD1 ＋ TIM3 ＋ 35. The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 34, characterized in that the composition has a phenotype of:

37. The immunosuppressive T cells are CD3 ＋ PD1 ＋ TIM3 － and CD3 ＋ PD1 － TIM3 － The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 34, further having any one or more phenotypes selected from the group consisting of:

38. CD3 produced by the method for mass-producing immunosuppressive T cells according to claim 1 ＋ PD1 ＋ TIM3 ＋ A cell therapy composition for preventing or treating autoimmune diseases, comprising immunosuppressive T cells having the phenotype of

39. The immunosuppressive T cells are CD3 ＋ PD1 ＋ TIM3 － and CD3 ＋ PD1 － TIM3 － The cell therapeutic composition for preventing or treating an autoimmune disease according to claim 38, further having any one or more phenotypes selected from the group consisting of:

40. A method for preventing or treating an autoimmune disease, comprising: administering to an individual immunosuppressive T cells produced by the method for mass-producing immunosuppressive T cells according to claim 1.

41. The autoimmune diseases include graft-versus-host disease, rheumatoid arthritis, asthma, dermatitis, psoriasis, cystic fibrosis, post-transplantation late and chronic solid organ rejection, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, and the like. syndrome, chronic thyroiditis (Hashimoto thyroiditis), polymyositis, scleroderma, Addison's disease, vitiligo, pernicious anemia, glomerulonephritis and pulmonary fibrosis, inflammatory bowel diseases, autoimmune diabetes, diabetic retinopathy retinopathy, rhinitis, ischemia-reperfusion injury, post-angioplasty restenosis, chronic obstructive pulmonary disease (COPD), Graves' disease, gastrointestinal allergies, conjunctivitis, atherosclerosis, and coronary artery disease.

41. The method for preventing or treating an autoimmune disease according to claim 40, wherein the autoimmune disease is one or more diseases selected from the group consisting of angina, cancer metastasis, and arteriolar disease.

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