JP2000143534A - Dendritic cell vaccine and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a safe and effective dendritic cell vaccine having antigen presentation activity and useful for leukemia, hepatic cancer, etc., by using a cell obtained by the cultivation and differentiation of a precursor cell of a dendritic cell as an active component. SOLUTION: A dendritic cell having antigen presentation activity and produced by culturing and differentiating (A) a cell suspension containing a precursor cell of a dendritic cell in the presence of (B) a differentiation inducing agent and (C) a chemokine is used as a main component of the objective vaccine. Preferably, the component B is a combination of granular macrophage colony stimulation factor (abbreviated as GM-CSF) and interleukin 4 (abbreviated as IL-4), a combination of GM-CSF, IL-4 and tumor necrosis factor (abbreviated as TNF-α) or a combination of GM-CSF, stem cell factor and TNF-α, the component A is marrow cell, cell originated from umbilical cord blood or peripheral blood monocyte and the component C is CC chemokine.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a dendritic cell vaccine and a safe and simple method for producing the vaccine.

[0002]

2. Description of the Related Art Conventional tumor immunotherapy includes immunostimulation therapy for administering interleukin-2 (IL-2) and the like, lymphokine-activated killer cells (LAK) derived from peripheral lymphocytes of a patient, and intratumoral tumors. Infiltrating lymphocytes (TI
Various immunization methods, such as adoptive immunotherapy by transfusing L) or missile therapy using an anti-tumor antigen monoclonal antibody, have been attempted, but satisfactory therapeutic effects have not been obtained except in some cases.

[0003] Recently, gene therapy for cancer has also been actively performed. In particular, cell vaccine therapy for inducing immunity against cancer by transplanting cancer cells into which a cytokine gene such as granulocyte macrophage colony stimulating factor (GM-CSF) has been introduced, and treating cancer has attracted attention. Macrophages and dendritic cells are known as typical antigen presenting cells. Macrophages present antigen to activated T cells and B cells (Sornase et a
l. (1992) J.I. Exp. Med. 175: 15-
21). However, activation of helper T cells depends on antigen presentation by dendritic cells. Dendritic cells are thought to capture antigens in peripheral blood, migrate to the lymph, and eventually mature in the paracortical T cell portion of the lymph node. Macrophages and dendritic cells can present antigen to already activated T cells. It is reported that only mature dendritic cells can sensitize naive T cells that have not been activated. (Mehta-Dama
ni et al. (1994) J. Am. Immunolo
gy153: 996).

[0004] Immature dendritic cells generally have a high ability to take in foreign substances serving as antigens into cells, a high ability to digest foreign substances, and a low antigen presenting ability. Conversely, more differentiated mature dendritic cells are characterized by low antigen uptake ability and high antigen presentation ability (Ebner et al. (1998)).
Immunobiology 198: 568). From this, in vivo, immature dendritic cells take up foreign substances, digest them in the cells, and mature dendritic cells differentiated by the stimulation present a part of the digested foreign substances outside the cells, It is thought to convey specific antigenic species to lymphocytes.

In vivo, dendritic cells are derived from bone marrow hematopoietic stem cells. Dendritic cell precursors and immature dendritic cells are present in blood and lymph, and fully mature dendritic cells are present in the spleen and lymph nodes. In playing a role in antigen presentation, dendritic cells overexpress MHC class I and class II proteins. Recently, dendritic cells can be prepared in large quantities in vitro (Romani et al. (1994) J. Ex.
p. Med. 180: 83-93). Undifferentiated CD34-positive cells derived from bone marrow cells or cord blood or peripheral blood monocytes are precursor cells of dendritic cells, and GM-CSF, interleukin-4 (IL-
4) It is known that differentiation into dendritic cells is induced by stimulation of tumor necrosis factor (TNF).

[0006] By stimulating with GM-CSF, IL-4, dendritic cell precursor cells can differentiate into immature dendritic cells. The cells can be differentiated into mature dendritic cells by further acting on TNFα. Progenitor cells of dendritic cells
GM-CSF, IL-4, and TNFα can also be differentiated into mature dendritic cells by simultaneously acting them in advance.

In general, cancer cells of cancer patients are expected to be degenerated by administration of an anticancer drug, and the degenerated cancer cells are expected to be completely eliminated by immunization of the patient. However, in many cases, administration of an anticancer drug to a patient also reduces the function of its own immunocompetent cells, and as a result, phenomena such as recurrence and metastasis are often observed. In order to solve such a situation, development of adjuvant therapy for immune cells is expected.

[0008]

DISCLOSURE OF THE INVENTION The present invention provides a method for differentiating dendritic cell precursor cells into dendritic cells by culture.
It is an object of the present invention to provide a method for producing a safe and effective cell vaccine containing the cells as an active ingredient.

[0009]

As a method for preparing a large amount of dendritic cells, which are antigen-presenting cells, differentiation is induced by culturing from hematopoietic undifferentiated cells of bone marrow or cord blood or peripheral blood monocytes. Coming technologies have been reported. Efficient antigen presentation of dendritic cells depends on the major histocompatibility complex of the presenting dendritic cells (Major Histcompati).
It is known that it is necessary that the MHC (bilityComplex: MHC) and the MHC such as T lymphocytes to be presented are the same. Therefore, attempts have been made to isolate dendritic cells of the same type or autologous MHC having substantially the same MHC from blood, and to pulse antigen peptides or the like to present a target antigen.

[0010] The present inventors, when inducing differentiation of immature or mature dendritic cells from dendritic cell precursor cells in vitro, add a chemokine to efficiently add antigen presenting ability. Thus, a cell preparation method that can be used as an effective cell vaccine was devised. According to the present invention, progenitor cells of dendritic cells cultured with the addition of GM-CSF and IL-4 differentiate into immature dendritic cells. During this culture, RANTES (regulated)
on activation, normalT ex
pressed and secreted) or M
IP-1α (macrophage inflamma)
By adding a chemokine such as tory protein-1α), cells having the same level of antigen-presenting ability as adding TNF can be produced. Also,
According to the present invention, differentiation and induction into mature dendritic cells can be induced by simultaneously stimulating and culturing GM-CSF, IL-4 and TNF from the beginning.
Alternatively, by adding a chemokine such as MIP-1α, dendritic cells having higher antigen-presenting ability can be produced from mature dendritic cells cultured and differentiated by adding GM-CSF, IL-4 and TNF.

At present, immature dendritic cells include RANTES, M
It is considered that the migration ability is increased by stimulation with chemokines such as IP-1α and MCP-3, and that CC chemokines have no effect on mature dendritic cells (Ya
nagihara et al. (1998) J. Am. Im
munol. 161, 3096). However, the present inventors have conducted numerous studies to produce a dendritic cell preparation suitable for treating cancer patients. As a result, it has been shown that the antigen-presenting ability is increased by using the production method of the present invention. Obtained.

The method of the present invention for producing a cell vaccine containing dendritic cells as an active ingredient specifically activates autologous lymphocytes and induces lymphocytes capable of specifically attacking cancer cells. Furthermore, the cell vaccine containing the dendritic cells of the present invention as an active ingredient was found to activate lymphocytes non-specifically to MHC, and was found to be usable not only as an autologous vaccine but also as an allogeneic vaccine. . The vaccine can be administered directly into the body, but can also be used in vitro to produce antitumor lymphocytes against cancer cells. In this case, it has been found that lymphocytes can be derived from autologous cells, but can be efficiently used even from the same species.

The present invention provides (1) a dendritic cell having an antigen-presenting ability obtained by differentiating a cell suspension containing dendritic cell precursor cells by culturing the cell suspension in the presence of a differentiation inducer and a chemokine. Cell vaccine as a main component, (2) differentiation inducer is granulocyte macrophage colony stimulating factor (GM-
CSF), a combination of interleukin-4 (IL-4), GM-CSF, IL-4, tumor necrosis factor (TNF
-Α) or a combination of GM-CSF, stem cell factor (SCF) and TNF-α, (3) dendritic cell precursor cells derived from bone marrow cells and cord blood The cell vaccine according to the above (1) or (2), which is a cell or a peripheral blood mononuclear cell; (4) the cell vaccine according to the above (1) to (3), wherein the chemokine is a CC chemokine; The cell vaccine according to the above (4), wherein the CC chemokine is RANTES or MIP-1α.

The present invention also provides (6) dendritic cells having antigen-presenting ability by culturing a cell suspension containing dendritic cell precursor cells in the presence of a differentiation inducer and a chemokine. (7) the differentiation inducer is a combination of granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL-4), GM-CS
F, IL-4, a combination of tumor necrosis factor (TNF-α), or GM-CSF, stem cell factor (SC
F), the method according to (6) above, which is a combination of TNF-α, (8) the method according to (6) or (6), wherein the progenitor cells of dendritic cells are bone marrow cells, cord blood-derived cells, or peripheral blood mononuclear cells The method according to (7), (9) the method according to (6) to (8), wherein the chemokine is a CC chemokine, (1)
0) CC chemokine is RANTES or MIP-1α
(9).

By culturing undifferentiated cells derived from cord blood or mononuclear cells derived from peripheral blood, which are precursor cells of dendritic cells, a large amount of functional dendritic cells is provided as an active ingredient of a cancer vaccine. Differentiated dendritic cells can be used as a source of antigen presenting cells for use in in vitro assays and immunomodulatory treatments, particularly for sensitizing naive T cells.

As a source of dendritic cell precursor cells, umbilical cord blood, peripheral blood, bone marrow fluid, and the like can be used. A large amount can be collected from peripheral blood by apheresis, but can be obtained by syringe. It is desirable that bone marrow cells, undifferentiated cells derived from cord blood, or peripheral blood-derived mononuclear cells, which are precursor cells of dendritic cells, be separated and purified from the collected blood. Any method of separating nucleated cells from red blood cells can be employed. Methods that utilize Ficoll fractionation or Ficoll-Paque density gradients or elution are commonly used. Alternatively, the blood cells are dissolved in a solution that selectively lyses adult red blood cells, such as ammonium chloride-potas.
sium), ammonium oxalate (ammoni)
(moxalate) or the like, and can be separated.

Further separation can be carried out using a suitable surface antigen. For example, an antibody against the CD34 antigen may be used. The separation method can be performed using magnetic beads, antibody-immobilized beads, a column filled with these, and a flow cytometer. Immature dendritic cells are HLA
-DR (low), CD1a (high), CD14
(-), CD25 (-), CD83 (-), CD86
The expression pattern of a cell surface marker such as (-) is shown. Mature dendritic cells have extended dendrites characteristic of dendritic cells, appear dendritic, and have HLA-DR (high), CD1a.
(Low), CD14 (-), CD25 (+), CD8
3 shows the expression pattern of surface markers such as 3 (+) and CD86 (high) (Yanagihara et a
l. (1998) J. Am. Immunol. 161,309
6). In general, dendritic cells are most lymphocyte and monocyte specific cell markers such as CD3, CD11b, C
Lack of expression of D14, CD16, and CD19.

For culture for inducing differentiation of dendritic cells, RPMI
-1640 medium, Dulbecco's modified Eagle medium (DME
M), an appropriate commercially available medium such as Iskov medium (IMDM) may be used. Clinically, X-VIVO15 medium (Biowittaker) and the like are more preferable. As the serum, about 5 to 20% of bovine serum, fetal calf serum, human serum or the like can be used. It is preferably used without serum, but if necessary, bovine albumin (BSA), human albumin (HSA) and the like can be added. If necessary, it may contain an appropriate antibiotic, antibody, pyruvic acid (0.1-5 mM), glutamine (0.5-5 mM), and 2-mercaptoethanol (10-100 μM). A differentiation inducer is added to these media, and the cells are cultured at about 37 ° C. in an atmosphere of about 5% carbon dioxide for about 5 to 21 days. Culture for about 7 to 14 days is desirable. Culture temperature (34-38 ° C),
The mixture ratio of the gas (2 to 10% of carbon dioxide gas, and further, nitrogen gas or oxygen gas can be appropriately mixed) can be set under appropriate conditions.

In order to induce the differentiation of dendritic cells into dendritic cells from a cell group containing progenitor cells, it is necessary to use an appropriate inducer. The inducer can be selected from cytokines and used. Suitable cytokines may be used, and in particular, granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL-IL) having a concentration ranging from about 1 to 1000 ng / ml.
4), stem cell factor (SCF), interleukin-13 (IL-13), tumor necrosis factor (TNF-
α), Flt3-Ligand and the like can be used efficiently. Interleukin-1 (IL-1), Interleukin-2 (IL-2), Interleukin-3 (IL-
Additions such as 3) can also be used. Desirably GM-CS
F, a combination of IL-4, GM-CSF, IL-4,
Combination of TNF-α, GM-CSF, SCF, TN
The combination of F-α is more efficient. By adding a chemokine to a culture system using these combinations, it becomes possible to produce a cell group that can exhibit antigen more effectively.

As the cytokines and chemokines used in the culture, there may be used factors derived from xenogeneic animals such as mice, and preferably, factors derived from humans.
Many of these cytokines and chemokines are commercially available from a large number of companies such as Boehringer, PeproTech (treated by Immune Biological Laboratory),
Each can be used equally. The factors used may be of natural origin or synthetic, for example those prepared by genetic recombination. Mononuclear cells, lymphocytes, or culture supernatants of certain cell lines can also be used as a source of cytokines and chemokines. The concentration of the chemokine in the culture is preferably used in a concentration range of 1 ng / ml to 10 μg / ml, more preferably 10 to 1000 ng / ml.

Chemokines are known to be involved in inflammatory actions, mainly IL-8, and as factors that mainly cause leukocyte migration, and more than 30 new molecules have been known. That receptor is known as a G-protein coupled receptor. A number of reviews have been published in detail on these molecules (Baggiolini et al.
(1997) Annu. Rev .. Immunol. 1
5: 675. Nobuyuki Kouchi Molecular Medi
cine Vol. 34 Special Issue Immune 1997-9
8 Nakayama Bookstore. Osamu Yoshie Molecular Medic
ine Vol. 35 Special Issue Immune 1998-99
Nakayama Bookstore. ). Chemokines are derived from the structure of the first two cysteine residues of their amino acid sequence, CXC, CC, C,
It is divided into four subfamilies of CXXXC. CX
IL-8, GROα, GROβ, G as C chemokines
ROγ, NAP-2, ENA-78, GCP-2, PF
4, IP-10, Mig, PBSF / SDF-1, BC
A-1 and the like are known. As a CC chemokine, RAN
TES, MIP-1α, MCAF / MCP-1, MCP
−2, MCP-3, MCP-4, eotaxin, MI
P-1β, HCC-1, MIP-3α / LARC, MI
P-3 / ELC, I-309, TARC, MIPF-
1, MIPF-2 / eotaxin-2, MDC, DC
-CK / PARC, SLC, LD78β, leukot
actin-1, LEC, TECK and the like are known. C
Lymphotoactin as a chemokine, SCM-
1β, and as a CXXXC chemokine, fracta
Ikine is known. RANTES, MIP-1
α has been analyzed in many ways as a factor acting on lymphocytes (Roth et al. (1995) Eu).
r J Immunol 25: 3482). The chemokine used in the present invention is more preferably a CC chemokine.

Various methods for detecting the antigen-presenting activity of dendritic cells are known and can be used. For example, as a mixed lymphocyte reaction test, isolated T cells stimulated with sub-optimal concentrations of anti-CD3 antibodies
The ability to promote cell proliferation allows the ability of the cell to be measured (Thomas (1994) J. Im.
munol. 153: 4016-4028).

The group of dendritic cells during or after culture is
It can be used as an antigen presenting cell to stimulate T cells in vivo or in vitro. The protein antigen or its peptide is incorporated into cells and displayed on the cell surface after modification, or is directly displayed on the cell surface. Antigenic peptides usually consist of about 6 to 20 amino acids in length, more usually about 10 to 18 amino acids. This peptide has sequences derived from a wide variety of different proteins. In many cases, it is desirable to use a peptide that acts as a T cell antigenic determinant, usually the major antigenic determinant.

The cell suspension containing dendritic cells, which is an active ingredient of the vaccine produced according to the present invention, is inoculated as a therapeutic vaccine into the human body. is there. It is known that undifferentiated cells derived from umbilical cord blood or mononuclear cells derived from peripheral blood greatly reduce the proliferation ability by inducing differentiation.However, heat treatment and radiation treatment are required for safer use as a cell vaccine. Alternatively, the treatment can be performed under conditions such that the protein of cancer cells is denatured while leaving the function as a vaccine, such as treatment with mitomycin C. For example, X
When using X-ray irradiation, a flask containing dendritic cells is placed under the tube of the X-ray irradiator, and the total radiation dose is 1000 to 330.
Irradiate at 0 Rad. The mitomycin C treatment method includes, for example, suspending dendritic cells at a density of 1 to 3 × 10 ⁷ cells / ml, and mitomycin C25 / ml of cell suspension.
The mixture is added at a ratio of 50 μg, and is kept at 37 ° C. for 30 to 60 minutes. The cell treatment method using heat can be prepared, for example, by subjecting a centrifuge tube containing a cell suspension adjusted to a living cell concentration of 1 × 10 ⁷ cells / ml at 50 to 65 ° C. for 20 minutes.

[0025] The cell vaccine of the present invention can be used for leukemia, liver cancer,
It can be used for various cancers such as lung cancer, stomach cancer, and colon cancer, and infectious diseases caused by various viruses and bacteria. The dose of the cell vaccine of the present invention varies depending on the age, weight, sex, type of cancer, degree of progression of the cancer, symptoms, etc. of the patient, and cannot be unconditionally determined. The same amount as described above may be administered to the patient. The dendritic cell vaccine according to the present invention can be used for patients themselves, but can be administered to a large number of MHC-compatible patients of the same type due to the development of bone marrow banks and cord blood banks.

In preparing dendritic cells from dendritic cell progenitor cells, an antigen peptide may be additionally added and used as a presenting cell for the antigen. The addition may be performed at the time of culture for inducing differentiation, or may be performed after differentiation into dendritic cells. Cancer patients can be protected against viruses and bacteria, such as cytomegalovirus, because their immunity has been reduced by anticancer drugs. Dendritic cells are stimulated with the antigen by placing the cells in a physiologically acceptable buffer containing the antigen at a concentration of at least about 0.1 μM to 1 mM. Peptide antigens can usually be effective at lower concentrations than whole protein antigens or cell lysates. Antigenic peptides usually consist of about 6 to 20 amino acids in length, and more usually about 1 to about 20 amino acids.
Consists of 0 to 18 amino acids. The dendritic cells can be incubated with the antigen, generally at 37 ° C., for a time sufficient for the antigen to bind to the cells. Peptide antigens are usually available for at least about 1 hour, and 6
The incubation is performed for hours or longer.
For intact protein antigens, usually at least about 3 hours
Preferably, the incubation is performed for about 12 hours or more.

The dendritic cells stimulated with the antigen or the membrane components thereof may be used as an immunoadsorbent for obtaining antigen-specific T cells. The cell vaccine of the present invention migrates cells,
It is desirable to keep it in a container that is effective for preservation. For example, a cryovial, a blood bag for freezing, a blood bag for blood transfusion, and the like are useful. By adding dimethyl sulfoxide or the like and preparing a preservation solution for freezing, it is also possible to freeze and preserve. By using a blood bag capable of gas-phase exchange for culturing, it is possible to efficiently prepare and use a cell preparation in a closed system, such as culturing, washing, collecting, and storing.

[0028]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in more detail with reference to embodiments. It is not necessarily limited to these.

[0029]

Example 1 A buffy coat was collected from 400 ml of peripheral blood of a healthy subject, and cells in a mononuclear cell layer were obtained by Ficoll fractionation. From the obtained cells, a culture dish (Falco
(Company n) to remove adherent cells. The obtained mononuclear cells were cultured at 37 ° C. in a 5% carbon dioxide atmosphere for 11 days.
Alternatively, the cells were cultured for 12 days and differentiated into dendritic cells. The medium is 10% fetal bovine serum (FBS: Intergen)
And antimycotic-antibiotics
RPMI-164 to which (Gibco BRL) was added.
0 medium, 100 ng / ml human granulocyte macrophage colony stimulating factor (GM-CSF) and 50 ng / ml
ml human interleukin-4 (IL-4) was added. When culturing with addition of tumor necrosis factor (TNFα), TNFα and chemokines, or chemokines, 100 ng / ml human GM-CSF and 5 ng for 7 days after the start of culturing
0 ng / ml human IL-4 was added, and for culture after 7 days, 10 ng / ml human TNFα, 10 ng / ml human TNFα and 500 ng / ml chemokine, or 5
00 ng / ml chemokine was added. As chemokines, human RANTES (regulated on)
activation, normal T expr
esed and secreted), human MIP
-1α (macrophage inflammato)
ry protein-1α), or human MCP-1
(Monocyte chemotactic pro
tein-1) was used. The medium was replaced as needed. Thus, dendritic cells were prepared from peripheral blood.

[0030]

Example 2 The surface antigen of the dendritic cells prepared in Example 1 was detected using a flow cytometer. Cells before and after differentiation were measured. The cells to be measured were stained at 4 ° C. for 30 minutes with the target antibody added to a phosphate saline solution containing normal mouse serum (DAKO). The stained antibody is P
E-labeled or FITC-labeled CD1a (Cult
er), CD4, CD11c, CD13, CD14,
CD19, CD33, CD34 (HPCA-1), CD
45, HLA-DR (above, Becton-Dick
son), CD40, CD86 (Pharminge)
n), CD83 (Immunotech). Isotype matched antibodies were used as negative control antibodies. After washing the stained cells, FACS calibu
r (Beckton-Dickson).
As a result, cells cultured in IL-4 and GM-CSF
It has a surface marker for immature dendritic cells and has IL-
4. Cells induced to differentiate by GM-CSF and TNFα
D1a, CD11c, CD40, CD83, CD86,
It was confirmed that it was an HLA-DR positive mature dendritic cell population. In addition, immature dendritic cells showed little change in their cell surface markers even when they were cultured with the addition of chemokines.

[0031]

Example 3 The antigen-presenting ability of peripheral blood-derived dendritic cells prepared in Example 1 was measured and evaluated in vitro by a lymphocyte mixed culture reaction test. The mononuclear cell layer was collected from peripheral blood of a healthy individual different from that of Example 1 by Ficoll and cultured in a culture flask (Falcon) to remove adherent cells. T lymphocytes were isolated by the E rosette method. 1 × 10 ⁵ isolated lymphocytes and dendritic cells irradiated with 15 to 30 Gy were spread on a 96-well plate and cultured for 4 days. Thereafter, tritium-labeled thymidine was incorporated, and the proliferation of lymphocytes was measured. The measurement of tritium-labeled thymidine was performed after 18 hours of uptake culture by using MicroBetaTRILUX1450β-scin.
tiltation counter (Wallac)
I went in. FIG. 1 is a bar graph showing the antigen-presenting ability of dendritic cells by a lymphocyte mixing test. The vertical axis indicates radioactivity as the amount of tritium-labeled thymidine incorporated. From the left of the horizontal axis, IL-4 and GM-C were used as negative controls.
Immature dendritic cells obtained by culturing in SF, mature dendritic cells cultured for 4 days after adding TNFα at the time of culturing for 7 days by the same method, mature dendritic cells added with RANTES simultaneously with the addition of TNFα, and TNFα added At the same time, mature dendritic cells to which MIP-1α was added were shown. The test was performed under the condition that 1 × 10 ⁵ T lymphocytes were mixed with 5000 cells.

As a result of the main culture reaction test, dendritic cells obtained by inducing differentiation can be used for lymphocytes that do not match HLA.
The proliferation of lymphocytes was significantly promoted as compared with the control. It was confirmed that the cell vaccine obtained by adding chemokines of RANTES and MIP-1α according to the method of the present invention exhibited higher antigen-presenting ability to cells obtained by culturing with addition of TNFα.

[0033]

Example 4 The antigen-presenting ability of dendritic cells derived from peripheral blood prepared in Example 1 was measured and evaluated in vitro by a mixed lymphocyte reaction test. The mononuclear cell layer was collected from peripheral blood of a healthy individual different from that of Example 1 by Ficoll and cultured in a culture flask (Falcon) to remove adherent cells. T lymphocytes were isolated by the E rosette method. 1 × 10 ⁵ isolated lymphocytes and dendritic cells irradiated with 15 to 30 Gy were spread on a 96-well plate and cultured for 4 days. Thereafter, tritium-labeled thymidine was incorporated, and the proliferation of lymphocytes was measured. The measurement of tritium-labeled thymidine was performed after 18 hours of uptake culture by using MicroBetaTRILUX1450β-scin.
tiltation counter (Wallac)
I went in. FIGS. 2 and 3 show bar graphs showing the antigen-presenting ability of dendritic cells in the lymphocyte mixed test. The vertical axis indicates radioactivity as the amount of tritium-labeled thymidine incorporated. From the left of the horizontal axis in FIG.
Immature dendritic cells obtained by culturing in L-4 and GM-CSF for 11 days, matured dendritic cells cultured in positive control for 7 days and then added with TNFα and cultured for 4 days, RANTES instead of TNFα Cells with the addition of
The values of uptake of tritium-labeled thymidine in cells to which MIP-1α was added instead of TNF are shown. FIG. 3 shows that MCP-1 was used instead of TNFα when a similar method was performed.
Was added. T for 5000 pieces each
The test was performed under the condition that 1 × 10 ⁵ lymphocytes were mixed.

As a result of the main culture reaction test, undifferentiated dendritic cells obtained by inducing differentiation promoted lymphocyte proliferation as compared with the negative control. The cell vaccine of the present invention,
It was confirmed that the cells had the same high antigen-presenting ability as cells obtained by culturing with the addition of TNFα. The cells to which TNFα is not added have the properties of immature dendritic cells from the analysis of the surface markers performed in Example 2 and have high antigen uptake ability, and thus can be considered to work as a more efficient cell vaccine.
As a result of the main culture reaction test, dendritic cells obtained by inducing differentiation
Lymphocytes inconsistent with HLA also promoted lymphocyte proliferation significantly compared to controls. From this, it was confirmed that the cell vaccine of the present invention has higher antigen presenting ability than control immature dendritic cells. Further, as shown in the third embodiment, TNFα and RANTE
In the case of mature dendritic cells differentiated with S or TNFα and MIP-1α, higher antigen-presenting ability was confirmed than mature dendritic cells obtained by culturing with the addition of TNFα. From this, it was confirmed that cells to which chemokines such as RANTES or MIP-1α were added had higher antigen-presenting ability. Furthermore, it was shown that not only autologous cells but also dendritic cells with inconsistent MHC activate lymphocytes.

[0035]

Example 5 Acute myeloid leukemia cells and peripheral blood were collected from patients with myelodysplastic syndrome, and dendritic cells were prepared in the same manner as in Example 1. The antigen presenting ability of dendritic cells was measured in the same manner as in Example 3, and as a result, R
The cell group to which ANTES and MIP-1α had been added showed higher antigen-presenting ability than the cell group without addition.

[0036]

Example 6 A method for preparing dendritic cells from bone marrow fluid of a healthy subject was performed. By treating the mononuclear cells obtained by Ficoll fractionation with Dynabeads (Dynal: M-450), C
D34 positive cells were isolated. The separation method followed the attached manual. The obtained cells were cultured at 37 ° C. in a 5% carbon dioxide atmosphere for 14 days to differentiate into dendritic cells. The medium is 10% fetal bovine serum (FBS: Intergen)
And antimycotic-antibiotics
(Gibco BRL), 100 ng / ml human granulocyte macrophage colony stimulating factor (GM-CSF) and 50 ng / ml human interleukin-4 (IL-
4) 10 ng / ml human tumor necrosis factor (TNFα) and 500 ng / ml human RANTES were used. The medium was replaced as needed. The cell group thus obtained was measured for the antigen-presenting ability of dendritic cells by a method similar to the method described in Example 3. TNFα, RANTE
In the S-added group, it was confirmed that the antigen-presenting ability was higher than that in the TNFα-added group.

[0037]

According to the present invention, dendritic cells having high antigen-presenting ability can be prepared from dendritic cell precursor cells present in peripheral blood and the like. Since these dendritic cells can efficiently activate lymphocytes, an excellent cell vaccine can be obtained.

[Brief description of the drawings]

FIG. 1 is a bar graph showing the antigen-presenting ability of dendritic cells by a lymphocyte mixing test.

FIG. 2 is a bar graph showing the antigen-presenting ability of dendritic cells by a lymphocyte mixing test.

FIG. 3 is a bar graph showing the antigen-presenting ability of dendritic cells by a lymphocyte mixing test.

Claims (10)

[Claims] 1. A cell mainly composed of dendritic cells having antigen-presenting ability obtained by culturing a cell suspension containing dendritic cell precursor cells in the presence of a differentiation inducer and a chemokine. vaccine. 2. The differentiation inducer is granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-4.
(IL-4) combination, GM-CSF, IL-4,
Combination of tumor necrosis factor (TNF-α) or GM
-CSF, Stem Cell Factor (SCF), TNF-
The cell vaccine according to claim 1, which is a combination of α. 3. The cell vaccine according to claim 1, wherein the dendritic cell precursor cells are bone marrow cells, cord blood-derived cells, or peripheral blood mononuclear cells. 4. The cell vaccine according to claim 1, wherein the chemokine is a CC chemokine. 5. The method according to claim 5, wherein the CC chemokine is RANTES or M.
The cell vaccine according to claim 4, which is IP-1α. 6. A method for obtaining a dendritic cell having an antigen-presenting ability by culturing a cell suspension containing dendritic cell precursor cells in the presence of a differentiation inducer and a chemokine. 7. The differentiation inducer is granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-4.
(IL-4) combination, GM-CSF, IL-4,
Combination of tumor necrosis factor (TNF-α) or GM
-CSF, Stem Cell Factor (SCF), TNF-
7. The method according to claim 6, which is a combination of α. 8. The method according to claim 6, wherein the dendritic cell precursor cells are bone marrow cells, cord blood-derived cells, or peripheral blood mononuclear cells. 9. The method according to claim 6, wherein the chemokine is a CC chemokine. 10. The method according to claim 9, wherein the CC chemokine is RANTES or MIP-1α.