JP2018007599A - Methods for preparing dendritic cell population - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide methods for obtaining a large amount of dendritic cells from blood cells taken from a subject.SOLUTION: Disclosed is a method for preparing a population of dendritic cells comprising the steps of: (a) isolating monocytes from collected blood cells; (b) removing cells expressing CD3 on the cell surface from the monocytes isolated in the step (a); and (c) culturing the monocytes obtained in the step (b) in a culture medium containing GM-CSF and SCF using a culture vessel coated with a novel coating agent.SELECTED DRAWING: None

Description

  The present invention relates to a method for culturing dendritic cells. Specifically, the present invention relates to a method for culturing dendritic cells, which comprises using a culture vessel coated with a novel coating agent.

Dendritic cells can stimulate or suppress the immune system, including both acquired and innate immunity, as antigen-presenting cells. 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. 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 1 and 2). 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 collected from one person even when apheresis is performed.

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, since immunotherapy is often attempted when other treatments are ineffective at present, the order of 10 ⁹ necessary for effective treatment is possible even if apheresis is used due to side effects of other treatments. The inability to prepare dendritic cells or their progenitor cells prevents the clinical trial from expanding.

  As a method for culturing a large amount of dendritic cells from dendritic cell precursor cells, mononuclear cells are separated from blood cells collected from a subject, T cells are removed from the separated mononuclear cell population, and the rest A method for culturing dendritic cells by culturing these cells in a predetermined medium has been proposed (Patent Document 1).

JP2011-239701A

Harada, Y .; Et al., PLoS ONE, 4: e6674 (2009) Tatsuta, K .; Et al., Gene Ther. 16: 240-251 (2009)

  The method for culturing dendritic cells described in Patent Document 1 is a very excellent method for obtaining a large amount of dendritic cells from blood cells collected from a subject in order to perform dendritic cell transplantation therapy. is there. The present inventors examined a more efficient method for culturing dendritic cells from various angles based on the method described in Patent Document 1.

  As described above, the present inventors examined a more efficient method for culturing dendritic cells from various angles based on the method described in Patent Document 1. Then, the inventors of the present invention surprisingly more efficiently than the method described in Patent Document 1 by using a culture vessel coated with a novel coating agent in examining various culture conditions. Thus, the inventors have found that dendritic cells can be cultured, and have completed the present invention.

That is, the present invention, in one embodiment, is a method for preparing a dendritic cell population,
(A) separating the mononuclear cell population from the collected blood cell population;
(B): removing a cell population expressing CD3 on the cell surface from the mononuclear cell population separated in the step (a);
(C): A cell having a coating layer formed from a fluoropolymer containing a polymer unit based on a monomer having a biocompatible group as a culture vessel using the mononuclear cell population obtained in the step (b). The present invention relates to an adjustment method including a step of culturing in a medium containing GM-CSF and SCF using a culture vessel.

Moreover, in one embodiment of this invention, the fluorine atom content rate in the said fluoropolymer is 5-60 mass%, and the ratio P represented by the following Formula is 0.1-4.5 mass%. It is characterized by being.
(Ratio P) = [(Ratio of units having a biocompatible group to all units of fluoropolymer (mass%)) / (Fluorine atom content of fluoropolymer (mass%))] × 100

  In one embodiment of the present invention, the coating layer has a thickness of 1 nm to 100 μm.

  In one embodiment of the present invention, the cell population that expresses CD3 on the cell surface is a T cell population.

  In one embodiment of the present invention, the step (b) further comprises a cell population expressing CD19 on the cell surface from the mononuclear cell population separated in the step (a), and CD56 on the cell surface. It is a step of removing a cell population to be expressed.

In one embodiment of the present invention, the cell population expressing CD19 on the cell surface is a B cell population, and the cell population expressing CD56 on the cell surface is an NK cell population. To do.

  In one embodiment of the present invention, after step (c), (d): the cell population cultured in step (c) is further cultured in a medium containing GM-CSF and IL-4. A step.

  In one embodiment of the present invention, the culture period in step (c) is 2 to 5 weeks, and the culture period in step (d) is 2 to 14 days.

  Moreover, in one embodiment of the present invention, after the step (d), the method includes (e): stimulating the cell population cultured in the step (d) with OK-432. To do.

  In one embodiment of the present invention, the collected blood cell population is a collected blood cell population derived from peripheral blood, umbilical cord blood, bone marrow and / or lymph node.

  In one embodiment of the present invention, the collected blood cell population is a blood cell population collected from the collected peripheral blood by an apheresis method.

  Further, it goes without saying that an invention in which one or more features of the present invention listed above are arbitrarily combined is also included in the scope of the present invention.

FIG. 1 shows a schematic diagram of a cell culture process (New Coat, MPC, Adhesion) performed in the examples of the present application. FIG. 2 shows the composition of the mononuclear cell population after separation by Ficoll, and the mononuclear cell population after further removal of CD3-expressing cells, CD19-expressing cells, and CD56-expressing cells by flow cytometry. Results are shown. FIG. 3 shows changes in the number of cells under each culture condition (Day 0 to 29). FIG. 4 shows the transition of the number of CD11c positive cells under each culture condition (Day 0 to 29). FIG. 5 shows the amplification factor of the number of CD11c positive cells at Day 0 to 29 in each culture condition. FIG. 6 shows the expression of cell surface markers in the final product at the end of culture (CD11c, HLA-ABC, CD83). FIG. 7 shows the expression of cell surface markers in the final product at the end of culture (CD80, CD86, HLA-DR).

<Step (a)>
The origin of the blood cell population used in step (a) of the method of the present invention may be, for example, collected peripheral blood, collected bone marrow, collected lymph nodes, or collected umbilical cord blood. As long as the method of the present invention can be performed, the present invention is not limited to these, and blood cell populations derived from various origins can be used. For example, an embryonic stem cell, an adult stem cell, or a hematopoietic stem cell derived from an induced pluripotent stem (iPS) cell (or a blood cell population prepared from the hematopoietic stem cell) or the like is used in the step (a ) In “collected blood cell population”. However, the origin of the blood cell population used in step (a) of the method of the present invention is preferably peripheral blood.

  In step (a) of the method of the present invention, the method for separating the mononuclear cell population from the blood cell population is not limited, and those skilled in the art can implement it in various ways. For example, a mononuclear cell population can be separated from a blood cell population by density gradient centrifugation.

<Step (b)>
In the step (b) of the method of the present invention, the method for removing the cell population expressing CD3 on the cell surface from the mononuclear cell population is not limited, and those skilled in the art can implement it in various ways. For example, Dyna3 by Dynale (trademark) sold by Life Technologies, CliniMACS (trademark) from Miltenyi Biotech and CD3 microbeads are used to place CD3 on the cell surface. Cell populations expressed in can be removed. Alternatively, a cell population that expresses CD3 on the cell surface may be removed using flow cytometry or other immunological separation means, for cell populations that express CD3 on the cell surface (eg, T cells). Specific binding partners may be utilized to remove by selectively injuring or killing only the cell population that expresses CD3 on the cell surface.

  In addition, in step (b) of the method of the present invention, in addition to a cell population expressing CD3 on the cell surface, a cell population expressing CD19 on the cell surface and a cell population expressing CD56 on the cell surface are simply used. It may be removed from the nucleus population. A method for removing a cell population that expresses CD19 on the cell surface and a method for removing a cell population that expresses CD56 on the cell surface are not limited, and those skilled in the art can implement them in various ways. For example, a cell population that expresses CD19 on the cell surface and a cell population that expresses CD56 on the cell surface can be removed using CD19 microbeads or CD56 microbeads manufactured by Miltenyi Biotech. Alternatively, the cell population expressing CD19 on the cell surface and the cell population expressing CD56 on the cell surface may be removed using flow cytometry or other immunological separation means, and CD19 is expressed on the cell surface. Utilizing specific binding partners for cell populations (eg, B cells) and cell populations that express CD56 (eg, NK cells), CD19 is expressed on the cell surface and CD56 is expressed on the cell surface Only cell populations that do so may be removed by selectively injuring or killing them.

  In the method of the present invention, cells that express CD3 on the cell surface from the mononuclear cell population before the step of culturing the mononuclear cell population in a medium containing GM-CSF and SCF (ie, step (c)). The removal of the population (especially T cells) is not simply to remove lymphocytes and enrich the mononuclear cell population. For example, as shown in JP2011-239701A, when a mononuclear cell population is cultured in a medium to which a cytokine cocktail containing GM-CSF and SCF is added, the proliferation of T cells is significantly promoted rather than the dendritic cells themselves proliferating. A cell population with a low proportion of dendritic cells is formed. This is presumably because the interaction with T cells prevents the growth and / or differentiation of the dendritic cells themselves. Therefore, the T cells are removed so that the dendritic cells do not unnecessarily interact with the T cells. By including this step, as shown in the Examples of the present application, it is possible to prepare a dendritic cell population containing dendritic cells at a rate of almost 100% and having a high proliferation efficiency.

<Step (c)>
In the method of the present invention, a culture vessel coated with a coating agent comprising a fluoropolymer having a polymer unit based on a monomer having a bioaffinity group is prepared from the mononuclear cell population prepared by the above method. Culture. By culturing a mononuclear cell population using this vessel, for example, by a cell low adsorption culture vessel coated with a non-fluorine compatible polymer coating containing 2-methacryloyloxyethyl phosphorylcholine (MPC), or by corona discharge treatment. Compared with the case of culturing using a polystyrene cell adhesion culture vessel whose surface is hydrophilically treated (for example, Nunclon ™ Delta Surface, Thermo Fisher Scientific Co., Ltd.), dendritic cells can be prepared extremely efficiently. Yes (see examples in this application). An example of such a culture container is EZ-BindShut (registered trademark) SP (manufactured by AGC Techno Glass Co., Ltd.).

A preferred bioaffinity group is selected from the group consisting of the following groups (1), (2) and (3) from the viewpoint of easily forming a coating layer having a high protein adsorption preventing effect. At least one is preferred. As the bioaffinity group, the group (1) alone, or either one or both of the group (2) and the group (3) are preferable from the viewpoint that an effect of preventing protein adsorption is easily obtained, and the group (1), Any one of group (2) or group (3) is particularly preferred. When the fluoropolymer (A) contains the groups (1) to (3), it is excellent in biocompatibility.

  However, in said formula, n is an integer of 1-10, m is an integer of 1-100 when group (1) is contained in a side chain in a fluoropolymer (A), and is contained in a principal chain. R1 to R3 are each independently an alkyl group having 1 to 5 carbon atoms, a is an integer of 1 to 5, b is an integer of 1 to 5, and R4 And R5 are each independently an alkyl group having 1 to 5 carbon atoms, X- is the following group (3-1) or the following group (3-2), and c is an integer of 1 to 20. Yes, d is an integer of 1-5.

Group (1):
The group (1) has high mobility in blood or the like, and it is difficult to adsorb proteins to be adsorbed on the surface of the coating layer.
The group (1) may be contained in the main chain of the fluoropolymer (A) or in the side chain.
In the group (1), n is preferably an integer of 1 to 6 and particularly preferably an integer of 1 to 4 from the point that protein is difficult to adsorb.
The group (1) may be linear or branched. The group (1) is preferably linear because it has a higher protein adsorption inhibitory effect.
When group (1) is contained in the side chain of the fluoropolymer (A), m in group (1) is preferably from 1 to 40, particularly preferably from 1 to 20, from the viewpoint of excellent water resistance.
When group (1) is contained in the main chain of the fluoropolymer (A), m in group (1) is preferably from 5 to 300, particularly preferably from 10 to 200, from the viewpoint of excellent water resistance.
When m is 2 or more, (CnH2nO) of the group (1) may be one or two or more. In the case of two or more types, the arrangement may be random, block, or alternating. When n is 3 or more, it may be a straight chain structure or a branched structure.
When the fluoropolymer (A) has a group (1), the group (1) of the fluoropolymer (A) may be one type or two or more types.

Group (2):
Group (2) has a strong affinity for phospholipids in blood, but has a weak interaction force with plasma proteins. Therefore, by using the fluoropolymer (A) having the group (2), for example, in blood, phospholipid is preferentially adsorbed on the coating layer, and the phospholipid self-assembles to form an adsorption layer. It is thought that it is done. As a result, since the surface has a structure similar to the vascular endothelial surface, adsorption of proteins such as fibrinogen is suppressed.
The group (2) is preferably contained in the side chain of the fluoropolymer (A).
R1 to R3 in the group (2) are each independently an alkyl group having 1 to 5 carbon atoms, and an alkyl group having 1 to 4 carbon atoms is preferable, and a methyl group is particularly preferable from the viewpoint of availability of raw materials. .
A in the group (2) is an integer of 1 to 5, and an integer of 2 to 5 is preferable from the viewpoint of availability of raw materials, and 2 is particularly preferable.
In the group (2), b is an integer of 1 to 5, and an integer of 1 to 4 is preferable and 2 is particularly preferable from the viewpoint that protein is difficult to adsorb.
When the fluoropolymer (A) has a group (2), the group (2) of the fluoropolymer (A) may be one type or two or more types.

Group (3):
By using the fluoropolymer (A) having the group (3), protein adsorption is suppressed for the same reason as in the case of using the fluoropolymer (A) having the group (2).
The group (3) is preferably contained in the side chain of the fluoropolymer (A).
R4 and R5 in the group (3) are each independently an alkyl group having 1 to 5 carbon atoms, and an alkyl group having 1 to 4 carbon atoms is preferable, and a methyl group is particularly preferable from the viewpoint that protein is difficult to adsorb.
C in the group (3) is an integer of 1 to 20, preferably an integer of 1 to 15, more preferably an integer of 1 to 10, and more preferably 2 from the viewpoint that the fluoropolymer (A) is excellent in flexibility. Particularly preferred.
D in the group (3) is an integer of 1 to 5, and an integer of 1 to 4 is preferable and 1 is particularly preferable from the viewpoint that protein is difficult to adsorb.

Preferred fluorinated polymer The preferred fluorinated polymer is any one of the following 1) to 3).
1) The unit (m1) derived from the monomer represented by the following formula (m1), the unit (m2) derived from the monomer represented by the following formula (m2), and the following formula (m3) And a fluorine-containing polymer having at least one selected from the group consisting of units derived from the monomer (m3).

(Wherein R ⁶ is a hydrogen atom, a chlorine atom or a methyl group, e is an integer of 0 to 3, and R ⁷ and R ⁸ are each independently a hydrogen atom, a fluorine atom or a trifluoromethyl group. R ^f1 is a C 1-20 perfluoroalkyl group, R ⁹ is a hydrogen atom, a chlorine atom or a methyl group, and Q ¹ is —C (═O) —O— or C (═O). —NH—, R ^{1 to} R ³ are each independently an alkyl group having 1 to 5 carbon atoms, a is an integer of 1 to 5, b is an integer of 1 to 5, and R ¹⁰ is hydrogen. An atom, a chlorine atom or a methyl group, Q ² is —C (═O) —O— or C (═O) —NH—, and R ⁴ and R ⁵ are each independently an alkyl having 1 to 5 carbon atoms. a group, X ^- is a group represented by the group or the following formula represented by the following formula (3-1) (3-2), c is 1 to 20 integer Ri, d is an integer of 1-5.)

2) A fluoropolymer having a unit (m1) derived from a monomer represented by the following formula (m1) and a unit (m4) derived from a monomer represented by the following formula (m4).

(Wherein R ⁶ is a hydrogen atom, a chlorine atom or a methyl group, e is an integer of 0 to 3, and R ⁷ and R ⁸ are each independently a hydrogen atom, a fluorine atom or a trifluoromethyl group. R ^f1 is a C 1-20 perfluoroalkyl group, R ¹¹ is a hydrogen atom, a chlorine atom or a methyl group, and Q ³ is —COO— or COO (CH ₂ ) _h —NHCOO— (wherein , H is an integer of 1 to 4, and R ¹² is a hydrogen atom or (CH ₂ ) _i -R ¹³ (wherein R ¹³ is an alkoxy group having 1 to 8 carbon atoms, a hydrogen atom, a fluorine atom, A trifluoromethyl group, a hydroxy group or a cyano group, i is an integer of 1 to 25), f is an integer of 1 to 10, and g is an integer of 1 to 100.)

3) A segment (I) containing a unit (m6) derived from a monomer represented by the following formula (m6), and a molecule derived from a polymer azo initiator having a structure represented by the following formula (6) A fluorine-containing polymer having a segment (II) containing a chain.

(Wherein R ¹⁶ is a hydrogen atom, a chlorine atom or a methyl group, Q ⁵ is a single bond or a divalent organic group, and R ¹⁷ is an etheric oxygen atom between the carbon atoms. And a polyfluoroalkyl group having 1 to 6 carbon atoms, α is an integer of 5 to 300, and β is an integer of 1 to 20.)

The fluorine atom content rate in the said fluoropolymer can be 5-60 mass%, for example, it is preferable that it is 10-50 mass%, and it is more preferable that it is 15-40 mass%. Further, the ratio P represented by the following formula is more preferably 0.1 to 4.5% by mass.
(Ratio P) = [(Ratio of units having a biocompatible group to all units of fluoropolymer (mass%)) / (Fluorine atom content of fluoropolymer (mass%))] × 100

  The thickness of the coating layer can be, for example, 1 nm to 100 μm, preferably 5 nm to 50 μm, and more preferably 10 nm to 10 μm.

  In the method of the present invention, the medium for culturing cells includes, in addition to IMDM medium, MEM, DMEM, RPMI-1640, and X-VIVO15 medium, but not limited thereto, 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 method of the present invention includes culturing cells using a medium containing GM-CSF and SCF. In this step, the medium containing GM-CSF and SCF contains GM-CSF and SCF, but may be a medium substantially free of cytokines other than these.

<Step (d)>
The method of the present invention may comprise the step of culturing cells using a medium comprising GM-CSF and IL-4. In this step, the medium containing GM-CSF and IL-4 may be a medium containing GM-CSF and IL-4 but not containing SCF, and may contain GM-CSF and IL-4. However, a medium substantially free of cytokines other than these may be used.

<Step (e)>
In the method of the present invention, OK-432, TNF-α, IL-1β, IL-6 are used to acquire the T cell stimulating ability of the amplified dendritic cells after the culture step for amplifying the dendritic cells. Stimulating with a pro-inflammatory mediator such as PGE ₂ (preferably OK-432) may further be included.

<Culture conditions for each step>
In the method of the present invention, the cytokine added to the medium preferably has a human amino acid sequence, and is preferably produced by recombinant DNA technology for safety. The concentration of GM-CSF in the medium may range from 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, It may range from 10 to 150 ng / mL, 20 to 120 ng / mL or 30 to 100 ng / mL. The concentration of SCF or IL-4 in the medium may range from 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. It may range from mL, 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, the medium substantially free of a certain cytokine may be, for example, a medium containing the cytokine within a range not significantly exceeding the concentration of the cytokine in normal serum. More specifically, in a medium containing the cytokine at 3 times or less, 2 times or less, 1 time or less, 1/2 or less, 1/3 or less, or 1/5 or less of the concentration of the cytokine in normal serum. It may be. Alternatively, the cytokine is 50 ng / mL or less, 40 ng / mL or less, 30 ng / mL or less on the condition that it behaves substantially the same as the behavior of dendritic cells or their precursor cells when the concentration of the cytokine is 0. , 20 ng / mL or less, 10 ng / mL or less, 5 ng / mL or less, 3 ng / mL or less, 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, more dendritic cells can be obtained when the period of culturing in a medium containing GM-CSF and SCF is 2 to 5 weeks. This is advantageous. On the other hand, when the culture period is less than 2 weeks and exceeds 5 weeks, the number of dendritic cells obtained may not be sufficient.

Actually, as shown in Examples of the present application, CD11c positive cells increase 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 and the number of precursor cells of dendritic cells does not increase even when cultured in a medium containing GM-CSF and SCF. It is time to become.

  Therefore, in the method of the present invention, the culture period in the step of culturing the cells in a medium containing GM-CSF and SCF may be 2 to 5 weeks, more preferably 2 to 3 weeks. In addition, the culture period of the culture period in the step of culturing the cells in the medium containing GM-CSF and IL-4 after switching from the medium containing GM-CSF and SCF to the medium containing GM-CSF and IL-4 May be from 2 days to 14 days, more preferably from 2 days to 7 days.

<Embodiment>
In one embodiment of the present invention, the mononuclear cell population separated from the collected blood cell population may be once stored frozen and then thawed at a preferred timing for use in the next step. In one embodiment of the present invention, after the cells expressing CD3 on the surface are removed from the mononuclear cell population, the remaining mononuclear cell population is cryopreserved, and then at a preferred timing. It may be melted and used for the next step. For cell freezing, for example, a bun bunker (trademark) manufactured by Lymphoctech Co., Ltd., other commercially available cell cryopreservation solutions or self-prepared cell cryopreservation solutions may be used.

<Uses of dendritic cells>
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 dendritic cells prepared by the method of the present invention may be further prepared as a pharmaceutical composition (including regenerative medicine products such as cellular pharmaceutical compositions) for the treatment of diseases. For example, an agent for other therapy, such as a chemotherapeutic agent or a radiation therapy agent, may be combined with the dendritic cells prepared by the method of the present invention. 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.

  A pharmaceutical composition containing dendritic cells prepared by the method of the present invention (including regenerative medicine products such as cellular pharmaceutical compositions) may contain a pharmaceutically acceptable carrier. Examples of the carrier include any solution capable of suspending living cells, such as physiological saline, phosphate buffered saline (PBS), medium, serum, and the like. When dendritic cells are used as a vaccine, an immunogenicity enhancing agent may be used in combination in order to enhance immunogenicity. Examples of the immunogenicity enhancer include immunostimulators such as cytokines, bacterial toxins, and Mycobacterium tuberculosis cell components, and alum, Freund's adjuvant, and other adjuvant agents.

  The terms used in this specification are used to describe specific embodiments, unless otherwise defined, and are not intended to limit the invention.

  In addition, the term “comprising” as used herein is intended to mean that there is a matter (member, step, element, number, etc.) described, unless the context clearly requires a different understanding. It does not exclude the presence of other items (members, steps, elements, numbers, etc.).

  Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms used herein should be interpreted as having a meaning consistent with the meaning in this specification and the related technical field, unless otherwise defined, idealized, or overly formal. It should not be interpreted in a general sense.

  In the following, the present invention will be described in more detail with reference to examples. However, the invention can be embodied in various ways and should not be construed as limited to the embodiments set forth herein.

<Materials and methods>

  An outline of the cell culture process performed in this example is shown in FIG.

Peripheral blood collection 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 Committee (approval date: August 21, 2013), and written consent was obtained from the volunteer subjects. Yes. Blood collection was performed using a vacuum blood collection tube (TERUMO Venoject II EDTA-2Na) equipped with a 22-23G needle or by apheresis.

Separation of mononuclear cells from peripheral blood The obtained blood was diluted 2-fold with diluent 1 (D-PBS) kept at room temperature, and 20 mL to 30 mL of diluted blood was added to 20 mL of Ficoll Paque in each centrifuge tube. Overlaid with PLEMIUM (specific gravity 1.077). Centrifugation was performed at 500 × 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, the volume was adjusted to 50 mL with the diluent 1, and centrifugation was further performed twice. Thereafter, the supernatant was removed, and the pellet was suspended in Diluent 2 (PBS to which 2 mM EDTA and 2% fetal calf serum were added) (hereinafter referred to as “mononuclear cell suspension”).

Removal of CD3-expressing cells, CD19-expressing cells, and CD56-expressing cells Each magnetic bead (CD3 microbead, human, Cat. No. 130-050) to which an anti-CD3 antibody, anti-CD19 antibody, or anti-CD56 antibody is immobilized. CD19 microbeads, human, Cat.no.130-050-301; CD56 microbeads, human, Cat.no.130-050-401 (Milteny Biotech KK)) Added to the suspension. The mononuclear cell suspension containing the beads was allowed to stand at 4 ° C. for 15 minutes. Thereafter, the magnetic beads are separated from the suspension by an LD column and a manual cell separator, and cells expressing CD3 on the surface (CD3 expressing cells), cells expressing CD19 on the surface (CD19 expressing cells), and Cells expressing CD56 on the surface (CD56 expressing cells) were removed.

  FIG. 2 shows the results of examining the composition of the mononuclear cell population after separation by Ficoll and the composition of the mononuclear cell population after removing CD3-expressing cells, CD19-expressing cells, and CD56-expressing cells by the flow cytometry method. It was shown to.

Cultivation of cell population after removal: Examination of influence of surface treatment of culture vessel on cell proliferation The cells remaining in the suspension (hereinafter referred to as “CD3 ^- CD19 ^- CD56 ^- cells”) were 10 ng / mL. IMDM (hereinafter referred to as “GM”) to which human GM-CSF, ng / mL recombinant human SCF, 10% fetal bovine serum, and antibiotics (penicillin G100 units / mL, streptomycin sulfate 100 μg / mL) were added. Cell-adsorbing culture vessel (EZ-BindShut (registered trademark) SP) coated with a fluoropolymer containing polymerized units based on a monomer having a biocompatible group and diluted with a / SCF medium. AGC Techno Glass Co., Ltd. (hereinafter referred to as “New Coat”), 2-methacryloyloxyethyl phosphorylcholine (MPC) Cell adsorption low culture container (EZ-Bindshut (registered trademark) II, AGC Techno Glass Co., Ltd., 6 well plate) (hereinafter referred to as MPC) coated with a body compatible polymer coating, and the surface is made hydrophilic by corona discharge treatment The cells were cultured in a polystyrene cell adhesion culture vessel (Nunclon ™ Delta Surface, manufactured by Thermo Fisher Scientific Co., Ltd., 6 well plate) (hereinafter referred to as Adhesion).

Medium exchange Medium exchange was performed every 3 to 4 days. The cells were collected, the medium was removed by centrifugation at 500 × g for 5 minutes, and the pelleted cells were suspended in the GM / SCF medium and continuously cultured in New Coat, MPC, and Adhesion, respectively. .

Monitoring of cells in culture Every week from the start of culture, the expression of cell surface markers CD11c and CD14 is analyzed by flow cytometry in addition to counting the number of cells according to reagents, devices and procedures well known to those skilled in the art. It was.

Dendritic cell maturation by switching medium After CD3 ⁻ CD19 ⁻ CD56 ⁻ cells are cultured in the GM / SCF medium for 3 weeks in New Coat, MPC, Adhesion, the precursor cells of dendritic cells are differentiated into dendritic cells. The medium was switched to make it happen. The new medium is CellGro (registered trademark) DC Medium (hereinafter referred to as “GM / IL-4 medium”) supplemented with 50 ng / mL recombinant human GM-CSF and 50 ng / mL recombinant human IL-4. )), After 3 weeks from the start of the culture, the GM / SCF medium was switched to the GM / IL-4 medium, and further cultured for 1 week.

Examination of Dendritic Cell Differentiation Markers CD3 ⁻ CD19 ⁻ CD56 ⁻ cells 4 weeks after the start of culture were examined using CellGro DC (hereinafter referred to as “OK”) to which OK-432 was added in order to examine the response to stimulation by OK-432. -432 medium ") and further cultured overnight. The cells were subjected to analysis by a flow cytometry method according to a conventional method for measuring dendritic cell differentiation markers. The cell surface markers used were CD11c, HLA-ABC, CD83, CD80, CD86, and HLA-DR.

<Result>

Number of cells during amplification culture (day 0-29)
FIG. 3 is a growth curve of cells obtained by culturing CD3 ⁻ CD19 ⁻ CD56 ⁻ cells with New Coat, MPC, and Adhesion, respectively. In either case, the number of cells increased in the GM / SCF medium for 3 weeks, and continued to increase until switched to GM / IL-4 medium.

CD11c positive rate transition during amplification culture period (day 0-29)
In the cells at the start of culture (day 0), the proportion of CD11c-positive and CD14-positive cells in the cell population from which CD3, CD19, and CD56-expressing cells on day 0 of culture were removed was 52.3% (common to all groups). CD11c-positive and CD14-positive cells are dendritic cells or their precursor cells. As these cultures continue in the GM / SCF medium, CD14 gradually becomes negative. For each of New Coat, MPC, and Adhesion, the CD11c positive rate transition during the amplification culture period (day 0 to 29) was monitored. In all cases, the positive rate decreased slightly from the second week to the third week of culture in GM / SCF medium. Although it was observed, after switching to GM / IL-4 medium, the positive rate increased to about 90% by culturing for 1 week (that is, at day 29) (data not shown).

  FIG. 4 (Transition of cell number during amplification culture period (day 0 to 29)) shows the transition of the number of CD11c positive cells during the amplification culture period. FIG. 5 shows the amplification factor of the number of CD11c positive cells during day 0 to 29. Is shown. In any case, the number of CD11c positive cells was determined at the start of culture, the total number of cells at the 7th day, the 14th day, the 21st day, the 28th day, and the 29th day. Calculated as the product of the percentage of CD11c positive cells. The number of CD11c positive cells continued to increase until the 3rd week in the order of New Coat, Adhesion, and MPC, and became 10.7 times, 9.4 times, and 7.0 times from the beginning of the culture at the 3rd week stage, respectively. . Thereafter, when switching to GM / IL-4 medium and entering the differentiation induction stage of dendritic cell precursor cells into dendritic cells, the rate of increase decreases or stops, and the final number of CD11c positive cells is New Coat, MPC. , 13.3 times, 11.9 times, and 4.6 times in the order of Adhesion. Adhesion may have hindered proliferation and / or differentiation of dendritic cells per se due to the effect of cell adhesion to the culture vessel, or may have caused proliferation of cells other than dendritic cells. Thus, in the culture of the present invention, the coating of the culture vessel is important, and it was considered that the culture vessel provided with the low cell adsorption coating, particularly the coating by “New Coat” was most suitable.

Analysis of Dendritic Cell Differentiation Markers FIGS. 6 and 7 are graphs comparing flow cytometry analysis results of cell surface markers of cells cultured in New Coat, MPC, and Adhesion. The cells used for the analysis were prepared by switching from GM / SCF medium to GM / IL-4 medium 3 weeks after the start of culture, further culturing for 1 week, and then culturing overnight in OK-432 medium. It was. As shown in FIGS. 6 and 7, in New Coat, expression was confirmed for both CD83 and CD86, which are known to increase in expression when dendritic cells differentiate. In addition, when dendritic cells function as antigen-presenting cells, HLA molecules forming a complex with antigen fragments were also expressed without problems. From this, it was shown that the cell prepared by New Coat expresses a differentiation marker as a dendritic cell and has a function as an antigen-presenting cell.

  According to the standard definition of dendritic cells (Clin Cancer Res 2011; 17: 3064-3076), 70% or more of cells express MHC class II and CD86 by flow cytometry. Is described as a requirement for dendritic cells, and it was also confirmed that the cells obtained by the method of the present invention satisfy this condition.

  From the above results, according to the method of the present invention, it is possible to obtain a large number of dendritic cells as compared with the prior art. That is, by using the method of the present invention, it becomes easier to secure the number of dendritic cells for use in dendritic cell transplantation therapy. In addition, by using the method of the present invention, a certain number of dendritic cells can be obtained in a shorter period of time, and earlier treatment is possible for patients who need dendritic cell transplantation therapy. In dendritic cell transplantation therapy, the preparation of dendritic cells used for treatment becomes a bottleneck, so the necessary amount of dendritic cells can be prepared as soon as possible, especially for patients who require early treatment. Can be a great advantage in therapy.

  In addition, since the culture vessel used in the method of the present invention has less adhesion of dendritic cells to the culture vessel as compared with the conventional culture vessel, if the method of the present invention is used, it exceeds the value shown as actual data. Many dendritic cells can be recovered. Furthermore, if the method of the present invention is used, damage to cells due to detachment from the culture vessel can be reduced, and thus more normal (or higher biological activity) dendritic cells can be recovered.

Claims (10)

A method for preparing a dendritic cell population comprising:
(A) separating the mononuclear cell population from the collected blood cell population;
(B): removing a cell population expressing CD3 on the cell surface from the mononuclear cell population separated in the step (a);
(C): a cell culture vessel having a coating layer formed from a fluoropolymer containing a polymer unit based on a monomer having a bioaffinity group from the mononuclear cell population obtained in step (b). Culturing in a medium containing GM-CSF and SCF,
A preparation method comprising: The preparation method of Claim 1 whose fluorine atom content rate in the said fluoropolymer is 5-60 mass%, and the ratio P represented by the following Formula is 0.1-4.5 mass%.
(Ratio P) = [(Ratio of units having a biocompatible group to all units of fluoropolymer (mass%)) / (Fluorine atom content of fluoropolymer (mass%))] × 100   The preparation method according to claim 1 or 2, wherein the coating layer has a thickness of 1 nm to 100 µm.   The preparation method according to any one of claims 1 to 3, wherein the cell population expressing CD3 on the cell surface is a T cell population.   The step (b) is a step of removing a cell population expressing CD19 on the cell surface and a cell population expressing CD56 on the cell surface from the mononuclear cell population separated in the step (a). The preparation method of any one of Claims 1-4. The cell population expressing CD19 on the cell surface is a B cell population,
The cell population expressing CD56 on the cell surface is an NK cell population.
The preparation method according to claim 5. After step (c),
(D): a step of further culturing the cell population cultured in the step (c) in a medium containing GM-CSF and IL-4;
The preparation method of any one of Claims 1-6 containing this. The culture period in the step (c) is 2 to 5 weeks,
The culture period in the step (d) is 2 days to 14 days,
The preparation method according to claim 7.   The preparation method according to any one of claims 1 to 8, wherein the collected blood cell population is a collected peripheral blood, cord blood, bone marrow and / or lymph node-derived blood cell population.   The preparation method according to claim 9, wherein the collected blood cell population is a blood cell population collected from the collected peripheral blood by an apheresis method.