Method for mass production of immunosuppressive T cells by using human serum albumin

Abstract

The invention relates to a method for mass production of immunosuppressive T cells by using human serum albumin. According to the present invention, it is confirmed that when a peripheral blood stem cell (PBSC) obtained from an individual administered with a granulocyte colony stimulating factor (G-CSF) is cultured in a culture medium containing human serum albumin (HSA) or HSA and mercaptoethanol, an immunosuppressive T cell having an immunosuppressive effect can be produced in a large scale, and the immunosuppressive T cell having an immunosuppressive effect can be produced in a large scale. The immunosuppressive T cell shows an excellent acute graft-versus-host disease treatment effect, so that the immunosuppressive T cell is expected to be effectively applied to treatment of immune diseases such as acute graft-versus-host diseases.

Description

Technical Field

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

[0002] Acute graft-versus-host disease (aGVHD) is an immune response caused by a major histocompatibility complex (MHC) mismatch between transplanted immune cells and cells in the patient's body after allogeneic hematopoietic stem cell transplantation. Approximately 20% of patients with GVHD do not respond to commonly used steroid treatments, leading to long-term use of various immunosuppressants. The resulting side effects not only reduce quality of life but also ultimately lead to infection and death. To prevent organ damage caused by GVHD and improve survival rates, cell therapy that can correct the imbalance between immune cells is the most ideal, rather than the combined use of various immunosuppressants. However, mesenchymal stem cells (MSCs), currently used as cell therapy agents, have not been widely used in clinical practice due to their short-lived and weak therapeutic effects.

[0003] On the other hand, according to recent research reports, it is known that T lymphocytes that express surface antigens such as PD1, TIM3, TIGIT, and LAG3 on their cell surfaces, unlike other T lymphocytes, have the effect of suppressing immune responses. + PD1 + TIM3 + The cells are present in very small amounts in the blood (CD3 + 3.3±2.9% in lymphocytes), thus posing a problem of difficulty in clinical use as a cell therapy agent.

[0004] Therefore, in order to mass-produce T cells with excellent immunosuppressive effects, it was confirmed that when CD3 + Lymphocytes, surface antigens such as CD3 + PD1 + TIM3 + Lymphocytes and CD3 + PD1 + TIM3 - The present invention was completed by finding that immunosuppressive T lymphocytes such as lymphocytes were significantly increased. Summary of the Invention

[0005] Technical issues

[0006] The present invention aims to provide a method for mass production of immunosuppressive T cells. Another object of the present invention is to provide a method for inducing T cells to have a gene selected from CD3 + PD1 + TIM3 + 、CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - A method for composing a panel of T cells of more than one phenotype.

[0007] Another object of the present invention is to provide a method for inducing T cells to have CD3 + PD1 + TIM3 + Methods for phenotyping T cells.

[0008] Another object of the present invention is to provide a culture 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 provide a method for proliferating a gene selected from the group consisting of CD3 + PD 1 + TIM3 + 、CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - A culture medium composition for immunosuppressive T cells of one or more phenotypes in a group consisting of:

[0011] Another object of the present invention is to provide a method for proliferating CD3 + PD1 + TIM3 + Culture medium composition for phenotyping immunosuppressive T cells.

[0012] Another object of the present invention is to provide a method for proliferating a gene selected from the group consisting of CD3 + PD 1 + TIM3 + 、CD3 + PD1+ TIM3 - and CD3 + PD1 - TIM3 - A kit comprising a panel of immunosuppressive T cells of more than one phenotype.

[0013] Another object of the present invention is to provide a method for proliferating CD3 + PD1 + TIM3 + A kit to phenotype immunosuppressive T cells.

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

[0015] Another object of the present invention is to provide a cell therapy composition for preventing or treating autoimmune diseases.

[0016] Another object of the present invention is to provide a method for preventing or treating autoimmune diseases.

[0017] Technical Solution

[0018] In order to achieve the above object, the present invention provides a method for mass production of immunosuppressive T cells, comprising the steps of: sorting CD3 T cells from a sample isolated from an individual; + cells; and culturing the CD3 + cell.

[0019] In order to achieve the further object, the present invention provides a method for mass production of immunosuppressive T cells, comprising the steps of: sorting CD3 T cells from a sample separated from an individual administered with granulocyte colony stimulating factor (G-CSF); + cells; and + The cells were cultured in a medium containing human serum albumin (HSA) and mercaptoethanol for 1 to 7 days.

[0020] Furthermore, in order to achieve the other object, the present invention provides a method for inducing T cells to have a gene selected from the group consisting of CD3 + PD1 + TIM3 + 、CD3 + PD1 + TIM3- and CD3 + PD1 - TIM3 - A method for detecting T cells of one or more phenotypes in a group consisting of the above-mentioned cells, the method comprising the steps of: sorting CD3 T cells from a sample isolated from an individual administered granulocyte colony stimulating factor (G-CSF) + cells; and + The cells were cultured in a medium containing human serum albumin (HSA) and mercaptoethanol for 1 to 3 days.

[0021] Furthermore, in order to achieve another object, the present invention provides a method for inducing T cells to have CD3 + PD1 + TIM3 + A method for phenotyping T cells, comprising the steps of: sorting CD3 T cells from a sample isolated from an individual administered granulocyte colony stimulating factor (G-CSF); + cells; and + The cells were cultured in a medium containing human serum albumin (HSA) and mercaptoethanol for 4 to 7 days.

[0022] Furthermore, in order to achieve the further object, the present invention provides a culture medium composition for mass production of immunosuppressive T cells, comprising human serum albumin (HSA).

[0023] Furthermore, to achieve the further object, the present invention provides a kit for mass production of immunosuppressive T cells, comprising granulocyte colony stimulating factor (G-CSF) and human serum albumin (HSA).

[0024] Furthermore, to achieve the further object, the present invention provides a culture medium composition for proliferating immunosuppressive T cells having one or more phenotypes selected from the group consisting of CD3+PD1+TIM3+, CD3+PD1+TIM3-, and CD3+PD1-TIM3-, comprising human serum albumin (HSA) and culturing peripheral blood stem cells (PBSC) for 1 to 3 days.

[0025] Furthermore, in order to achieve the further object, the present invention provides a method for proliferating cells having CD3 + PD1 + TIM3 + The culture medium composition for immunosuppressive T cells of a specific phenotype comprises human serum albumin (HSA), and peripheral blood stem cells (PBSC) are cultured for 4 to 7 days.

[0026] Furthermore, in order to achieve the further object, the present invention provides a method for proliferating a gene having a gene selected from the group consisting of CD3 + PD1 + TIM3 + 、CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - A kit for producing immunosuppressive T cells of one or more phenotypes in a group of cells comprises granulocyte colony stimulating factor (G-CSF) and human serum albumin (HSA), and culture peripheral blood stem cells (PBSC) for 1 to 3 days.

[0027] Furthermore, in order to achieve the further object, the present invention provides a method for proliferating cells having CD3 + PD1 + TIM3 +A kit for producing immunosuppressive T cells of different phenotypes comprises granulocyte colony stimulating factor (G-CSF) and human serum albumin (HSA), and peripheral blood stem cells (PBSC) are cultured for 4 to 7 days.

[0028] Furthermore, to achieve the further object, the present invention provides a pharmaceutical composition for preventing or treating autoimmune diseases, comprising immunosuppressive T cells prepared by the method for mass production of immunosuppressive T cells of the present invention.

[0029] Furthermore, in order to achieve the above-mentioned further object, the present invention provides a cell therapy composition for preventing or treating autoimmune diseases, which comprises a cell therapy composition having CD3 T cells prepared by the method for mass production of immunosuppressive T cells of the present invention. + PD1 + TIM3 + Phenotype of immunosuppressive T cells.

[0030] Furthermore, to achieve the further object, the present invention provides a method for preventing or treating an autoimmune disease, comprising administering immunosuppressive T cells prepared by the method for mass production of immunosuppressive T cells of the present invention to an individual.

[0031] Effects of the Invention

[0032] The present invention has confirmed that when peripheral blood stem cells (PBSC) 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 an immunosuppressive effect can be produced in large quantities, and that the immunosuppressive T cells exhibit excellent therapeutic effects for acute graft-versus-host disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The results showed that CD3 T cells isolated from G-CSF mobilized peripheral blood stem cells (G-PBSC) were cultured in a medium containing human serum albumin (HSA) or a medium containing HSA and mercaptoethanol. + Lymphocytes are a result of the number of cells.

[0034] Figure 2 The results show that CD3 isolated from normal human blood + Lymphocytes (CD3 + PB SC), CD3 before culture + G-PBSC (DOCD3 + G-PBSC) and CD3 + G-PBSC (D4CD3 + CD3 in G-PBSC + PD1+, CD3 + PD1 + TIM3 + 、CD3 + PD1 + LAG3 + and CD3 + PD1 + TIGIT + The result is the number of lymphocytes.

[0035] Figure 3 The results show that CD3 + CD3 isolated from G-PBSC + PD1 + TIM3 + and CD3 + PD1 - TIM3 - Mixed lymphocyte reaction results measured in lymphocytes.

[0036] Figure 4 To confirm the CD3 + PD1 + TIM3 + Diagram of TIGIT and LAG3 surface antigens expressed in lymphocytes.

[0037] Figure 5 To confirm the CD3 + G-PBSC (control group) and CD3 T cells cultured for 2 or 4 days by the method of the present invention + Graph of mixed lymphocyte reactions measured in G-PBSC.

[0038] Figure 6 To show the CD3 + G-PBSC (control group), CD3 + CD3 in G-PBSC cells + PD1 +TIM3 + 、CD3 + PD1 + TIM3 - 、CD3 + PD1 - TIM3 - Graph of the number of lymphocytes.

[0039] Figure 7 The results show that the CD3 + CD3 + PD1 + TIM3 + 、CD3 + PD1 - TIM3 - and CD3 + PD1 + TIM3 - The results of mixed lymphocyte reactions were measured in each lymphocyte after lymphocyte isolation, and the results of mixed lymphocyte reactions were measured after treating isolated lymphocytes with anti-PD1 mAb (anti-PD1 mAb) or anti-Tim3 mAb (anti-Tim3mAb) according to the expression of cell surface antigens.

[0040] Figure 8 CD3 before culture + G-PBSCs were isolated with only CD3 + PD1-TIM3- and CD3 + PD1 + TIM3 - Lymphocytes were cultured for 4 days and the CD3 + PD1 - TIM3 - and CD3 + PD1 + TIM3 - CD3 lymphocyte differentiation + PD1 + TIM3 + The result is the number of lymphocytes.

[0041] Figure 9 To evaluate the effect of CD3 T cells cultured for 4 days using the method of the present invention, a xenograft aGVHD mouse model (NOD-scidIL-2Rγnull) was used. + Graph showing the results of the treatment effects on G-PBSC cells.

[0042] Figure 10To evaluate the effect of CD3 T cells cultured for 2 days using the method of the present invention using a xenograft aGVHD mouse model (NOD-scid IL-2Rγnull). + Graph of the therapeutic effects on G-PBSC cells. DETAILED DESCRIPTION

[0043] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, detailed descriptions of technologies well known to those skilled in the art may be omitted. Furthermore, in the process of describing the present invention, when it is determined that a detailed description of a related known function or structure may unnecessarily obscure the gist of the present invention, such detailed descriptions may be omitted. Furthermore, the terminology used in this specification is used to appropriately express preferred embodiments of the present invention and may be changed according to the intention of the user, operator, or the custom of the field to which the present invention belongs.

[0044] Therefore, these terms should be defined based on the contents of the entire specification. Throughout the specification, when a part "includes" a certain structural element, unless otherwise specifically stated to the contrary, it means that other structural elements may also be included, rather than excluding other structural elements.

[0045] The present invention provides a method for mass production of immunosuppressive T cells, comprising the steps of: sorting CD3 T cells from a sample isolated from an individual; + cells; and culturing the CD3 + cell.

[0046] In the present invention, the term "CD3" is a T cell co-receptor, and refers to a molecule involved in the activity of cytotoxic T cells and helper T cells.

[0047] In the present invention, the term "human serum albumin" refers to a water-soluble protein that constitutes the majority of human serum proteins and can regulate the osmotic pressure of blood and transport substances through the blood.

[0048] In the present invention, the sample may be 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 one or more selected from the group consisting of blood and peripheral blood stem cells (PBSC), more preferably, PBSC.

[0049] In the present invention, the term "peripheral blood stem cell (PBSC)" refers to hematopoietic stem cells present in peripheral blood vessels. Hematopoietic stem cells are stem cells that produce blood, and refer to stem cells that produce all cells in the blood, such as red blood cells, white blood cells, platelets, T cells, etc.

[0050] Alternatively, the individual may be a healthy individual of the same species. Preferably, the individual may be a healthy individual of the same species as the individual to whom the immunosuppressive T cells of the present invention are to be administered.

[0051] Additionally, the individual is characterized by CD3 + Cells are mobilized into the peripheral blood. The mobilization may refer to CD3 + In the present invention, the CD3 + Cell mobilization into peripheral blood In one embodiment of the present invention, leucocyte growth factor is administered to the individual to achieve the mobilization.

[0052] Thus, the subject may be one to whom a leucocyte growth factor is administered, and the leucocyte growth factor may be granulocyte colony stimulating factor (G-CSF).

[0053] 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 survival ability of neutrophils, and therefore can be used to prevent and treat neutropenia.

[0054] On the other hand, according to one embodiment of the present invention, in the method for mass production of immunosuppressive T cells of the present invention, it was confirmed that CD3 + During the process of lymphocytes, when cultured in a medium containing HSA, the cell number increased by 0.4±0.1 times compared to before culture. In addition, when cultured in a medium containing HSA and mercaptoethanol, the cell number increased by 1.0±0.6 times compared to before culture, confirming that CD3 + The yield of lymphocytes was significantly increased.

[0055] Therefore, in the method for mass-producing immunosuppressive T cells of the present invention, the culture medium may further contain mercaptoethanol.

[0056] In the present invention, the term "mercaptoethanol" refers to a compound that is used as a bioreductant and antioxidant because it can reduce disulfide bonds and remove hydroxyl radicals.

[0057] The concentration of the mercaptoethanol may be 1 μM to 200 μM, but is not limited thereto. Preferably, the concentration of the mercaptoethanol may be 20 μM to 100 μM, more preferably, the concentration of the mercaptoethanol may be 30 μM to 70 μM, but is not limited thereto. Even more preferably, the concentration of the mercaptoethanol may be 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, but is not limited thereto.

[0058] Furthermore, the concentration of human serum albumin (HSA) may be 1 to 10 weight percent, but is not limited thereto. Preferably, the concentration of human serum albumin may be 1 to 8 weight percent, more preferably, the concentration of human serum albumin may be 2 to 8 weight percent, but is not limited thereto. More preferably, the concentration of human serum albumin may be 2 to 7 weight percent, 2 to 6 weight percent, 2 to 5 weight percent, 3 to 8 weight percent, 3 to 7 weight percent, 3 to 6 weight percent, 3 to 5 weight percent, 4 to 8 weight percent, 4 to 7 weight percent, 4 to 6 weight percent, 4 to 5 weight percent, 5 to 8 weight percent, 5 to 7 weight percent, or 5 to 6 weight percent, but is not limited thereto.

[0059] Human serum albumin is a homologous protein, rather than a xenogeneic protein like fetal bovine serum (FBS), making it a safer cell therapy agent in clinical practice. Furthermore, when the concentration of human serum albumin is between 1 and 10 weight percent, it can significantly increase the productivity of immunosuppressive T cells, allowing for efficient mass production of immunosuppressive T cells.

[0060] On the other hand, according to another embodiment of the present invention, in the peripheral blood of normal people, every 1×10 3 CD3 +CD3 measured in G-PBSC cells + PD1 + TIM3 + The number of lymphocytes was very low, 2 ± 1. However, CD3 + G-PBSCs were cultured in a medium containing 5% HSA and mercaptoethanol for 4 days, and the number of cells per 1×10 3 CD3 + CD3 in cells + PD1 + TIM3 + The number of lymphocytes was 865±88. That is, it was confirmed that the method of the present invention can increase the number of CD3 + PD1 + TIM3 + The number of lymphocytes increased 433 times, and compared with that before culture, it could be increased by about 13 times or more.

[0061] In addition, according to another embodiment of the present invention, when CD3 + G-PBSC cells, CD3 + PD1 + TIM3 + The number of cells increases with the time of culture. Before culture, every 1×10 3 CD3 + The number of cells in G-PBSC was 68 ± 8, but it increased to 212 ± 44 after 2 days of culture and to 865 ± 88 after 4 days of culture. + PD1 + TIM3 - Before culture, 1×10 lymphocytes were 3 CD3 + The number of G-PBSC cells was 197 ± 52, but it decreased to 172 ± 64 after 2 days of culture, and almost all cells disappeared after 4 days of culture. + PD1 - TIM3 - Before culture, 1×10 lymphocytes were 3 CD3 + The number in G-PBSCs was 735 ± 105, but decreased to 616 ± 192 after 2 days of culture and to 67 ± 38 after 4 days of culture.

[0062] In particular, according to another embodiment of the present invention, from CD3 + Isolation of CD3 from G-PBSC + PD1- TIM3 - and CD3 + PD1 + TIM3 - Lymphocytes were cultured for 4 days using the method of the present invention, and 70.8±3.1% of CD3 + PD1 - TIM3 - Lymphocytes and 80.7±5.2% CD3 + PD1 + TIM3 - Lymphocyte differentiation into CD3 + PD1 + TIM3 + Lymphocytes. Based on the above results, it is speculated that CD3 + PD1 - TIM3 - Lymphocytes and CD3 + PD1 + TIM3 - Lymphocytes differentiate into CD3 + PD1 + TIM3 + Lymphocytes.

[0063] Therefore, in the present invention, the term "mass production" refers to the number of cells required to ensure the therapeutic dose. The method of the present invention for mass production of immunosuppressive T cells is to produce a large number of cells from the CD3 + The cell fraction produced approximately 87% (86.5 ± 8.8%) of CD3 cytokines with excellent immunosuppressive effects. + PD1 + TIM3 + Lymphocyte cells.

[0064] The terms "PD1" and "Tim3" are co-inhibitory molecules. T cells expressing these molecules can block excessive immune responses and maintain immune tolerance.

[0065] In the present invention, the term "immune tolerance" refers to a state in which a person does not exhibit an immune response to a specific antigen. Therefore, the immunosuppressive T cells of the present invention are T cells that induce tolerance to self-antigens and suppress T cell proliferation. For the purposes of the present invention, the immunosuppressive T cells of the present invention are T cells that induce immune tolerance to self-antigens that cause autoimmune diseases, preferably, graft-versus-host disease.

[0066] In the present invention, the culture can be carried out 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 have CD3 + PD1 + TIM3 + Phenotype.

[0067] On the other hand, when the culture is performed for 1 to 3 days, the immunosuppressive T cells may have a gene selected from the group consisting of CD3 + PD1 + TIM3 + 、CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - More than one phenotype in a group.

[0068] In addition, the present invention provides a method for mass-producing immunosuppressive T cells, comprising the steps of: isolating CD3 T cells from a sample isolated from an individual administered with granulocyte colony stimulating factor (G-CSF); + cells; and culturing the CD3+ cells in a culture medium comprising human serum albumin (HSA) and mercaptoethanol for 1 to 7 days.

[0069] Furthermore, the present invention provides a method for inducing T cells to have a gene selected from CD3 + PD1 + TIM3 + 、CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - A method for expressing T cells of one or more phenotypes in a group consisting of the above-mentioned cells, the method comprising the steps of: sorting CD3 T cells from a sample isolated from an individual administered granulocyte colony stimulating factor (G-CSF) + cells; and + The cells were cultured in a medium containing human serum albumin (HSA) and mercaptoethanol for 1 to 3 days.

[0070] Furthermore, the present invention provides a method for inducing T cells to have CD3 + PD1 + TIM3 + A method for phenotyping T cells, comprising the steps of: sorting CD3 T cells from a sample isolated from an individual administered granulocyte colony stimulating factor (G-CSF); + cells; and + The cells were cultured in a medium containing human serum albumin (HSA) and mercaptoethanol for 4 to 7 days.

[0071] The granulocyte colony stimulating factor (G-CSF) can be administered at a concentration of 1 μg / kg to 50 μg / kg, but is not limited thereto. Preferably, it can be administered at a concentration of 1 μg / kg to 30 μg / kg, and more preferably, it can be administered at a concentration of 5 μg / kg to 20 μg / kg, but is not limited thereto. More preferably, the dosage may be 5μg / kg to 15μg / kg, 5μg / kg to 10μg / kg, 6μg / kg to 20μg / kg, 6μg / kg to 15μg / kg, 6μg / kg to 10μg / kg, 7μg / kg to 20μg / kg, 7μg / kg to 15μg / kg, 7μg / kg to 10μg / kg, 8μg / kg to 20μg / kg, 8μg / kg to 15μg / kg, 8μg / kg to 10μg / kg, 9μg / kg to 20μg / kg, 9μg / kg to 15μg / kg, 9μg / kg to 10μg / kg, 10μg / kg to 20μg / kg or 10μg / kg to 15μg / kg, but is not limited thereto.

[0072] Furthermore, the present invention provides a culture medium composition for mass production of immunosuppressive T cells, comprising human serum albumin (HSA).

[0073] The medium composition may further comprise mercaptoethanol.

[0074] The concentration of the mercaptoethanol may be 1 μM to 200 μM, but is not limited thereto. Preferably, the concentration of the mercaptoethanol may be 20 μM to 100 μM, more preferably, the concentration of the mercaptoethanol may be 30 μM to 70 μM, but is not limited thereto.

[0075] Furthermore, the concentration of human serum albumin (HSA) may be 1 to 10 weight percent, but is not limited thereto. Preferably, the concentration of HSA may be 1 to 8 weight percent, and more preferably, the concentration of HSA may be 2 to 8 weight percent, but is not limited thereto.

[0076] The culture medium composition of the present invention can be used to culture blood or peripheral blood stem cells (PBSC), more preferably, can be used to culture PBSC.

[0077] The immunosuppressive T cells cultured using the medium composition of the present invention have CD3 + PD1 + Tim3 + Immunophenotype.

[0078] In the present invention, culture medium (media) refers to a culture medium that can support cell growth and survival in vitro, including conventional culture medium suitable for culturing cells used in all areas of the art. Culture medium and culture conditions can be selected according to the type of cell. Preferably, the culture medium for cultivating is cell culture minimum medium (CCMM), which generally comprises a carbon source, a nitrogen source and trace element components. As the culture medium, any conventional culture medium for cultivating animal cells can also be used. Preferably, a culture medium comprising serum (for example, fetal bovine serum, horse serum or human serum) can be used. Culture media that can be used in the present invention include, for example, RPMI series (e.g., RPMI 1640), Eagle's minimum essential medium (Eagle's MEM), α-MEM, Iscove's MEM, 199 medium, CMRL 1066, F12, F10, Dulbecco's modification of Eagle's medium (DMEM), a mixture of DMEM and F12, Way-mouth's MB752 / 1, McCoy's 5A, and MCDB series, but are not limited thereto. The culture media may contain other components, for example, antioxidants or antibiotics such as penicillin, streptomycin, and gentamicin.

[0079] In addition, the present invention provides a kit for mass production of immunosuppressive T cells, comprising granulocyte colony stimulating factor (G-CSF) and human serum albumin (HSA). The kit may further comprise mercaptoethanol.

[0080] The concentrations of G-CSF, HSA, and mercaptoethanol contained in the kit are as described above.

[0081] The kit is not limited to the above kits and may include other reagents or tools. For example, it may include a culture plate for culturing cells of the cultured subject or reagents required for immunosuppressive activity analysis (e.g., mixed lymphocyte reaction (MLR)), and may also include PBSC of the cultured subject.

[0082] The kit of the present invention can be provided in the form of a single container containing a culture medium containing G-CSF and HSA or G-CSF, HSA and mercaptoethanol, other components required for culture, and other reagents in appropriate doses and / or forms, or can be provided in separate containers.

[0083] The kit of the present invention may include instructions describing procedures for carrying out the above-mentioned method of the present invention.

[0084] Furthermore, the present invention provides a method for proliferating a gene having a gene selected from the group consisting of CD3 + PD1 + TIM3 + 、CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - A culture medium composition for immunosuppressive T cells of one or more phenotypes in the group consisting of the above, characterized in that it contains human serum albumin (HSA) and is used to culture peripheral blood stem cells (PBSC) for 1 to 3 days.

[0085] In addition, the present invention provides a method for proliferating cells harboring CD3 + PD1 + TIM3 +The culture medium composition for immunosuppressive T cells of a specific phenotype is characterized by comprising human serum albumin (HSA) and culturing peripheral blood stem cells (PBSC) for 4 to 7 days.

[0086] The culture medium composition for proliferating immunosuppressive T cells of the present invention may further contain mercaptoethanol.

[0087] The specific description of the medium composition including the concentrations of HSA and mercaptoethanol is as described above.

[0088] Furthermore, the present invention provides a method for proliferating a gene having a gene selected from the group consisting of CD3 + PD1 + TIM3 + 、CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - A kit for producing immunosuppressive T cells of one or more phenotypes in a group of cells, characterized in that the kit comprises granulocyte colony stimulating factor (G-CSF) and human serum albumin (HSA), and culture peripheral blood stem cells (PBSC) for 1 to 3 days.

[0089] In addition, the present invention provides a method for proliferating cells harboring CD3 + PD1 + TIM3 + A kit for producing immunosuppressive T cells with a specific phenotype, characterized by comprising granulocyte colony stimulating factor (G-CSF) and human serum albumin (HSA), and culturing peripheral blood stem cells (PBSC) for 4 to 7 days.

[0090] The kit for proliferating immunosuppressive T cells of the present invention may further comprise mercaptoethanol.

[0091] In the kit of the present invention, the specific specifications including the concentrations of G-CSF, HSA, and mercaptoethanol are as described above.

[0092] According to another embodiment of the present invention, all CD3 + The results of mixed lymphocyte reaction of G-PBSC confirmed that CD3 + In the case of G-PBSC (control group), 0.5±0.1% of T cells did not proliferate against allogeneic monocytes, and CD3 + In the case of G-PBSC, 13.9±6.4% of T cells did not proliferate against allogeneic monocytes. + In the case of G-PBSC, 43.2±10.1% of T cells did not proliferate against allogeneic monocytes. + G-PBSC has the best immune response suppression effect, which is better than CD3 isolated after 4 days of culture. + PD1 + TIM3 + That is, it was confirmed that T cells cultured by the method of the present invention exhibited an excellent immune response suppressive effect, and that T cells cultured for 2 days had a more excellent effect.

[0093] Furthermore, according to another embodiment of the present invention, the CD3 + The results of injecting G-PBSC cells into a xenograft aGVHD mouse model showed that the survival rate of mice was significantly increased. + G-PBSC were 1×10 7 In the case of the experimental group injected with a dose of 100 cells / kg, 100% of the test mice survived at the 80th day after cell injection, thus showing a significant improvement over the CD3 + Therapeutic Effects of G-PBSC: Specifically, it was confirmed that T cells cultured by the method of the present invention exhibited an excellent therapeutic effect on graft-versus-host disease, and that T cells cultured for 2 days exhibited even more excellent such effects.

[0094] Therefore, the present invention provides a pharmaceutical composition for preventing or treating an autoimmune disease, comprising immunosuppressive T cells prepared by the method for mass production of immunosuppressive T cells of the present invention.

[0095] In the pharmaceutical composition of the present invention, the immunosuppressive T cells have CD3 + PD1 + TIM3 +Phenotype. In addition, the immunosuppressive T cells may also have a phenotype selected from CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - More than one phenotype in a group.

[0096] In the pharmaceutical composition of the present invention, the autoimmune disease can be selected from graft-versus-host disease, rheumatoid arthritis, asthma, dermatitis, psoriasis, cystic fibrosis, post transplantation late and chronic solid organ rejection, multiple sclerosis, systemic lupus erythematosus, Sjögren syndrome, Hashimoto thyroiditis, polymyositis, scleroderma, Addison disease, vitiligo, pernicious anemia, glomerulonephritis, pulmonary fibrosis, inflammatory bowel disease, autoimmune diabetes, The present invention may include at least one disease selected from the group consisting of: diabetes, diabetic retinopathy, rhinitis, ischemia-reperfusion injury, post-angioplasty restenosis, chronic obstructive pulmonary disease (COPD), Graves' disease, gastrointestinal allergies, conjunctivitis, atherosclerosis, coronary artery disease, angina, cancer metastasis and arteriolar disease, preferably, graft-versus-host disease, but is not limited thereto.

[0097] For the purposes of this invention, the term "graft-versus-host disease" refers to a condition in which the injected lymphocytes attack the immune-compromised host (the recipient's body) during hematopoietic stem cell transplantation, causing symptoms such as fever, rash, liver dysfunction, diarrhea, and pancytopenia (a condition in which white blood cells, red blood cells, and platelets are all decreased). There are two types of graft-versus-host disease that occur after transplantation: acute and chronic. In addition to cases occurring during hematopoietic stem cell transplantation, graft-versus-host disease can occur during blood transfusion, although it is extremely rare. Once a graft-versus-host reaction occurs after a blood transfusion, it can be fatal.

[0098] The pharmaceutical composition of the present invention can be administered to an "individual" who has developed or is likely to develop an autoimmune disease. The "individual" may refer to all animals 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.

[0099] The term "prevention" used in the present invention refers to all actions to suppress the symptoms of a specific disease or delay its progression by administering the composition of the present invention.

[0100] The term "treatment" as used in the present invention refers to all actions to improve or beneficially change the symptoms of a specific disease by administering the composition of the present invention.

[0101] In addition to the active ingredients, the pharmaceutical composition of the present invention may further comprise an adjuvant. Any adjuvant known in the art may be used as the adjuvant without limitation. For example, the effect of the adjuvant may be enhanced by further comprising Freund's complete adjuvant or incomplete adjuvant.

[0102] The pharmaceutical composition of the present invention can be prepared in the form of a mixture of an active ingredient and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes 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 may include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinyl pyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil, but are not limited thereto.

[0103] The pharmaceutical composition of the present invention can be prepared according to conventional methods into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions for use.

[0104] In the case of formulation, conventional diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants can be used for preparation. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules. Such solid preparations can be prepared by mixing one or more excipients such as starch, calcium carbonate, sucrose, lactose, and gelatin with the active ingredient. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, internal liquids, emulsions, and syrups. In addition to conventional diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate can be used as non-aqueous solvents and suspensions. Semi-synthetic fatty acid ester (witepsol), Tween 61, cocoa butter, laurin, glycerin gelatin, etc. can be used as the base of the suppository.

[0105] The pharmaceutical composition of the present invention can be administered to an individual by various routes. All modes of administration are contemplated, for example, administration can be by oral, intravenous, intramuscular, subcutaneous, or intraperitoneal injection.

[0106] The dosage of the pharmaceutical composition of the present invention is selected based on the individual's age, weight, sex, and physical condition. Obviously, the concentration of the active ingredient in the pharmaceutical composition can be selected based on the subject, preferably at a concentration of 0.01 to 5000 μg / ml. When the concentration is less than 0.01 μg / ml, pharmacological activity may not be observed, while when it exceeds 5000 μg / ml, it may be toxic to the human body.

[0107] In the present invention, in addition to immunosuppressive T cells as an active ingredient, the pharmaceutical composition for preventing or treating autoimmune diseases may also contain any compound or natural extract with proven safety and known therapeutic efficacy, thereby enhancing the therapeutic effect of the 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 for the autoimmune disease, preferably graft-versus-host disease.

[0108] In addition, the present invention provides a cell therapy composition for preventing or treating autoimmune diseases, which comprises cells having CD3 T cells prepared by the method for mass production of immunosuppressive T cells of the present invention. + PD1 + TIM3 +In addition, the immunosuppressive T cells may also have a phenotype selected from CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - More than one phenotype in a group.

[0109] In the present invention, the term "cellular therapeutic agent" refers to a drug (as defined by the U.S. FDA) that uses cells and tissues isolated, cultured, and prepared through special procedures from the human body for therapeutic, diagnostic, and preventive purposes. It refers to a drug that uses these cells for the treatment, diagnosis, and prevention of diseases through a series of actions, such as in vitro proliferation, screening of living autologous, allogeneic, or xenogeneic cells, or other methods to alter the biological properties of cells in order to restore their function.

[0110] The cell therapy composition of the present invention can be directly administered to the lesion site, in which case it can be administered in the form of an injection. In particular, the cell therapy composition of the present invention can be formulated into an injection form to directly administer the cell therapy composition having CD3 T cells prepared by the preparation method of the present invention. + PD1 + TIM3 + Phenotypic immunosuppressive T cells, in this case, can also be included in the hydrogel to make it suitable for injection.

[0111] The hydrogels that can be used in the cell therapy agent of the present invention may include, but are not limited to, hydrogels suitable for injections known in the art, and may be one or more selected from the group consisting of small intestinal submucosal tissue, hyaluronic acid, carboxymethylcellulose (CMC), alginate, chitosan, polyacrylamide, poly(N-isopropylacrylamide), β-glycerophosphate, Pluronic (Poly(ethylene oxide)poly(propylene oxide)poly(ethyleneoxide), Pluronic), fibrin, polyethylene oxide (PEO), and a mixture of carboxymethylcellulose (CMC) and polyethyleneimine (PEI), but are not limited thereto.

[0112] The preferred dosage of the cell therapy composition of the present invention varies depending on the individual's condition and weight, the severity of the disease, the drug form, the route of administration, and the dosing cycle, but can be appropriately selected by one of ordinary skill in the art. Administration can be once daily or in divided doses, and the dosage does not in any way limit the scope of the present invention.

[0113] Furthermore, the present invention provides a method for preventing or treating an autoimmune disease, comprising the step of administering immunosuppressive T cells prepared by the method for mass production of immunosuppressive T cells of the present invention to an individual.

[0114] The "individual" refers to a subject who has developed or may develop an autoimmune disease, and the "individual" may refer to all animals including humans.

[0115] The autoimmune disease may be 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's thyroiditis, polymyositis, scleroderma, Addison's disease, vitiligo, pernicious anemia, glomerulonephritis, pulmonary fibrosis, inflammatory bowel disease, autoimmune diabetes mellitus, and leukemia. The present invention may include at least one disease selected from the group consisting of: acute myocardial ischemia, diabetic retinopathy, rhinitis, ischemia-reperfusion injury, post-angioplasty restenosis, chronic obstructive pulmonary disease (COPD), Graves' disease, gastrointestinal allergies, conjunctivitis, atherosclerosis, coronary artery disease, angina, cancer metastasis and arteriolar disease, preferably, graft-versus-host disease, but is not limited thereto.

[0116] Hereinafter, the present invention will be described in more detail by way of examples. These examples are only used to describe the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

[0117] Example 1: Cell collection and proliferation method

[0118] G-CSF (neutrogin, 10 μg / kg) was subcutaneously injected into normal peripheral blood stem cell (PBSC) donors for 5 days, and then G-CSF mobilized peripheral blood stem cells (G-PBSC) were collected using COBE spectra cell separator. TM CD3 T cells were isolated from G-PBSCs by magnetic-activated cell sorting (MACS). + Lymphocytes. The isolated CD3 + Lymphocytes were cultured in 5% human serum albumin (HSA) at a concentration of 5×10 5 Cells were cultured at a concentration of 10 cells / mL for 2 or 4 days using RPMI 1640 medium containing 2 mM L-glutamine, 25 mM hydroxyethylpiperazine ethane sulfonic acid (HEPES), 1 mM sodium pyruvate, and 50 μM mercaptoethanol.

[0119] CD3 + When G-PBSC lymphocytes were cultured in a medium containing HSA and mercaptoethanol for 4 days, the total number of cells increased by 1.0±0.6 times compared with that before culture. When cultured with HSA alone, the number of cells increased by 0.4±0.1 times (refer to Figure 1 ). Through the above experiment, it was confirmed that CD3 + Lymphocytes to increase CD3 + Lymphocyte yield.

[0120] Example 2: Confirmation of the phenotype of proliferating cells

[0121] Flow cytometry analysis using a fluorescence-activated cell sorter (FACS) was performed to confirm whether the cells cultured in Example 1 expressed the co-inhibitory molecules PD1, TIM3, LAG3, and TIGIT. Furthermore, the expression of PD1, TIM3, LAG3, and TIGIT in the peripheral blood of healthy individuals (normal controls) was also confirmed.

[0122] As a result, in the peripheral blood of normal people, every 1×10 3 CD3 + CD3 measured in G-PBSC cells + PD1 + TIM3 + 、CD3 + PD1 + LAG3 + and CD3 + PD1 + TIGIT + The number of lymphocytes was very low, 2 ± 1, 3 ± 1, and 3 ± 1, respectively. In contrast, G-CSF was injected subcutaneously for 5 days, and then G-PBSC were collected and CD3 + The results of lymphocytes were confirmed to be per 1×10 3 CD3 in CD3+G-PBSC + PD1 + TIM3 + 、CD3 + PD1 + LAG3 + and CD3 + PD1 + TIGIT + The number of lymphocytes increased to 68±8, 105±18 and 38±6 respectively. + The results of G-PBSC cultured in 5% HSA for 4 days were as follows: 1×10 3 CD3 + CD3 measured in G-PBSC + PD1 + LAG3 + and CD3 + PD1 + TIGIT +The number of lymphocytes was 865±89, 295±36 or 5±4 respectively. + PD1 + TIM3 + The number of lymphocytes increased 433 times compared to that in peripheral blood and about 13 times more than that before culture (ref. Figure 2 ). In addition, in cultured CD3 + PD1 + TIM3 + 48.9±5.2% of lymphocytes expressed LAG3 surface antigen, and TIGIT was almost not expressed (ref. Figure 4 ).

[0123] In particular, CD3 + G-PBSC differentiated into CD3 + PD1 + TIM3 + and CD3 + PD1 - TIM3 - Two groups of lymphocytes with surface antigens (ref Figure 3 、 Figure 6 ). In contrast, CD3 + G-PBSC differentiated into CD3 + PD1 + TIM3 + 、CD3 + PD + TIM3 - and CD3 + PD1 - TIM3 - Three groups of lymphocytes with surface antigens (ref Figure 6 ).

[0124] CD3 cells were cultured in a medium containing 5% HSA and 50 μM mercaptoethanol. + CD3 in G-PBSC + PD1 + TIM3 + The number of cells increased with the passage of time. Before culture, every 1×10 3 CD3 +The number of cells in G-PBSC was 68 ± 8, but it increased to 212 ± 44 after 2 days of culture and to 865 ± 88 after 4 days of culture (refer to Figure 6 ). In contrast, CD3 + PD1 + TIM3 - Before culture, 1×10 lymphocytes were 3 CD3 + The number of cells in G-PBSC was 197 ± 52, but after 2 days of culture, the number was 172 ± 64, and almost all cells disappeared after 4 days of culture (refer to Figure 6 ). And, CD3 + PD1 - TIM3 - The number of lymphocytes also decreased with the passage of time. Before culture, every 1×10 3 CD3 + The number of G-PBSC cells was 735 ± 105, but decreased to 616 ± 192 after 2 days of culture and to 67 ± 38 after 4 days of culture (ref. Figure 6 ). Based on the above results, it can be inferred that CD3 + PD1 - TIM3 - Lymphocytes and CD3 + PD1 + TIM3 - Lymphocytes differentiate into CD3 + PD1 + TIM3 + To demonstrate this, CD3 + Isolation of CD3 from G-PBSC + PD1 - TIM3 - and CD3 + PD1 + TIM3 - Lymphocytes were cultured for 4 days. As a result, it was confirmed that 70.8 ± 3.1% of CD3 + PD1 - TIM3 - Lymphocytes and 80.7±5.2% CD3 + PD1 + TIM3 - Lymphocyte differentiation into CD3 + PD1 + TIM3 + Lymphocytes (refer to Figure 8 ).

[0125] Example 3: Confirmation of the immune response suppressive effect of cultured lymphocytes using mixed lymphocyte reaction

[0126] The immune response suppression effect of cells cultured by the method of the present invention was confirmed by 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, when T lymphocytes and monocytes (CD14 + When mixed culture of mononuclear cells (monocytes), due to the inconsistency of MHC between the two cells, the T lymphocytes in culture recognize the monocytes of another person as alloantigens and proliferate. In this case, the immune response of T lymphocytes to alloantigens is measured by measuring the proliferation degree of T lymphocytes. In order to prevent the proliferation of allogeneic monocytes other than T lymphocytes, the allogeneic monocytes are irradiated. In this case, the proliferation degree of T lymphocytes is measured by staining T lymphocytes with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE). The stained CFSE penetrates the cell membrane and stays by non-specifically binding to proteins in the cell, and the fluorescence amount of CFSE is halved each time the cell divides. Taking advantage of this, when the CFSE fluorescence intensity in the cells after culture is measured, the proliferation degree of the lymphocytes can be confirmed. On this basis, in order to perform a mixed lymphocyte reaction, the T lymphocytes cultured by the method of the present invention are separated into CD3 + PD1 + TIM3 + 、CD3 + PD1 - TIM3 - and CD3 + PD1 + TIM3 - Lymphocytes were isolated and compared with allogeneic monocytes (CD14 + The cells were mixed at a 1:2 cell ratio and cultured for 3 days. All cultured cells were then harvested and the CSFE fluorescence intensity was measured. The percentage of lymphocytes that did not proliferate in response to alloantigens was also determined. Anti-PD1 mAb and anti-Tim3 mAb were added to 5% HSA cell culture medium at concentrations of 20 μg / mL and 40 μg / mL, respectively.

[0127] CD3 cells were cultured in a medium containing 5% HSA and 50 μM mercaptoethanol for 4 days. + G-PBSC differentiated into CD3+ PD1 + TIM3 + and CD3 + PD1 - TIM3 - Two groups of lymphocytes (ref. Figure 3 ). + PD1 + TIM3 + and CD3 + PD1 - TIM3 - The results of mixed lymphocyte reaction of lymphocytes showed that CD3 + PD1 + TIM3 + In the case of lymphocytes, 34.7±1.8% of T cells did not proliferate against allogeneic monocytes. + PD1 - TIM3 - In the case of lymphocytes, 18.7±0.6% of T cells did not proliferate (ref. Figure 3 ). Thus, it was confirmed that CD3 + CD3 in G-PBSC-differentiated lymphocytes + PD1 + TIM3 + Lymphocytes have a high immune response suppressive effect.

[0128] Furthermore, to confirm CD3 + The immunosuppressive effect of G-PBSC cells over time was observed in CD3 T cells cultured in a medium containing 5% HSA and 50 μM mercaptoethanol for 2 or 4 days. + G-PBSC cells were subjected to mixed lymphocyte reaction. The results showed that the CD3 + In the case of G-PBSC, 0.5±0.1% of T cells did not proliferate against allogeneic monocytes, but CD3 + In the case of G-PBSC, 43.2±10.1% of T cells did not proliferate against allogeneic monocytes, and CD3 + In the case of G-PBSC, 13.9±6.4% of T cells did not proliferate against allogeneic monocytes (refer to Figure 5 ). Based on this result, it was confirmed that CD3 + G-PBSC has the best immunosuppressive effect (refer to Figure 5), the immunosuppressive effect of the cells was higher than that of the CD3 isolated after 4 days of culture. + PD1 + TIM3 + Lymphocytes (refer to Figure 3 ).

[0129] In addition, CD3 + G-PBSC differentiated into the following three groups: CD3 + PD1 + TIM3 + 、CD3 + PD1 - TIM3 - and CD3 + PD1 + TIM3 - Lymphocytes (refer to Figure 6 ). The results of mixed lymphocyte reaction after separation of three groups of lymphocytes confirmed that CD3 + PD1 + TIM3 + In the case of lymphocytes, 19.5±3.0% of T cells did not proliferate against allogeneic monocytes. + PD1 + TIM3 - In the case of lymphocytes, 29.1±12.6% of T lymphocytes did not proliferate against allogeneic monocytes. + PD1 - TIM3 - In the case of lymphocytes, 20.3±7.9% of T lymphocytes did not proliferate against allogeneic monocytes (ref. Figure 7 ). In mixed lymphocyte reaction, CD3 + Compared with the results of 43.2±10.1% T lymphocytes in G-PBSC that did not proliferate against allogeneic monocytes (ref. Figure 5 ), isolated CD3 + PD1 + TIM3 + 、CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - The immunosuppressive capacity of lymphocytes is significantly reduced (refer to Figure 7 ). Based on the above results, it was concluded that the complete CD3 + The conclusion is that the G-PBSC method is the most effective in suppressing immune response.

[0130] Furthermore, it was confirmed that CD3 + CD3 in G-PBSCs + PD1 + TIM3 + The number of lymphocytes was 86.5±8.8%, which was higher than 21.2±4.4% after 2 days of culture. However, the number of CD3 + The immunosuppressive effect of G-PBSC is significantly reduced (ref. Figure 5 、 Figure 6 ). This indicates that CD3 + PD1 + TIM3 + Lymphocytes may express the same surface antigens but have different immunosuppressive abilities. To demonstrate this, CD3 + PD1 + TIM3 + Cells were isolated and treated with monoclonal antibodies against PD1 (anti-PD1 mAb) and monoclonal antibodies against Tim3 (anti-Tim3 mAb), respectively, and then mixed lymphocyte reaction was performed. Generally, it is expected that when treated with anti-PD1 mAb or anti-Tim3 mAb, the inhibitory effect of allogeneic immune responses of immunosuppressive T lymphocytes will be offset, thereby reducing the number of T lymphocytes that do not react to allogeneic monocytes. As described above, CD3 isolated after 4 days of culture was treated with anti-PD1 mAb and anti-Tim3 mAb, respectively. + PD1 + TIM3 + The results of lymphocytes showed that 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. Therefore, it was confirmed that anti-PD1 mAb and anti-Tim-3 mAb offset the inhibitory effect of allogeneic immune response. + PD1 + TIM3 + When the cells were treated with anti-PD1 mAb, the percentage of T lymphocytes that did not proliferate 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 from 19.7±3.6%, respectively. The expected effect of increasing the immune response by anti-PD1 mAb and anti-Tim-3 mAb was not observed. + CD3 isolated from G-PBSC + PD1+ TIM3 - When cells were detected, the percentage of T lymphocytes that did not proliferate against allogeneic monocytes decreased from 29.1±12.6% to 23.0±11.1%, confirming that CD3 + PD1 + TIM3 - The immune response of cells to anti-PD1 mAb increased (refer to Figure 7 ). Based on these results, it can be confirmed that the CD3 + PD1 + TIM3 + Lymphocytes, cultured for 2 days CD3 + PD1 + TIM3 + Lymphocytes are cells whose immune response suppression effects are not counteracted by anti-PD1 mAb and anti-Tim3 mAb. Although these two cells express the same surface antigens, they are different types of cells with different functions.

[0131] Example 4: Confirmation of the immune response suppression effect of cultured lymphocytes using a xenotransplantation GVHD mouse model

[0132] Experiments were performed using an immunodeficient xenograft aGVHD mouse model (NOD-scid IL-2Rγnull) to confirm that cultured CD3 + The therapeutic effect of G-PBSC cells. NOD-scid IL-2Rγnull mice are mice that lack the function of T lymphocytes and B lymphocytes. NOD-scid IL-2Rγnull mice were irradiated with 3Gy of radiation, and then 3.5±1.8×10 7 PBMCs of 1000 cells / kg were injected into the peritoneal cavity of mice to induce aGVHD. Then, the CD3 + The number of G-PBSC lymphocytes (control group vs. 1×10 6 cells / kg vs. 1×10 7 cells / kg vs. 5×10 7 cells / kg vs.10×10 7 The cells (cells) / kg) were injected into the tail vein of mice, and then the survival of the mice was confirmed to evaluate the cultured CD3 + The therapeutic effect of G-PBSC lymphocytes.

[0133] As a result, in the control group (n=7), only 14% of the mice survived at 100 days after peripheral mononuclear cell injection. In contrast, 14% of the mice survived after being cultured in a medium containing 5% HSA and 50 μM mercaptoethanol for 4 days. + G-PBSC lymphocytes were expressed at 1×10 6 In the group (n=5) injected with 1×10 cells / kg, 20% of the mice survived. 7 In the group (n=7) injected with a dose of 10 cells / kg, 71% of the mice survived, confirming that CD3 + The appropriate therapeutic dose of G-PBSC is 1×10 7 cells / kg (reference Figure 9 ). And, when the CD3 + G-PBSC lymphocytes were increased to 5×10 7 cells / kg (n=3) or 10 times the dose of 10×10 7 When 3 cells / kg (n=3) were injected, most mice died within 20 days (ref. Figure 9 ).

[0134] CD3 cells cultured in the same manner for 2 days were injected + G-PBSC is used to evaluate the therapeutic effect on aGVHD (refer to Figure 10 ). Confirmed that at 1×10 7 In the group (n=4) injected with 100 cells / kg, 100% of the mice survived at 80 days after cell injection (refer to Figure 10 ). In addition, it was confirmed that when CD3+G-PBSC lymphocytes cultured for 2 days were counted at 1×10 7 cells / kg, a 5-fold dose of 5×10 7 When 10 cells / kg (n=2) were injected, 50% of the mice survived, and 10×10 7 All mice in the group injected with 10 cells / kg (n=2) survived without dying (ref. Figure 10 ). Thus, it was confirmed that CD3 + G-PBSC lymphocytes effectively suppress immune responses even in vivo, and it was confirmed that CD3 + G-PBSC lymphocytes were expressed at 1×10 7The method of administering the drug at a dose of 10 cells / kg can most effectively inhibit the graft-versus-host reaction.

[0135] From the results of the above examples, it was confirmed that when CD3 + When G-PBSCs were cultured in a medium containing HSA and mercaptoethanol for 4 days, most cells (86.5±8.8%) differentiated into CD3 + PD1 + TIM3 + Lymphocytes and differentiated CD3 + PD1 + TIM3 + Lymphocytes have excellent immune response suppression effects. + G-PBSCs contain a large number of CD3 + PD1 + TIM3 - Lymphocytes and CD3 + PD1 + TIM3 + lymphocytes and showed a higher expression of CD3 + PD1 + TIM3 + Therefore, the CD3 + G-PBSC is the most ideal cell therapy agent and is expected to be effectively used in the treatment of immune diseases such as graft-versus-host disease in the future.

Claims

1. A method for mass production of immunosuppressive T cells, characterized in that: The steps include: Isolation of CD3 from samples isolated from individuals + cells; and The CD3 + cell.

2. The method for mass production of immunosuppressive T cells according to claim 1, characterized in that The sample is one or more selected from the group consisting of blood, tissue, cells, plasma, feces, urine and semen.

3. The method for mass production of immunosuppressive T cells according to claim 1, wherein The sample is one or more selected from the group consisting of blood and peripheral blood stem cells.

4. The method for mass production of immunosuppressive T cells according to claim 1, wherein The individuals are healthy individuals of the same species.

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

6. The method for mass production of immunosuppressive T cells according to claim 1, wherein The individual is a subject to whom leukocyte growth factor is administered.

7. The method for mass production of immunosuppressive T cells according to claim 6, characterized in that: The leukocyte growth factor is granulocyte colony stimulating factor.

8. The method for mass production of immunosuppressive T cells according to claim 1, wherein The culture medium also contains mercaptoethanol.

9. The method for mass production of immunosuppressive T cells according to claim 8, characterized in that: The concentration of the mercaptoethanol is 1 μM to 200 μM.

10. The method for mass production of immunosuppressive T cells according to claim 1, wherein The concentration of the human serum albumin is 1 weight percent to 10 weight percent.

11. The method for mass production of immunosuppressive T cells according to claim 1, wherein The culturing is performed for 1 to 7 days.

12. The method for mass production of immunosuppressive T cells according to claim 1, wherein The immunosuppressive T cells have CD3 + PD1 + TIM3 + Phenotype.

13. The method for mass production of immunosuppressive T cells according to claim 1, wherein When the culture is performed for 1 to 3 days, the immunosuppressive T cells have a gene selected from the group consisting of CD3 + PD1 + TIM3 + 、CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - More than one phenotype in a group.

14. A method for mass production of immunosuppressive T cells, characterized in that: The steps include: CD3 was isolated from samples isolated from individuals who received granulocyte colony-stimulating factor. + cells; and The CD3 + The cells were cultured in a medium containing human serum albumin and mercaptoethanol for 1 to 7 days.

15. A method for inducing T cells in vitro to have a gene selected from the group consisting of CD3 + PD1 + TIM3 + 、CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - A method for expressing T cells of one or more phenotypes in a group consisting of: The steps include: CD3 was isolated from samples isolated from individuals who received granulocyte colony-stimulating factor. + cells; and The CD3 + The cells were cultured in a medium containing human serum albumin and mercaptoethanol for 1 to 3 days.

16. A method for inducing T cells to have CD3 + PD1 + TIM3 + A method for phenotyping T cells, characterized in that The steps include: CD3 was isolated from samples isolated from individuals who received granulocyte colony-stimulating factor. + cells; and The CD3 + The cells were cultured in a medium containing human serum albumin and mercaptoethanol for 4 to 7 days.

17. The method for mass production of immunosuppressive T cells according to claim 14, characterized in that: The granulocyte colony stimulating factor is administered at a concentration of 1 μg / kg to 50 μg / kg.

18. A culture medium composition for mass production of immunosuppressive T cells, characterized in that: Contains human serum albumin.

19. The culture medium composition for mass production of immunosuppressive T cells according to claim 18, characterized in that The medium composition also includes mercaptoethanol.

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

21. The culture medium composition for mass production of immunosuppressive T cells according to claim 18, characterized in that The concentration of the human serum albumin is 1 weight percent to 10 weight percent.

22. The culture medium composition for mass production of immunosuppressive T cells according to claim 18, characterized in that The culture medium composition is used for culturing blood or peripheral blood stem cells.

23. The culture medium composition for mass production of immunosuppressive T cells according to claim 18, characterized in that The immunosuppressive T cells have CD3 + PD1 + Tim3 + Immunophenotype.

24. A kit for mass production of immunosuppressive T cells, characterized in that: Contains granulocyte colony-stimulating factor and human serum albumin.

25. The kit for mass production of immunosuppressive T cells according to claim 24, characterized in that The kit also contains mercaptoethanol.

26. A method for proliferating a cell line having a gene selected from the group consisting of CD3 + PD1 + TIM3 + 、CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - A culture medium composition for immunosuppressive T cells of one or more phenotypes selected from the group consisting of: Human serum albumin is included, and peripheral blood stem cells are cultured for 1 to 3 days.

27. The method for proliferating a gene having a gene selected from the group consisting of CD3 + PD1 + TIM3 + 、CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - A culture medium composition for immunosuppressive T cells of one or more phenotypes selected from the group consisting of: The medium composition also includes mercaptoethanol.

28. A method for proliferating CD3 + PD1 + TIM3 + The culture medium composition for immunosuppressive T cells of a phenotype, characterized in that Human serum albumin is included, and peripheral blood stem cells are cultured for 4 to 7 days.

29. The method for proliferating cells having CD3 + PD1 + TIM3 + The culture medium composition for immunosuppressive T cells of a phenotype, characterized in that The medium composition also includes mercaptoethanol.

30. A method for proliferating a gene selected from the group consisting of CD3 + PD1 + TIM3 + 、CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - A kit for immunosuppressive T cells of one or more phenotypes in a group consisting of: Contains granulocyte colony-stimulating factor and human serum albumin, and culture peripheral blood stem cells for 1 to 3 days.

31. The method for proliferating a gene having a gene selected from the group consisting of CD3 + PD1 + TIM3 + 、CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - A kit for immunosuppressive T cells of one or more phenotypes in a group consisting of: The kit also contains mercaptoethanol.

32. A method for proliferating CD3 + PD1 + TIM3 + A kit for phenotyping immunosuppressive T cells, characterized in that Contains granulocyte colony-stimulating factor and human serum albumin, and culture peripheral blood stem cells for 4 to 7 days.

33. The method for proliferating cells having CD3 + PD1 + TIM3 + A kit for phenotyping immunosuppressive T cells, characterized in that The kit also contains mercaptoethanol.

34. A pharmaceutical composition for preventing or treating autoimmune diseases, characterized in that: The method comprises immunosuppressive T cells prepared by the method for mass production of immunosuppressive T cells according to claim 1.

35. The pharmaceutical composition for preventing or treating autoimmune diseases according to claim 34, characterized in that The autoimmune disease is one or more diseases selected from the group consisting of graft-versus-host disease, rheumatoid arthritis, asthma, dermatitis, psoriasis, cystic fibrosis, late and chronic solid organ rejection after transplantation, multiple sclerosis, systemic lupus erythematosus, Sjögren's syndrome, Hashimoto's thyroiditis, polymyositis, scleroderma, Addison's disease, vitiligo, pernicious anemia, glomerulonephritis, pulmonary fibrosis, inflammatory bowel disease, autoimmune diabetes, diabetic retinopathy, rhinitis, ischemia-reperfusion injury, restenosis after angioplasty, chronic obstructive heart disease, Graves' disease, gastrointestinal irritability, conjunctivitis, atherosclerosis, coronary artery disease, angina pectoris, cancer metastasis and arteriole disease.

36. The pharmaceutical composition for preventing or treating autoimmune diseases according to claim 34, characterized in that The immunosuppressive T cells have CD3 + PD1 + TIM3 + Phenotype.

37. The pharmaceutical composition for preventing or treating autoimmune diseases according to claim 34, characterized in that The immunosuppressive T cells also have a gene selected from the group consisting of CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - More than one phenotype in a group.

38. A cell therapy composition for preventing or treating autoimmune diseases, characterized in that: Comprising a CD3 T cell prepared by the method for mass production of immunosuppressive T cells according to claim 1 + PD1 + TIM3 + Phenotype of immunosuppressive T cells.

39. The cell therapy composition for preventing or treating autoimmune diseases according to claim 38, characterized in that The immunosuppressive T cells also have a gene selected from the group consisting of CD3 + PD1 + TIM3 - and CD3 + PD1 - TIM3 - More than one phenotype in a group.

40. A method for preventing or treating an autoimmune disease, characterized in that: The method comprises the step of administering the immunosuppressive T cells prepared by the method for mass production of immunosuppressive T cells according to claim 1 to an individual.

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 graft-versus-host disease, rheumatoid arthritis, asthma, dermatitis, psoriasis, cystic fibrosis, late and chronic solid organ rejection after transplantation, multiple sclerosis, systemic lupus erythematosus, Sjögren's syndrome, Hashimoto's thyroiditis, polymyositis, scleroderma, Addison's disease, vitiligo, pernicious anemia, glomerulonephritis, pulmonary fibrosis, inflammatory bowel disease, autoimmune diabetes, diabetic retinopathy, rhinitis, ischemia-reperfusion injury, restenosis after angioplasty, chronic obstructive heart disease, Graves' disease, gastrointestinal irritability, conjunctivitis, atherosclerosis, coronary artery disease, angina pectoris, cancer metastasis and arteriole disease.

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