WO2009054556A1

WO2009054556A1 - Method for preparation of therapeutic b cells against autoimmune diseases which are induced by autoimmune cytotoxicity

Info

Publication number: WO2009054556A1
Application number: PCT/KR2007/005245
Authority: WO
Inventors: Sang Yun Nam; Sang Mok Lee; Jong Cheon Ha
Original assignee: Sang Yun Nam; Sang Mok Lee; Jong Cheon Ha
Priority date: 2007-10-24
Filing date: 2007-10-24
Publication date: 2009-04-30

Abstract

Disclosed is a method for the preparation of a cell therapeutic agent for autoimmune immune diseases based on the ability of B cells to control the differentiation of T cells, which are responsible for self tissue destruction. The method comprises (A) separating B cells from blood or bone marrow of an individual suffering from an autoimmune disease selected from among multiple sclerosis, type 1 diabetes mellitus, rheumatoid arthritis, and Hashimoto's thyroiditis; (B) primarily culturing the B cells in a culture medium supplemented with a B cell proliferation-inducing factor in the presence of an antigen causing the autoimmune disease! and (C) secondarily culturing the B cells in a fresh culture medium supplemented with a B cell proliferation-inducing factor in the presence of an antigen causing the autoimmune disease. A cell therapeutic agent therefor can be prepared. The cell therapeutic agent shows highly persistent therapeutic efficacy against autoimmune diseases, such as multiple sclerosis, type 1 diabetes mellitus, rheumatoid arthritis, and Hashimoto' s thyroiditis without side effects.

Description

[DESCRIPTION]

[Invention Title]

METHOD FOR PREPARATION OF THERAPEUTIC B CELLS AGAINST AUTOIMMUNE DISEASES WHICH ARE INDUCED BY AUTOIMMUNE CYTOTOXICITY

[Technical Field]

The present invention relates to a cell therapeutic agent for the treatment of autoimmune diseases based on the ability of B cells to control the differentiation of T cells, which are responsible for the destruction of self tissues, and a method for the preparation thereof.

[Background Art]

(1) General Properties of the Immune System and B cells

Immunity refers to a self-defense system of an organism against all exogenous matter (antigens) which invades or enters the organism. Lymphocytes, playing an important role in the body' s defenses, are a type of white blood cell which originate from the bone marrow and circulate in blood and lymph and migrate to lymphoid tissues or organs, particularly, lymph node venules, the spleen and the tonsils. B cells, when stimulated by a suitable antigen, rapidly proliferate to form clones from which antibodiesCimmunoglobulin) for neutralizing the antigen are produced. Circulating in the blood, the antibodies produced by B cells function to perform humoral immunity. Once mature, T cells emigrate from the thymus and migrate to lymphoid tissues. Mature T cells, responsible for cell-mediated immunity, attack antigens.

(2) Aberration of Immune System and Arousal of autoimmune Disease

One of the most important immunological properties in all normal individuals is to show an immune response against self antigens to a sufficiently low degree to avoid harm, but to recognize and attack non-self antigens. On this basis, the process by which the immune system does not attack an antigen is called immunological unresponsiveness or tolerance. A problem afflicting the establishment or maintenance of self-tolerance results in an immune response against self antigens. Any disease that results from such an aberrant immune response is termed an autoimmune disease. Prominent examples include multiple sclerosis, diabetes mellitus type 1 (IDDM), Hashimoto's thyroiditis, and the like.

(3) Conventional Treatments for Autoimmune Diseases

Resulting from an aberrant immune response to self cells, autoimmune diseases are currently treated with drugs for suppressing immunity. However, such current immune suppressants are difficult to use for a long period of time due to their serious side effects, and suffer from the inability to sufficiently suppress the recurrence of autoimmune diseases. For the treatment of multiple sclerosis, beta interferon is usually employed due to its weak side effects. However, interferon therapy is expensive, requires injections for a patient' s entire life and cannot sufficiently suppress recurrence. Also used is a method of suppressing intercellular interactions by injecting an antibody to the CD40 ligand, the success thereof is not satisfactory. Various immunotherapies have been developed, but none of them have been recognized as a promising treatment.

(4) Mechanisms of Autoimmune Disease Induced by Aberration of Immune System: Function of Thl/2 Cells

When exposed to antigens from antigen-presenting cells (APCs), such as macrophages, dendritic cells, Langerhans' cells and B cells, nahelper T cells (Th cells) are differentiated into type 1 helper T cells (hereinafter abbreviated to "ThI" ) and type 2 helper T cells (hereinafter abbreviated to "Th2" )(1). It is inferred that the direction of the differentiation is determined depending on various factors, such as antigen amount, cytokines, APC signals, etc. However, the mechanism has to be proven further (5, 6). ThI is responsible for cellular immune response while Th2 promotes humoral immune response (Table 1). [Table 1] Functional Classification of T Cells

It was reported that some autoimmune disease are attributed to excessive differentiation into or excessive activation of ThI cells, induced by some poorly known factors, which results in the promotion of cellular immunity (4). That is, excessive ThI responses cause the destruction of self cells, which leads to autoimmune diseases, such as multiple sclerosis, type 1 diabetes mellitus, rheumatoid arthritis, Hashimoto's thyroiditis, etc. On the other hand, excessive differentiation into Th2 is known to allow the excessive production of immunoglobulin E (hereinafter abbreviated as "IgE" ), incurring diseases such as allergies or asthma (2, 3).

(5) Treatment of Autoimmune Disease by Controlling Functions of Thl/2 Cells

As described above, diseases occur when the Thl/Th2 differentiation balance is broken. Thus, the maintenance of a proper Thl/Th2 differentiation ratio (e.g. to control T cell differentiation) by suppressing the excess differentiation of T cells into ThI is necessary for the treatment of autoimmune diseases. To this end, it is important to select proper APC which is easily obtainable and can effectively induce/control T cell differentiation.

(6) Control of Thl/2 Cell Function Using Dendritic Cells Dendritic cells are known as effective APC which can induce T cell differentiation. However, dendritic cells are very difficult to apply to clinical practice because the low dendritic cell level in the peripheral blood of about 1% makes it difficult to obtain the cells in an amount necessary for use in cell therapy. Also, dendritic cells are likely to induce an immune response to self antigens because they present all antigens to T cells.

(7) Control of Thl/2 Cell Function Using B Cell

In addition to producing antibodies, B cells are known to have the functions of presenting antigens to T cells, like dendritic cells, and controlling T cell differentiation (7-11). Also, B cells are reported to regulate autoimmunity. For example, B cells relieve arthritis symptoms by producing interleukin-10 (hereinafter abbreviated to "IL-IO" ) (12). Also, the administration of B cells induces the down regulation of autoimmunity in experimental autoimmune encephalomyelitis (EAE) (13) and diabetes (14).

(8) Limitations of Conventional B Cell Application to Control of Thl/2 Cell Function

B cells, as described above, were reported to have the function of controlling Thl/2 cells, but their clinical practice has not yet been realized for the following reasons.

CD Ease in obtaining B cells:

Many B cells are necessary for clinical practice. Thus, they must be easily obtained. Some reports suggest that B cells can be obtained by being cultured in the presence of T cells (11). After being cultured in combination with T cells, the B cells, capable of controlling T cell differentiation, are obtained merely by removing the T cells. This removal process is very difficult and may entail the loss of B cells. Furthermore, if T cells are not completely removed, autoimmune diseases, when treated with the obtained B cells, may worsen due to the presence of the T cells. Therefore, it is very dangerous to apply these methods to clinical practice.

(2) Acquirement of large amount of B cells:

Despite the importance of obtaining many B cells for clinical practice, none of the above-mentioned reports suggest a method of obtaining B cells in a large amount effective for clinical application. Reported is only the efficacy of B cells (12) or the preparation of B cells through co-culture with T cells (11). In the case of B lymphocytes non-specifically activated with lipopolysaccharide (hereinafter abbreviated to "LPS" ), they are likely to incur side effects because they have an influence on responses to all antibodies of the body in addition to exhibiting low efficacy (even the administration of ten million cells in six doses results only in a delay of symptom development, but cannot suppress the autoimmune disease completely) (14). In the present invention, other cells, e.g., T cells, are not employed for the cultivation of B cells and ThI response T cell two culture rounds of B cells results not only in an improvement in suppressing the differentiation of T cells into ThI, but also in cell proliferation by 1.5 times per round, by 2.2 or higher times in total.

(3) Efficacy of B cell therapeutic agent:

Potent B cell therapeutic agent, having high medicinal efficacy, are required for clinical practice application. In some studies, B cells are obtained from autoimmune disease-induced mice and administered to other mice to treat autoimmune diseases, but with very low efficacy (13). For example, when as many as ten million cells were administered, symptoms similar to those of a control were induced and maintained for 10 days or longer. On the other hand, the B cells prepared according to the present invention were found to prevent symptoms completely even when five million cells were administered. Also, no therapeutic effects were found from B cells cultured only once for 4 days (see FIG. 1). Further, tens of millions of B cells cultured in the presence of LPS were administered in six doses to mice suffering from diabetes, but the symptoms could not be completely suppressed,

The therapeutic effects of B cells on autoimmune diseases, as mentioned above, have been identified in recent studies, but nowhere was any method applicable to clinical practice reported in the prior art. The present invention is different from the prior art in various aspects, as summarized in Table 2, below.

[Table 2] Comparison between Prior Art and Present Invention

[Disclosure]

[Technical Problem]

It is therefore an object of the present invention to provide a cell therapeutic agent for autoimmune diseases, which uses B cells in the function of T cells responsible for autoimmune diseases.

It is another object of the present invention to provide a clinically applicable method for the preparation of a cell therapeutic agent for autoimmune diseases.

More particularly, it is another object of the present invention to provide a cell therapeutic agent for autoimmune diseases caused by excessive cell destructive immune responses, comprising B cells capable of suppressing cellular immunity against an autoimmune disease-causing antigen, that is, the function of ThI cells.

[Technical Solution]

In order to accomplish the above objects, the present invention provides a cell therapeutic agent for autoimmune diseases caused as a result of the destruction of self tissues by T cells, comprising B cells, regulating the differentiation of T cells, and a method for preparing the same, comprising (A) a B cell separation step, (B) a primary culture step (pre- culturing step) and (C) a secondary culture step (post-culturing step). In the B cell separation step (A), B cells may be separated from an individual suffering from an autoimmune disease using a well-known process. For example, after blood is allowed to stand for 30 min or longer at 37²C in a plastic culture dish, it is washed with a culture medium at 37²C to remove, in part, non-adherent cells (T cells) and is then washed at 37^s C with a culture medium by pipetting to separate weakly adherent cells from strongly adherent cells, macrophages and granulocytes. The resulting cell suspension is exposed to antibodies against T cells, NK cells and granulocytes and reacted with magnetic microbeads binding to the antibodies, followed by extracting B cells alone, which freely float in the magnetic field.

The primary culture step (pre-culturing step) (B) is to culture the separated B cells through activation with an antigen. In this regard, a material for inducing B cell proliferation is added together with the antigen. The antigen useful in the present invention is an autoimmune disease-indueing antigen. Culturing is performed for 3 days in the presence of the antigen and the B cell proliferation agent.

In the secondary culture step (post-culturing step) (C), the B cells collected after the primary culture step are activated with the antigen again. This step is performed for 2 days or longer in the presence of both an autoimmune disease-causing antigen and a B cell proliferation factor. Through this secondary culture step, essential for the present invention, B cells are strongly induced to have the function of suppressing the activity of self-cell destructive T cells. This function of the B cells cultured according to the present invention, in the knowledge of the present inventors, serves to greatly increase the production of cytokines suppressive of the activity of cell-destructive T cells (16-fold increase in IL-IO production) while decreasing the production of cytokines promotive of the activity of cell-destructive T cells (50-fold decrease in IFNy production).

The B cell proliferation-inducing agent suitable for use in the present invention means not only non-specific activators of B cells, but also autoimmune disease-indueing specific antigens, and is termed B cell mitogen. Examples of the B cell proliferation-inducing agent include anti-CD40, LPS (lipopolysaccharide), PWM (pokeweed mitogen), and anti-human immunoglobulin. The type of mitogen used is determined depending on the animal species and the autoimmune disease.

In accordance with the present invention, a combination of interleukin- 2 (hereinafter abbreviated as "IL-2" ) and IL-4 is used as a B cell proliferation-inducing factor.

As will be identified in the following Examples, the cell therapeutic agent according to the present invention is useful for the treatment of autoimmune diseases because the B cells cultured in a two-step process convert the T cell function from a ThI type to a Th2 type (suppressing ThI but activating Th2).

In accordance with the present invention, B cells, after being extracted from autoimmune disease-afflicted individuals (patients), are differentiated by themselves to ones (B2) which can convert the function of T lymphocytes from type 1 to type 2 during the two-step culture process in which the B cells are exposed to the B cell proliferation-inducing factors IL-2 and IL-4 and cultured in the present of a target antigen. Generally, naB lymphocytes and primarily cultured B cells (Bl) serve as simple APCs that induce the differentiation of T cells into both ThI and Th2 (see Table 1). In the contrast, the B cells (B2) which undergo the secondary culture step significantly suppress the ThI response, but highly promote the Th2 response (see Table 1). The present invention is based on this (see FIG. 5). The ability of the B cells, obtained after the post-culturing step of the present invention, to strongly promote the Th2 response alone is found to be attributable to the fact that the B cells produce the cytokines (IL-4, IL-6 and IL-IO) which are suppressive of the ThI response and promotive of the Th2 response while the cytokine (IFNγ), promotive of the ThI response, is restrained from being produced (see Table 3). [Table 3] Change in Cytokine Production of B Cell by Secondary Culture

Examples of the autoimmune diseases targeted by the cell therapeutic agent of the present invention include multiple sclerosis, type 1 diabetes mellitus, rheumatoid arthritis and Hashimoto' s thyroiditis, but are not limited thereto. As long as it is attributed to the destruction of self tissues induced by the promotion of ThI response, any autoimmune disease may be a target of the cell therapeutic agent of the present invention.

In the present invention, the B cell separation step (A) may be conducted by leaving a suspension of white blood cells separated from the splenocytes or blood of autoimmune disease individuals in a culture medium at 37° C for 1 hour to remove adherent cells (macrophages), followed by the removal of T cells and NK cells using magnetic beads conjugated with anti-T cell antibodies and anti-NK (natural killer) cell antibodies (Miltenyi, S.; Muller, W.; Weichel, W. Radbruch, A. High gradient magnetic cell separation with MACS. Cytometry. 1990, 11 (2), 231-238).

In addition to B cells, various kinds of blood cells are present in splenocytes and white blood cells, and must be removed to isolate B cells. Macrophages, which form adherent colonies, can be removed, for example, by allowing a cell suspension to stand for a period of time. As for T cells and NK cells, their removal is performed using antibodies specific therefor. Such white blood cells may be removed using a different method or in a different order.

Autoimmune diseases arise from the failure of an organism to recognize its own constituent parts (e.g., peptides, glycoproteins, etc.) as exogenous antigens, which results in an immune response against its own cells and tissues. Thus, the term "autoimmune disease inducing antigen" as used in the pre-culturing step (B) and the post-culturing step (C) is intended to refer to an antigen which is derived from an individual suffering from an autoimmune disease and is directly responsible for the autoimmune disease.

In the pre-culturing step (B), for example, concentration conditions may be set at 1-100 μg/ml for the antigen, at 0.5-5.0X10 cells/ml for B cells, at 0.01~1.0μg/ml for anti~CD40, and at 0.1-10 μg/ml for LPS for proliferating B lymphocytes. Other conditions include 0.1-10 ng/ml for the concentration of IL-2, 0.1-10 ng/ml for the concentration of IL-4, 37±2°C for culture temperature, and 60-90 hours for a culture time period. Preferably, the culture conditions of the pre-culturing step are set at 40-60 μg/ml for the concentration of the antibody (upon incubation for antigen attachment), 0.8-1.2X10 cells/ml for the concentration of B cells, 0.1~1.0μ g/ml for the concentration of anti^~CD40, 0.1-10 μg/ml for the concentration of LPS, 4~6ng/ml for the concentration of IL-2,4~6ng/ml for the concentration of IL-4, 37°C for culture temperature, and 60-84 hours for culture time period. In the post-culturing step (C), a concentration is set at 1-100 μ g/ml for the antigen, 1-10X10 cells/ml for B cells, 0.1~10ng/ml for IL-2, and

0.1-10 ng/ml for IL-4. The culture is conducted at 37±2°C for 24-90 hours. Preferably, culture conditions are set at 40-60 μg/ml for the concentration of the antigen, 4-6X105 cells/ml for the concentration of B cells, 4-6 ng/ml for the concentration of IL-2, 4-6 ng/ml for the concentration of IL-4, 37°C for culture temperature, and 38-58 hours for culture time period. However, since both the pre-culture step and the post-culture step are designed to activate B cells, the culture conditions are not strict, but may vary depending on the properties of the individual target, the kind of autoimmune diseases, and the state of health and amounts of the separated B cells.

As long as it allows B cells to grow therein, any culture medium may be used in the present invention. Preferable is human serum, bovine serum or a serum-free culture medium.

For the cell therapeutic agent according to the present invention, the B cells may be suspended in a proper aqueous solution (for example, phosphate buffered saline, aqueous solution for injection, etc.). In addition, the cell therapeutic agent according to the present invention may be administered to individuals, for example, by intravenous injection at a dosage depending on the kinds and properties (body weight, age, health state) of patients, type and properties (severity) of disease, etc.

The cell therapeutic agent according to the present invention are found not only to completely prevent the development of symptoms of autoimmune diseases, but also to relieve symptoms that have already developed, as will be noted in the following Examples using mice suffering from autoimmune diseases.

A better understanding of the present invention may be obtained through the following Examples. In the following Examples, although mice suffering from experimental autoimmune encephalomyelitis were employed, it should be understood to those skilled in the art that similar results can be obtained by applying the present invention to other autoimmune diseases when the spirit of the present invention is taken into consideration. Therefore, the examples are set forth to illustrate the technical spirit of the present invention, but are not intended to be construed to limit the present invention to a cell therapeutic agent for experimental autoimmune encephalomyelitis.

[Advantageous Effects]

According to the present invention, a cell therapeutic agent for autoimmune diseases induced by self T cells, such as multiple sclerosis, type 1 diabetes mellitus, rheumatoid arthritis, Hashimoto' s thyroiditis can be prepared. The cell therapeutic agent shows very persistent therapeutic efficacy without side effects because it uses self cells and has no influence on the immune response to other antigens. [Description of Drawings]

FIGS. 1 to 3 are graphs showing the preventive effect of the cell therapeutic agent prepared according to the present invention on experimental autoimmune encephalomyelitis (hereinafter abbreviated as "EAE" ).

FIG. 4 is a graph showing the suppression of already developed EAE symptoms by the cell therapeutic agent prepared according to the present invention.

FIG. 5 is a graph showing a significant increase in the level of ThI type cytokines and a significant decrease in the level of ThI type cytokines upon treatment with the cell therapeutic agent according to the present invention.

FIG. 6 is a graph showing the functional switching of T cells directly responsible for the induction of autoimmune diseases by the B cell therapeutic agent according to the present invention.

FIG. 7 is a graph showing the relationship between the functional switching of T cells and the cell therapeutic agent according to the present invention. [Best Mode]

1. Preparation of Cell therapeutic agent According to the Present invention

(1) Immunity Induction in Mice

Oligodendrocytes are a variety of neuroglia and their main function is o wrap axons in the central nervous system and to produce the Myelin sheath, which insulates axons. EAE arises as a result of the recognition of M0G35-55 (myelin oligodendrocyte glycoprotein) as a foreign antigen.

C57BL/6 female mice were abdominally injected with 100 μg of a mixture comprising equal amounts of M0G35-55 and a complete Freund' s adjuvant (CFA) for boosting the immune response to the antigen, so as to induce the activation and proliferation of the B cells recognizing M0G35-55.

(2) Preparation of B cells and Activation of B cells through Two Serial Culture Rounds

Hereinafter, the term "B 2 cells" refers to B cells obtained after two serial culture rounds.

(D Isolation of B cells

10 days after immunization, splenocytes were extracted from the mice and suspended in a culture medium. They were left for 1 hour at 37° C to allow adherent cells to attach to a culture dish, followed by removing the suspension (free of macrophages). To this suspension were added magnetic beads (MACS microbeads, Miltenyi Biotec GmbH, Germany) conjugated with an anti-T cell antibody and an anti-NK cell antibody, so that the remaining T cells and NK cells were bound to the magnetic beads. The removal of T cells and NK cells by a cell sorting device (MACS, supra) left pure anti-M0G35-55 B cells. (Miltenyi, S.; MuI ler, W.; Weichel, W.Radbruch, A. High gradient magnetic cell separation with MACS. Cytometry. 1990, 11 (2), 231-238.)

(2) Pre-culture of B cells

M0G35-55 was plated at a concentration of 50 μg/ml onto 48 well plates to which the B cells were then added at a density of Ix

10 cells/mlandincubatedforSdays. Together with the B cells, anti-CD40 (0.5 μ g/ml), IL-2 (5 ng/ml) and IL-4 (5 ng/ml) were also added. In the case of LPS, it was used at a concentration of 1 μg/ml.

φ Post-culture of B cells (Repeated culture) After being harvested, the B cells of the pre-culture step were washed

5 twice with a culture medium and added at a density of 5X10 cells/ml to them icroplates overwhich the antigen M0G35-55 had been plated at a concentration of 50 μg/ml, followed by incubation for two days. At this time, IL-2 (5 ng/ml) and IL-4 (5 ng/ml) were added to the culture medium.

After harvest, the B cells induced into activation were washed twice with PBS through centrifugation and suspended at a density of 5X10 cells/ml in PBS and stored until use as a "celltherapeutic agent" according to the present invention.

2. Preparation of model mice of disease (EAE induction in mice)

Model mice were prepared by inducing experimental autoimmune encephalomyelitis (EAE) therein.

M0G35-55 (1.5 mg/ml), an EAE inducer, and tubercle bacillus (4 mg/ml) were mixed in CFA to give an antigen solution. Separately, a solution of Bordetella pertussis toxin in PBS (4 μg/ml) was prepared.

On Day 1 and 2, C57BL/6 female mice were subcutaneousIy injected at the opposite sides with 100 μl of the antigen solution and abdominally injected with 0.1 ml of the pertussis toxin solution to induce EAE therein.

3. Effect of the cell therapeutic agent according to the present invention

1 hour or 14 days after EAE induction, a predetermined amount of the cell therapeutic agent according to the present invention was injected into the tail vein of the EAE-induced mice.

Under observation with the naked eyes, the EAE mice were analyzed for symptoms and graded according to the standards shown in Table 4, below.

[Table 4]

In the following examples, the data shown for each group represent means of six separate measurements.

(1) Effect of treatment of the cell therapeutic agent just after EAE induction

1 hour after EAE induction in mice, B cells listed in Table 5 were

6 administered at a density of 7.5 XlO cells into them ice, which were then observed for symptoms(FIG.l). [Table 5]

GrouDs B Cells

Control Non-treated

Fresh B Treated with B cells seoerated from the spleen

Bl Treated with Dre-cultured B eel Is

B2 Treated with the cell therapeut ic agent

As shown in FIG. 1, the B2 group that was treated with the cell therapeutic agent of the present invention was completely protected from the occurrence of EAE. On the other hand, suppression effects were found neither in the fresh B group treated with the fresh B cells separated from the spleen nor in the Bl group treated with the pre-cultured B cells, as in the control group.

(2) Determination of proper dosage

The cell therapeutic agent according to the present invention was analyzed for proper dosages. 1 hour after EAE induction in mice, the B2

6 6 cells were administered at densities of 2.5X10 cells ,5.0x10 cells and 7.5 6

XlO cells and them ice were monitored for symptoms(FIG.2).

6

At a dosage of 5.0X10 cells, the B2cells were found to sufficiently suppress EAE. Thus, the B2 cells were used at a dosage of 5.0X10 cells in the following experiments.

This dosage was, however, set only for EAE mouse models and may vary depending on the kinds and properties (severity) of autoimmune diseases and the kinds and properties (body weight, age, health state) of patients.

(3) Efficacy maintenance of cell therapeutic agent in mice re-exposed to antigen after treatment

After being treated with the B2 cells according to the present invention, the mice were exposed again to the EAE antigen and monitored for symptoms. The results are graphed in FIG. 3.

In spite of the passage of 50 days from the administration thereof to suppress the occurrence of EAE, as can be seen, the B cells according to the present invention were found to significantly prevent the recurrence of EAE in the mice re-immunized with the antigen. In FIG. 3, EAE was re-induced in the control and the treated group (B2-tx) by immunization with the same antigen on Day 50 after treatment.

(4) Therapeutic effect on mice suffering from EAE

The cell therapeutic agent of the present invention was examined for therapeutic effectiveness in mice already suffering from EAE.

From about 10 days following administration with the EAE antigen, EAE started to be induced, as shown in FIGS. 1 and 2. The mice which showed apparent EAE symptoms 14 days after antigen administration were treated with the cell therapeutic agent according to the present invention (FIG. 4).

As can be seen, even when the cell therapeutic agent of the present invention was administered after the development of EAE symptoms, definite suppressive (therapeutic) effects were observed with the passage of 10 days. 4. Operation Mechanism of the Cell therapeutic agent

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

The mechanism in which the cell therapeutic agent according to the present invention can suppress/treat EAE was investigated in vitro. Splenocytes were taken from mice 10 days (FIG. 5) and 50 days (FIG. 6) after treatment with the B2 cells immediately subsequent to EAE induction and cultured in vitro for 3 days in the presence of the same antigen(M0G35-55). The levels of Th2 type cytokines IL-4, IL-6, and IL-IO were measured to be significantly higher than those of the ThI type cytokine IFN Y (FIGS. 5 and 6).

These results indicate that treatment with the cell therapeutic agent (B2 cells) according to the present invention renders T cells to undergo polarization to Th2, meaning that when introduced to autoimmune disease individuals, the B2 cells of the present invention induce the differentiation of T cells into Th2, thereby suppressing the autoimmune disease. Also, this polarization effect was observed to be maintained for 50 days after the injection of the B2 cells.

(2) Th2 polarization of T cells by B2 cells! invivo

The therapeutic effect attributed to the polarized differentiation was examined in vivo.

50 days after treatment with the B2 cells immediately after EAE induction, only T cells were isolated from the control (non-treated, EAE outbreak) and the B2 group (treated with the B2 cells, no symptoms) (the B2 cells were removed). Normal mice were administered with the isolated T cells and immediately immunized with the antigen (EAE induction), followed by monitoring symptoms over time (FIG. 7). The control and the experimental groups used in this in vivo experiment are summarized in Table 6, below.

[Table 61

As seen in FIG. 7, when the T cells of the control mice were introduced (Control T), the autoimmune disease symptoms were rapidly developed, leading to the death of the mice 21 days after the introduction thereof, while only slight symptoms were found in the mice (B2-tx T) into which the T cells taken from the B2 cell-injected mice exhibiting no symptoms were introduced. Taken together, the results demonstrate that treatment with the cell therapeutic agent according to the present invention (introduction of B2 cells) induces T cells to differentiate into Th2 in a polarization manner in vivo, implying that the therapeutic effect of the B2cells on autoimmune diseases depends on the functional switching of T cells.

Reference Lists

1. Mosmann, T.R. ; Coffman, R.L. THl and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol. 1989, 7 145-173.

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

3.Romagnani ,S.Thl and Th2 in human diseases. Clin Immunol Immunopatho1.1996,80(3Pt1) ,225-235.

4. Moss, R.B.; Moll, T.; El-Kalay, M.; Kohne, C; Soo Hoo, W.; Encinas, J.Carlo, D.J. Thl/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 ThI and Th2 cells: functional properties, mechanisms of regulation, and role in disease. Lab Invest. 1994, 70, 299-306.

6. Szabo, S.J.; Sullivan, B.M.; Peng, S.L.; Glimcher, L.H. Molecular mechanisms regulating ThI immune responses. Annu Rev Immunol. 2003, 21, 713- 758.

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

8. Moser, M.; Murphy, K.M. 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 lymphnodes.J. Immunol .1987,1382848-2856.

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Claims

[CLAIMS] [Claim 1]

A method for preparing a cell therapeutic agent for the treatment of an autoimmune disease, comprising:

(A) separating B cells from blood or bone marrow of an individual suffering from an autoimmune disease selected from among multiple sclerosis, type 1 diabetes mellitus, rheumatoid arthritis, and Hashimoto's thyroiditis;

(B) primarily culturing the B cells in a culture medium supplemented with a B cell proliferation-inducing agent in presence of an antigen causing the autoimmune disease! and

(C) secondarily culturing the B cells in a fresh culture medium supplemented with a B cell proliferation-inducing factor in the presence of an antigen causing the autoimmune disease.

[Claim 2]

The method according to claim 1, wherein the B cell proliferation- inducing agent used for the primarily culturing (B) is selected from a group consisting of an anti-CD40 antibody, LPS, IL-2 IL-4 and a combination thereof . [Claim 3]

The method according to claim 1, wherein the B cell proliferation- inducing factor used for the secondarily culturing (C) is selected from a group consisting of IL-2, IL-4 and a combination thereof. [Claim 4]

The method according to claim 1, wherein the primarily culturing (B) is conducted at a culture temperature of 37±2°C for 60-90 hours with the B

6 cells at a density of 0.5-5.0X10 cells/ml , the antigen at a concentration of l~100μg/ml, the anti-CD40 at a concentration of 0.01-1.Oμg/ml , the LPS at a concentration of 0.1-10 ug/ml,the IL-2 at a concentration of 0.1-lOng/ml ,and the IL-4 at a concentration of 0.1-lOng/ml and the secondarily culturing(C) is conducted at a culture temperature of 37±2°C for 24-90 hours with the B cells at a density of 1-10X10 cells/ml , the antibody at a concentration of

1-100 μ g/ml ,the IL-2 at a concentration of 0.1-lOng/ml , and the IL-4 at a concentration of 0.1-lOng/ml . [Claim 5]

A cell therapeutic agent, comprising the B cells prepared according to the method of one of claims 1 to 4 as an active ingredient.