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WO2009054556A1 - Procédé de préparation de cellules b thérapeutiques contre des maladies auto-immunes qui sont induites par la cytotoxicité auto-immune - Google Patents

Procédé de préparation de cellules b thérapeutiques contre des maladies auto-immunes qui sont induites par la cytotoxicité auto-immune Download PDF

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Publication number
WO2009054556A1
WO2009054556A1 PCT/KR2007/005245 KR2007005245W WO2009054556A1 WO 2009054556 A1 WO2009054556 A1 WO 2009054556A1 KR 2007005245 W KR2007005245 W KR 2007005245W WO 2009054556 A1 WO2009054556 A1 WO 2009054556A1
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Prior art keywords
cells
cell
therapeutic agent
concentration
autoimmune disease
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PCT/KR2007/005245
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English (en)
Inventor
Sang Yun Nam
Sang Mok Lee
Jong Cheon Ha
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Sang Yun Nam
Sang Mok Lee
Jong Cheon Ha
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Application filed by Sang Yun Nam, Sang Mok Lee, Jong Cheon Ha filed Critical Sang Yun Nam
Priority to PCT/KR2007/005245 priority Critical patent/WO2009054556A1/fr
Publication of WO2009054556A1 publication Critical patent/WO2009054556A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4612—B-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46433—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0635—B lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/05—Adjuvants
    • C12N2501/052—Lipopolysaccharides [LPS]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/52—CD40, CD40-ligand (CD154)

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • autoimmune diseases are currently treated with drugs for suppressing immunity.
  • drugs for suppressing immunity 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.
  • beta interferon is usually employed due to its weak side effects.
  • interferon therapy is expensive, requires injections for a patient' s entire life and cannot sufficiently suppress recurrence.
  • 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.
  • Th cells When exposed to antigens from antigen-presenting cells (APCs), such as macrophages, dendritic cells, Langerhans' cells and B cells, justifylper 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
  • ThI immunoglobulin E
  • Thl/Th2 differentiation balance 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.
  • Thl/Th2 differentiation ratio e.g. to control T cell differentiation
  • Dendritic cells are known as effective APC which can induce T cell differentiation.
  • 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.
  • dendritic cells are likely to induce an immune response to self antigens because they present all antigens to T cells.
  • 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).
  • IL-IO interleukin-10
  • B cells are necessary for clinical practice. Thus, they must be easily obtained.
  • T cells other cells, e.g., T cells
  • 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.
  • 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,
  • 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.
  • 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).
  • B cells may be separated from an individual suffering from an autoimmune disease using a well-known process.
  • T cells non-adherent cells
  • a culture medium for example, after blood is allowed to stand for 30 min or longer at 37 2 C in a plastic culture dish, it is washed with a culture medium at 37 2 C 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.
  • 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.
  • 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.
  • 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 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.
  • B cell mitogen examples 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.
  • IL-2 interleukin-2
  • IL-4 interleukin-4
  • 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).
  • 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.
  • B2 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).
  • 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).
  • 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.
  • 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).
  • B cells 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 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.
  • concentration conditions may be set at 1-100 ⁇ g/ml for the antigen, at 0.5-5.0X10 cells/ml for B cells, at 0.01 ⁇ 1.0 ⁇ g/ml for anti ⁇ CD40, and at 0.1-10 ⁇ g/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.
  • the culture conditions of the pre-culturing step are set at 40-60 ⁇ g/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 ⁇ 1.0 ⁇ g/ml for the concentration of anti ⁇ CD40, 0.1-10 ⁇ g/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.
  • a concentration is set at 1-100 ⁇ g/ml for the antigen, 1-10X10 cells/ml for B cells, 0.1 ⁇ 10ng/ml for IL-2, and
  • the culture is conducted at 37 ⁇ 2°C for 24-90 hours.
  • culture conditions are set at 40-60 ⁇ g/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.
  • 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.
  • any culture medium may be used in the present invention.
  • Preferable is human serum, bovine serum or a serum-free culture medium.
  • the B cells may be suspended in a proper aqueous solution (for example, phosphate buffered saline, aqueous solution for injection, etc.).
  • a proper aqueous solution for example, phosphate buffered saline, aqueous solution for injection, etc.
  • 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.
  • 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.
  • 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.
  • 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]
  • 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 ⁇ g 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.
  • CFA complete Freund' s adjuvant
  • B 2 cells refers to B cells obtained after two serial culture rounds.
  • 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 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.)
  • M0G35-55 was plated at a concentration of 50 ⁇ g/ml onto 48 well plates to which the B cells were then added at a density of Ix
  • 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.
  • mice were prepared by inducing experimental autoimmune encephalomyelitis (EAE) therein.
  • EAE experimental autoimmune encephalomyelitis
  • M0G35-55 (1.5 mg/ml), an EAE inducer, and tubercle bacillus (4 mg/ml) were mixed in CFA to give an antigen solution.
  • CFA a solution of Bordetella pertussis toxin in PBS (4 ⁇ g/ml) was prepared.
  • mice were subcutaneousIy injected at the opposite sides with 100 ⁇ l of the antigen solution and abdominally injected with 0.1 ml of the pertussis toxin solution to induce EAE therein.
  • a predetermined amount of the cell therapeutic agent according to the present invention was injected into the tail vein of the EAE-induced mice.
  • the B2 group that was treated with the cell therapeutic agent of the present invention was completely protected from the occurrence of EAE.
  • 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.
  • the cell therapeutic agent according to the present invention was analyzed for proper dosages. 1 hour after EAE induction in mice, the B2
  • the B2cells were found to sufficiently suppress EAE.
  • 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.
  • mice 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.
  • 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.
  • EAE was re-induced in the control and the treated group (B2-tx) by immunization with the same antigen on Day 50 after treatment.
  • the cell therapeutic agent of the present invention was examined for therapeutic effectiveness in mice already suffering from EAE.
  • EAE 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).
  • 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).
  • the therapeutic effect attributed to the polarized differentiation was examined in vivo.
  • mice 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.

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Abstract

La présente invention concerne un procédé de préparation d'un agent thérapeutique à base de cellules pour des maladies auto-immunes basé sur la capacité des cellules B pour contrôler la différenciation des lymphocytes T, qui sont responsables de l'autodestruction tissulaire. Le procédé comprend : (A) la séparation des cellules B du sang ou de la moelle osseuse d'un sujet atteint d'une maladie auto-immune parmi la sclérose en plaques, le diabète de type 1, la polyarthrite rhumatoïde et la thyroïdite chronique de Hashimoto; (B) la culture primaire des cellules B dans un milieu de culture supplémenté d'un facteur induisant la prolifération des cellules B en présence d'un antigène provoquant la maladie auto-immune; et (C) la culture secondaire de cellules B dans un nouveau milieu de culture supplémenté d'un facteur induisant la prolifération des cellules B en présence d'un antigène provoquant la maladie auto-immune, permettant de préparer un agent thérapeutique à base de cellules contre ladite maladie. L'agent thérapeutique à base de cellules présente une efficacité thérapeutique hautement persistante contre des maladies auto-immunes, telles que la sclérose en plaques, le diabète de type 1, la polyarthrite rhumatoïde et la thyroïdite chronique de Hashimoto sans effets secondaires.
PCT/KR2007/005245 2007-10-24 2007-10-24 Procédé de préparation de cellules b thérapeutiques contre des maladies auto-immunes qui sont induites par la cytotoxicité auto-immune WO2009054556A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4402934A (en) * 1980-10-30 1983-09-06 Teodorescu Marius C Diagnostic technique for rheumatoid arthritis
WO2000074718A1 (fr) * 1999-06-09 2000-12-14 Immunomedics, Inc. Immunotherapie de troubles auto-immuns a l'aide d'anticorps ciblant les cellules b
WO2001055216A1 (fr) * 2000-01-26 2001-08-02 Raven Biotechnologies, Inc. Methodes et compositions pour generer des anticorps monoclonaux humains
US6465251B1 (en) * 1996-11-13 2002-10-15 Dana-Farber Cancer Institute, Inc. Method of promoting b-cell proliferation and activation with CD40 ligand and cyclosporin
US20030099650A1 (en) * 2001-07-25 2003-05-29 Ho Alice Suk-Yue Treatment of immune disorders and B cell disorders

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4402934A (en) * 1980-10-30 1983-09-06 Teodorescu Marius C Diagnostic technique for rheumatoid arthritis
US6465251B1 (en) * 1996-11-13 2002-10-15 Dana-Farber Cancer Institute, Inc. Method of promoting b-cell proliferation and activation with CD40 ligand and cyclosporin
WO2000074718A1 (fr) * 1999-06-09 2000-12-14 Immunomedics, Inc. Immunotherapie de troubles auto-immuns a l'aide d'anticorps ciblant les cellules b
WO2001055216A1 (fr) * 2000-01-26 2001-08-02 Raven Biotechnologies, Inc. Methodes et compositions pour generer des anticorps monoclonaux humains
US20030099650A1 (en) * 2001-07-25 2003-05-29 Ho Alice Suk-Yue Treatment of immune disorders and B cell disorders

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