US20100291677A1 - Reducer of immunosuppression by tumor cell and antitumor agent using the same - Google Patents

Reducer of immunosuppression by tumor cell and antitumor agent using the same Download PDF

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US20100291677A1
US20100291677A1 US12/674,881 US67488108A US2010291677A1 US 20100291677 A1 US20100291677 A1 US 20100291677A1 US 67488108 A US67488108 A US 67488108A US 2010291677 A1 US2010291677 A1 US 2010291677A1
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protein
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mcp1
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Chie Kudo
Yutaka Kawakami
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Keio University
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Definitions

  • the present invention is related to reducers of immunosuppression by tumor cells and antitumor agents using the same.
  • Regulatory T cells are known to play roles in the maintenance of self-tolerance and immunological homeostasis by suppressing immune responses both pathologically and physiologically (NPL 1).
  • the cells which were initially CD4+CD25 ⁇ are activated by stimulation with various factors to become CD4+CD25+ regulatory T cells, and eventually account for about 5 to 10% of peripheral CD4+ T cells.
  • the CD4+ CD25+ regulatory T cells start to express FoxP3 protein along with the differentiation of the CD4+ T cells (NPL 2, 3).
  • Intercellular interactions as well as humoral factors such as TGF-beta (herein also referred to as TGF-b) and IL-10 have been shown to play important roles in this process (NPL 4).
  • the FoxP3 protein is considered to serve as a specific marker for the activation of regulatory T cells because its expression can be observed not only in CD4+CD25+ T cells but also in CD8+CD25+ T cells (NPL 5). Further, FoxP3 protein plays an important role in manifestation of functions of the regulatory T cell, because a naive T cell in which FoxP3 is forced to be expressed starts to show a phenotype like a regulatory T cell (NPL 6). Thus, FoxP3 gene is believed to be a master gene to regulate the differentiation and function of regulatory T cells (NPL 1).
  • the regulatory T cells are known to suppress immune responses (NPL 1) by exceptionally suppressing function of other cells (NPL 7). Its mechanism is yet to be known, but it has been suggested that the suppression of other cells functions is dependent on the intercellular interaction and that CTLA-4 is involved in the suppression (NPL 8). In particular, CTLA-4 was shown to be also involved in the differentiation of regulatory T cells (NPL 8).
  • NPL 17 While a host immunity is present in the body of a cancer patient to attack and eliminate the cancer, the cancer cells have a system to evade the defense by the host immunity. For example, it has been shown both in vitro and in vivo that the immune responses against cancer cells were changed when regulatory T cells were deleted in the presence of the cancer cells (NPL 17). Since increases in the number of regulatory T cells have been observed in stomach cancer (NPL 9, 10), rectal cancer (NPL 11), pancreatic cancer (NPL 12, 13), lung cancer (NPL 14) and glioma (NPL 17), they are considered to be involved in a system of the cancer cells to evade the immunity. However, its mechanism is yet to be known, and the way how the cytokines derived from the regulatory T cells affect the system is still a matter of controversy (NPL 17).
  • NPL 15 Since a deficiency in regulatory T cells causes severe autoimmune diseases (NPL 15), a mechanism common to the autoimmunity and the cancer immunity is considered to be present (NPL 16).
  • the regulatory T cells are known to be involved in the suppression of immunological reactions to the cancer cells as well as the hyperimmune responses such as autoimmunity and allergic reactions through the suppression of immune responses (NPL 1).
  • a development of effective treatment for the diseases involving regulatory T cells is anticipated to be achieved by revealing the molecular mechanism of the immunosuppression by the regulatory T cells, thereby bringing it to a target of the treatment for the diseases involving regulatory T cells.
  • the present invention was made to provide the followings: a gene expression enhancer for enhancing expression of FoxP3 gene in a cell; a cell differentiation inducer for inducing differentiation of a cell into a regulatory T cell; an immunosuppressor for suppressing immunity and an agent for treating hyperimmune diseases based on the abovementioned actions; an inhibitor of enhancement of gene expression for inhibiting enhancement of FoxP3 gene expression in a cell; an inhibitor of induction of cell differentiation for inhibiting induction of differentiation of a cell into a regulatory T cell; a reducer of immunosuppression for reducing immunosuppression, a stimulator of tumor immunity and an antitumor agent based on the abovementioned actions; and the like.
  • Snail a Zinc-finger transcription factor
  • Snail is a malignant transformation factor of cancer, which has been known to progress as the expression of Snail becomes higher (Nature Rev Cancer 7, 415-428, 2007).
  • EMT epithelial-mesenchymal transition
  • the inventors of the present invention endeavored to reveal the mechanism of malignant transformation of a cancer by Snail, and discovered by a forced expression of Snail in cultured cells that Snail protein enhances expression of MCP1 (monocyte chemoattractant protein-1) gene, TSP1 (thrombospondin-1) gene, FSTL1 (Follistatin-like 1) gene or IL-13Ra2 (interleukin 13 alpha 2 receptor) gene in a cancer cell, and that their gene products enhance the expression of FoxP3, a marker for activation of a regulatory T cell, in CD4+ T cells and CD8+ T cells, thereby accomplishing the present invention.
  • MCP1 monocyte chemoattractant protein-1 gene
  • TSP1 thrombospondin-1) gene
  • FSTL1 Follistatin-like 1 gene
  • IL-13Ra2 interleukin 13 alpha 2 receptor
  • FIG. 1 is a table showing phenotypes of the Panc-1 cells in which snail gene is forced to be expressed in one example of the present invention.
  • FIG. 2A shows induction of expression of FoxP3 in CD4+ cells by coculturing PBMCs with the Hs294T cells treated with TGF-beta in one example of the present invention.
  • FIG. 2B shows the induction of expression of FoxP3 in CD4+ cells by coculturing PBMCs with Panc-1 cells or F3 cells in one example of the present invention.
  • FIG. 2C shows suppression of proliferation of T cells by the CD4+ cells cocultured with Panc-1 cells, F3 cells or D10 cells in one example of the present invention.
  • FIG. 3 shows an induction of expression of FoxP3 in CD4+ cells by coculturing PMBCs with HCT116 cells in one example of the present invention.
  • FIG. 4 shows the induction of expression of FoxP3 in CD4+ cells by culture supernatants from the cell clones where snail gene was forced to be expressed in one example of the present invention.
  • FIG. 5 shows the genes whose expression was increased by forced expression of Snail protein in each of Panc-1, HCT116 and Hs294T cell lines in one example of the present invention.
  • FIG. 6 shows suppression of enhancement of FoxP3 protein expression in CD4+ cells by F3 clone using an anti-MCP1 antibody, an anti-TSP1 antibody, an anti-FSTL1 antibody or an anti-IL-13Ra2 antibody in one example of the present invention.
  • FIG. 7A shows suppression of enhancement of FoxP3 protein expression in CD4+ cells, CD4+CD25+ cells and CD4+CD25 ⁇ cells by F3 clone using the anti-MCP1 antibody or the anti-TSP1 antibody in one example of the present invention.
  • FIG. 7B shows suppression of enhancement of FoxP3 protein expression in CD4+ cells, CD4+CD25+ cells and CD4+CD25 ⁇ cells by F3 clone using the anti-FSTL1 antibody in one example of the present invention.
  • FIG. 8 shows suppression of enhancement of FoxP3 protein expression using the anti-IL-13Ra2 antibody in one example of the present invention.
  • FIG. 9 shows suppression of enhancement of FoxP3 protein expression in mouse melanoma B16-F10 using the anti-MCP1 antibody or the anti-IL-13Ra2 antibody in one example of the present invention.
  • FIG. 10 shows the action of MCP1, TSP1, FSTL1 and secretory IL-13Ra2 to enhance the expression of FoxP3 protein in one example of the present invention.
  • FIG. 11 shows a direct action and an indirect action of Snail-expressing cells to enhance the expression of FoxP3 protein in one example of the present invention.
  • FIG. 12 shows induction of the FoxP3 expression in CD8+ cells by a culture supernatant of a cell clone in which snail gene is forced to be expressed in one example of the present invention.
  • FIG. 13 shows suppression of the action of F3 cells to enhance the expression of FoxP3 protein in CD8+ cells using the anti-MCP1 antibody or the anti-TSP1 antibody in one example of the present invention.
  • FIG. 14 shows suppression of the action of B11 cells to enhance the expression of FoxP3 protein in CD8+ cells using the anti-IL-13Ra2 antibody in one example of the present invention.
  • FIG. 15 shows suppression of proliferation of Snail-expressing tumor cells using the anti-MCP1 antibody or the anti-TSP1 antibody in one example of the present invention.
  • FIG. 16 shows suppression of infiltration of Snail-expressing tumor cells using the anti-MCP1 antibody or the anti-FSTL1 antibody in one example of the present invention.
  • FIG. 17A shows results of analyses by RT-PCR for the expression of snail in leukemia cell lines in one example of the present invention.
  • FIG. 17B shows suppression of the infiltration of leukemia cells using snail gene-specific siRNAs in one example of the present invention.
  • FIG. 18 shows that the anti-MCP1 antibody, the anti-IL-13Ra2 antibody, an anti-IL-13 antibody, an anti-IL-4 antibody, an anti-CCR2 antibody and an anti-IL-10 antibody reduce suppression of tumor immunity by Snail-expressing tumor cells in one example of the present invention.
  • FIG. 19 shows results of an experiment of in vivo treatment using siRNAs specific for snail gene or MCP1 gene (A: measured tumor volumes, B: numbers of metastatic lung nodules, C: flow cytometry analysis for intratumoral infiltrated cells).
  • FIG. 20 shows results of an experiment of in vivo treatment using the anti-TSP1 antibody (A: measured tumor volumes, B: numbers of metastatic lung nodules, C: flow cytometry analysis for intratumoral infiltrated cells).
  • the gene expression enhancer for enhancing expression of MCP1 (monocyte chemoattractant protein-1) gene, TSP1 (thrombospondin-1) gene, FSTL1 (Follistatin-like 1) gene or IL-13Ra2 (interleukin 13 alpha 2 receptor) gene in the cells to be treated according to the present invention may contain a substance for enhancing Snail protein activity.
  • the substance for enhancing Snail protein activity may be any of substances that enhances the intrinsic activity of a Snail protein molecule, as well as any of substances that enhances overall Snail protein activity in a cell, and the examples include expression vectors of NBS1 which provide an enhancement of expression of Snail protein (Yang et al. Oncogene, 26, 1459-1467, 2007), expression vectors of snail gene, and the like.
  • the method for using the gene expression enhancer maybe chosen in accordance with the characteristics of the substance for enhancing Snail protein activity as an effective ingredient.
  • An administration of the substance from outside of the cells to be treated should be chosen when it acts on the membrane of the cells, whereas an introduction of the substance into the cell should be chosen when the substance acts inside the cell.
  • the cells to be treated by the enhancement of gene expression are not particularly limited, the preferred examples are tumor cells, in particular Panc-1 cell.
  • the gene expression enhancer for enhancing the expression of FoxP3 gene in the cells to be treated according to the present invention may contain a substance for enhancing Snail protein activity.
  • the substance for enhancing Snail activity may be any of substances that enhances the intrinsic activity of a Snail protein molecule, as well as any of substances that enhances overall Snail protein activity in the cell, and the examples include the expression vectors for NBS1 that enhance the Snail expression (Yang et al. Oncogene, 26, 1459-1467, 2007), the expression vectors for snail gene, and the like.
  • a cell in which the Snail activity has been enhanced by the substance for enhancing Snail activity, or the culture supernatant of the cell may be administered to the cells to be treated to enhance the expression of FoxP3 gene.
  • both cells may be co-cultured in vivo or in vitro. If the cells to be treated are located in a living body, the cell with the enhanced Snail protein activity or its culture supernatant maybe injected to the vicinity of the cells to be treated.
  • the cells to be treated are not particularly limited, the preferred examples are T cells, and more preferred are naive T cells, CD4+ T cells and CD8+ T cells.
  • the gene expression enhancer for enhancing the expression of FoxP3 gene in the cells to be treated according to the present invention may activate the MCP1 signaling in the cells to be treated and/or in other cells that coexist with the cells to be treated.
  • the effective ingredient to be contained in the gene expression enhancer may be, for example, cells expressing Snail protein, cells expressing MCP1, MCP1 protein or MCP1 receptor activating substance, and more specifically tumor cells secreting MCP1, cells to which an expression vector for MCP1 gene has been introduced, culture supernatants of cells secreting MCP1 protein, purified MCP1 protein, anti-MCP1 receptor antibody which activates MCP1 receptor, and the like.
  • the gene expression enhancer for enhancing the expression of FoxP3 gene in the cells to be treated according to the present invention may contain a cell expressing Snail protein, FSTL1 protein, membrane IL-13Ra2 protein or secretory IL-13Ra2 protein, the FSTL1 protein or the secretory IL-13Ra2 protein.
  • genes that may be used as the gene expression enhancer include tumor cells expressing Snail protein, FSTL1 protein, membrane IL-13Ra2 protein or secretory IL-13Ra2 protein, cells to which an expression vector for the gene encoding Snail, FSTL1, membrane IL-13Ra2 or secretory IL-13Ra2 has been introduced, culture supernatants of cells expressing Snail protein, culture supernatants of cells secreting FSTL1 protein or secretory IL-13Ra2 protein, purified FSTL1 proteins, and the secretory IL-13Ra2 proteins.
  • the gene expression enhancer may contain one or more than one of the abovementioned substances.
  • the enhancer may be, for example, directly administered to the cells to be treated.
  • another cell may coexist, and the preferred examples of the cell to coexist are antigen-presenting cells such as dendritic cells and macrophages that can induce proliferation of T cells by presenting antigens.
  • the gene expression enhancer may be administered to cells other than the cells to be treated, and then culture supernatants of this cell maybe administered to the cells to be treated.
  • the cell to be used in this case is preferably antigen-presenting cells such as dendritic cells or macrophages.
  • the cells to be treated are not particularly limited, the preferred examples are T cells, and more preferred are naive T cells, CD4+ T cells or CD8+ T cells.
  • FoxP3 is a master gene to regulate the differentiation and function of regulatory T cells which possess the function of immunosuppression (Miyara and Sakaguchi Trends Mol. Med. 13, 108-116, 2007; Hori et al. Science 299, 1057-1061, 2003; Jiang and Chess J. Clin. Invest. 114, 1198-1208, 2004). Therefore, the abovementioned gene expression enhancer for enhancing the expression of FoxP3 gene can be used as a cell differentiation inducer for inducing differentiation of the cells to be treated into regulatory T cells, as well as an immunosuppressor.
  • the fact that the FoxP3 gene expression enhancer which contains cells expressing snail gene can provide cocultured CD4+ cells with the proliferation suppressing capability against T cells also indicates that the gene expression enhancer for FoxP3 can be used as a cell differentiation inducer as well as an immunosuppressor.
  • the cells to be treated are not particularly limited, the preferred examples are T cells, and more preferred are naive T cells, CD4+ T cells or CD8+ T cells.
  • the cell differentiation inducer and the immunosuppressor may be used either in vivo or in vitro.
  • the immunosuppressor may suppress hyperimmunity and/or normal immunity.
  • hypoimmune diseases such as autoimmunity and allergic diseases (Miyara and Sakaguchi Trends Mol. Med. 13, 108-116, 2007; Turk et al. Immunol. Rev. vol. 188, p. 122-135, 2002). Therefore, these hyperimmune diseases can be treated by inducing differentiation of the T cells to be treated into regulatory T cells, thereby suppressing immunity. Accordingly, the abovementioned cell differentiation inducer as well as the immunosuppressor can be used as an agent for treating hyperimmune diseases to treat autoimmunity, allergic diseases and the like.
  • a hyperimmune disease as used herein means a disease caused by the hypofunction of regulatory T cells, including but not limited to autoimmunity and allergic diseases. While the cells to be treated are not particularly limited, the preferred examples are T cells, and more preferred are naive T cells, CD4+ T cells or CD8+ T cells.
  • the agent is preferably administered to patients by either a systemic administration or a topical administration to the site of hyperimmunity.
  • the inhibitor of enhancement of FoxP3 gene expression for inhibiting the enhancement of expression of FoxP3 gene in the cells to be treated according to the present invention may suppress MCP1 signaling in the cells to be treated and/or cells coexisting with the cells to be treated.
  • the effective ingredient to be contained in the inhibitor of enhancement of gene expression may suppress function of, for example, MCP1 protein or MCP1 receptor, and the specific examples include inhibitory antibodies to inhibit the function, hybridomas secreting the inhibitory antibodies, small compounds with the inhibiting activity, and the like.
  • the inhibitor of enhancement of FoxP3 gene expression according to the present invention may also suppress function of FSTL1 protein, membrane IL-13Ra2 protein or secretory IL-13Ra2 protein.
  • the effective ingredient to be contained in the inhibitor of enhancement of gene expression may be, for example, anti-FSTL1 antibody with FSTL1 protein inhibiting activity, anti-membrane IL-13Ra2 antibody with membrane IL-13Ra2 protein inhibiting activity or anti-secretory IL-13Ra2 antibody with secretory IL-13Ra2 protein inhibiting activity.
  • the inhibitor may be administered in vivo or in vitro to the cells to be treated or its vicinity.
  • the inhibitor of enhancement of FoxP3 gene expression exerts its effect by suppressing MCP1 signaling in a cell which coexists with the cells to be treated by inhibiting the enhancement of expression of FoxP3 gene
  • the inhibitor of enhancement of gene expression is administered so that it can act on the coexisting cells.
  • the coexisting cells are preferably antigen-presenting cells, such as dendritic cells or macrophages.
  • the cells to be treated are not particularly limited, the preferred examples are T cells, and more preferred are naive T cells, CD4+ T cells or CD8+ T cells.
  • FoxP3 is a master gene to regulate differentiation and function of regulatory T cells that possess function of immunosuppression (Miyara and Sakaguchi Trends Mol. Med. 13, 108-116, 2007; Hori et al. Science 299, 1057-1061, 2003; Jiang and Chess J. Clin. Invest. 114, 1198-1208, 2004). Therefore, by suppressing the enhancement of expression of FoxP3, an induction of differentiation of the cells to be treated into regulatory T cells can be inhibited, and in turn, an immunosuppression by the regulatory T cell can be reduced.
  • the abovementiond inhibitor of enhancement of gene expression for suppressing the enhancement of expression of FoxP3 gene can be used as an inhibitor of induction of cell differentiation for inhibiting induction of differentiation of the cells to be treated into regulatory T cells, as well as a reducer of immunosuppression.
  • the inhibitor or reducer may be administered in vivo or in vitro to the cells to be treated by inhibiting the induction of differentiation into regulatory T cells, or to the vicinity of the cells to be treated.
  • the inhibitor or the reducer exerts its effect by suppressing MCP1 signaling in cells which coexist with the cells to be treated
  • the inhibitor or the reducer is administered so that it can act on the coexisting cells.
  • the coexisting cells are preferably antigen-presenting cells, such as dendritic cells or macrophages.
  • the cells to be treated are not particularly limited, the preferred examples are the T cells, and more preferred are naive T cells, CD4+ T cells or CD8+ T cells.
  • the inhibitor of induction of cell differentiation and the reducer of immunosuppression may be used either in vivo or in vitro.
  • Snail protein enhances expression of MCP1 gene, TSP1 gene, FSTL1 gene and IL-13Ra2 gene in tumor cells, and that their gene products act directly or indirectly on CD4+ T cells or CD8+ T cells to enhance the expression of FoxP3, a marker for activation of regulatory T cells.
  • tumor cells suppress the tumor immunity of their host by acting on the surrounding immune cells via mediating proteins for suppression of tumor immunity such as MCP1 protein, TSP1 protein, FSTL1 protein, IL-13Ra2 protein, IL-13 protein, IL-4 protein, CCR2 protein and IL-10 protein, thereby inducing differentiation of the immune cells into regulatory T cells.
  • the inhibitor of enhancement of FoxP3 gene expression according to the present invention which inhibits the action of these mediating proteins, can reduce the suppression of tumor immunity by tumor cells, thereby stimulating the tumor immunity.
  • the stimulation of the patient's own tumor immunity can bring a therapeutic effect against the tumor in the patient. Therefore, the inhibitor of enhancement of FoxP3 gene expression according to the present invention can be used as a stimulator of tumor immunity as well as an antitumor agent.
  • the inhibitory substances for inhibiting function of the mediating protein for suppression of tumor immunity include antibodies and competitive inhibitory molecules such as dominant-negative mutants against the mediating protein for suppression of tumor immunity.
  • tumor cells are usually digested and eliminated via phagocytosis by phagocytes including the antigen-presenting cells such as dendritic cells, but Snail-expressing tumor cells inhibit the phagocytosis.
  • the inhibitory substance for inhibiting the action of the mediating proteins can suppress the inhibition of phagocytosis by the Snail-expressing tumor cells.
  • the method for using the stimulator of tumor immunity and the antitumor agent maybe chosen appropriately, they are preferably administered to patients by a topical administration to the site of the tumor or its vicinity.
  • the suppressor of tumor growth according to the present invention may suppress the proliferation of tumor cells by suppressing MCP1 signaling or function of TSP1 protein.
  • the suppressor of tumor growth may contain as an effective ingredient, for example, an inhibitory antibody for inhibiting function of MCP1 protein, MCP1 receptor or TSP1 protein, or a hybridoma secreting the inhibitory antibody.
  • the method for using the suppressor of tumor growth may be appropriately chosen, it is preferably administered to patients by either systemic administration or topical administration to the site of the tumor or its vicinity.
  • the suppressor of tumor cell infiltration according to the present invention may suppress infiltration of tumor cells by suppressing MCP1 signaling or function of FSTL1 protein.
  • metastasis of tumor cells During the metastasis of tumor cells, they infiltrate between normal cells or cells forming vascular walls. Therefore, metastasis of tumors can be suppressed by suppressing the infiltration of tumor cells. Accordingly, the suppressor of tumor cell infiltration can be used as a suppressor of tumor metastasis.
  • the suppressor of tumor cell infiltration and the suppressor of tumor metastasis according to the present invention may contain as an effective ingredient, for example, an inhibitory antibody for inhibiting function of MCP1 protein, MCP1 receptor or FSTL1 protein, a hybridoma secreting the inhibitory antibody or a dominant-negative mutant of MCP1 (7ND).
  • an inhibitory antibody for inhibiting function of MCP1 protein, MCP1 receptor or FSTL1 protein a hybridoma secreting the inhibitory antibody or a dominant-negative mutant of MCP1 (7ND).
  • the method for using the suppressor of tumor metastasis may be appropriately chosen, it is preferably administered to patients by either systemic administration or topical administration to the site of the tumor or its vicinity.
  • the tumor to be treated is not particularly limited, and it may be either a solid cancer or a blood cancer, and may be either an epithelial cancer or any other type of malignant cancer.
  • the anticancer agent for blood cancers may contain an inhibitory substance for inhibiting Snail function.
  • the inhibitory substance for inhibiting Snail function as used herein means a small compound or a protein (such as a dominant-negative mutant) which inhibits intrinsic activity of the Snail protein molecule, as well as any of substances that inhibits overall function of Snail protein in the cell, and the examples include nucleic acids (antisense RNA, siRNA, shRNA, etc.) which inhibit the expression of Snail protein, expression vectors for these nucleic acids, and competitive inhibitory proteins.
  • the anticancer agent preferably exerts its function by inhibiting the infiltration of malignantly-transformed blood cells.
  • blood cancers are not particularly limited, the examples include leukemia, malignant lymphoma and multiple myeloma.
  • Cell clones D6, D10, F3 and F5 having enhanced expression of Snail were obtained from Panc-1 cell, a strain of human pancreatic cancer cell, by forced expression of snail gene, and their phenotypes were examined with regard to cellular shape, expression levels of mRNA and protein of Snail and E-cadherin, cellular growth capability, cellular adhesion capability, cellular mobility and cellular infiltration capability.
  • a snail cDNA (CDS 71-865, 795 bp) was amplified by PCR from the Panc-1 cell which had been stimulated by TGF-beta, known as one of the inducers for EMT, and was inserted into an EcoR I-Xho I restriction site of a pcDNA3.1(+) plasmid vector (Invitrogen) having a G418 resistance gene.
  • the vector was then introduced into a tumor cell strain by electroporation, and after 2 weeks of culturing, cells that acquired drug resistance were selected using G418 (2 mg/mL) and cloned.
  • RNAs were extracted from the human tumor cell lines by using RNeasy (QIAGEN) and reverse-transcribed by using AMV (incubated at 42° C. for 50 min and at 70° C. for 15 min), and cDNAs thus obtained were used in the following PCR (iCycler, BIO-RAD).
  • the sequences of primers for snail cDNA were as follows.
  • the tumor cells (5 ⁇ 10 4 cells) were cultured in slide chambers overnight (37° C., 5% CO 2 ), and then fixed with 4% paraformaldehyde. After blocking of non-specific staining with using normal goat serum, cells were treated with Cytofix/Cytoperm (BD Phermingen) for intracellular staining (4° C., 20 min), then stained with various antibodies (e.g.
  • FIG. 1A summarizes the phenotypes of the cell lines introduced with snail gene.
  • oligonucleotides of either the snail gene-specific siRNAs (Invitrogen) or their scrambled sequences as a negative control were added to the culture media for culturing human melanoma Hs294T cells in a 6-well plate at 2 ⁇ g/3 ⁇ 10 5 /2 mL, and the cells were cultured for 2 days. The cells were then washed, added with TGF-beta (5 ng/mL), and cultured for 3 more days.
  • the tumor cells were recovered and the expression of snail gene was measured by RT-PCR according to ⁇ 1 ⁇ above.
  • the expression of GAPDH gene as a control for quantifying the expression was also measured by using the following primers.
  • a blood obtained from a healthy individual was added with 1/10 volume of 4% sodium citrate, overlaid on Ficoll (specific gravity of 1.090) and centrifuged (1500 rpm, 20 min, room temperature), and a fraction of cells present in the interphase was used as “(bulk) PBMCs”.
  • the tumor cells obtained as above were first inactivated by either a treatment with MMC (100 ⁇ g/mL, 2 hours at 37° C.) or an irradiation of X-ray (20K rad).
  • the PBMCs were seeded and cocultured in a plate along with the tumor cells at a ratio of 1:10 (for example, 1 ⁇ 10 5 of PBMCs and 1 ⁇ 10 4 tumor cells in a 96-well plate, or 5 ⁇ 10 5 of PBMCs and 5 ⁇ 10 4 of tumor cells in a 24-well plate) at 37° C. in 5% CO 2 for 3 to 5 days, and then the PBMCs were recovered.
  • the PBMCs thus obtained were first incubated with a commercially available anti-CD4 antibody (BD PharMingen) and an anti-CD25 antibody (BD PharMingen) for 1 hour. Afterwards, the cells were treated with Cytofix/Cytoperm (BD Pharmingen) for intracellular staining (4° C., 20 min), then incubated with an anti-FoxP3 antibody (eBioscience) at 4° C. for 1 hour, and subjected to a FACScan flow cytometer (Becton Dickinson) with gating set for CD4+ or CD4+CD25+ cell fraction to analyze the expression level of FoxP3.
  • BD PharMingen commercially available anti-CD4 antibody
  • BD PharMingen anti-CD25 antibody
  • FACScan flow cytometer Becton Dickinson
  • Panc-1 cells or F3 cells were coculturd with PBMCs in the same method as in ⁇ 2 ⁇ above.
  • the PBMCs having been cocultured with tumor cells for 3 to 5 days were overlaid on Ficoll (specific gravity of 1.090) and centrifuged (1500 rpm, 20 min, room temperature); a fraction of cells present in the interphase was separated and washed, then mixed with an magnetic bead-bound anti-CD4 antibody (MACS Antibody, Miltenyi Biotec) and incubated at 4° C. for 30 minutes; and CD4+ cells were isolated by using a MACS automated cell sorter (Miltenyi Biotec).
  • Ficoll specific gravity of 1.090
  • T cells (CD4+ cells or CD8+ cells) were isolated in the same method as above with using the MACS antibody (the anti-CD4 antibody or the anti-CD8 antibody).
  • Premix WST-1 Solution (Takara Bio)
  • the cells were further cultured for 24 hours.
  • optical density (at 450-655 nm) was measured by a microplate reader and the measured values were taken as the amounts of proliferation of the T cells.
  • FIG. 2B shows the results of the FACS analyses.
  • CD4+CD25+ in lymphocytes show the results of the fractionation of cells according to expressions of CD4 and CD25, and the numbers in the panels indicate the contents (%) of CD4+CD25 ⁇ cells (left) or CD4+CD25+ cells (right) in the lymphocyte fraction.
  • the cells expressing snail gene can enhance the expression of FoxP3 protein in the cocultured CD4+ cells.
  • the forced expression of snail gene to increase the expression level of Snail protein also increases the expression enhancing capability for FoxP3 protein.
  • FIG. 2C shows the results of the measurements for proliferation of respective T cells (CD4+ T cells in the left-hand panel, CD8+ T cells in the right-hand panel).
  • “None” indicates a background value for the proliferation of T cells solely without addition of the anti-CD3 antibody or the CD4+ cells.
  • the proliferation was measured in the T cells to which only the anti-CD3 antibody was added but no CD4+ cells, and both CD4+ cells and CD8+ cells registered values greater than 2.0 (not shown).
  • the cells expressing snail gene are capable of allowing the cocultured CD4+ cells to acquire the proliferation suppressing capability against T cells, i.e., capable of inducing differentiation into regulatory T cells.
  • FIG. 3 shows the results of the FACS analyses.
  • the expression of FoxP3 was not enhanced by coculturing with the HCT116 cells in which Snail was not forced to be expressed (“Parent” in the figure: the expression level of FoxP3 was 18.30) in comparison to the cells without the coculturing with tumor cells (“No tumor” in the figure: 20.42).
  • the expression of FoxP3 was enhanced by coculturing with the B11 Clone where snail gene was forced to be expressed (“B11” in the figure: 44.79).
  • 1 ⁇ 10 5 of tumor cells were cultured in a 25 cm 2 flask for 3 to 4 days, and then a supernatant from the culture was transferred to a test tube and centrifuged (3000 rpm, 20 min, 4° C.), from which a supernatant was taken as the culture supernatant, and stored at 4° C. until used in the following experiment.
  • a suspension of 5 ⁇ 10 5 of PBMCs (or a fraction thereof) was mixed with an equal volume of the culture supernatant from the tumor cells in a 24-well plate and cultured therein, i.e., in the 2-fold diluted culture supernatant from the tumor cells, at 37° C. and 5% CO 2 for 3 to 4 days. Afterwards, the PBMCs were recovered. As for the negative control, a similar experiment was conducted with using a medium which was not used for a culture, in place of the culture supernatant from the tumor cells.
  • FIG. 4 shows the results of the FACS analyses.
  • the culture supernatant from the cells expressing Snail protein can be also used as an enhancer for the expression of FoxP3 protein.
  • CD4+CD25+ cells are at an advanced stage in the course of differentiation into regulatory T cells, in some cases FoxP3 protein are being expressed at the maximum level, and thus the effect of the supernatant from the Snail-expressing cells might not be observed (see FIG. 6 below).
  • ELISA kits were used to detect the protein expression of each of MCP1 (by ELISA kit #EHMCP1 from ENDOGEN), TSP1 (by ELISA kit #CYT168 from CHEMICON) and secretory IL-13Ra2 (by ELISA kit #ab46112 from ABCAM) with following their attached protocols.
  • MCP1 by ELISA kit #EHMCP1 from ENDOGEN
  • TSP1 by ELISA kit #CYT168 from CHEMICON
  • secretory IL-13Ra2 by ELISA kit #ab46112 from ABCAM
  • FSTL1 gene expression was measured by the RT-PCR method as described in ⁇ 1 ⁇ above.
  • the following oligonucleotides were used as the primers.
  • NC negative control
  • FIG. 5 shows the results of comparisons of protein expression levels of MCP1, TSP1 and sIL-13Ra2 (secretory form), and (B) shows the results of a comparison of mRNA expression levels of FSTL-1. (C) shows the localization of IL-13Ra2 in respective types of tumor cells.
  • an enhancement of the activity of Snail protein can enhance the expression of MCP1, TSP1, IL-13Ra2 and FSTL-1.
  • an anti-MCP1 antibody (BD PharMingen #551226), an anti-TSP1 antibody (ABCAM #ab3131), an anti-FSTL1 antibody (R&D #MAB1694), an anti-TGF-beta1 antibody (R&D #MAB246), an anti-IL-10 antibody (R&D #MAB2171), an anti-IL-13Ra2 antibody (R&D #AF146) and a mouse IgG antibody (BD Pharmingen #557273) were used.
  • the anti-MCP1 antibody, the anti-TSP1 antibody, the anti-FSTL1 antibody or the anti-IL13Ra2 antibody was added at a final concentration of 1 to 5 ⁇ g/mL either directly to inactivated tumor cells or into their culture medium, then the cells were cocultured with PBMCs for 3 days, and the PBMCs were recovered.
  • the anti-TGF-beta1 antibody and the anti-IL-10 antibody were used as the positive control, and the mouse IgG was used as the negative control.
  • FIG. 6 shows the results of the measurements for the expression levels of FoxP3 protein in the CD4+ cells with which the respective antibodies were used.
  • FIG. 7 shows the results of measurements for the expression levels of FoxP3 protein in the CD4+ cells, CD4+CD25+ cells and CD4+CD25 ⁇ cells with which the respective antibodies were used.
  • TGF-b and IL-10 are known to be involved in the induction of expression of FoxP3 protein, and the anti-TGF-b antibody and the anti-IL-10 antibody were indeed capable of suppressing the expression enhancing capability for FoxP3 protein of the culture supernatant of F3 clone in each of CD4+ cells, CD4+CD25+ cells and CD4+ CD25 ⁇ cells.
  • the administration of the anti-TGF-b antibody (“Anti-TGF-b” in the figures) reduced the expression levels of FoxP3 protein from 35.34 to 23.43 ( FIG. 6 ) or from 38.39 to 30.63 ( FIG.
  • Anti-MCP1 also reduced the expression levels of FoxP3 protein to 16.27 ( FIG. 6 ) or to 22 . 89 ( FIG. 7 ) (in CD4+ cells), to 22.08 (in CD4+CD25+ cells) and to 19.02 (CD4+CD25 ⁇ cells), and the anti-TSP1 antibody (“Anti-TSP1” in the figures) similarly reduced the expression levels of FoxP3 protein to 9.26 ( FIG. 6 ) or to 21.35 ( FIG. 7 ) (in CD4+ cells), to 27.78 (in CD4+CD25+ cells) and to 16.56 (in CD4+CD25 ⁇ cells).
  • the administration of the anti-FSTL1 antibody (“Anti-FSTL1” in the figures) showed smaller effect, but still it reduced the expression levels of FoxP3 protein to 14.86 ( FIG. 6 ) or to 34.67 ( FIG. 7 ) (in CD4+ cells), to 29.85 (in CD4+ CD25+ cells) and to 32.64 (in CD4+CD25 ⁇ cells).
  • the administration of the anti-IL13Ra2 antibody (“Anti-IL13Ra2” in the figure) reduced the expression level from 35.34 to 11.86 ( FIG. 6 ) (in CD4+ cells).
  • the anti-MCP1 antibody, the anti-TSP1 antibody, the anti-FSTL1 antibody and the anti-IL13Ra2 antibody are shown to be capable of suppressing the expression enhancing capability for FoxP3 protein of the culture supernatant.
  • a substance that inhibits function of MCP1, TSP1 or an FSTL1 can inhibit the expression enhancing capability for FoxP3 protein of the Snail-expressing cells.
  • anti-IL-13Ra2 antibody inhibits the activity of B11 clone to enhance the expression of FoxP3 protein.
  • FIG. 8 shows the results with using the anti-MCP1 antibody and the anti-IL-13Ra2 antibody.
  • the anti-MCP1 antibody reduced the expression of FoxP3 protein which had been enhanced by the culture supernatant of B11 clone from 23.69 (no antibody; “No mAbs” in the figure) to 4.69 (with antibody administration; “Anti-MCT” in the figure), and the anti-IL-13Ra2 antibody reduced the expression to 7.60 (with antibody administration; “Anti-IL13Ra2” in the figure).
  • these antibodies can inhibit the activity for enhancement of FoxP3 protein expression by the culture supernatant of B11 clone. It should be noted that the effect by the anti-IL-13Ra2 antibody was not observed in the case of the parent cell of HCT116 because the HCT116 cells do not express Snail.
  • a substance that inhibits function of not only MCP1 but also IL-13Ra2 can inhibit the expression enhancing capability for FoxP3 protein of Snail-expressing cells.
  • anti-MCP1 antibody and the anti-IL-13Ra2 antibody inhibit the activity for enhancement of the FoxP3 protein expression in mouse melanoma B16-F10.
  • Mouse spleen cells were used as the immune cell in place of the bulk PBMCs. First, a spleen was removed from a mouse and homogenized, then the cell suspension was separated by using Ficoll similarly to the case of human bulk PBMCs, and a fraction of cells present in the interphase was used.
  • the experiment was conducted basically in the same method as the case of human cultured cells, but the coculturing or the culturing in the presence of culture supernatant was conducted for 5 to 6 days in the case of the mouse melanoma, in contrast to 3 to 4 days in the case of the human cells.
  • FIG. 9 shows the results with using the anti-MCP1 antibody, the anti-TSP1 antibody and the anti-IL-13Ra2 antibody.
  • the mouse IgG was used as the negative control, and the anti-TGF-b antibody and the anti-IL-10 antibody were used as the positive control.
  • the anti-TSP1 antibody did not suppress the enhancing action on the FoxP3 protein expression in mouse melanoma B16-F10, but the anti-MCP1 antibody and the anti-IL-13Ra2 antibody suppressed the enhancing action on the FoxP3 protein expression by about 28 to 45% as shown in the figure.
  • each of the cytokines MCP1, TSP1, FSTL1 and secretory IL-13Ra2 is useful as an agent for enhancing the expression of FoxP3 protein.
  • the expression level of FoxP3 protein in the CD4+ cells was examined in each of the following cases: (1) F3 cells were added to CD4+ cells only; (2) F3 cells were added to CD4+ cells plus dendritic cells (DCs); (3) F3 cells were added to CD4+ cells plus other cells (other cells, “others”, are remaining cells after removal of CD4+ cells and dendritic cells from bulk PBMCs); (4) culture supernatant of F3 cells was added to CD4+ cells only; (5) coculture supernatant of F3 cells/DCs was added to CD4+ cells only; (6) coculture supernatant of F3 cells/others was added to CD4+ cells only; and (7) coculture supernatant of F3 cells/DCs/others was added to CD4+ cells only.
  • CD4+ cells and CD11c+ cells were isolated from the bulk PBMCs by using MACS Antibodies (Miltenyi Biotec) in the same method as in (2) above. Other experimental methods were also the same as above.
  • FIG. 11 shows the results of the measurements for the expression of FoxP3 protein.
  • the panels in the middle of the figure show the results of experiments where the cells were added, and those at the bottom show the results of experiments where the culture supernatants were added.
  • CD4+F3 cells yielded 10.93
  • CD4+F3sup yielded 5.48
  • CD4+DC+F3 cells yielded 25.72
  • CD4+sup (Tu+DC) yielded 14.9
  • CD4+others+F3 cells yielded 16.15
  • CD4+sup (Tu+others) yielded 6.37. Therefore, it is considered that not only the action via a humoral factor but also the direct intercellular interaction contributes to the enhancement of expression of FoxP3 protein.
  • FIG. 12 shows the results of the measurements for the expression of FoxP3 protein.
  • the cell culture supernatant from the parent cell line Panc-1 (“Parent (Snail-)” in the figure) increased the expression of FoxP3 protein only slightly in comparison to the case where no supernatant from tumor cells was added (“No Tumor” in the figure), whereas an enhanced expression of FoxP3 protein was observed in CD8+ cells by the addition of the cell culture supernatant from F3 (“F3(Snail+)” in the figure).
  • a culture supernatant of Snail-expressing cells can also enhance the expression of FoxP3 protein in CD8+ cells.
  • the anti-TSP1 antibody and the anti-IL-13Ra2 antibody also suppress the enhancement of expression of FoxP3 protein in CD8+ cells.
  • FIG. 13 shows the results of the measurements for the expression of FoxP3 protein where the anti-MCP1 antibody and the anti-TSP1 antibody were used for F3 cells
  • FIG. 14 shows the results where the anti-IL-13Ra2 antibody was used for B11 cells.
  • the enhancing action of the cell culture supernatant on the FoxP3 protein expression was inhibited in each of the following cases where the anti-MCP1 antibody was used ( FIG. 13 ; 14.71 by the culture supernatant only (mIgG) vs. 11.97 by the anti-MCP1 antibody (Anti-MCP1)); the anti-TSP1 antibody was used ( FIG. 13 ; 14.71 by culture supernatant only (mIgG) vs. 5.28 by the anti-TSP1 antibody (Anti-TSP1)); and the anti-IL-13Ra2 antibody was used ( FIG. 14 ; 14.87 by B11 culture supernatant (No mAbs) vs.
  • At least MCP1, TSP1 and IL-13Ra2 also have the enhancing action on the FoxP3 protein expression in CD8+ cells.
  • the anti-MCP1 antibody, the anti-TSP1 antibody or the anti-TGF-b antibody as a control was added at 2 ⁇ g/mL to the culture medium for Panc-1 cells or F3 cells, and the cells were cultured for 3 days, then for additional 4 hours after an addition of 1/10 volume of Premix WST-1 Solution (Takara Bio). Then optical density (at 450-655 nm) was measured by the microplate reader and the measured values were taken as the amounts of proliferation of the cells. Inhibition rates of the proliferation were also calculated from the measured results by bringing the value obtained with using the control mIgG to 100%.
  • FIG. 15 shows the results.
  • the anti-MCP1 antibody as well as the anti-TSP1 antibody inhibited the proliferation of both Panc-1 cells (“Parent-snail( ⁇ )” in the figure) and F3 cells (“F3-snail(+)” in the figure) by 34% to 77%.
  • the proliferation capability of tumor cells can be reduced by inhibiting function of MCP1 or TSP1.
  • FIG. 16 shows the results of the measurements for the cellular infiltration capability.
  • the cellular infiltration capability of F3 cells was enhanced in comparison to the parent cell line of Panc-1 cells (designated as “parent” in the figure).
  • the anti-TSP1 antibody was added to these F3 cells, the cellular infiltration capability was not changed, whereas the cellular infiltration capability was reduced by about 45% when the anti-MCP1 antibody or the anti-FSTL1 antibody was added.
  • the anti-MCP1 antibody and the anti-FSTL1 antibody have the action to reduce the cellular infiltration capability of Snail-expressing tumor cells.
  • each of the leukemia cell lines (Molt-4, EL4, Daudi and KT1) in a culture medium added with the same siRNAs specific for snail gene or the control siRNAs as those used in ⁇ 2 ⁇ (1) above (2 ⁇ g/1 ⁇ 10 6 cells/2 mL, Invitrogen) were cultured in a 6-well plate.
  • the cells were placed in the matrigel-coated Transwell Chamber (pore size 8 ⁇ m, BD bioscience) and cultured for 4 hours. Then the number of cells infiltrated through the filter was counted.
  • FIG. 17B shows the number of infiltrated cells counted for each of the leukemia cell lines.
  • the infiltration capability of the leukemia cells was significantly suppressed (P ⁇ 0.001 to 0.05) by treating with the snail gene-specific siRNAs in comparison to the leukemia cells without the treatment with the snail gene-specific siRNAs (“None” or “Control” in the figure).
  • the infiltration capability of leukemia cells can be reduced by suppressing the expression of Snail in the leukemia cells. Accordingly, a substance that suppresses function of Snail is useful as an antileukemic agent.
  • a Snail-expressing tumor cell inhibits the phagocytotic action of a phagocytotic cell by which the tumor cell would be digested/eliminated via phagocytosis, and that the antibodies to MCP1 protein, IL-13Ra2 protein, IL-13 protein, IL-4 protein, CCR2 protein and IL-10 protein reduce the inhibitory action of the Snail-expressing tumor cells against the phagocytosis.
  • An anti-MCP-1 antibody BD Phamingen; an anti-CCR2 antibody: Abcam; the anti-IL-13 antibody: Abcam; the anti-IL-13Ra2 antibody: R&D; an anti-IL-4 antibody: BD Biosciences; and the anti-IL-10 antibody: R&D.
  • Human colon cancer cells HCT116 or the B11 clones in which snail gene had been forced to be expressed were allowed to contact with human PBMCs by coculturing for 3 days in the presence of each of the antibodies (1 ⁇ g/mL), and the PBMCs were then labeled with red fluorescence of PKH26.
  • These PBMCs were added with the HCT116 cells labeled with green fluorescence of CSFE at a ratio of 1:1 and cultured for 2 hours at 37° C. Contents of the cells labeled with both the red and green fluorescence (i.e., the cells phagocytosing cancer cells among the PBMCs) were analyzed by the flow cytometry (FCM). In order to estimate naturally occurring phagocytosis (background), cells were cultured at low temperature (4° C.) for 2 hours.
  • PBMCs treated with the HCT116 cells not expressing snail gene (“Mock-treated PBMCs” in FIG. 18 ) were labeled with both fluorescence.
  • B11-treated PBMCs were labeled with both fluorescence.
  • a Snail-expressing tumor cell is capable of suppressing the action of phagocytotic cells among PBMCs.
  • the content of cells labeled with both fluorescence was increased to 15% ⁇ 35%, i.e., the decrease of the content of the cells labeled with both fluorescence due to the contact with Snail-expressing tumor cell was prevented.
  • the suppression of tumor immunity by tumor cells can be reduced, thereby stimulating the tumor immunity.
  • the cells in which snail gene was forced to be expressed were generated from mouse melanoma B16-F10 in the same method as in ⁇ 1 ⁇ above, and transplanted to C57BL/6N mice (a subcutaneous injection with 1 ⁇ 10 6 cells and an intravenous injection with 2 ⁇ 10 5 cells were both applied to one individual simultaneously).
  • the siRNAs specific for Snail or MCP1 gene or the control siRNAs (5 ⁇ g/mouse, Invitrogen) formed in lipid complexes by using PEI (Polyplus Transfection) were injected into the subcutaneously transplanted tumor.
  • intratumoral infiltrated cells were analyzed by the FACScan flow cytometer.
  • the intratumoral infiltrated cells were collected by homogenizing the solid tumor in a culture medium, incubated with either of the antibodies to mouse antigens (anti-CD4 antibody, anti-CD8 antibody, anti-CD11c antibody and anti-I-A(b) antibody from BD Phamingen; anti-FoxP3 antibody from eBioscience) or an H-2K(b) restrictive gp70 tetramer (peptide sequence: KSPWFTTL; from MBL, SEQ ID NO:21) at 4° C. for 1 hour, and subjected to the analysis.
  • FIG. 19 shows the results of the measurements for the tumor volumes (A), the numbers of metastatic lung nodules (B), and the flow cytometry analysis for the intratumoral infiltrated cells (C).
  • an in vivo suppression of function of Snail protein or MCP1 signaling by using siRNAs specific for snail gene or MCP1 gene can reduce the suppression of tumor immunity by tumor cells, producing various antitumor effects such as a suppression of increase in tumor volume, a suppression of tumor metastasis, and an enhancement of infiltration of cells into the tumor.
  • mice 1 ⁇ 10 6 cells of mouse melanoma B16-F10 or H6-snail+ were transplanted subcutaneously into C57BL/6N mice, and after 7 days, an anti-TSP-1 antibody (5 ⁇ g/mouse, Calbiochem) was injected into the subcutaneously transplanted tumor, and the following assays were conducted after 1 week.
  • an anti-TSP-1 antibody 5 ⁇ g/mouse, Calbiochem
  • FIG. 20 shows the results of the measurements for the tumor volumes (A), numbers of metastatic lung nodules (B), and the flow cytometry analysis for the intratumoral infiltrated cells (C).
  • an inhibition of the action of TSP-1 by using an anti-TSP-1 antibody can reduce the suppression of tumor immunity by tumor cells, producing various antitumor effects such as a suppression of increase in tumor volume, a suppression of tumor metastasis, and an enhancement of infiltration of cells into the tumor.
  • the present invention can provide the followings: a gene expression enhancing method for enhancing expression of FoxP3 gene in a cell; a cell differentiation inducer for inducing differentiation of a cell into a regulatory T cell; an immunosuppressor for suppressing immunity and an agent for treating hyperimmune diseases based on the abovementioned actions; an inhibitor of enhancement of gene expression for inhibiting enhancement of expression of FoxP3 gene in a cell; an inhibitor of induction of cell differentiation for inhibiting induction of differentiation of a cell into a regulatory T cell; a reducer of immunosuppression for reducing immunosuppression, a stimulator of tumor immunity and an antitumor agent based on the abovementioned actions; and the like.
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