KR20230002080A - Composition for preventing or treating Immune Related Disease comprising immune Inhibitory T cells - Google Patents

Abstract

The present invention relates to a composition, containing T cells exhibiting an immunosuppressive effect, for preventing or treating immune-related diseases, and more specifically, relates to a composition for preventing or treating immune-related diseases, wherein the composition contains cells having immune phenotypes of CD3+, PD1+, and Tim3+. According to the present invention, it is possible to easily collect large quantities of cells having immune phenotypes of CD3+, PD1+, and Tim3+ having an immunosuppressive effect. Due to having an immunosuppressive effect, the cells are expected to be widely used as a therapeutic agent for immune-related diseases for immunosuppression.

Description

Composition for preventing or treating immune related disease comprising immune suppressive T cells {Composition for preventing or treating Immune Related Disease comprising immune Inhibitory T cells}

The present invention relates to a composition for preventing or treating immune-related diseases including T cells exhibiting an immunosuppressive effect, and more specifically, to preventing or treating immune-related diseases including T cells having CD3+, PD1+ and Tim3+ immune phenotypes. It relates to therapeutic compositions.

As the demand for cell therapy for immunosuppression increases, immune cell therapy that can control the alloimmune response while correcting the balance between cells is evaluated as the most ideal, rather than immunosuppressive agents that unilaterally suppress the immune response. However, MSC (Mesenchymal Stem Cells), a cell therapy for immunosuppression, requires a multi-step culture process to secure the number of cells, and cell function is damaged due to active oxygen generated during this process.

Antigen invasion signals are transmitted by antigen presenting cells through the TCR of T cells and co-stimulatory molecules, resulting in an immune response. When the immune response is terminated, T cells differentiate into central memory cells, whereas under continuous stimulation, T cells produce co-inhibitory molecules such as PD-1, Tim3, LAG-3, and TIGIT. expression, and the surface antigen-converted T cells block excessive immune responses and maintain a tolerance state. Therefore, if a cell therapy agent capable of suppressing an immune response by inducing T cells transformed with a surface antigen can be developed, it will be useful for various purposes in various immune diseases. Although studies on selective inactivation of T cell surface antigens by DNA cleavage are in progress (Korean Patent Publication No. 10-2016-0029017), most studies are only on non-alloreactive immunosuppression, Studies on T cells and their use for suppressing the alloimmune response are still insignificant.

On the other hand, graft-versus-host disease (GvHD) is an allogeneic immune response caused by a mismatch between donor and patient histocompatibility antigens (HLA) after transplantation, with a frequency ranging from 10% to 80%. Severe GvHD (grade 3-4) with a high mortality rate is also known to occur in about 20% of patients. Various immunosuppressants such as immunoglobulin, thalidomide, and cyclosporine are used for the treatment of graft-versus-host disease, but most patients die from infection or long-term The problem of deteriorating quality of life due to impaired function has emerged. Therefore, there is an urgent need to develop cell therapy agents for preventing or treating graft-versus-host disease.

In order to solve the above problems, the present inventors confirmed that T cells having CD3+, PD1+, and Tim3+ immune phenotypes exhibit significant immunosuppressive effects in allogeneic monocytes, and thus cells having CD3+, PD1+, and Tim3+ immunophenotypes. The purpose of preventing or treating immune-related diseases was identified.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating immune-related diseases, including T cells having CD3+, PD1+ and Tim3+ immune phenotypes.

In addition, an object of the present invention is to provide a pharmaceutical preparation for preventing or treating immune-related diseases, including the above composition.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating immune-related diseases, including T cells having an immune phenotype of CD3+, PD1+ and Tim3+.

In one embodiment of the present invention, the T cells may further have an immunophenotype of TIGIT+ or TIGIT-.

In another embodiment of the present invention, the composition can suppress an immune response.

In another embodiment of the present invention, the immune-related disease is an autoimmune disease; graft-versus-host disease; organ transplant rejection; asthma; atopy; or an acute or chronic inflammatory disease.

In another embodiment of the present invention, the autoimmune disease is rheumatoid arthritis, type 1 diabetes, systemic scleroderma, systemic lupus erythematosus, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behçetsi disease , Sjogren's syndrome, Gilia-Barre syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibromyalitis, and polyarteritis nodosa.

In another embodiment of the present invention, the T cells may be prepared by culturing a CD3+ cell fraction and IL-2+ in a mixture.

In addition, the present invention provides a method for treating immune-related diseases comprising administering the pharmaceutical composition to a subject.

In addition, the present invention provides a therapeutic use of the pharmaceutical composition for immune-related diseases.

In addition, the present invention provides a pharmaceutical preparation for preventing or treating immune-related diseases, including the above composition.

In one embodiment of the present invention, the pharmaceutical preparation can suppress the immune response.

In another embodiment of the present invention, the immune-related disease is an autoimmune disease; graft-versus-host disease; organ transplant rejection; asthma; atopy; or an acute or chronic inflammatory disease.

In another embodiment of the present invention, the autoimmune disease is rheumatoid arthritis, type 1 diabetes, systemic scleroderma, systemic lupus erythematosus, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behçetsi disease , Sjogren's syndrome, Gilia-Barre syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibromyalitis, and polyarteritis nodosa.

According to the present invention, cells having immunosuppressive CD3+, PD1+, and Tim3+ immune phenotypes can be easily collected in large quantities, and since the cells have an immunosuppressive effect, they can be used as a treatment for immune-related diseases for immunosuppression. It is expected to be widely used.

Figure 1 shows G-CSF mobilize PBSC CD3+ lymphocytes not cultured (PBSC T cells), cultured with IL-2 (50 U/mL) for 4 days (IL-2(+) PBSC T cells), or treated with IL-2 After culturing for 4 days (IL-2(-) PBSC T cells) and analyzing each cell, Figure 1a shows IL-2(+) PBSC T cells and IL-2(-) PBSC T cells as CD14+ allogeneic monocytes, respectively. (mononuclear cells; MNCs) and the result of inducing an immune response (mixed lymphocyte reaction, MLR) is shown, Figure 1b confirms the expression level of co-inhibitory molecules on the surface of each cell, It shows the result of comparing cell fractions.
FIG. 2 analyzes the immunosuppressive effect of CD3+PD1+Tim3+ cells, and FIG. 2a shows the result of CD3+PD1+ cell fractionation and the induction of an immune response (mixed lymphocyte reaction, MLR) using only PD1- cells after removing PD1+ cells. One result is shown, and FIG. 2b shows the results of CD3+PD1+Tim3+ cell fractionation and the induction of a mixed lymphocyte reaction (MLR) after removing Tim3+ cells.
3 shows the result of inducing a mixed lymphocyte reaction (MLR) according to the expression of TIGIT in the CD3+PD1+Tim3+ cell fraction.
Figure 4a shows the number of CD3 + PD1 + Tim3 + TIGIT- cells with excellent alloimmune response suppression effect IL-2 (+) PBSC T cells after culture based on the number of CD3 + PD1 + Tim3 + TIGIT- cells in PBSC T cells before culture Cells and IL-2 (-) PBSC T cells CD3 + PD1 + Tim3 + TIGIT- cell number and cell ratio comparison results, Figure 4b is a CD3 + that can be obtained by collecting PBSC once per unit weight in actual clinical practice. It is a result of comparing the number of PD1+Tim3+TIGIT- cells in PBSC T cells before culture, IL-2(+) PBSC T cells, and IL-2(-) PBSC T cells after culture.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.

The present inventors confirmed that cells with CD3+, PD1+, and Tim3+ immune phenotypes exhibit significant immunosuppressive effects in allogeneic monocytes, thereby preventing or treating immune-related diseases of T cells with CD3+, PD1+, and Tim3+ immune phenotypes. identified.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating immune-related diseases, including T cells having CD3+, PD1+ and Tim3+ immune phenotypes.

As used herein, the term "prevention" refers to any activity that suppresses or delays the onset of immune-related diseases by administration of the composition according to the present invention.

As used herein, the term "treatment" refers to any activity that improves or beneficially changes the symptoms of an immune-related disease by administration of the composition according to the present invention.

As used herein, the term "immune-related disease" refers to a disease caused by dysfunction of the immune system, including autoimmune disease; graft-versus-host disease; organ transplant rejection; asthma; atopy; Or it may be an acute or chronic inflammatory disease, but is not limited thereto.

As used herein, the term "autoimmune disease" refers to a disease that exhibits a pathological response to a self antigen, and the autoimmune disease in the present invention includes rheumatoid arthritis, type 1 diabetes, systemic scleroderma, systemic lupus erythematosus, Systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Bechetsy's disease, Sjogren's syndrome, Guillain-Barré syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibromyalgia, and polyarteritis nodosa It may be one or more selected from, but is not limited thereto.

As used herein, the term "graft-versus-host disease" is a disease that occurs when transplanted lymphocytes attack the body of a patient with reduced immune function. Graft-versus-host disease in the present invention may include acute graft-versus-host disease. However, it is not limited thereto.

In the present invention, the T cell may have a TIGIT+ or TIGIT- immune phenotype, but is not limited thereto.

In addition, in the present invention, the composition may suppress an immune response, more preferably suppress an alloimmune response, but is not limited thereto.

In addition, the T cells may be prepared by culturing a CD3+ cell fraction and IL-2+ in a mixture.

The present inventors confirmed through specific examples that the cells of the present invention are effective in preventing or treating immune-related diseases.

In one embodiment of the present invention, as a result of confirming the alloimmune response of IL-2(+) PBSC T cells expressing co-inhibitory molecules such as PD1, Tim3 and Lag3, in the case of IL-2(+) PBSC T cells, The percentage of cells that did not proliferate in response to allo-MNCs was 16.1±4.7%, showing a clear immunosuppressive effect compared to PBSC T cells (0%) before culture, and IL-2(-) PBSC T cells (11.0±4.7%). ± 0.1%), it was confirmed that the immunosuppressive effect was increased 1.5 times (see Example 2).

In another embodiment of the present invention, as a result of inducing a mixed lymphocyte reaction (MLR) after removing PD1 or Tim3 from CD3+PD1+ cells, the effect of suppressing the allogeneic immune response is lost, resulting in CD3+PD1+ It was confirmed that T lymphocytes suppressing the immune response exist in Tim3+ cells (see Example 3).

In addition, most of the CD3+PD1+Tim3+ cells were TIGIT-, and as a result of confirming the alloimmune response according to TIGIT expression, CD3+PD1+Tim3+TIGIT- cells did not proliferate in response to allo-MNCs. The percentage of cells that did not respond was 24.3±9.4%, which was 1.7 times higher than that of CD3+PD1+Tim3+TIGIT+ cells (14.3±17.0%). It was confirmed that the % was still maintained (see Example 4).

From the results of the above examples, it is expected that the T cells of the present invention can be used as cell therapy agents for the treatment of immune-related diseases by inducing immunosuppression through the expression of co-inhibitory molecules.

The pharmaceutical composition according to the present invention may include cells having immunophenotypes of CD3+, PD1+, and Tim3+ alone in a pharmaceutically effective amount, or may include one or more pharmaceutically acceptable carriers. At this time, the pharmaceutically acceptable carrier is one commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose , polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto. In addition to the above components, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like may be further included.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically applied) depending on the desired method, and the dosage depends on the patient's condition, body weight, and disease. Depending on the degree, drug form, administration route and time, it can be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level depends on the type of disease, severity, activity of the drug, and drug sensitivity, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitantly used drugs, and other factors well known in the medical arts. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, body weight, absorption rate, inactivation rate and excretion rate of the active ingredient in the body, type of disease, and concomitant drugs, generally Depending on the body weight, an appropriate dose may be administered daily or every other day, or divided into 1 to 3 times a day. In addition, the dosage may be increased or decreased depending on the route of administration, gender, age, and the like.

In addition, as another aspect of the present invention, the present invention provides a pharmaceutical formulation for preventing or treating immune-related diseases, including the above composition.

In the present invention, the pharmaceutical agent may suppress an immune response, and more preferably suppress an alloimmune response, but is not limited thereto.

As another aspect of the present invention, the present invention provides a method for preventing or treating an immune-related disease comprising administering T cells having an immune phenotype of CD3+, PD1+, and Tim3+ to a subject in need thereof.

The “CD3+”, “PD1+”, “Tim3+”, “immunophenotype”, “T cell”, “immune-related disease”, “administration”, “prevention” or “treatment” may be within the aforementioned range.

In the present invention, the "individual" may be a mammal such as a rat, livestock, mouse, human, etc., and specifically may be a companion dog, racehorse, human, etc. in need of treatment for a metabolic disease, and preferably may be a human.

In addition, in the above method, the T cells may further have an immunophenotype of TIGIT+ or TIGIT-.

As another aspect of the present invention, the present invention provides the use of T cells having CD3+, PD1+ and Tim3+ immune phenotypes for the manufacture of a drug for preventing or treating immune-related diseases.

The “CD3+”, “PD1+”, “Tim3+”, “immunophenotype”, “T cell”, “immune-related disease”, “prevention” or “treatment” may be within the aforementioned range.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.

[Example]

Example 1. Cell collection and propagation method

Peripheral hematopoietic stem cell donors are subcutaneously injected with G-CSF (neutrogin; 10 μg/kg) for 5 days, and then G-CSF mobilized peripheral blood stem cells (PBSC) are collected using COBE spectra. Afterwards, monocytes were separated with Ficoll-Plaque plus (GE healthcare, 17-1440-02) and then MACS magnetic, MS column (Miltenyibiotec. 130-042-201) containing CD3 MicroBeads, human (miltenyibiotec. 130-050-101). ) to sort CD3+ lymphocytes. Thereafter, 0.5 X 10 ⁷ CD3+ cells were loaded on a 100π dish with 15 mL of serum-free medium containing IL-2 (50 U/ml, human IL-2 (peprotech, 200-02)) as a culture medium and cultured for 4 days. .

Example 2. Verification of phenotype and immunosuppressive effect of proliferated cells

G-CSF mobilize PBSC CD3+ lymphocytes without culture (PBSC T cells), with IL-2 (50 U/mL) for 4 days (IL-2(+) PBSC T cells), or without IL-2 treatment for 4 days. Cultured for a day (IL-2(-) PBSC T cells), the level of expression of co-inhibitory molecules on the surface of each cell was confirmed.

As a result, the same IL-2(+) PBSC T cells and IL-2(-) PBSC T cells as described above were mixed with CD14+ allogeneic mononuclear cells (MNCs) and an immune response (mixed lymphocyte reaction, MLR) was induced. As a result, as shown in FIG. 1a, in the case of IL-2(+) PBSC T cells, the proportion of cells that did not proliferate in response to allo-MNCs was 16.1±4.7%, compared to PBSC T cells (0%) before culture. It showed a clear immunosuppressive effect compared to IL-2 (-) PBSC T cells (11.0 ± 0.1%), and it was confirmed that the immunosuppressive effect was increased 1.5 times.

In addition, in the case of PBSC T cells cultured for 4 days with IL-2 (50 U/mL), as shown in Figures 1b and 1c, PBSC T cells cultured without IL-2 treatment and uncultured PBSC It was confirmed that the expression of TIM3 and TIGIT was increased compared to that of TIM3.

Example 3. Confirmation of immunosuppressive effect of CD3+PD1+Tim3+ cells

As a result of confirming the ratio of PD1+ cells in cells cultured with G-CSF mobilized PBSC CD3+ lymphocytes with IL-2 (50 U/mL) for 4 days (IL-2(+) cultured PBSC T cells), the results shown on the left of FIG. 2a As shown, the percentage of PD1+ cells was 34.2±21.1%, and as a result of inducing a mixed lymphocyte reaction (MLR) using only PD1- cells after removing PD1+ cells from CD3+PD1+ cells, FIG. 2a right side As shown in , it was confirmed that most of the cells proliferated in response to allo-MNC. From the above results, it was confirmed that T cells suppressing the immune response were present in the CD3+PD1+ cell population.

In addition, as shown in the left side of FIG. 2B , it was confirmed that 20.0±10.5% of CD3+PD1+Tim3+ cells were present in total IL-2(+) PBSC T cells.

In addition, to confirm whether the expression of Tim3 affects immunosuppression, Tim3+ cells were removed from the CD3+PD1+ cell group and then a mixed lymphocyte reaction (MLR) was induced. As a result, Tim3+ cells were removed from the CD3+PD1+ cell group. In this case, the effect of suppressing the allogeneic immune response was confirmed to be lost. Therefore, it was confirmed from the above results that T lymphocytes suppressing the allogeneic immune response exist in CD3+PD1+Tim3+ cells.

Example 4. Confirmation of immunosuppressive effect of CD3+PD1+Tim3+TIGIT- cells

As a result of analyzing the CD3+PD1+Tim3+ cells (20.0±10.5%) described in Example 2b, in the case of TIGIT, as shown in the left side of FIG. It was confirmed that the expression of Also, about 80% of the CD3+PD1+Tim3+ cells were TIGIT−.

In addition, most of the CD3+PD1+Tim3+ cells were TIGIT-, and as a result of confirming the alloimmune response according to TIGIT expression, CD3+PD1+Tim3+TIGIT- cells did not proliferate in response to allo-MNCs. The percentage of cells that did not respond was 24.3±9.4%, which was 1.7 times higher than that of CD3+PD1+Tim3+TIGIT+ cells (14.3±17.0%). It was confirmed that the % was still maintained.

Furthermore, as shown in FIG. 4a of the present invention, the number of CD3+PD1+Tim3+TIGIT-cells with excellent immunosuppressive effect was about 10 times higher in the IL-2(+) PBSC T cell group compared to PBSC 0 day (control group). Increased. In contrast, in the IL-2(-) PBSC T cell group, the number of CD3+PD1+Tim3+TIGIT- cells increased by about 6.6 times compared to that of PBSC T cells (control group).

In addition, as shown in FIG. 4b of the present invention, CD3+PD1+Tim3+TIGIT- cells account for 15.8±10.2% of total CD3+ cells, which is necessary for treatment related to immunosuppressive response by collecting and culturing PBSCs once. It was found that a sufficient number of cells (a total of 1.5±1.1 x 10 ⁸ CD3+PD1+Tim3+TIGIT- cells per unit weight) could be obtained.

From the results of the above examples, by culturing the CD3+ cell fraction mixed with IL-2+, it is possible to obtain a sufficient number of CD3+PD1+Tim3+TIGIT-cells excellent in suppressing the alloimmune response, and through the culture process CD3+ lymphocytes that have acquired co-inhibitory molecule expression are expected to be used as cell therapy agents to treat graft-versus-host disease by suppressing the alloimmune response.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

Claims (10)

A pharmaceutical composition for preventing or treating immune-related diseases, comprising T cells having immunophenotypes of CD3+, PD1+ and Tim3+.
According to claim 1,
Characterized in that the T cells further have an immunophenotype of TIGIT+ or TIGIT-, the pharmaceutical composition.
According to claim 1 or 2,
The pharmaceutical composition, characterized in that the composition inhibits the immune response.
According to claim 1 or 2,
The immune-related diseases include autoimmune diseases; graft-versus-host disease; organ transplant rejection; asthma; atopy; Or characterized in that the acute or chronic inflammatory disease, the pharmaceutical composition.
According to claim 1 or 2,
The autoimmune disease is rheumatoid arthritis, type 1 diabetes, systemic scleroderma, systemic lupus erythematosus, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Bechetsy's disease, Sjogren's syndrome, Gilia-Barre syndrome , Chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibrous tissue and polyarteritis nodosa, characterized in that at least one selected from the group consisting of, a pharmaceutical composition.
According to claim 1 or 2,
The T cells are characterized in that prepared by mixing the CD3 + cell fraction and IL-2 + culture, the pharmaceutical composition.
A pharmaceutical preparation for preventing or treating immune-related diseases, comprising the composition of claim 1 or 2.
According to claim 7,
The pharmaceutical formulation is characterized in that for suppressing the immune response, pharmaceutical formulations.
According to claim 7,
The immune-related diseases include autoimmune diseases; graft-versus-host disease; organ transplant rejection; asthma; atopy; Or characterized in that the acute or chronic inflammatory disease, pharmaceutical preparations.
According to claim 7,
The autoimmune disease is rheumatoid arthritis, type 1 diabetes, systemic scleroderma, systemic lupus erythematosus, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Bechetsy's disease, Sjogren's syndrome, Gilia-Barre syndrome , Chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibromyalgia and polyarteritis nodosa, characterized in that at least one selected from the group consisting of, pharmaceutical preparations.

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