JP2017110023A - Methods for culturing dendritic cells - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technology for preparing, from blood cells taken from a patient, dendritic cells in a number optimum for the immune therapy by proliferating dendritic cells or precursor cells thereof in vitro.SOLUTION: The pharmaceutical composition for dendritic cell transplantation therapy of the invention comprises dendritic cells prepared by a process comprising the steps of: removing mononuclear cells from the blood cells taken from a subject; removing T cells from the mononuclear cells; culturing the rest of the cells after the removal of T cells from the mononuclear cells in a culture medium containing GM-CSF and SCF. The step of culturing the rest of the cells after the removal of T cells from the mononuclear cells in a culture medium containing GM-CSF and SCF is included in the process in some cases. The pharmaceutical composition of the invention for dendritic cell transplantation therapy may be used for treating cancers or infectious diseases.SELECTED DRAWING: None

Description

  The present invention relates to a method for culturing dendritic cells and a pharmaceutical composition containing dendritic cells obtained by the method, and specifically, a large amount of precursor cells of dendritic cells from peripheral blood collected from a patient. The present invention relates to a culture method for selective growth and a pharmaceutical composition comprising dendritic cells prepared by the method.

Dendritic cells can stimulate or suppress the immune system including both acquired immunity and innate immunity as antigen-presenting cells (Non-patent Document 1). Therefore, the patient's immune system is controlled by transplanting dendritic cells previously treated outside the body to present specific antigens related to cancer, infectious diseases, autoimmune diseases, allergic diseases, etc. Dendritic cell transplantation therapy has been attempted (Non-patent Document 2). However, such dendritic cell transplantation therapy requires large amounts of dendritic cells. For example, according to studies using experimental animal model systems for skin tumors and lung metastases, the optimal number of dendritic cells to be transplanted in any model system is at least 10 ⁶ per 30 g body weight of experimental animals. The result that it was was obtained (nonpatent literature 3 and 4). This corresponds to at least about 10 ⁹ dendritic cells in a single dose per patient when converted to humans. However, clinically, only 10 ⁶ to 10 ⁸ dendritic cells can be recovered from one person even when apheresis is performed.

In the method for preparing dendritic cells established in the 1990s, monocytes derived from peripheral blood, umbilical cord blood, lymph nodes and the like are cultured in the presence of GM-CSF and IL-4 for 5 to 7 days. A mature dendritic cell that has been stimulated with a microbial or T cell-derived factor, for example, a pro-inflammatory mediator such as TNF-α, IL-1β, IL-6, PGE ₂ or the like, and has acquired T cell stimulating ability is obtained. (Patent Documents 1 to 3). Thereafter, a forcing preparation method was developed in which culturing was carried out for 1 day in the presence of the pro-inflammatory mediator after culturing for 1 day in the presence of GM-CSF and IL-4 (Patent Document 4). However, these preparation methods only differentiate the dendritic cell progenitor cells into dendritic cells, and do not substantially increase the number of dendritic cell progenitor cells themselves.

Many patients who are candidates for immunotherapy by dendritic cell transplantation have weak hematopoietic potential due to the disease itself or the side effects of other treatments. In particular, at present, immunotherapy is often tried when there is no effect on other treatments. Therefore, due to side effects of other treatments, the order of 10 ⁹ necessary for effective treatment is possible even if apheresis is used. The inability to prepare dendritic cells or their progenitor cells prevents the clinical trial from expanding.

Lipscomb, M.M. F. And Masten, B .; J. et al. Physiol. Rev. 82: 97-130 (2002). Steinman, R.M. M.M. And Banchereau, J. et al. Nature 449: 419-426 (2007). Harada, Y .; Et al., PLoS ONE, 4: e6674 (2009) Tatsuta, K .; Et al., Gene Ther. 16: 240-251 (2009) Caux, C.I. Et al., Nature, 360: 258-261 (1992). Romani, N.M. S. Et al. Exp. Med. 180: 83-93 (1994). Sallusto, F.M. And Lanzavecchia, A .; J. et al. Exp. Med. 179: 1109-1118 (1994) Dauer, M.M. Et al. Immunol. 170: 4069-4076 (2003)

  Therefore, it is necessary to develop a technique for preparing an optimal number of dendritic cells for the immunotherapy by growing dendritic cells or their precursor cells in vitro from blood cells collected from patients.

  The present invention provides a method for preparing dendritic cells. The method for preparing dendritic cells of the present invention comprises the steps of separating mononuclear cells from blood cells collected from a subject and removing T cells from the mononuclear cells.

  In the method for preparing a dendritic cell of the present invention, the step of removing the T cell from the mononuclear cell may be achieved by removing a cell expressing CD3.

  The method for preparing dendritic cells of the present invention may include a step of culturing the remaining cells from which the T cells have been removed in a medium containing GM-CSF and SCF.

  In the method for preparing a dendritic cell of the present invention, the step of culturing in a medium containing GM-CSF and SCF is performed for at least 3 weeks, and thereafter, the culture is performed by switching to a medium containing GM-CSF and IL-4. May be included.

  In the method for preparing a dendritic cell according to the present invention, the step of culturing in a medium containing GM-CSF and SCF is performed for at least 4 weeks, and then the culture is performed by switching to a medium containing GM-CSF and IL-4. May be included.

  In the method for preparing dendritic cells of the present invention, the blood cells may be collected from peripheral blood, umbilical cord blood, bone marrow and / or lymph nodes.

  In the method for preparing dendritic cells of the present invention, the blood cells may be collected from peripheral blood by an apheresis method.

  The present invention provides a pharmaceutical composition for dendritic cell transplantation therapy comprising dendritic cells prepared by the method for preparing dendritic cells of the present invention.

  The pharmaceutical composition for dendritic cell transplantation therapy of the present invention may be used for treating cancer or infectious diseases.

  The pharmaceutical composition for dendritic cell transplantation therapy of the present invention may be used to treat autoimmune diseases, allergic diseases or type 1 diabetes.

  The pharmaceutical composition for dendritic cell transplantation therapy of the present invention may be transplanted to a patient having an HLA genotype different from the dendritic cell prepared by the preparation method of the present invention.

  The present invention provides a method for treating a disease in a patient comprising the step of transplanting dendritic cells prepared by the method of the present invention into the patient.

  The treatment method of the present invention may be used to treat cancer or infection.

  The treatment methods of the present invention may be for treating autoimmune diseases, allergic diseases or type 1 diabetes.

  The treatment method of the present invention may comprise the step of transplanting into a patient having an HLA genotype different from the dendritic cells prepared by the preparation method of the present invention.

  The technical scope of the present invention is defined based on the description of the appended claims. Modifications of the present invention, for example, addition, deletion, and replacement of the configuration requirements of the present invention can be made on the condition that the gist of the present invention is not deviated.

  The source of dendritic cells or progenitor cells thereof of the present invention may be tissues including but not limited to peripheral blood, bone marrow, lymph nodes and umbilical cord blood. It is preferable that the dendritic cell or its precursor cell of the present invention is prepared from peripheral blood. However, it may be prepared from hematopoietic stem cells generated from embryonic stem cells, adult stem cells and induced pluripotent stem (iPS) cells.

  The dendritic cells of the present invention or progenitor cells thereof may be derived from a patient who has undergone dendritic cell transplantation therapy or a human having the same genotype as the patient. Alternatively, it may be derived from a human having a genotype different from that of the patient, provided that the effectiveness of the immunotherapy is not impaired.

  In order to prepare the dendritic cells of the present invention, various procedures known to those skilled in the art can be used. For example, when separating leukocytes from peripheral blood, Ficoll specific gravity centrifugation can be used. When separating and removing T cells from the leukocytes, immunomagnetic beads including, but not limited to, Dynabeads (trademark) manufactured by Dynal and CliniMACS (trademark) manufactured by Miltenyi Biotech are used. Thus, cells expressing the cell surface antigen CD3 can be separated and removed.

  In the method of the present invention, removing T cells is not merely removing and enriching lymphocytes. As shown in the Examples of the present specification, when mononuclear cells are cultured in a medium to which a cytokine cocktail containing GM-CSF and SCF of the present invention is added, the proliferation of T cells is promoted rather than the proliferation of dendritic cells themselves. Significantly, dendritic cells are very diluted. This is thought to be because dendritic cells interact with T cells, but the growth and / or differentiation of the dendritic cells themselves is hindered. Therefore, the T cells are removed so that the dendritic cells do not unnecessarily interact with the T cells.

  In the method of the present invention, the step of removing T cells from the mononuclear cells may be performed using any cell marker that is expressed in T cells but not in dendritic cells or their precursor cells. A preferred cell marker is CD3 or a combination of CD4 and CD8. Further, in order to remove T cells, when the cell marker is a cell surface marker, the antibody against the cell surface marker or other specific binding partner immobilized on a magnetic bead or other solid phase is used. May be isolated from a mononuclear cell population. Alternatively, flow cytometry methods or other immunological separation means may be used. In some cases, only T cells are selectively injured or killed using specific binding partners for T cells. The step of removing the T cells from the mononuclear cells may be a step of removing other mononuclear cell types such as B cells and / or NK cells together with T cells.

  As a medium for culturing the obtained cells, in addition to IMDM medium, MEM, DMEM, RPMI-1640, and X-VIVO15 medium are used, but not limited to these, a medium suitable for the growth of blood cells is used. . Fetal calf serum may be added to these media. The concentration of fetal calf serum may be 5-20%, more preferably 10%. As an alternative, human AB serum available from BioWhittaker et al. Or donated human serum albumin available from Japanese Red Cross may be added. Human AB serum is preferably added at a concentration of 0 to 15%, and blood donated human serum albumin is preferably added at a concentration of 0 to 10%.

  The medium contains GM-CSF and SCF, but a cytokine cocktail that does not contain any other cytokine or a cytokine cocktail that contains GM-CSF and IL-4 but does not contain SCF. These cytokines preferably have human amino acid sequences, and for safety, are preferably produced by recombinant DNA technology. The concentration of GM-CSF may be used in the range of 1 to 500 ng / mL, 1 to 200 ng / mL, or 1 to 100 mg / mL, more preferably 2 to 300 ng / mL, 5 to 200 ng / mL, 10 It is used in the range of from 150 to 150 ng / mL, from 20 to 120 ng / mL, or from 30 to 100 ng / mL. The concentration of SCF or IL-4 may be used in the range of 0.5 to 500 ng / mL, 0.5 to 100 ng / mL or 0.5 to 50 ng / mL, more preferably 1 to 300 ng / mL. Used in the range of 2 to 200 ng / mL, 5 to 100 ng / mL, 10 to 70 ng / mL, 20 to 60 ng / mL or 25 to 50 ng / mL.

  In the present specification, a medium not containing a certain cytokine is a range that does not significantly exceed the concentration of the cytokine in normal serum, that is, three times, two times, or less than the concentration in normal serum. Preferably, it is 1/2, 1/3, or 1/5 or less. Alternatively, 50 ng / mL, 40 ng / mL, 30 ng / mL, 20 ng / mL, 10 ng, on the condition that it behaves substantially the same as the behavior of dendritic cells or their progenitor cells when the concentration of the cytokine is 0 / ML, 5 ng / mL, 3 ng / mL or 1 ng / mL or less.

In the method of the present invention, dendritic cell progenitor cells grown in a medium containing GM-CSF and SCF can be changed to a dendritic cell subset by switching to a medium containing GM-CSF and IL-4. . Here, cells that grow in a medium containing GM-CSF and SCF are cells that have been determined to differentiate into dendritic cells at any time by GM-CSF and IL-4. Therefore, switching from a medium containing GM-CSF and SCF to a medium containing GM-CSF and IL-4 may be performed at any time after the start of culture. However, since the object of the present invention is to prepare a large amount of dendritic cells, a longer period of culturing in a medium containing GM-CSF and SCF is advantageous because more dendritic cells can be obtained. On the other hand, a short culture period is useful because dendritic cell transplantation therapy can be performed for more patients. Actually, as shown in Example 1, the number of CD11c positive cells increases steadily until 3 weeks after the start of culture in a medium containing GM-CSF and SCF, but the growth rate decreases after 4 weeks. Therefore, a compromise between obtaining a larger amount of dendritic cells and obtaining dendritic cells in a shorter period of time is that the growth in a medium containing GM-CSF and SCF is necessary for the therapeutic effect. When the number of dendritic cells (about 10 ⁹ ) can be achieved, or the proliferation rate decreases, the number of precursor cells of dendritic cells does not increase so much even when cultured in a medium containing GM-CSF and SCF. It is time to enter a state. Therefore, switching from a medium containing GM-CSF and SCF to a medium containing GM-CSF and IL-4 is performed at any time after the start of culture, provided that the cells grow to the number of dendritic cells necessary for the therapeutic effect. It doesn't matter. However, it is preferably after 3 weeks after the growth rate in the medium containing GM-CSF and SCF begins to drop, and more preferably after 4 weeks when the growth in the medium containing GM-CSF and SCF almost stops. .

Growth-stimulated cells may be cultured at 37 ° C. in the presence of 5-7% CO ₂ . Cells are seeded at a concentration of 5 × 10 ⁵ cells / mL, medium changed after 2 to 4 days, and seeded at a concentration of 2.5 × 10 ⁵ cells / mL to 1 × 10 ⁶ cells / mL in fresh media. There is a case. A preferred seeding concentration is 5 × 10 ⁵ cells / mL. In the case of mass culture of 1 × 10 ⁸ or more in total, an apparatus such as WAVE Bioreactor 2/10 (trademark) manufactured by GE Healthcare may be used.

  In the method of the present invention, mononuclear cells isolated from collected peripheral blood may be cryopreserved. In addition, after the CD3-expressing cells are removed, the remaining monocyte cells may be cryopreserved. Any method known to those skilled in the art may be used for freezing and thawing blood cells. In order to freeze the cells, a commercially available cell cryopreservation solution such as bun bunker (trademark) manufactured by Lymphtec Corporation may be used.

Dendritic cells prepared by the method of the present invention were stimulated with pro-inflammatory mediators such as OK-432, TNF-α, IL-1β, IL-6, and PGE ₂ to acquire T cell stimulating ability. Later, it may be transplanted into the patient. For the treatment of cancer, there are cases where stimulation is caused by an antigen specifically expressed in each cancer. For example, prostate cancer, non-small cell lung cancer, and high-risk melanoma may be stimulated with prostate acid phosphatase (PAP), tumor antigen MAGE-A3, and cancer testis antigen NY-ESO-1, respectively. For the treatment of a single cancer, combine dendritic cells that are individually stimulated with multiple cancer-specific antigens, or use dendritic cells that are simultaneously stimulated with multiple cancer-specific antigens. In some cases.

  The pharmaceutical composition containing the dendritic cell of the present invention may be mixed with a dendritic cell prepared by the method of the present invention together with another therapeutic agent such as a chemotherapeutic agent or a radiation therapeutic agent. . Such agents include recombination of agents that suppress the growth of cancer cells such as CDDP, TS-1, and Gemzar, and factors that enhance the activity of cytotoxic T cells that attack cancer cells such as IL-12p70 and PGE2. Examples include, but are not limited to, inhibitors for proteins and factors that suppress the activity of cytotoxic T cells such as IL-10, such as antibodies and short interfering RNAs.

  The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. Representative examples are any solution capable of suspending living cells, such as physiological saline, phosphate buffered saline (PBS), medium, serum, and the like. In addition, when dendritic cells are used as vaccines, in order to enhance immunogenicity, immunosuppressants such as cytokines, bacterial toxins, Mycobacterium tuberculosis, and other adjuvants such as alum, Freund's adjuvant, etc. However, the pharmaceutical composition of the present invention may contain an immunogenicity enhancer, which is not limited thereto.

FIG. 3 is a graph showing the results of analyzing a flow cytometry method after mono-nuclear cells isolated from peripheral blood were cultured in a GM / SCF medium for 1 week and then double-stained with antibodies against CD3 and CD11c. FIG. 3 is a graph showing the results of analyzing a mononuclear cell isolated from peripheral blood by culturing in an GM / IL-4 medium for 1 week and then double-staining with an antibody against CD3 and CD11c and analyzing by a flow cytometry method. FIG. 3 is a graph showing the results of analyzing a flow cytometry method after mononuclear cells isolated from peripheral blood were cultured in a GM / SCF medium for 2 weeks and then double-stained with antibodies against CD3 and CD11c. FIG. 3 is a graph showing the results of analyzing a flow cytometry method after mononuclear cells isolated from peripheral blood were cultured in GM / IL-4 medium for 2 weeks and then double-stained with antibodies against CD3 and CD11c. FIG. 3 is a graph showing the results of analyzing mononuclear cells isolated from peripheral blood in GM / SCF medium for 2 weeks, gating only CD11c-negative cells, staining with an antibody against CD3, and analyzing by flow cytometry. FIG. 2 is a graph showing the results of analyzing mononuclear cells isolated from peripheral blood in GM / IL-4 medium for 2 weeks, gating only CD11c-negative cells, staining with an antibody against CD3, and analyzing by flow cytometry. CD11c positive in the case where the remaining cells from which CD3 expressing cells have been removed from mononuclear cells in peripheral blood are cultured (solid line) and in the case where the mononuclear cell population is cultured as it is without removing CD3 expressing cells (broken line) Cell number growth curve After removing CD3-positive cells from mononuclear cells isolated from peripheral blood, the cells were cultured in GM / SCF medium for 4 weeks and further in GM / IL-4 medium for 1 week, and then stained with an antibody against CCR5 and flow cytometry The result figure analyzed by. FIG. 3 is a diagram showing the results of analysis by flow cytometry after removing CD3-positive cells from mononuclear cells isolated from peripheral blood, culturing in GM / SCF medium for 5 weeks, staining with an antibody against CCR5. After removing CD3-positive cells from mononuclear cells isolated from peripheral blood, the cells were cultured in GM / SCF medium for 4 weeks and further in GM / IL-4 medium for 1 week, and then stained with an antibody against CCR7 and flow cytometry The result figure analyzed by. FIG. 3 is a diagram showing the results of analysis by flow cytometry after removing CD3 positive cells from mononuclear cells isolated from peripheral blood, culturing in GM / SCF medium for 5 weeks, staining with an antibody against CCR7. After removing CD3-positive cells from mononuclear cells isolated from peripheral blood, the cells were cultured in GM / SCF medium for 4 weeks and further in GM / IL-4 medium for 1 week, and then stained with an antibody against CD40 and flow cytometry. The result figure analyzed by. FIG. 3 is a graph showing the results of analysis by flow cytometry after removing CD3-positive cells from mononuclear cells isolated from peripheral blood, culturing in GM / SCF medium for 5 weeks, staining with an antibody against CD40. After removing CD3-positive cells from mononuclear cells isolated from peripheral blood, the cells were cultured in GM / SCF medium for 4 weeks and further in GM / IL-4 medium for 1 week, and then stained with an antibody against CD54 and flow cytometry. The result figure analyzed by. FIG. 3 is a graph showing the results of analysis by flow cytometry after removing CD3-positive cells from mononuclear cells isolated from peripheral blood, culturing in GM / SCF medium for 5 weeks, staining with an antibody against CD54. After removing CD3-positive cells from mononuclear cells isolated from peripheral blood, the cells were cultured in GM / SCF medium for 4 weeks and further in GM / IL-4 medium for 1 week, and then stained with an antibody against CD80 and flow cytometry. The result figure analyzed by. FIG. 3 is a graph showing the results of analysis by flow cytometry after removing CD3-positive cells from mononuclear cells isolated from peripheral blood, culturing in GM / SCF medium for 5 weeks, staining with an antibody against CD80. After removing CD3-positive cells from mononuclear cells isolated from peripheral blood, the cells were cultured in GM / SCF medium for 4 weeks and further in GM / IL-4 medium for 1 week, and then stained with an antibody against CD83 and flow cytometry The result figure analyzed by. FIG. 3 is a graph showing the results of analysis by flow cytometry after removing CD3-positive cells from mononuclear cells isolated from peripheral blood, culturing in GM / SCF medium for 5 weeks, staining with an antibody against CD83. After removing CD3-positive cells from mononuclear cells isolated from peripheral blood, the cells were cultured in GM / SCF medium for 4 weeks and further in GM / IL-4 medium for 1 week, and then stained with an antibody against CD86 and flow cytometry. The result figure analyzed by. FIG. 3 is a graph showing the results of analysis by flow cytometry after removing CD3-positive cells from mononuclear cells isolated from peripheral blood, culturing in GM / SCF medium for 5 weeks, staining with an antibody against CD86. After removing CD3-positive cells from mononuclear cells isolated from peripheral blood, the cells were cultured in GM / SCF medium for 4 weeks and further in GM / IL-4 medium for 1 week, and then stained with an antibody against HLA-ABC. The result figure analyzed by cytometry. FIG. 3 is a diagram showing the results of analysis by flow cytometry after removing CD3-positive cells from mononuclear cells isolated from peripheral blood, culturing in GM / SCF medium for 5 weeks, staining with an antibody against HLA-ABC. After removing CD3-positive cells from mononuclear cells isolated from peripheral blood, the cells were cultured in GM / SCF medium for 4 weeks and further in GM / IL-4 medium for 1 week, and then stained with an antibody against HLA-DR. The result figure analyzed by cytometry. FIG. 3 is a diagram showing the results of analysis by flow cytometry after removing CD3-positive cells from mononuclear cells isolated from peripheral blood, culturing in GM / SCF medium for 5 weeks, staining with an antibody against HLA-DR. After removing CD3-positive cells from mononuclear cells isolated from peripheral blood, culture conditions (GM / IL-4, white) for 4 weeks in GM / SCF medium and 1 week in GM / IL-4 medium The graph which compared the average fluorescence intensity (MFI) of the flow cytometry analysis result dye | stained with each cell surface marker after culture | cultivating on GM / SCF culture | cultivation conditions (GM / SCF, black) for 5 weeks. After removing CD3-positive cells from mononuclear cells isolated from peripheral blood, culture conditions (G4) for 4 weeks in GM / SCF medium and further for 1 week in GM / IL-4 medium or in GM / SCF medium The graph which compared the density | concentration of IL-1b released in the culture medium in 2 days after culture | cultivating on culture conditions (GS) for 5 weeks, and stimulating with OK432. After removing CD3-positive cells from mononuclear cells isolated from peripheral blood, culture conditions (G4) for 4 weeks in GM / SCF medium and further for 1 week in GM / IL-4 medium or in GM / SCF medium The graph which compared the density | concentration of IL-6 released in the culture medium in 2 days after culture | cultivating on culture conditions (GS) for 5 weeks, and stimulating with OK432. After removing CD3-positive cells from mononuclear cells isolated from peripheral blood, culture conditions (G4) for 4 weeks in GM / SCF medium and further for 1 week in GM / IL-4 medium or in GM / SCF medium The graph which compared the density | concentration of IL-8 which was stimulated with OK432 and was released | released in the culture medium for 2 days after culture | cultivation on culture conditions (GS) for 5 weeks. After removing CD3-positive cells from mononuclear cells isolated from peripheral blood, culture conditions (G4) for 4 weeks in GM / SCF medium and further for 1 week in GM / IL-4 medium or in GM / SCF medium The graph which compared the density | concentration of IL-10 which was stimulated with OK432 and was released | released in the culture medium for 2 days after culture | cultivation on culture conditions (GS) for 5 weeks. After removing CD3-positive cells from mononuclear cells isolated from peripheral blood, culture conditions (G4) for 4 weeks in GM / SCF medium and further for 1 week in GM / IL-4 medium or in GM / SCF medium The graph which compared the density | concentration of IL-12p70 which was stimulated with OK432 and was released | released in the culture medium in two days after culture | cultivation on culture conditions (GS) for 5 weeks. After removing CD3-positive cells from mononuclear cells isolated from peripheral blood, culture conditions (G4) for 4 weeks in GM / SCF medium and further for 1 week in GM / IL-4 medium or in GM / SCF medium The graph which compared the density | concentration of TNF- (alpha) released in the culture medium in 2 days after culture | cultivating on the culture condition (GS) for 5 weeks, stimulating with OK432.

  The embodiments of the present invention described below are for illustrative purposes only and are not intended to limit the technical scope of the present invention. The technical scope of the present invention is limited only by the appended claims. Modifications of the present invention, for example, addition, deletion, and replacement of the configuration requirements of the present invention can be made on the condition that the gist of the present invention is not deviated.

Blood collection from peripheral blood Peripheral blood was collected from healthy volunteer subjects. This experiment was conducted with the approval of the Kyushu University Medical District Department Clinical Research Ethics Review Board (approval date: March 12, 2010), and written consent was obtained from the volunteer subjects. Yes. For blood collection, a vacuum blood collection tube (TERUMO Venoject II EDTA-2Na) equipped with a 22-23G needle was used.

Separation of mononuclear cells from peripheral blood The obtained blood was diluted 2-fold with a diluent kept at room temperature (PBS supplemented with 1 mM EDTA and 2% fetal calf serum), and diluted blood was added to each centrifuge tube. 20 to 35 mL was overlaid on 10 to 15 mL of Ficoll Paque (specific gravity 1.077). Centrifugation was performed at 580 × g for 20 minutes at room temperature and stopped without braking. The centrifugal supernatant (plasma part) was removed leaving a few mL, and the intermediate layer was recovered. The intermediate layer collected from one or two centrifuge tubes was collected in one new centrifuge tube, and the volume was adjusted to 50 mL with the diluent. The second centrifugation was performed under the conditions of 420 × g, room temperature, 5 minutes or 15 minutes. The supernatant was removed and the pellet was suspended in 30 mL of the diluent. The third centrifugation was performed under conditions of 250 × g and room temperature for 10 minutes. The supernatant was removed, and the pellet was suspended in PBS supplemented with 2 mM EDTA and 0.1% BSA so that the cell concentration was 1 × 10 ⁷ cells / mL (hereinafter referred to as “mononuclear”). Sphere suspension ”).

CD3 magnetic beads removal of cells expressing the anti-CD3 antibody is immobilized (Dynabeads CD3), after being washed once with 0.1% BSA was added PBS, the cells 10 in the monocyte suspension ⁷ 50 μL was added per piece. The mononuclear cell suspension containing the beads was stirred with a rotator at 4 ° C. for 30 minutes. Thereafter, the magnetic beads were separated from the suspension by a magnet, and cells expressing CD3 on the surface were removed.

Culture of cell population from which CD3-expressing cells have been removed Cells remaining in the suspension (hereinafter referred to as “CD3 ^- cells”) were recombinant with 100 ng / mL recombinant human GM-CSF and 50 ng / mL recombinant. 5 × 10 ⁵ IMDM (hereinafter referred to as “GM / SCF medium”) supplemented with human SCF, 10% fetal bovine serum, and antibiotics (penicillin G100 units / mL, streptomycin sulfate 100 μg / mL). The cells were cultured in a 6-well plate (Lipidure Coated Plate (registered trademark), NOF Corporation) coated with a biocompatible polymer containing 2-methacryloyloxyethyl phosphorylcholine (MPC).

Culture of mononuclear cell population containing CD3 expressing cells As a control experiment, a mononuclear cell population containing CD3 expressing cells was cultured. The mononuclear cell suspension 200μL (2 × 10 ⁶ cells) are centrifuged at 400 to 800 × g, 5 min, the resulting pellet is suspended in the GM / SCF medium 3 mL, CD3 ^- cells Incubated in 6-well plates coated with the same MPC polymer used for culture.

Medium change Medium change was performed every 3 to 4 days. Cells are collected, the medium is removed by centrifugation at 400 × g for 5 minutes, and the pellet cells are suspended in the GM / SCF medium to a concentration of 2.5 to 5 × 10 ⁵ cells / mL. Incubated in new 6-well plates coated with MPC polymer.

Monitoring of cells in culture Every week after the start of culture, the expression of cell surface markers CD3, CD11c and CD14 is determined by flow cytometry in addition to the measurement of cell number according to reagents, devices and procedures well known to those skilled in the art. Was analyzed.

Results FIG. 1A and FIG. 2A show the results obtained by analyzing mononuclear cells isolated from peripheral blood in GM / SCF medium for 1 and 2 weeks, respectively, and then double-staining with antibodies against CD3 and CD11c and analyzing by flow cytometry. FIG. FIGS. 1B and 2B show the results obtained by culturing mononuclear cells isolated from peripheral blood in GM / IL-4 medium for 1 and 2 weeks and then double-staining with antibodies against CD3 and CD11c and analyzing by flow cytometry. It is. In the condition of FIG. 1A, the CD11c positive and CD3 negative cells were 27.9%, and the CD11 negative and CD3 positive cells were 44.29%, but in the condition of FIG. 1B, 10.66% and 55 respectively. It was 31%. On the other hand, in the condition of FIG. 2A, the CD11c positive and CD3 negative cells were 5.16% and the CD11 negative and CD3 positive cells were 75.31%. 32% and 74.29%. CD11c positive and CD3 negative cells are dendritic cells or their progenitor cells. As is clear from these results, the ratio of dendritic cells or their precursor cells was higher in the GM / SCF medium than in the GM / IL-4 medium. However, in any medium, the proportion of dendritic cells or their progenitor cells decreased sharply for 2 weeks rather than 1 week, and conversely, the proportion of CD11-negative and CD3-positive cells became much higher. CD3-positive cells are T cells, and it is known that dendritic cells can interact with T cells and stimulate T cells even in vitro. Thus, from these results, when T cells coexist, dendritic cells stimulate T cells, but the proliferation and / or differentiation of dendritic cells themselves may be hindered. In fact, from the results of FIGS. 3A and B, among CD11c negative cells, 87.07% in FIG. 3A and 88.27% in FIG. 3B, about 90% were CD3 positive, that is, T cell lineage cells. It was. Therefore, by removing T cells from mononuclear cells derived from peripheral blood before the start of culture, it is not possible to concentrate on the proliferation and differentiation of dendritic cells themselves without being bothered by the interaction with T cells. I got the idea.

FIG. 4 shows the number of CD11c positive cells when CD3 ⁻ cells are cultured from mononuclear cells in peripheral blood (solid line) and when the mononuclear cell population is cultured as it is without removing CD3 expressing cells (broken line). It is a growth curve. In any case, the number of CD11c-positive cells per mL of peripheral blood collected from 3 subjects was measured at the start of culture, 1 week, 2 weeks, ..., 5 weeks later. Was calculated as the product of the total number of cells and the percentage of CD11c positive cells at each time point. When the remaining cells from which CD3-expressing cells are removed from mononuclear cells in peripheral blood are cultured, the number of CD11c positive cells continues to increase until 4 weeks after the start of culture, and reaches a maximum after 5 weeks. During this time, the rate of increase continues to increase until after 3 weeks, but the rate of increase decreases after 3 weeks, and there is little increase between 4 and 5 weeks. The proportion of CD11c positive cells was about 20% at the start of culture, but exceeded 95% after 4 weeks of culture. In contrast, when the mononuclear cell population was cultured as it was without removing CD3-expressing cells, no increase in the number of CD11c positive cells was observed from the beginning of the culture, as shown in FIGS. 1A and 2A. Similarly, it decreased significantly after 2 weeks. From these results, it was shown that by removing CD3-expressing cells, ie, T cells, and culturing in the GM / SCF medium, only dendritic cells can be selectively amplified 10 to 100 times. .

Maturation of dendritic cells by switching of medium In order to differentiate the precursor cells of dendritic cells into dendritic cells after CD3 ^- cells were cultured for 4 weeks in the GM / SCF medium according to the protocol of Example 1, Has been switched. The new medium is 100 ng / mL recombinant human GM-CSF, 50 ng / mL recombinant human IL-4, 10% fetal bovine serum, and antibiotics (penicillin G100 units / mL, streptomycin sulfate 100 μg / mL) And IMDM (hereinafter referred to as “GM / IL-4 medium”) to which 4 and 4 were added, 4 weeks after the start of the culture, the GM / SCF medium was switched to the GM / IL-4 medium, and further cultured for 1 week. . In control experiments, CD3 ^- cells were cultured in GM / SCF media for 5 consecutive weeks without switching media after 4 weeks.

Examination of Dendritic Cell Differentiation Marker Five weeks after the start of culture, cells cultured under each experimental condition were subjected to analysis by a flow cytometry method according to a conventional method. The cell surface markers used were CCR5, CCR7, CD40, CD54, CD80, CD83, CD86, HL-ABC and HLA-DR. In addition, in order to examine the response to stimulation by OK-432, the cells after 5 weeks of culture were supplemented with 0.5KE / mL of OK-432 at a concentration of 1 × 10 ⁶ cells / mL. It was suspended in IMDM and further cultured for 48 hours. Thereafter, the production amounts of IL-1b, IL-6, IL-8, IL-10, IL-12p70, and TNF-α were measured.

  5A to 13B show graphs comparing the results of flow cytometry analysis for each cell surface marker. Moreover, the measured value of the mean fluorescence intensity (MFI) of the flow cytometry analysis result of each cell surface marker is shown in FIG. In these figures, “GM / IL-4” was prepared by the method of the present invention, which was switched from the GM / SCF medium to the GM / IL-4 medium after 4 weeks from the start of the culture, and further cultured for 1 week. Represents the analysis results in cells. “GM / SCF” represents the result of analysis in cells of a control experiment cultured in GM / SCF medium for 5 consecutive weeks. As is clear from FIG. 2, for CD83 and CD86, the cells prepared by the method of the present invention were expressed more strongly than the cells of the control experiment. CD83 and CD86 are known to increase in expression when dendritic cells are differentiated. In addition, when dendritic cells function as antigen-presenting cells, HLA molecules that form a complex with antigen fragments were also expressed more strongly in cells prepared by the method of the present invention than in control experiments. . That is, as is apparent from these figures, it was shown that the cells prepared by the method of the present invention expressed differentiation markers as dendritic cells and had a function as antigen-presenting cells.

  15A to 15F are graphs showing the T cell stimulating ability of dendritic cells prepared by the method of the present invention. 15A to 15F, “G4” is a cell prepared by the method of the present invention, which was switched from the GM / SCF medium to the GM / IL-4 medium after 4 weeks from the start of the culture, and further cultured for 1 week. Represents the analysis result. “GS” represents the result of analysis in cells of a control experiment cultured in GM / SCF medium for 5 consecutive weeks. As is apparent from FIGS. 15A to 15F, IL-12p70 is expressed only in cells prepared by the method of the present invention, and conversely, IL-1b, IL-6, and IL-8 are expressed only in control experimental cells. It was. From this, it was proved that the cells prepared by the method of the present invention were mature dendritic cells also from the viewpoint of the ability to respond to stimulation by OK-432.

  When the remaining cells from which CD3 expressing cells were removed from mononuclear cells in peripheral blood were cultured, CD11c positive cells grew 10 to 100 times in 5 weeks, but CD3 expressing cells were not removed. The CD11c positive cells did not grow at all, but rather decreased to 1/10 of a few minutes after 1 week and 1/10 after 1 week. As is clear from FIG. 1, CD11c positive cells could be proliferated in large quantities by removing CD3-expressing cells from peripheral blood-derived mononuclear cells. Moreover, the cell prepared by the method of the present invention has the same ability as the dendritic cell just recovered from the living body, as proved by the experimental result of Example 2. In the method of preparing dendritic cells currently employed, the number of CD11c positive cells does not increase in vitro because it is cultured only for 2 days in the forcing method and 5 to 7 days in the normal method. It is known to decrease. On the other hand, according to the method of the present invention, it can be increased 10 times to 100 times, so the method of the present invention is very useful.

Claims (1)

  A step of separating mononuclear cells from blood cells collected from a subject, a step of removing T cells from the mononuclear cells, and a remaining cell population from which the T cells have been removed are designated as GM-CSF and SCF. A pharmaceutical composition for dendritic cell transplantation therapy, comprising: a dendritic cell population prepared by a preparation method comprising: culturing in a medium containing