JPWO2016024628A1 - Method for promoting anticancer immunostimulatory ability of dendritic cells and use thereof - Google Patents

Abstract

Provided is a method capable of easily promoting immunostimulatory ability for dendritic cells. A method for promoting the anti-cancer immunostimulatory ability of dendritic cells, comprising a treatment step of inhibiting TGF-β of dendritic cells. The inhibition of TGF-β is, for example, inhibition of expression of Smad2 involved in TGF-β signal.

Description

  The present invention relates to a method for promoting anticancer immunostimulatory ability of dendritic cells, a method for producing dendritic cells with enhanced anticancer immunostimulatory ability, dendritic cells with enhanced anticancer immunostimulatory ability, The present invention relates to an accelerator that promotes anti-cancer immunostimulatory ability, an anti-cancer agent preparation kit, and a method for treating cancer.

  In recent years, attention has been focused on methods for activating immune functions and improving anticancer effects for cancer patients whose immune functions are suppressed as cancer treatment methods. This method uses, for example, cells such as NK (natural killer) cells, CTL (cytotoxic T cells), dendritic cells, etc., and is also called immune cell therapy. Among immune cells, dendritic cells are cells that present antigens to lymphocytes (for example, CTL, helper T cells, etc.) and control immune defenses, and activate cellular immunity that eliminates cancer and viruses. It is known.

  However, since these immune cells promote activation of immunostimulatory capacity, it is necessary to add cytokines and culture, which is costly and is considered to be low in cost effectiveness. In addition, NK cell therapy has, for example, a problem of non-cancer specific reaction, and CTL therapy has a problem that the effect is limited to a specific antigen.

  In addition, the therapy using dendritic cells is, for example, autologous cancer cells that return cancer cells prepared by surgical resection from cancer patients to dendritic cells collected from cancer patients and then return the dendritic cells to the body. There is a peptide-sensitized dendritic cell vaccine therapy in which a sensitized or artificially synthesized cancer antigen peptide is pulsed into dendritic cells collected from cancer patients and then the dendritic cells are returned to the body. However, such therapies have the problem that they are limited to surgical indications and cancer indications for which cancer antigen peptides have been identified.

  Then, this invention aims at provision of the method which can accelerate | stimulate immunostimulatory ability simply about a dendritic cell.

  In order to achieve the above object, the method for promoting the anticancer immunostimulatory ability of dendritic cells of the present invention comprises a treatment step of inhibiting TGF-β of dendritic cells.

  The method for producing a dendritic cell with enhanced anti-cancer immunostimulatory ability of the present invention is characterized by including a step of treating dendritic cells by the antigen immunostimulatory ability of the present invention.

  The dendritic cell with enhanced anticancer immunostimulatory ability of the present invention is obtained by the production method of the present invention.

  The promoter for promoting the anticancer immunostimulatory ability of dendritic cells of the present invention is characterized by containing a TGF-β inhibitor. The present invention is also a TGF-β inhibitor for use in a method for promoting the anticancer immunostimulatory ability of dendritic cells.

  The anticancer agent preparation kit of the present invention is characterized by containing a TGF-β inhibitor.

  The cancer treatment method of the present invention is characterized by administering the dendritic cells of the present invention to a patient.

  According to the present invention, the ability of dendritic cells to stimulate anti-cancer immunity can be promoted only by applying treatment that inhibits TGF-β.

FIG. 1 shows the result of Example A1 of the present invention. FIG. 2 shows the result of Example A1 of the present invention. FIG. 3 shows the results of Example A1 of the present invention. FIG. 4 shows the result of Example A2 of the present invention. FIG. 5 shows the results of Example A2 of the present invention. FIG. 6 shows the result of Example A3 of the present invention. FIG. 7 shows the result of Example A3 of the present invention. FIG. 8 shows the result of Example A3 of the present invention. FIG. 9 shows the results of Example A3 of the present invention. FIG. 10 shows the result of Example A3 of the present invention. FIG. 11 shows the result of Example A4 of the present invention. FIG. 12 shows the result of Example A4 of the present invention. FIG. 13 shows the result of Example A4 of the present invention. FIG. 14 shows the result of Example A4 of the present invention. FIG. 15 shows the results of Example A4 of the present invention. FIG. 16 shows the result of Example A4 of the present invention. FIG. 17 shows the result of Example A5 of the present invention. FIG. 18 shows the result of Example A5 of the present invention. FIG. 19 shows the result of Example A5 of the present invention.

  In the promotion method of the present invention, for example, the treatment step is a step of treating dendritic cells with a TGF-β inhibitor.

  In the promotion method of the present invention, for example, the TGF-β inhibitor is a Smad2 inhibitor.

  In the promotion method of the present invention, for example, the Smad2 inhibitor is an expression suppressor of Smad2.

  In the promotion method of the present invention, for example, the expression inhibitor is a nucleic acid molecule that suppresses transcription of Smad2 mRNA.

  In the promotion method of the present invention, for example, the nucleic acid molecule is siRNA.

  In the promotion method of the present invention, for example, the dendritic cells are derived from bone marrow or monocytes.

  In the promotion method of the present invention, for example, the monocytes are peripheral blood monocytes.

  In the promotion method of the present invention, for example, the dendritic cell is derived from a human or a non-human animal.

In the promotion method of the present invention, for example, the processing step is performed in vitro .

  The anticancer agent preparation kit of the present invention further includes, for example, a culture reagent for dendritic cells.

  In the anticancer agent preparation kit of the present invention, for example, the culture reagent contains a medium.

  In the anticancer preparation kit of the present invention, for example, the culture medium contains the TGF-β inhibitor.

  The terms used in this specification can be used in the meaning normally used in the art unless otherwise specified.

  The present invention is described in detail below.

(1) Method for promoting anticancer immunostimulatory ability of dendritic cells The method for promoting anticancer immunostimulatory ability of dendritic cells of the present invention includes, for example, a treatment step of inhibiting TGF-β of dendritic cells. Features.

  In the present invention, the treatment method for inhibiting TGF-β is not particularly limited, and the treatment step is, for example, a step of treating dendritic cells with a TGF-β inhibitor. In the present invention, the inhibition of TGF-β is inhibition of TGF-β signaling, specifically, for example, inhibition of Smad2 signal, and preferably inhibition of Smad2 expression. For this reason, the said TGF- (beta) inhibitor is a Smad2 inhibitor, for example, and is specifically an expression inhibitor of Smad2.

  In the promotion method of the present invention, for example, the expression inhibitor is a nucleic acid molecule that suppresses transcription of Smad2 mRNA.

  The Smad2 inhibitor is not particularly limited, and for example, an expression-suppressing nucleic acid molecule that suppresses the expression of Smad2 can be used. Examples of the expression-suppressing acid molecule include siRNA and microRNA. The suppression of the expression of Smad2 is, for example, suppression of expression of the Smad2 gene, that is, suppression of translation of the Smad2 protein encoded by the Smad2 gene. The suppression of the expression of the Smad2 gene can be achieved, for example, by reducing the production amount of the transcription product from the Smad2 gene, reducing the activity of the transcription product (Smad2 mRNA), or the production amount of the translation product (Smad2 protein) from the Smad2 gene. It can be confirmed by a decrease or a decrease in the activity of the translation product.

  In the present invention, the origin of the dendritic cells is not particularly limited, and examples thereof include origins of bone marrow, monocytes, lymph nodes, spleen and the like. The monocytes are, for example, peripheral blood monocytes.

  In the present invention, the biological origin of the dendritic cells is not particularly limited, and examples thereof include those derived from humans or non-human animals. Examples of the non-human animals include non-human mammals such as mice, rats, rabbits, sheep, cows, horses, dogs, goats and camels.

In the promotion method of the present invention, for example, the treatment step may be performed in vitro or in vivo . In the case of in vitro , the dendritic cell may be, for example, a cell isolated from a living body or a cultured cell cultured after isolation.

  The treatment step can be performed, for example, by culturing the dendritic cell in a medium containing the TGF-β inhibitor. The medium is not particularly limited, and can be appropriately set according to, for example, the type of dendritic cells. Moreover, the culture conditions of the dendritic cells are not particularly limited, and can be appropriately set according to, for example, the type of the dendritic cells. The TGF-β inhibitor may be added to the medium in advance, for example, in the culture of the dendritic cells, or may be added to the medium after the start of the culture.

  The addition ratio of the TGF-β inhibitor in the medium is not particularly limited, and can be appropriately set according to, for example, the type of dendritic cells and the type of TGF-β.

  When the TGF-β inhibitor is an expression-suppressing nucleic acid molecule, the treatment step can be performed, for example, under the following conditions. The addition ratio of the expression-suppressing nucleic acid molecule is, for example, 1 to 1000 nmol per 1000 cells. The expression-suppressing nucleic acid molecule is preferably transfected into the dendritic cell in the treatment step. Examples of the transfection include a method using a transfection reagent and an electroporation method. As the transfection reagent, for example, a commercially available transfection reagent can be used.

  According to the promotion method of the present invention, dendritic cells with enhanced anti-cancer immunostimulatory ability can be obtained. Such dendritic cells are useful for the treatment of cancer, as will be described later.

  The promotion method of the present invention is not limited to dendritic cells, for example, and can also be applied to immune cells such as NK cells and CTLs. The same applies hereinafter.

(2) Method for producing dendritic cells with enhanced anticancer immunostimulatory ability As described above, the production method of the present invention is a method for producing dendritic cells with enhanced anticancer immunostimulatory ability. The method for promoting dendritic cells is characterized by the method for promoting antigen-immunity activation ability of the invention. The present invention is characterized in that dendritic cells are treated by the method for promoting the antigen immunostimulatory ability, and other steps and conditions are not particularly limited. Moreover, the description of the promotion method of the said this invention can be used for the manufacturing method of this invention.

(3) Dendritic cells with enhanced anti-cancer immunostimulatory ability As described above, the dendritic cells of the present invention are dendritic cells with enhanced anti-cancer immunostimulatory ability. It is characterized by being obtained.

(4) Accelerator for anticancer immunostimulatory activity As described above, the promoter of the present invention is a promoter that promotes the anticancer immunity activation capability of dendritic cells and contains the TGF-β inhibitor. Features.

  The promoter of the present invention can be used, for example, in the promotion method of the present invention and the production method of the present invention. In the present invention, the TGF-β inhibitor is the same as described above.

  The promoter of the present invention may contain, for example, only the TGF-β inhibitor or may contain other components. Examples of the other components include components that can be used when dendritic cells are treated with the TGF-β inhibitor in the promotion method of the present invention. As a specific example, the other component includes, for example, a culture reagent for dendritic cells. Examples of the culture reagent include a medium, and the medium may contain the TGF-β inhibitor. When the TGF-β inhibitor is the expression-suppressing nucleic acid molecule, examples of the other components include the transfection reagent in addition to the medium.

  The promoter of the present invention may be, for example, a form in which the TGF-β inhibitor and the other components are mixed, or may be in a separately separated form and mixed at the time of use.

(5) Anticancer agent preparation kit As described above, the anticancer agent preparation kit of the present invention comprises a TGF-β inhibitor.

  The promoter of the present invention can be used, for example, in the promotion method of the present invention and the production method of the present invention. In the present invention, the TGF-β inhibitor is the same as described above, and the description of the accelerator of the present invention can be incorporated.

  The anticancer agent preparation kit of the present invention may further contain instructions for use, for example.

(6) Method for treating cancer As described above, the method for treating cancer is a method for treating cancer, characterized in that the dendritic cells of the present invention are administered to a patient.

  In the treatment method of the present invention, the dendritic cell administered to a patient is the dendritic cell of the present invention, and the description in the description of the dendritic cell of the present invention can be incorporated.

  In the present invention, the treatment includes, for example, the meanings of prevention, improvement, and prognosis, and may be any. In the present invention, the cancer to be treated is not particularly limited. For example, breast cancer, lung cancer, stomach cancer, colon cancer, liver cancer, pancreatic cancer, esophageal cancer, prostate cancer, gallbladder cancer, endometrium Examples include cancer, cervical cancer, ovarian cancer, osteosarcoma, and leukemia.

  In the present invention, examples of the method for administering dendritic cells of the present invention include parenteral administration. Specifically, for example, administration can be performed by placing the dendritic cells of the present invention in a lesion.

  Hereinafter, although an example etc. explain the present invention in detail, the present invention is not limited to these.

  First, reagents and methods used in Example A are shown below.

(1) Mouse
C57BL / 6 mice with conditional Smad2 allele homozygous (Smad2 loxp / loxp) are mated with Mx-1Cre mice or Cd11cCre recombinase Tg mice (Jackson Laboratory), and Mx-1Cre; Smad2 ^{fl / fl} mice, or Cd11cCre Smad2 ^{fl / fl} mice were generated. Mx-1Cre mice used were those in which PolyI: C was intraperitoneally administered at 2-3 weeks of age. In the following examples, mice of the same age (8-16 weeks) were used.

(2) Generation of mouse BMDCs Bone marrow cells (BM cells) were collected from the femur and tibia of mice and cultured in a medium. The culture conditions were 7 days at 37 ° C. The medium consists of 10% inactivated fetal bovine serum (FBS) (Gibco), 50 μM 2-mercaptoethanol, 1% penicillin / streptomycin, mouse granulocyte macrophage colony stimulating factor (GM-CSF) (20 ng / mL, PeproTech) and Use RPMI 1640 with IL-4 (20 ng / mL, PeproTech), with or without TGF-β1 (5 ng / mL, R & D Systems) or with or without Activin A (10 ng / mL, R & D Systems) It was. The ratio of the BM cells to the medium was 3 × 10 ⁶ cells / 3 mL.

(3) Generation of human MoDCs Peripheral mononuclear cells were prepared from human blood by density gradient centrifugation using a reagent (Histopaque-1077, Sigma-Aldrich). From human blood, CD14 ⁺ cells were isolated using a reagent kit (Human Monocyte Isolation kit II, Miltenyi Biotec), and this was isolated from recombinant human GM-CSF (20 ng / mL, PeproTech) and IL-4 (10 ng / (7 mL) and PeproTech) at 37 ° C. for 7 days to generate CD14 ⁻ CD1a ⁺ MoDCs (> 90%).

(4) Transfection of siRNA 200 nmol / L of various nucleic acid molecules were transfected into isolated CD14 ⁺ cells (2-3 × 10 ⁶ cells) in a buffer. As the nucleic acid molecule, a non-target siRNA pool, Smad2 siRNA (Dharmacon RNA Technologies, GenBank Accession No. NM_005901) or Smad3 siRNA (GenBank Accession No. NM_005902) was used. Transfection was performed using a Human Monocyte Nucleofector kit (Amaxa) according to the instruction manual.

(5) Immunocytochemistry
BM cells and monocytes were cultured as described above on glass slides coated with poly-L-lysine in wells of 6-well plates (NUNC). These cells and CD4 ⁺ T cells were fixed with phosphate buffered saline (PBS) containing 4% formaldehyde, and May-Grunwald / Giemsa staining was performed. The slide after fixation was permeabilized with TBS containing 0.1% Triton X-100 for proximity ligation using a kit (Duolink II in situ PLA kits, OLINK). Anti-Smad2 rabbit antibody, anti-Smad3 rabbit antibody, anti-phosphorylated-Smad2 rabbit antibody (S465 / 467), and anti-phosphorylated-Smad3 rabbit antibody (S423 / 425) were used as the antibodies (all are Cell Signaling Technology ). The nuclei were stained with DAPI (diamidinophenylindole). The slides were observed using an optical microscope (Imager Z1, Carl Zeiss) or a confocal microscope (LSM700, Carl Zeiss). The signal was quantified using BlobFinder software (Uppsala University).

(6) Isolation of cells Spleen and lymph nodes collected from mice were minced, and these were incubated at room temperature using 10% FBS-containing RPMI 1640 in which type III collagenase (Worthington Biomedical Corporation) and DNase I (Roche) were dissolved. Digested by enzymatic treatment for 25 minutes. At 20 minutes from the start of the enzyme treatment, more EDTA (5 mmol / L, Sigma-Aldrich) was added. Then, spleen CD11c ⁺ cells and CD115 ⁺ lineage marker ⁻ BM cells were concentrated by MACS system (Miltenyi Biotech). The purity of spleen CD11c ⁺ cells and CD115 ⁺ lineage marker ⁻ BM cells was confirmed to be 80%, respectively. CD3 ⁺ T cells were enriched (> 90%) from the spleen using T cell enrichment columns (R & D Systems).

(7) Flow cytometry Mouse cells were incubated with optimal concentration of antibody for 30 minutes on ice. The antibody is an anti-mouse CD16 / CD32 antibody, an anti-CD11c antibody conjugated with a fluorescent dye, an anti-MHC Class II (IA / IE) antibody, an anti-CD11b antibody, an anti-D40 antibody, an anti-CD80 antibody, an anti-CD86 antibody, Anti-B220 antibody, anti-CD115 antibody, anti-Gr-1 antibody, anti-CD3ε antibody, anti-CD8 antibody, anti-CD4 antibody, anti-CD19 antibody, anti-DX5 antibody, anti-CD25 antibody and anti-CD45RA antibody were used. Human cells were stained with anti-CD14 antibody, anti-CD1a antibody, anti-CD11c antibody, anti-HLA-DR / DP / DQ antibody, anti-CD80 antibody and anti-CD86 antibody conjugated with a fluorescent dye. To measure cytokine production, cells were analyzed using phorbol-12-myristate-13-acetate (2.5 ng / mL, Sigma-Aldrich) and ionomycin (2.5 ng / mL, Sigma-) using GolgiPlug (BD Pharmingen). Aldrich) for 4 hours. Then, the cells were fixed by Cyperm / Cytofix Kit (eBiosciences) and permeabilized. For intracellular staining, anti-Tbet antibody, anti-Eomes antibody, anti-FoxP3 antibody, anti-perforin antibody, anti-granzyme B antibody, anti-IFN-γ antibody and anti-TNF-α antibody conjugated with fluorescent dye were used. Each antibody was purchased from BD Pharmingen and eBiosciences. Flow cytometry data was acquired by LSRII (BD Bioscience) and analyzed by FlowJo (Tree Star).

(8) Quantitative RT-PCR
Total RNA was extracted using the reagent Trizol® according to the instructions for use. The extracted RNA was reverse transcribed using cDNA RT kit (Invitrogen). The obtained mouse cDNA was quantified by SYBR green (Applied Biosystems) using ABI 7900 (Applied Biosystems). The following RT-PCR primers were used.

GAPDH
Sequence number 1 5'-TGGTGAAGGTCGGTGTGAAC-3 '
Sequence number 2 5'-CCATGTAGTTGAGGTCAATGAAGG-3 '
Smad2
Sequence number 3 5'-GGAACCTGCATTCTGGTGTT-3 '
Sequence number 4 5'-ACGTTGGAGAGCAAGCCTAA-3 '
Smad3
Sequence number 5 5'-TTAGGCACCAGCCTGTTTCT-3 '
Sequence number 6 5'-TGGCGATACACCACCTGTTA-3 '
granzyme B
Sequence number 7 5'-GGACTGCAAAGACTGGCTTC-3 '
Sequence number 8 5'-ATAACATTCTCGGGGCACTG-3 '
Perforin
Sequence number 9 5'-TTTCGCCTGGTACAAAAACC-3 '
Sequence number 10 5'-AGGGCTGTAAGGACCGAGAT-3 '
FasL
Sequence number 11 5'-CATCACAACCACTCCCACTG-3 '
Sequence number 12 5'-GTTCTGCCAGTTCCTTCTGC-3 '
IFN-γ
SEQ ID NO: 13 5'-ACTGGCAAAAGGATGGTGAC-3 '
SEQ ID NO: 14 5'-GACCTGTGGGTTGTTGACCT-3 '
TNF-α
SEQ ID NO: 15 5'-TATGGCTCAGGGTCCAACTC-3 '
SEQ ID NO: 16 5'-CTCCCTTTGCAGAACTCAGG-3 ',
IL-6
Sequence number 17 5'-GAGGATACCACTCCCAACAGACC-3 '
Sequence number 18 5'-AAGTGCATCATCGTTGTTCATACA-3 '
IL-12p35
SEQ ID NO: 19 5'-CACCCTTGCCCTCCTAAACC-3 '
Sequence number 20 5'-CACCTGGCAGGTCCAGAGA-3 '
IL-12p40
Sequence number 21 5'-GCTCAGGATCGCTATTACAATTCC-3 '
Sequence number 22 5'-TCTTCCTTAATGTCTTCCACTTTTCTT-3 '
IL-15
SEQ ID NO: 23 5'-CATCCATCTCGTGCTACTTGTGTT-3 '
Sequence number 24 5'-CATCTATCCAGTTGGCCTCTGTTT-3 '

  As human cDNA, human Smad2 (Hs00183425_m1), human Smad3 (Hs00969210_m1) and human GAPDH (Hs99999905_m1) were quantified using pre-synthesized TaqMan Gene Expression Assays (Applied Biosystems). For each mRNA, the relative mRNA level relative to GAPDH was calculated by comparative Ct method.

(9) Western blotting Cells were lysed with a reagent (RIPA buffer) and electrophoresed on a 10% SDS-polyacrylamide gel. The protein in the gel is transferred to a nitrocellulose membrane (Millipore), and anti-Smad2 antibody, anti-p-Smad2 antibody and anti-Smad3 antibody (all are Cell Signaling Technology) and anti-β-actin antibody (Santa Cruz Biotechnology). Labeled and visualized with ECL kit (GE Healthcare).

(10) EL4 lymphoma
EL4 cells were transfected with a GFP-expressing FG12 lentiviral vector and cultured in DMEM (WelGene Inc.) containing 10% FBS and antibiotics. The cultured EL4 cells (1 × 10 ⁶ cells) were inoculated into the right lower abdomen of 8-week-old Cd11cCre; Smad2 ^{+ / +} mice or Cd11cCre; Smad2 ^{fl / fl} mice. The size of the cancer was measured with a digital caliper, and the cancer volume was calculated by the following formula.
([Minor axis] ² x major axis) / 2

  Then, the inoculated cancer, inflow region lymph node (right groin), non-inflow lymph node and spleen were collected and used for evaluation.

(11) Ex vivo T cell stimulation by pulsing EL4 lysate into DCs (dendritic cells) As described above, spleen of Cd11cCre; Smad2 ^{+ / +} mice or Cd11cCre; Smad2 ^{fl / fl} mice inoculated with EL From which DCs (dendritic cells) and T cells were isolated. The purified DCs are brought to a predetermined concentration (2 × 10 ⁵ , 4 × 10 ⁴ , 2 × 10 ⁴ cells / well), and are used together with T cells (2 × 10 ⁵ cells / well) labeled with CFSE (Molecular Probe). Cultured. The co-culture was performed for 5 days in the presence of EL4 cell lysate (7 × 10 ⁴ frozen cells / well) frozen in liquid nitrogen using a U-bottom 96 well plate. CFSE dilution and intracellular cytokines were measured by flow cytometry.

In co-culture, the ratio of DCs to T cells was 1: 1, 1: 5, and 1:10, and the combination of DCs and T cells (DC / T) was as follows. .
Cd11cCreSmad2 ^{+ / +} / Cd11cCre; Smad2 ^{+ / +}
Cd11cCre; Smad2 ^{+ / +} / Cd11cCre; Smad2 ^{fl / fl}
Cd11cCre; Smad2 ^{fl / fl} / Cd11cCre; Smad2 ^{+ / +}
Cd11cCre; Smad2 ^{fl / fl} / Cd11cCre; Smad2 ^{fl / fl}

(12) Cytotoxicity analysis
CD8 ⁺ T cells (0, 1 × 10 ⁴ , 2 × 10 ⁴ , 1 × 10 ⁵ , 2 × 10 ⁵ cells) derived from the spleen of EL4 inoculated mice and CD8 ⁺ (Ly-2) microbeads (Miltenyi Biotec) It concentrated by the used immunomagnetic bead method (MACS). In addition, all influx lymph node cells (0, 1 × 10 ⁴ , 2 × 10 ⁴ , 1 × 10 ⁵ cells) were mixed with EL4 cells (2 × 10 ³ cells) in a U-bottom 96-well plate for 3 days. Cultured. Cytotoxicity was calculated by measuring absorbance at 490 nm using LDH cytotoxicity detection assay kit (Promega). The specific lysis percentage was calculated according to the instructions for use.

(13) Histological analysis Primary tumors were collected and fixed with 10% neutral buffered formalin. The fixed sample was dehydrated with 70% ethanol and embedded in paraffin. This was cut into 3 μm sections and subjected to ematoxylin / eosin staining (HE staining). For immunohistochemistry, paraffin-fixed tumor sections were collected overnight at 60 ° C., and deparaffinized with xylene, dehydrated with ethanol, and blocked with peroxidase blocking reagent (Dako). Then, the slide of the section was reacted with anti-CD3 antibody, anti-CD8 antibody (abcam) or anti-CD11c antibody (eBioscience) overnight, and subsequently reacted with HRP-conjugated secondary antibody (Vector). For counterstaining, Mayer's hematoxylin (Sigma) was used.

(14) Statistics Various data were analyzed by unpaired Student's t-test and two-way repeated ANOVA test. In addition, P value <0.05 shows that it is statistically significant.

(Example A1)
The expression pattern of TGF-βR-Smads in dendritic cells was confirmed.

  Smad2 is a type of R-smad that conveys a specific signal in the TGF-β pathway and is known as the major TGF-β R-Smad expressed in mouse and human dendritic cells (DSc). Therefore, mouse spleen-derived dendritic cells, mouse bone marrow (BM) -derived dendritic cells (BMDCs) and human monocyte-derived dendritic cells (MoDCs) are differentiated by treatment with GM-CSF and IL-4 for 7 days, The basic expression pattern of TGF-β R-Smads was confirmed.

BM cells derived from C57BL / 6 mice were cultured for 7 days in the presence of mouse GM-CSF (20 ng / mL) and IL-4 (20 ng / mL) to generate BMDCs, and human monocytes were transformed into human GMCSF. MoDCs were generated by culturing for 7 days in the presence of (20 ng / mL) and IL-4 (10 ng / mL). Mouse CD115 ⁺ lineage marker ⁻ BM cells and CD11c ⁺ cells were purified from spleen and BM, respectively.

First, the expression levels of Smad2 mRNA and Smad3 mRNA were confirmed by quantitative RT-PCR (n = 3-5). These results are shown in FIGS. FIGS. 1 (A) and (B) show the results in mouse total BM cells, mouse CD115 ⁺ lineage marker ⁻ BM cells, mouse BMDCs and mouse spleen DCs. (A) is Smad2 mRNA, (B) is Smad3 The expression level of mRNA is shown. 1 (C) and (D) show the results in human monocytes and human MoDCs, (C) shows the expression level of Smad2 mRNA, and (D) shows the expression level of Smad3 mRNA.

Next, the expression of Smad2, phosphorylated Smad2, Smad3 and phosphorylated Smad3 was determined by a proximity ligation assay. Specifically, cell nuclei were stained with DAPI, and images were acquired with a confocal microscope (LSM700). In 10 fields of view, stained red dots (black) in the nucleus and blue dots (white) in the cytoplasm were quantified. These results are shown in the graphs of FIGS. 2 (E) and (F). Fig. 2 (E) shows the results of mouse total BM cells, mouse CD115 ⁺ lineage marker ^- BM cells, mouse BMDCs and mouse spleen DCs, and Fig. 2 (F) shows the results of human monocytes and human MoDCs. The expression of Smad2, phosphorylated Smad2, Smad3, and phosphorylated Smad3, respectively, is shown. Each graph shows an average value + SD. The P value was calculated by 2-tailed unpaired Student's t-test. In each graph, NS indicates not significant. Furthermore, the black bar in the figure indicates the number of dots in the nucleus, and the white bar indicates the number of dots in the cytoplasm.

  Furthermore, the expression of Smad2, phosphorylated Smad2, Smad3 and β-actin was detected by Western blot. These results are shown in the photograph of FIG. FIG. 3 (G) shows the results of mouse total BM cells, mouse BMDCs, human monocytes and human MoDCs.

As shown in FIGS. 1 (A) and (C), Smad2 mRNA normalized to GAPDH is highly expressed in DC progenitor cells, mouse total BM cells, mouse CD115 ⁺ lineage ⁻ BM cells and human monocytes. (Mouse total BM cells: 0.02037 ± 0.00208, mouse CD115 ⁺ lineage ⁻ BM cells: 0.01712 ± 0.00166, mouse BMDC: 0.01154 ± 0.00117, mouse spleen DC: 0.15405 ± 0.00216). In contrast, as shown in FIGS. 1 (B) and (D), normalized Smad3 mRNA was much lower than Smad2 mRNA in mouse spleen DCs and DC progenitor cells. Among them, mouse BMDCs and human MoDCs were reduced to undetectable levels (mouse total BM cells: 0.00098 ± 0.00009, mouse CD115 ⁺ lineage ^- BM cells: 0.00057 ± 0.00006, mouse BMDC: 0.00009 ± 0.00003, mouse spleen DC : 0.00015 ± 0.00001).

  Further, in the Proximity ligation assay, as shown in FIGS. 2E and 2F, it was found that Smad2 was significantly expressed relative to Smad3 in mouse DCs and human DCs. And as shown to FIG. 3 (G), the expression level of Smad2 protein was comparable between the said DC precursor cell and DCs. Further, as shown in FIG. 3 (G), the expression level of Smad3 protein in the DC precursor cells was lower than the expression level of Smad2 protein, and was further decreased in DCs. In the progenitor cells and DCs, Smad2 was mostly phosphorylated, but Smad3 phosphorylation was hardly detectable.

  Similarly, Western blots for Smad2 (60 kDa), phosphorylated Smad2 and Smad3 (50 kDa) confirmed that the expression level of Smad2 protein in DCs was significantly higher than that of Smad3 protein. .

  Thus, the expression of Smad2 in mouse spleen DCs, mouse DCs induced by GM-CSF / IL-4 and human DCs, which is more significant than Smad3, is Smad2 is the major TGF-β R-Smad in DCs. It suggests that there is.

(Example A2)
DCs express dendrites and costimulatory molecules to activate T cells. Therefore, we confirmed the role of Smad2 in the immunogenicity of DCs.

As DCs, mouse BMDCs obtained from Mx-1Cre; Smad2 ^{+ / +} mice (hereinafter referred to as "Smad2 ^{+ / +} ") and Mx-1Cre; Smad2 ^{fl / fl} mice (hereinafter referred to as "Smad2 ^-/- ") And human MoDCs in which Smad2 was knocked down by siRNA. It has been confirmed that Smad2 was knocked down by siRNA.

First, May-Grunwald / Giemsa staining was performed, and an image was acquired with an optical microscope (Imager Z1, Carl Zeiss), and it was confirmed that TGF-β suppresses the immunoimmunogenicity of DCs. These results are shown in FIGS. 4 (A) and (B). FIG. 4 (A) shows the results of mouse BMDC staining. The left column shows the results of BMDC of Smad2 ^{+ / +} mice, the right column shows the results of BMDCs of Smad2 ^{− / −} mice, and the lower row shows the upper row. FIG. Fig. 4 (B) shows the results of human MoDC staining. The left column is the result of treatment with control siRNA (control), the right column is the result of treatment with siRNA against Smad2 (siSmad2). It is an enlarged view of the upper stage. In each figure, the scale bar shows 100 μm at the top and 20 μm at the bottom.

As shown in FIG. 4 (A), BMDCs in Smad2 ^{− / −} mice have significantly developed dendrites as compared to BMDCs in Smad2 ^{+ / +} mice, and as shown in FIG. 4 (B), Dendritic formation was also enhanced in human MoDCs in which Smad2 was knocked down with siRNA.

Next, BM cells of Mx-1Cre; Smad2 ^{+ / +} mice and Mx-1Cre; Smad2 ^{fl / fl} mice were used in the presence or absence of TGF-β1 (5 ng / mL) or activin A (10 ng / mL). To generate BMDC. Flow cytometry histograms showing the expression of CD40, CD80 and CD86 in these BMDCs and a graph of the percentage of CD40 ⁺ , CD80 ⁺ and CD86 ⁺ in CD11c ⁺ MHC class II ⁺ cells are shown in FIG. 5 (C ), (D) and (F) (n = 5-7). In FIG. 5, each bar graph on the right side shows an average value + SD, and the P value was calculated by 2-tailed unpaired Student's t-test. In each bar graph, NS indicates not significant. FIG. 5 (C) shows the result of mouse BMDC in the presence or absence of TGF-β, and FIG. 5 (D) shows the result of mouse BMDC in the presence or absence of activin A. FIG. ) Shows the results of human MoDCs introduced with control siRNA or siSmad2.

In addition, as shown in FIG. 5 (C), the expression levels of CD40, CD80 and CD86 in BMDC of Smad2 ^{− / −} mice are markedly increased compared to BMDC of Smad2 ^{+ / +} mice. The inhibitory effect of TGF-β1 completely disappeared by deletion of Smad2. On the other hand, activin A, which is a TGF-β superfamily cytokine, also activates Smad2 and Smad3. However, as shown in FIG. 5 (D), unlike TGF-β1, activin A (5 ng / mL) did not affect the expression of CD40, CD80 and CD86 in BMDC. In addition, as shown in FIG. 5 (E), the expression of B7 family molecules (CD80 and CD86) increased in human MoDC in which Smad2 was knocked down by siRNA. From these results, it can be said that Smad2-mediated TGF-β signal inhibits the immunogenicity of DCs.

(Example A3)
Smad2 deletion enhances the immunogenicity of DCs, suggesting that Smad2 is a target for enhancing the efficacy of cancer immunotherapy using DC. Thus, using mouse EL4 T cell lymphoma model, it was confirmed whether DCs lacking Smad2 enhance anti-cancer immunostimulatory ability.

(1) Incidence and growth of tumors
Cd11cCre with Smad2; Smad2 ^{+ / +} mice and DCs-specific Smad2-deficient mice (Cd11cCre; Smad2 ^{fl / fl} mice) were inoculated with EL4 cells (1 × 10 ⁶ cells) in the lower right abdomen, and cancer after inoculation Was measured over time (n = 15 / genotype). These results are shown in FIGS. 6 (A) and (B). FIG. 6 (A) shows the ratio (%) of cancers having a diameter of 5 mm or more, and FIG. 6 (B) shows the results of cancer volume. In FIG. 6, each graph shows the average value + SD or the average value−SD, and the P value was calculated by a two-way repeated ANOVA test.

As shown in FIGS. 6 (A) and (B), the incidence and growth of EL4 tumors were significantly suppressed in Cd11cCre; Smad2 ^{fl / fl} mice compared to Cd11cCre; Smad2 ^{+ / +} mice.

(2) Increase in CD11c ⁺ MHC II ⁺ cells
For mice inoculated with EL4 tumor, the proportion of CD11c ⁺ MHC class II ⁺ cells in the draining lymph nodes was confirmed. These results are shown in FIG. In FIG. 7, the upper diagram shows the distribution of cells, and the lower diagram is a graph showing the proportion of CD11c ⁺ MHC class II ⁺ cells in the draining lymph nodes. In the lower part of FIG. 7, the white bar is the result of Cd11cCre; Smad2 ^{+ / +} mice inoculated with EL4 tumor, and the black bar is the result of Cd11cCre; Smad2 ^{fl / fl} mice inoculated with EL4 tumor. + SD is shown (n = 10 / genotype).

As shown in FIG. 7, CD11c ⁺ MHC II ⁺ cells were significantly increased in the draining lymph nodes of Cd11cCre; Smad2 ^{fl / fl} mice compared to Cd11cCre; Smad2 ^{+ / +} mice.

(3) Immunohistochemistry
Paraffin sections of Cd11cCre; Smad2 ^{+ / +} mice and Cd11cCre; Smad2 ^{fl / fl} mice were prepared, and immunohistochemical examination of CD11c ⁺ cells was performed. The slide was confirmed using an optical microscope (Imager Z1, Carl Zeiss). These results are shown in FIG. FIG. 8D is a photograph of immunohistochemistry, and the scale bar is 20 μm. As shown in FIG. 8 (D), it was confirmed that CD11c ⁺ cells having dendrites with mononuclear cells infiltrate lymphomas of Cd11cCre; Smad2 ^{fl / fl} mice. On the other hand, the infiltration was not confirmed in Cd11cCre; Smad2 ^{+ / +} mice.

(4) Expression of costimulatory molecules
The expression of CD40, CD80 and CD86 of CD11c ⁺ MHC class II ⁺ DCs was confirmed in the draining lymph nodes of Cd11cCre; Smad2 ^{+ / +} mice and Cd11cCre; Smad2 ^{fl / fl} mice. These results are shown in FIG. FIG. 9 (E) shows the percentage of CD40 ⁺ , CD80 ⁺ and CD86 ⁺ cells in CD11c ⁺ MHC class II ⁺ DCs. In FIG. 9E, the lower bar graph shows the average value of% + SD, and the P value was calculated by 2-tailed unpaired Student's t-test. In FIG. 9 (E), the white bars show the results of Cd11cCre; Smad2 ^{+ / +} mice, and the black bars show the results of Cd11cCre; Smad2 ^{fl / fl} mice.

As shown in FIG. 9 (E), Cd11cCre; Smad2 ^{fl / fl} mice showed that CD11c ⁺ MHC class II ⁺ DCs costimulatory molecules (CD40, CD80 and CD86) were expressed in Cd11cCre; Smad2 ⁺ Significantly increased compared to ^{/ +} mice.

(5) Cytokine expression
DCs secrete IL-12 and IL-15 that activate Th1 cells, CTLs and NK cells. The production of IL-12 by DCs is essential for the initiation of an immune response involving early T cells, and DCs produce high levels of IFN-γ in response to IL-12. DCs also secrete various inflammatory cytokines such as TNF-α and IL-6. Therefore, the expression of inflammatory cytokines in splenic DCs was measured by quantitative RT-PCR.

These results are shown in FIGS. 10 (F) and (G). FIGS. 10 (F) and (G) show the mRNA expression levels of various cytokines in splenic DCs of Cd11cCre; Smad2 ^{fl / fl} mice (black bars) and Cd11cCre; Smad2 ^{+ / +} mice (white bars) inoculated with EL4 lymphoma. Indicates. Each graph shows an average value + SD (n = 11 / genotype). The P value was calculated by 2-tailed unpaired Student's t-test. In each graph, NS indicates not significant. As shown in FIGS. 10 (F) and (G), the expression of IL-12 p35, IL-15, IL-6 and TNF-α mRNA in splenic DCs of Cd11cCre; Smad2 ^{fl / fl} mice was expressed as Cd11cCre; Smad2 ⁺ Significantly increased compared to ^{/ +} mice.

  These results indicate that DC-specific Smad2 deletion enhances the immunogenicity of DCs and thereby suppresses the development of orthotopic lymphoma.

(Example A4)
The effect of Smad2 deletion on anti-lymphoma effector T cell differentiation was confirmed.

Immunological phenotyping of draining lymph nodes of Cd11cCre; Smad2 ^{fl / fl} and Cd11cCre; Smad2 ^{+ / +} mice inoculated with EL4 lymphoma was performed by flow cytometry. These results are shown in FIG. FIG. 11A shows the average value + SD (n = 10 / genotype). As shown in FIG. 11 (A), CD8 ⁺ T cells and CD4 ⁺ T cells were significantly increased in Cd11cCre; Smad2 ^{fl / fl} mice compared to Cd11cCre; Smad2 ^{+ / +} mice.

Eomes and T-bet are T-box transcription factors essential for the anti-tumor function of CTL and Th1 effector cells. Therefore, by flow cytometry, CD8 ⁺ T cell Eomes ⁺ and CD4 + T cell T-bet ⁺ in Cd11cCre; Smad2 ^{fl / fl} mice and Cd11cCre; Smad2 ^{+ / +} mice inoculated lymph nodes inoculated with EL4 lymphoma. The percentage (%) was confirmed. These results are shown in FIG. In FIG. 12B, each bar graph on the lower side shows an average value + SD (n = 5 / genotype), and the P value was calculated by 2-tailed unpaired Student's t-test. As shown in FIG. 12 (B), Cd11cCre; Smad2 ^{fl / fl} mice (black bars) showed increased CD8 ⁺ T in the inflowing lymph nodes compared to Cd11cCre; Smad2 ^{+ / +} mice (white bars). Cells and CD4 ⁺ T cells expressed Eomes and T-bet significantly at very high levels, respectively.

Cytokine mRNA expression level was measured for RNA of splenic T cells of Cd11cCre; Smad2 ^{fl / fl} mice and Cd11cCre; Smad2 ^{+ / +} mice inoculated with EL4 lymphoma by quantitative RT-PCR. These results are shown in FIG. The graph in FIG. 13C shows the average value + SD (n = 5 / genotype), and the P value was calculated by 2-tailed unpaired Student's t-test. As shown in FIG. 13, Cd11cCre; Smad2 ^{fl / fl} mice (black bars) were compared to Cd11cCre; Smad2 ^{+ / +} mice (white bars), and splenic T cells showed IFN-γ mRNA and TNF-α mRNA. Was significantly expressed at very high levels.

Percentage of IFN-γ ⁺ and TNF-α ⁺ in CD8 ⁺ T cells in draining lymph nodes of Cd11cCre; Smad2 ^{fl / fl} and Cd11cCre; Smad2 ^{+ / +} mice inoculated with EL4 lymphoma by flow cytometry Was measured. These results are shown in FIG. In FIG. 14 (D), the lower graph shows the average value + SD (n = 5 / genotype), and the P value was calculated by 2-tailed unpaired Student's t-test. As shown in FIG. 14 (D), Cd11cCre; Smad2 ^{fl / fl} mice (black bars) were compared to Cd11cCre; Smad2 ^{+ / +} mice (white bars) with CD8 ⁺ T cells stimulated with PMA and ionomycin. The intracellular expression of IFN-γ and TNF-α, and the intracellular expression of IFN-γ in CD4 ⁺ T cells was significantly increased.

We confirmed the effect of Smad2 deletion in DCs on antigen-specific effector T cell responses. Specifically, in order to confirm EL4-specific T cell division by CFSE dilution, CD3 ⁺ T cells purified from the spleen of mice inoculated with EL4 and DCs were co-cultured for 5 days in the presence of EL4 frozen lysate. . Then, the ratio (%) of dividing cells in CD8 ⁺ T cells was determined by flow cytometry. These results are shown in FIG. In FIG. 15E, each lower bar graph represents an average value + SD (n = 5 / genotype), and the P value was calculated by 2-tailed unpaired Student's t-test. As shown in FIG. 15 (E), the most effective T cell proliferation is induced when the ratio of DC: T cells is 1: 1, compared to other ratios, Smad2 ^{− / −} DCs CD8 ⁺ T cells and CD4 ⁺ T cells proliferated significantly.

In addition, the ratio of IFN-γ ⁺ and TNF-α ⁺ in CD8 ⁺ T cells was determined by flow cytometry. These results are shown in FIG. In FIG. 16 (F), each bar graph represents an average value + SD (n = 5 / genotype), and the P value was calculated by 2-tailed unpaired Student's t-test. In each graph, the type of each bar is the same as in FIG. 15E, and NS is not significant. As shown in FIG. 16 (F), maximal cytokine production was induced when the DC: T cell ratio was 1:10 and was stimulated ex vivo by Smad2 ^{− / −} DCs and EL4 lysate. T cells significantly produced inflammatory cytokines compared to T cells stimulated by Smad2 ^{+ / +} DCs.

Thus, compared to Cd11cCre; Smad2 ^{+ / +} mice, well-differentiated CD8 ⁺ T cells and even CD4 ⁺ T cells from Cd11cCre; Smad2 ^{fl / fl} mice, ex vivo, Smad2 ^{+ / +} DCs When re-stimulated by, failed to produce excellent growth or production of many effector cytokines. In contrast, Smad2 ^{− / −} DCs, compared to Smad2 ^{+ / +} DCs, are not only derived from Cd11cCre; Smad2 ^{fl / fl} mice but also CD8 ⁺ T cells and CD4 ⁺ T cells derived from Cd11cCre; Smad2 ^{+ / +} mice. Induced cell proliferation and cytokine production. These results indicate that Smad2 signal inhibits the induction of effective anti-cancer T cell responses of DCs.

(Example A5)
The effect of DC-specific Smad2 deletion on antilymphoma cytolytic activity was confirmed.

For Cd11cCre; Smad2 ^{+ / +} and Cd11cCre; Smad2 ^{fl / fl} mice inoculated with EL4 lymphoma, paraffin sections were prepared for CD8 ⁺ T cell HE staining and immunohistochemistry. The slide of the paraffin section was observed with an optical microscope (Imager Z1, Carl Zeiss). These results are shown in FIGS. 17 (A) and (B). FIG. 17 (A) shows the results of HE staining, FIG. 17 (B) shows the results of immunohistochemical examination, and in both figures, the scale bar is 100 μm. As shown in FIG. 17 (A), compared to Cd11cCre; Smad2 ^{+ / +} mice, lymphoma inoculated into Cd11cCre; Smad2 ^{fl / fl} mice showed a marked increase in mononuclear cell infiltration. In addition, as shown in FIG. 17 (B), in line with the increase in CD8 ⁺ T cells in the draining lymph nodes, many cancer-infiltrating mononuclear cells were found to be CD3 ⁺ T cells and CD8 ⁺ T cells. It was also found to be a cell.

Next, in order to detect lysis of the target cancer cells by effector immune cells, all draining lymph nodes containing both NK cells and CTLs from mice inoculated with EL4 as effector cells, or spleen CD8 ⁺ T cells, Cells (EL4 cells) were co-cultured at various ratios (effector cells E: target cells T). Cell lysis was then evaluated by measuring LDH release in the supernatant. These results are shown in FIGS. 18 (C) and (D). 18 (C) and (D) are graphs showing the percentage of cell lysis. FIG. 18 (C) shows the result of using draining lymph nodes, and FIG. 18 (D) shows spleen CD8 ⁺ The results using T cells, both graphs show mean + SD or mean-SD (n = 5 / genotype), and P values were calculated by 2-tailed unpaired Student's t-test. As shown in FIGS. 18 (C) and (D), all draining lymph nodes and splenic CD8 ⁺ T cells derived from Cd11cCre; Smad2 ^{fl / fl} mice were compared with Cd11cCre; Smad2 ^{+ / +} mice, respectively. Significantly enhanced LDH release was confirmed.

Cytolytic effector molecules such as Perforin, granzyme B and FasL are responsible for the cytotoxicity of NK cells and CTLs. Thus, the expression of splenic T cells of Cd11cCre; Smad2 ^{+ / +} mice and Cd11cCre; Smad2 ^{fl / fl} mice inoculated with EL4 was confirmed by quantitative RT-PCR. In addition, the ratio (%) of perforin ⁺ and granzyme B ⁺ in CD8 ⁺ T cells in the draining lymph nodes was determined by flow cytometry. These results are shown in FIGS. 19 (E) and (F). FIG. 19 (E) is a graph showing the expression level of the cytolytic effector molecule, and FIG. 19 (F) is a graph showing the percentage (%) of CD8 ⁺ T cells. Each graph shows the average value + SD (n = 5 / genotype), and the P value was calculated by 2-tailed unpaired Student's t-test. As shown in FIGS. 19 (E) and (F), Cd11cCre; Smad2 ^{fl / fl} mice (black bars) show expression of mRNA for cytolytic effector molecules in splenic T cells and CD8 ⁺ T cells in draining lymph nodes The protein levels of peforin and granzyme B in were significantly increased compared to Cd11cCre; Smad2 ^{+ / +} mice (white bars). These results show that Smad2-deficient DCs effectively activate the anti-lymphoid tumor cell lytic ability in vivo.

  The present invention has been described above with reference to the embodiments. However, the present invention is not limited to the above embodiments. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

  This application claims the priority on the basis of Japanese application Japanese Patent Application No. 2014-165287 for which it applied on August 14, 2014, and takes in those the indications of all here.

  According to the present invention, for example, the ability of dendritic cells to activate anti-cancer immunity can be promoted only by performing treatment that inhibits TGF-β.

Claims (18)

A method for promoting the anti-cancer immunostimulatory ability of dendritic cells, comprising a treatment step of inhibiting TGF-β of dendritic cells. The promotion method according to claim 1, wherein the treatment step is a step of treating dendritic cells with a TGF-β inhibitor. The promotion method according to claim 2, wherein the TGF-β inhibitor is a Smad2 inhibitor. The method according to claim 3, wherein the Smad2 inhibitor is a Smad2 expression inhibitor. The promotion method according to claim 4, wherein the expression inhibitor is a nucleic acid molecule that suppresses transcription of Smad2 mRNA. The promotion method according to claim 5, wherein the nucleic acid molecule is siRNA. The promotion method according to any one of claims 1 to 6, wherein the dendritic cells are derived from bone marrow or monocytes. The promotion method according to claim 7, wherein the monocytes are peripheral blood monocytes. The promotion method according to any one of claims 1 to 8, wherein the dendritic cell is derived from a human or a non-human animal. The promotion method according to claim 1, wherein the treatment step is performed in vitro . A dendritic cell having enhanced anti-cancer immunostimulatory ability, comprising a step of treating dendritic cells by the method for promoting anti-cancer immunostimulatory ability according to any one of claims 1 to 10. Manufacturing method. A dendritic cell having an enhanced anti-cancer immunostimulatory ability obtained by the production method according to claim 11. A promoter for promoting the anti-cancer immunostimulatory ability of dendritic cells, comprising a TGF-β inhibitor. An anticancer agent preparation kit comprising a TGF-β inhibitor. Furthermore, the anticancer agent preparation kit of Claim 14 containing the culture reagent for dendritic cells. The anticancer agent preparation kit according to claim 15, wherein the culture reagent contains a culture medium. The anticancer agent preparation kit according to claim 16, wherein the medium contains the TGF-β inhibitor. A method for treating cancer, comprising administering the dendritic cell according to claim 12 to a patient.