KR20070121217A - Method for preparation of therapeutic b cells against autoimmune diseases which are induced by autoimmune cytotoxicity - Google Patents

Abstract

A cell therapeutic agent for treating autoimmune disease induced by autoimmune cytotoxicity is provided to inhibit function of T cells, especially Th1 cells by regulating differentiation of T cells causing damage of itself body with therapeutic B cells. A method for preparing a cell therapeutic agent for treating autoimmune disease induced by autoimmune cytotoxicity comprises the steps of: (a) separating B cells from an autoimmune disease individual; (b) culturing the separated B cells in a medium containing a material for inducing proliferation of B cell, and adding an antigen causing autoimmune disease into the medium to stimulate the B cells; and (c) collecting the firstly cultured B cells, culturing the collected B cells in a medium containing B cell growth factor, and stimulating them with the antigen for causing autoimmune disease, wherein the autoimmune disease is multiple sclerosis, type 1 diabetes, rheumatoid arthritis or Hashimoto's thyroiditis.

Description

Method for preparation of therapeutic B cells against autoimmune diseases which are induced by autoimmune cytotoxicity}

1 to 3 are diagrams showing that the symptoms of EAE did not appear when the cell therapy prepared according to the present invention was treated.

4 is a chart showing the disappearance (treatment) of EAE symptoms that have already occurred when the cell therapy prepared by the present invention is treated.

FIG. 5 is a chart showing that the amount of Th1 cytokine produced by T cells is significantly lowered than that of the control and the amount of Th2 cytokine is significantly increased when treated with the cell therapy according to the present invention.

Figure 6 is a chart showing that the function of the T cells directly involved in the induction of the disease when the treatment of the B cell therapy according to the present invention has been changed.

Figure 7 is a diagram showing the relationship between the treatment of cell therapy according to the present invention and changes in the function of T cells.

The present invention relates to a cell therapy that can treat autoimmune diseases by adjusting the differentiation of T cells damaging one's body using B cells, which are their immune cells, and a method of manufacturing the same.

(1) General characteristics of immune system and B cell

Immunity is one of the body's self-protection systems against all foreign polymers (antigens) that invade or are injected into living tissue. The main component of the immune system is lymphocytes, which are white blood cells that are made in the bone marrow and circulate along the blood into lymph tissues and organs, mainly lymph nodes, spleen, and tonsils. When stimulated by an appropriate antigen, B cells multiply rapidly to form clones that produce specific antibodies (immunoglobulins) that neutralize the antigen. Antibodies produced by B cells circulate in body fluids to perform humoral immunity. T cells are made in the thymus and travel to lymphoid tissue, responsible for cell-mediated immunity that directly attacks antigens.

(2) Development of abnormalities of the immune system and autoimmune diseases

One of the most important traits of all normal individuals is the ability to recognize and react to and eliminate many non-self antigens, while not harmfully reacting to the antigenic substances that make up self. Is to have. The non-response of the body to autoantigens is called immunologic unresponsiveness or tolerance.

If there is a problem in inducing or maintaining such self-tolerance, an immune response occurs to the self-antigen, which causes attack of one's own tissues, causing multiple sclerosis, type 1 diabetes, rheumatoid arthritis, Autoimmune diseases such as Hashimoto's thyroiditis occur.

(3) Method of treating existing autoimmune disease

 Autoimmune diseases are diseases caused by adverse reactions to magnetic cells, and thus drugs are mainly used to suppress autoimmune function. However, there are many side effects that are difficult to use continuously and do not prevent enough recurrences, so the treatment is limited. In the case of multiple sclerosis, beta interferon is used, but the side effects are less expensive, the treatment costs are high, the life-long injection is inconvenient, and the effect of preventing recurrence is minimal. In addition, the method of inhibiting the interaction between cells by the antibody administration to CD40 ligand, etc. has been used, but not very successful. There are many other immunotherapies, but no one has yet been proven to show definite therapeutic efficacy.

(4) Mechanism of development of autoimmune diseases due to abnormal immune system: Function of Th1 / 2 cell subgroup

When an antigen is provided from an antigen presenting cell (APC), such as macrophages, dendritic cells, Langerhans' cells, and B cells, helper T cells; Th is differentiated into Th1 cells (1 type helper T cell, hereinafter abbreviated "Th1") or Th2 cells (2 type helper T cell, hereinafter abbreviated "Th2") (1). At this time, the direction of differentiation is influenced by various factors such as the amount of antigen, cytokine and APC-transmitted signal, but the mechanism is still not fully understood (5, 6). Th1 plays a role in promoting a cellular immune response and Th2 plays a role in promoting a humoral immune response (see Table 1).

Table 1 Functional Classification of T Cells

Cell types main function Th1 cell IFNγ production, enhanced cellular immunity, destruction of cancer cells and virus infected cells Th2 cell IL-4 production, humoral immunity enhancement, antibody production, extracellular foreign material removal

Some autoimmune diseases have been found to be due to excessive differentiation into Th1 cells or excessive enhancement of Th1 cell function for some reason not yet elucidated (4). In other words, self-cell destruction occurs due to excessive Th1 reaction, and as a result, autoimmune diseases such as multiple sclerosis, type 1 diabetes, rheumatoid arthritis, and Hashimoto's thyroiditis develop. On the other hand, excessive Th2 differentiation is known to cause IgE overproduction, causing hypersensitivity reactions and diseases such as asthma (2, 3).

(5) Treatment of autoimmune diseases by regulation of the function of Th1 / 2 cell subgroups

As such, many diseases occur when Th1 / Th2 do not balance well. Therefore, in order to treat such autoimmune diseases, it is necessary to maintain proper Th1 / Th2 differentiation ratio (T cell differentiation control) by suppressing T cell overdifferentiation into Th1. For this purpose, it is important to select APCs that can be easily obtained and effectively induce / control the differentiation of T cells.

(6) Regulation of Th1 / 2 Cell Function Using Dendritic Cells

Dendritic cells are known to be excellent APCs that induce T cell differentiation. However, since dendritic cells are present in the peripheral blood at less than 1%, it is difficult to obtain the amount necessary for the manufacture of cell therapeutics, which is very difficult to apply clinically. In addition, since dendritic cells present all antigens to T cells, they may have side effects such as inducing a response to self antigens.

(7) Regulation of Th1 / 2 Cell Function Using B Cells

On the other hand, in addition to the function of antibody production, B cells have been shown to have the function of regulating antigen presentation and T cell differentiation like T dendritic cells (7 ~ 11). In autoimmune diseases, B-cells have also been shown to have disease-reducing effects. In other words, in the arthritis model, B cells alleviate symptoms through IL-10 production (12), and B cells are administered in an autoimmune encephalomyelitis (EAE) model, which is a multiple sclerosis model, and in an autoimmune diabetes model. Has been reported to relieve symptoms (14).

(8) Limitations of Attempt to Apply B Cells to Modulate Th1 / 2 Cell Function

As described above, it has already been reported that B cells have a function of modulating the function of Th1 / 2 cells, but clinical application of B cell treatment agent has not been realized yet, for the following reasons.

① Ease of obtaining B cell treatment agent: For clinical application, it is necessary to obtain a large number of cells, so the method of obtaining cells should be easy. Although some reports suggest that B cells can be obtained by culturing in the presence of T cells (11), obtaining B cells that can control T cell differentiation in the presence of T cells requires removal of T cells after mixed culture. Can be tricky and lose B cells In addition, if T cells are not completely removed, the possibility that the mixed T cells worsen the symptoms cannot be excluded. Therefore, it is very dangerous to apply such a method to clinical practice.

② Mass acquisition method of B cell treatment agent: It is important to obtain a large number of cells for clinical application, but the above reports do not suggest a method for obtaining a clinically effective amount of B cells in large quantities. That is, simply reporting the efficacy of B cells (12) or obtaining them by culturing B cells in the presence of T cells (11) has reported only a simple acquisition method. Using BPS lymphocytes that were nonspecifically activated using LPS was not only likely to cause side effects by affecting the response to all antigens in the body, but also had very low efficacy (10 million doses were administered 6 times. Can delay expression but not completely) (14). In this study, T cells do not use other T cells or other cells in the culture process. In addition, the T1 response inhibition function of T cells can be increased and the number of cells can be expanded to 2.2 times or more by 1.5 times.

③ Efficacy of B cell treatment agent: For the clinical application of B cell treatment agent, the efficacy of B cell treatment agent should be strong, but in other studies, if a patient wants to treat symptoms by obtaining B cells in the mice causing symptoms and administering them to other mice, The therapeutic efficacy was very low (13). That is, the researchers administered 10 million cells, but the symptoms persisted for 10 days or more after the symptoms were induced similarly to the control group, and the B cells manufactured by the method of the present invention were able to completely suppress the symptoms by administering 5 million cells. In addition, even by repeated experiments, B cells cultured once for 4 days could hardly observe the efficacy (see FIG. 1). In addition, B lymphocytes cultured with LPS were administered 6 million times over 6 times in the diabetic model, but they could delay the onset of symptoms but not completely inhibit them (14).

As described above, the efficacy of treatment for autoimmune diseases using B cells has been confirmed in recent studies, but no method has been reported for clinical application. The present invention is different from the existing research report and the new points can be summarized as shown in Table 2 below.

<Table 2> Comparison of features of the present invention with the existing research report

How content Original Research Report (Problem) References The present invention difference How to Obtain Cell Therapy Mixed culture with T cells 11 B cell alone culture Existing methods are almost impossible for clinical application Nonspecific Activation Using LPS 14 Antigen specific activation Significantly poor efficacy and possible side effects from regulating nonspecific T lymphocyte activity and using LPS How to obtain cell therapy mass None (Difficult to obtain large quantities by mixed culture with T cells) 11, 12, 13 has exist Possible to secondary culture only B cells) Cell Therapy Benefits Weak (10 million doses lasted for at least 10 days after symptoms were induced similarly to controls) 13 Strong (5 million total suppression: never occurs) The first culture alone is ineffective and maximizes the efficacy by the second culture

An object of the present invention is to prepare a cell therapeutic agent that can treat symptoms by regulating T cell function using autoimmune diseases caused by T cells damaging one's body using B cells, which are immune cells. It is invented a possible manufacturing method.

More specifically, in order to treat autoimmune diseases caused by an excess of the cytotoxic immune response with their B cells, it is possible to suppress the cellular immune response to the antigen causing autoimmune diseases, that is, the function of Th1 cells. It is to provide a cell therapy containing the B cells.

The present invention for achieving the above object, the differentiation of autoreactive T cells using B cells, which are another immune cells of the self against the autoimmune diseases caused by their T cells attack and destroy their cells. A cell therapy agent and a method for producing the same, which are capable of treating a symptom by adjusting, include (A) a B cell separation step, (B) a pre-culture step as a primary culture, and (C) a post-culture step after a secondary culture.

The B cell separation step (A) is a process for isolating B cells from an individual with an autoimmune disease. That is, the non-adherent cells (T cells) are partially removed by standing in a plastic culture plate at 37 ° C. for at least 30 minutes and then rinsing with 37 ° C. culture medium, and then adding 37 ° C. culture medium and pipetting to remove weak adherent cells. By removing the remaining strongly adherent macrophages and granulocytes. After collecting the cell suspension obtained by pipetting, the antibody binds to antigens of T cells, NK cells, and granulocytes other than B cells, and then acts on a magnetic microbead that binds to the antibodies. It is the process of extracting only B cells.

In step (B), the primary culture (preculture) is a step of stimulating the isolated B cells of the cells with an antigen and culturing them. At this time, a substance that induces the proliferation of B cells together with the antigen should be added, and the antigen is incubated for 3 days or more using the antigen causing the disease according to the autoimmune disease.

In step (C), the secondary culture (post-culture) is a step of collecting the primary cultured B cells and stimulating with the antigen again. At this time, B cell proliferation factor should be added together with the antigen causing autoimmune disease and cultured for 2 days or more. This process is the core of the present invention is a process of strongly inducing the function of B cells to mitigate the function of autologous destructive T cells through secondary culture. This B cell function greatly increases cytokine production (16-fold increase in the case of IL-10) in which B cells produced by the method of the present invention alleviate the function of cell-destructive T cells, while cell-destructive T cells. Significantly reduce cytokine production that enhances function (50-fold reduction for IFNγ) It is because of this.

In the present invention, the substance for inducing the proliferation of B cells refers to a non-specific activator of B cells in addition to a specific antigen for inducing autoimmune diseases, also referred to as B cell mitogen. Examples thereof include anti-CD40, lipopolysaccharide (LPS), pokweed mitogen (PWM), anti human immunoglobuline antibodies, and the like. Which mitogen to use may be appropriately determined depending on the species and the disease.

In the present invention, the B cell proliferation factor is used by mixing IL-2 and IL-4.

Cell therapy according to the present invention, as confirmed in the following examples, B cells cultured in two stages as an active ingredient converts the function of Th1 type T cells to Th2 type (Th1 function is inhibited and Th2 function is activated ) Has the function of treating autoimmune diseases.

According to the present invention, B cells are extracted from an autoimmune disease individual (patient), and B cells themselves type 1 T by adding IL-2 and IL-4, which are proliferation factors of B cells, and culturing in two stages by adding a target antigen. It can differentiate into B cells (B2), which have the function of converting the function of lymphocytes to type 2. Normally inactivated B lymphocytes and primary precultured B cells (B1) act as simple antigen presenting cells (APCs), inducing both type 1 (Th1) and type 2 (Th2) responses of T cells. (See Table 1). However, the inventors of the present application have shown that B cells (B2) that have undergone secondary post-culture have a function of regulating T cell response that significantly inhibits type 1 (Th1) response of T cells and strongly enhances type 2 (Th2) response. Newly discovered by (see Table 1). The present invention is based on this fact. (See FIG. 5) The reason why B cells undergoing post-culture by the method of the present invention strongly enhance the Th2 response is a cytokine (IL-4, IL-6, IL that inhibits the Th1 response of T cells and promotes the Th2 response). -10) is produced in large quantities, whereas the production of cytokines (IFNγ) that promotes the Th1 reaction is inhibited (see Table 3).

TABLE 3 Changes in B Cell Cytokine Production by Secondary Postculture

B cell activation method Cytokine Production (pg / ml) IL-4 IL-6 IL-10 IFNγ  Primary preculture Not detected 420 337 161  1st preculture + 2nd postculture 1532 1072 1963 56

Autoimmune diseases that are subject to the cell therapy of the present invention include multiple sclerosis, type 1 diabetes mellitus, rheumatoid arthritis, or Hashimoto's thyroiditis, but are not necessarily limited thereto. It can be used for all autoimmune diseases caused by destruction.

In the present invention, the (A) B cell separation step, the mononuclear leukocytes isolated from the spleen cells or blood of an individual with autoimmune diseases are suspended in the culture medium and left for 1 hour at 37 ° C. to form adherent cells (macrophages). Small step of removal and magnetic bead-magnetic cell separation with anti-T cell and anti-NK cell antibodies attached (Miltenyi, S .; Muller, W .; Weichel, W. Radbruch, A. High gradient magnetic cell separation with MACS Cytometry.1990, 11 (2), 231-238.) Can be done in small steps to remove T cells and NK cells.

Splenocytes or mononuclear leukocytes have a variety of blood cells other than B cells, so they need to be removed. For example, adherent macrophages are isolated by standing cell suspension, and T cells or NK cells are removed using an antibody that specifically binds to them. These small steps do not matter if you choose a different separation method or change their order.

Autoimmune diseases are due to the expression of the immune system that recognizes the macromolecules (eg peptides, glycoproteins, etc.) of the tissue cells in which the disease occurs as foreign antigens. Accordingly, the "antigen causing the autoimmune disease" in the (B) pre-culture step and (C) post-culture step of the present invention is an antigen derived from tissue cells of the target tissue of the autoimmune disease, and the antigen that is directly causing the symptoms. Say.

The present invention, for example, in the (B) pre-culture step (stage 1 culture), the concentration of the antigen is 1 ~ 100 μg / ml, the concentration of B cells is 0.5 ~ 5.0 × 10 ⁶ cells / ml, anti-CD40 The concentration is 0.01 ~ 1.0 μg / ml, the concentration of IL-2 is 0.1-10 ng / ml, the concentration of IL-4 is 0.1-10 ng / ml, the incubation temperature is 37 ± 2 ℃, and the incubation period is 60 ~ 90 Time, preferably, the antigen concentration is 40-60 μg / ml (incubation concentration for attachment), the B cell concentration is 0.8-1.2 × 10 ⁶ cells / ml, and the anti-CD40 concentration is 0.1-1.0 μg / ml, the concentration of IL-2 is 4-6 ng / ml, the concentration of IL-4 is 4-6 ng / ml, the incubation temperature is 37 ℃, the incubation period is 60-84 hours. In addition, in the (C) post-culture (step 2 culture), the concentration of antigen is 1-100 μg / ml, the concentration of B cells is 1-10 × 10 ⁵ cells / ml, and the concentration of IL-2 is 0.1-10. ng / ml, IL-4 concentration is 0.1-10 ng / ml, the incubation temperature is 37 ± 2 ℃, the incubation period is 24 to 90 hours, preferably the concentration of antigen is 40 ~ 60 μg / ml, B Cell concentration is 4-6 × 10 ⁵ cells / ml, IL-2 concentration is 4-6 ng / ml, IL-4 concentration is 4-6 ng / ml, incubation temperature is 37 ℃, incubation period is 38 It is ~ 58 hours.

However, in the present invention, the pre-culture step and the post-culture step is a step of activating the B cells, the culture conditions are not essential and suitable conditions depending on the characteristics of the subject, the type of disease, the health or quantity of the isolated B cells It may be possible to incubate at.

The medium used in the present invention may be a normal basal culture medium in which B cells can grow, human serum, bovine serum or serum-free culture medium.

In a cell therapy for the treatment of autoimmune diseases containing B cells as an active ingredient according to the present invention, the B cells are an aqueous solution containing an appropriate component as necessary (for example, a phosphate buffer solution, a conventional aqueous solution for injection, etc.). May be suspended at an appropriate concentration. In addition, the cell therapy according to the present invention may be a dose appropriately selected according to the type and characteristics (weight, age, health) of the individual with an autoimmune disease, the type and characteristics of the disease (progression of the disease, etc.) It can be put into diseased individuals.

As shown in the following examples of autoimmune disease model mice, the treatment of the cell treatment agent according to the present invention not only completely suppresses the symptoms of autoimmune diseases, but also treats the symptoms already developed. .

The present invention will be described in more detail with reference to the following examples. In the following examples, experiments were conducted using a model of autoimmune encephalomyelitis-induced mouse, but it will be apparent to those skilled in the art that the same results can be obtained for other autoimmune diseases with reference to the technical idea and the examples of the present invention. That is, the following examples are only intended to explain the technical idea of the present invention, and thus the present invention is not limited to the cell therapy for "autoimmune encephalomyelitis".

<Example>

1. Preparation of cell therapy according to the present invention

(1) Induction of Immunity in Mice

Oligodendocytes (oligodendrocyte) is a type of cell constituting the nervous system is a cell that plays an important role in myelin sheath production of neurons around the nerve fiber axon in the central nervous system and the peripheral nervous system. Autoimmune encephalomyelitis occurs as a result of the recognition of the glycoprotein (MOG _35-55 : myelin oligodendrocyte glycoprotein) of the oligodendrosite as an foreign antigen.

MOG _35-55 and as immune adjuvants to enhance an immune response to an antigen CFA (complete Freund's adjuvant) to which a mixture of 100 μg mixed with the same volume was injected into the abdominal cavity C57BL / 6 female mice recognize MOG _35-55 B cells Activation and proliferation were induced.

(2) 2 repeated cultures for preparation of B cells and activation of B2 cells

Hereinafter, "B2 cell" means "B cell cultured twice."

① Isolation of B cells

After 10 days of immunization, mouse splenocytes were isolated, suspended in cell culture, and allowed to stand at 37 ° C. for 1 hour to attach adherent cells (macrophages) to the culture surface and to separate suspension (macrophages removed). Was mixed with anti-T cell-antibody and anti-NK cell-antibody attached magnetic beads (MAC® microbeads, Miltenyi Biotec GmbH, Germany) to allow the remaining T and NK cells to bind to the magnetic beads. Anti-MOG _35-55 B cells were purified by removing T cells and NK cells using the following cell separation apparatus (MACS, manufactured by the same company as above). (Miltenyi, S .; Muller, W .; Weichel, W. Radbruch, A. High gradient magnetic cell separation with MACS.Cytometry. 1990, 11 (2), 231-238.)

② Preculture of B Cells

50 μg / ml antigen MOG _{35 -55} was attached to the wells of a 48 well plate and B cells suspended at a concentration of 1 × 10 ⁶ cells / ml were added and incubated for 3 days. Anti-CD40 (0.5 μg / ml) and IL-2 (5 ng / ml) and IL-4 (5 ng / ml) are added.

③ Post-culture of B cells (repeat culture)

After pre-cultured B cells were collected and washed twice with the cell culture solution, the solution was added to a microplate attached with 50 μg / ml antigen MOG _{35 -55} at a concentration of 5 × 10 ⁵ cells / ml for 2 days. Incubate further during. At this time, IL-2 (5 ng / ml) and IL-4 (5 ng / ml) are added to the culture.

Activity-induced B cells were harvested and centrifuged twice with phosphate buffer (PBS) and suspended in PBS at a concentration of 5 × 10 ⁵ cells / ml and used as “cell therapy” according to the present invention.

2. Construction of Disease Model Mice (Mouse EAE Induction)

Model mice were made by inducing experimental autoimmune encephalomyelitis (EAE) in mice.

MOG _{35 -55} (1.5 mg / ml) and Mycobacterium tuberculosis (4 mg / ml), which are antigens of autoimmune encephalomyelitis, were mixed with CFA to prepare an antigen solution. Separately, pertussis toxin solution (4 μg / ml) was prepared. The solvent was PBS.

EAE was induced by subcutaneous injection and 0.1 ml of pertussis toxin solution in both sides of C57BL / 6 female mice in 100 μl of antigen solution on Day 1 and Day 2, respectively.

3. Confirmation of the effect of the cell therapy according to the present invention

One hour or 14 days after the start of EAE induction, a predetermined amount of the therapeutic agent according to the present invention was injected through the tail vein of EAE mice (B2 introduction of B2 cells).

EAE mice were visually observed according to the criteria according to Table 4 daily, and the treatment effect of the cell therapy according to the present invention was confirmed by analyzing the symptoms of the disease.

TABLE 4

Judgment Symptom 0 No abnormality One Weak or flaccid tail 2 Hind limb weakness 3 Hind limb paralysis 4 Forelimb and hind limb paralysis (quadriplegia, moribund state) 5 Death of test animal

The data for each experimental section in the following experiments is the average of six mice.

(1) Effect of EAE induction and concurrent cell treatment

One hour after the start of EAE induction in mice, B cells according to Table 5 were treated with 7.5 × 10 ⁶ , respectively, and the symptoms of the disease over time were confirmed (FIG. 1).

TABLE 5

Experiment Type of B cells treated Control Do not kill Fresh b B cell isolated from spleen B1 B-cells only till preculture B2 Cell treatment agent according to the present invention

As can be seen in Figure 1, B2 cells treated with the cell treatment agent according to the present invention (B2 cell introduction) were completely suppressed the induction of EAE. However, B-cell treatment (Fresh B) isolated from the spleen and B-cell treatment (B1) only undergoing pre-culture showed the same results as the untreated treatment with little EAE inhibitory effect.

(2) Confirmation of appropriate dosage

Appropriate B2 cell treatment concentration was investigated in the cell treatment agent according to the present invention. One hour after the start of EAE induction in mice, predetermined B cells were treated with 2.5 × 10 ⁶ , 5.0 × 10 ^6, and 7.5 × 10 ⁶ , respectively, and the symptoms of the disease over time were confirmed (FIG. 2).

As a result, it was confirmed that EAE was sufficiently suppressed by B2 cell treatment of 5.0 × 10 ⁶ or more. Therefore, in the following experiment, B2 cells were treated by 5.0 × 10 ⁶ .

However, since the amount of treatment is for the mouse EAE model only, it will vary depending on the type and characteristics of autoimmune diseases (such as disease progression) and the type and characteristics of diseased individuals (weight, age, health).

(3) Confirmation of the effect of the cell treatment agent according to the present invention upon re-administration of antigen after treatment

After treating the B2 cell-containing cell treatment according to the present invention to alleviate the symptoms, it was confirmed in FIG. 3 to see if the effect appeared when exposed to the antigen over time.

As shown, recurrence was remarkably suppressed even when antigens were re-immunized after 50 days in mice whose symptoms were inhibited by proton introduction. That is, it was confirmed that the symptom suppression effect by B2 cells can be maintained for a very long time. Figure 3 shows the results of re-inducing symptoms by immunizing both control (Control) and the treatment (B2-tx) treated with the antigen again on the 50th day.

(4) Confirmation of Efficacy in Early-Earth EAE Treatment

The treatment of the cell therapeutic agent according to the present invention (protonal introduction of B2 cells) confirmed that the mice already developing EAE could be treated.

When EAE is induced in mice, as shown in FIGS. 1 and 2, EAE symptoms begin to appear after 10 days of induction (antigen treatment). Therefore, the disease treatment mouse which showed the EAE symptom clearly after 14 days of antigen treatment was treated with the cell processing agent by this invention (FIG. 4).

As shown, when the cell treatment agent according to the present invention is treated after the EAE symptom is initiated, it can be seen that the suppression (treatment) effect of the symptom is apparent after about 10 days.

4. Analysis of mechanism of action of cell therapy according to the present invention

(1) confirming Th2 polarization of T cells by B2 cells; in vitro

It was confirmed by in vitro experiments how the cell treatment agent according to the present invention to alleviate / treat EAE symptoms.

Simultaneous treatment with EAE induction and B2 cells 10 days (FIG. 5) and 50 days (FIG. 6), mouse splenocytes were harvested and stimulated incubated with the same antigen (MOG _35-55 ) for 3 days in vitro. It was confirmed that the production amount of the Cain IL-4, IL-6, IL-10 is significantly higher than the production amount of IFNγ which is a Th1 cytokine (Figs. 5 and 6).

This is the fact that T cells are biased toward Th2 when the cell treatment agent (B2 cells) according to the present invention is treated. It shows the therapeutic effect. In addition, it can be seen that the bias differentiation effect can last 50 days or more after B2 cell injection.

(2) confirming Th2 polarization of T cells by B2 cells; in vivo

It was confirmed whether the disease treatment effect due to the above-mentioned deflection differentiation is exhibited in vivo.

After 50 days of EAE induction and co-treatment with B2 cells, only T cells were isolated from the control group (untreated group expressing disease) and the experimental group in which both B2 cells were introduced (no symptoms) (quantitatively introduced B2 cells were removed). The isolated T cells were administered to normal mice, and simultaneously immunized (EAE induction) and analyzed the symptoms over time (FIG. 7). The control and experimental information is shown in Table 6 below.

TABLE 6

Source of T Cells T cell treatment target T cell throughput Control T (2.5 × 10 ⁶ ) EAE-induced (early diseased) controls 50 days ago Membrane EAE Induced Mice 2.5 × 10 ⁶ B2-tx T (2.5 × 10 ⁶ ) Experimental group treated with B2 cells (without symptoms) 50 days prior to EAE induction Same as above 2.5 × 10 ⁶ B2-tx T (5.0 × 10 ⁶ ) Same as above Same as above (5.0 × 10 ⁶ )

As shown in the figure, when control cells were protonated in T cells (Control T), symptoms suddenly appeared and all died 21 days later, but B2 cells were injected into B cells in which no symptoms were introduced (B2). -tx T) symptoms were minimal. This resulted in treatment of the cell treatment agent (proton introduction of B2 cells) according to the present invention, T cells are deflected to Th2 in vivo, and the therapeutic effect of autoimmune diseases due to proton introduction of B2 cells depends on the change of T cell function. This is a direct indication of that.

(references)

1. Mosmann, TR; Coffman, RL TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol. 1989, 7 145-173.

2. Spellberg, B .; Edwards, JE, Jr. Type 1 / Type 2 immunity in infectious diseases. Clin Infect Dis. 2001, 32, 76-102.

Romagnani, S. Th1 and Th2 in human diseases. Clin Immunol Immunopathol. 1996, 80 (3 Pt 1), 225-235.

4. Moss, RB; Moll, T .; El-Kalay, M .; Kohne, C .; Soo Hoo, W .; Encinas, J. Carlo, DJ Th1 / Th2 cells in inflammatory disease states: therapeutic implications. Expert Opin Biol Ther. 2004, 4, 1887-1896.

5. Del Prete, G .; Maggi, E .; Romagnani, S. Human Th1 and Th2 cells: functional properties, mechanisms of regulation, and role in disease. Lab Invest. 1994, 70, 299-306.

6. Szabo, SJ; Sullivan, BM; Peng, SL; Glimcher, LH Molecular mechanisms regulating Th1 immune responses. Annu Rev Immunol. 2003, 21, 713-758.

7. Maldonado-Lopez, R .; Moser, M. Dendritic cell subsets and the regulation of Th1 / Th2 responses. Semin Immunol. 2001, 13, 275-282.

8. Moser, M .; Murphy, KM Dendritic cell regulation of TH1-TH2 development. Nat Immunol. 2000, 1, 199-205.

9. Ron, Y., Sprent, J. T cell priming in vivo: a major role for B cells in presenting antigen to T cells in lymph nodes. J. Immunol. 1987, 138 2848-2856.

10. Yang, X., Brunham, RC Gene knockout B cell-deficient mice demonstrate that B cells play an important role in the initiation of T cell responses to Chlamydia trachomatis (mouse pneumonitis) lung infection. J Immunol. 1998, 161, 1439-1446.

11. Harris, DP; Haynes, L .; Sayles, PC; Duso, DK; Eaton, SM; Lepak, NM; Johnson, LL; Swain, SL; Lund, FE Reciprocal regulation of polarized cytokine production by effector B and T cells. Nat Immunol. 2000, 1, 475-482.

12. Mauri, C .; Gray, D .; Mushtaq, N .; Londei, M. Prevention of arthritis by interleukin 10-producing B cells. J Exp Med. 2003, 197, 489-501.

13. Fillatreau, S .; Sweenie, CH; McGeachy, MJ; Gray, D. Anderton, SM B cells regulate autoimmunity by provision of IL-10. Nat Immunol. 2002, 3, 944-950.

14. Tian, J .; Zekzer, D .; Hanssen, L .; Lu, Y .; Olcott, A .; Kaufman, D.L. Lipopolysaccharide-activated B cells down-regulate Th1 immunity and prevent autoimmune diabetes in nonobese diabetic mice. J Immunol. 2001, 167, 1081-1089

According to the present invention, it is possible to obtain a long-term therapeutic effect without any side effects on autoimmune diseases caused by one's T cells, such as multiple sclerosis, type 1 diabetes, rheumatoid arthritis, and Hashimoto's thyroiditis. The cell treatment agent according to the present invention does not affect the immune response to other antigens at all, and can be used as a therapeutic agent having no side effects since it uses its own cells.

Claims (6)

(A) B cell separation step of separating B cells from an autoimmune disease individual; (B) the pre-culture step of stimulating the isolated B cells of their own in a culture medium to which the substance for inducing B cells is added, and then stimulating with the antigen causing the autoimmune disease, followed by primary culture; And (C) a post-culture step of collecting the first cultured B cells and suspending them in a culture medium to which the B cell proliferation factor is added and then stimulating again with the antigen causing the autoimmune disease to secondary culture. Method for producing a cell therapy for the treatment of autoimmune diseases, containing B cells as an active ingredient. The method of claim 1, The autoimmune disease is multiple sclerosis, type 1 diabetes mellitus, rheumatoid arthritis or Hashimoto thyroiditis, characterized in that the method for producing a cell therapy for the treatment of autoimmune diseases. The method of claim 1, The pre-culture step (B) of the primary culture, characterized in that the anti-CD40, IL-2, IL-4 is added with the antigen, a method for producing a cell therapy for the treatment of autoimmune diseases. The method of claim 1, (C) the secondary culture in the post-culture step, characterized in that the addition of IL-2, IL-4 with the antigen, a method for producing a cell therapy for the treatment of autoimmune diseases. The method of claim 1, In the (B) step 1 culture, the concentration of antigen is 1 ~ 100 μg / ml, the concentration of B cells is 0.5 ~ 5.0 × 10 ⁶ cells / ml, the concentration of anti-CD40 is 0.01 ~ 1.0 μg / ml, IL- 2 concentration of 0.1-10 ng / ml, IL-4 concentration of 0.1-10 ng / ml, incubation temperature of 37 ± 2 ℃, incubation period of 60 ~ 90 hours, In the (C) step 2 culture, the concentration of antigen is 1 ~ 100 μg / ml, the concentration of B cells is 1 ~ 10 × 10 ⁵ cells / ml, the concentration of IL-2 is 0.1-10 ng / ml, IL- The concentration of 4 is 0.1-10 ng / ml, the incubation temperature is 37 ± 2 ℃, the incubation period is 24 to 90 hours, characterized in that the preparation of cell therapy for the treatment of autoimmune diseases. A cell therapeutic agent for the treatment of autoimmune diseases, comprising B cells prepared according to claim 1 as an active ingredient.

Images