U.S. patent application number 12/674881 was filed with the patent office on 2010-11-18 for reducer of immunosuppression by tumor cell and antitumor agent using the same.
This patent application is currently assigned to Keio University. Invention is credited to Yutaka Kawakami, Chie Kudo.
Application Number | 20100291677 12/674881 |
Document ID | / |
Family ID | 40387133 |
Filed Date | 2010-11-18 |
United States Patent
Application |
20100291677 |
Kind Code |
A1 |
Kudo; Chie ; et al. |
November 18, 2010 |
REDUCER OF IMMUNOSUPPRESSION BY TUMOR CELL AND ANTITUMOR AGENT
USING THE SAME
Abstract
In order to provide 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 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, for example, an agent containing at least
one of a cell expressing Snail protein, MCP1 protein, FSTL1
protein, membrane IL-13Ra2 protein or secretory IL-13Ra2 protein,
or MCP1 protein, FSTL1 protein or secretory IL-13Ra2 protein, is
used as a gene expression enhancer for FoxP3, an inducer of
regulatory T cell differentiation, an immunosuppressor or an agent
for treating hyperimmune diseases; and an agent containing an
anti-MCP1 antibody, an anti-FSTL1 antibody or an anti-IL-13Ra2
antibody is used as an inhibitor of enhancement of FoxP3 gene
expression, a reducer of immunosuppression, a stimulator of tumor
immunity, an antitumor agent, or the like.
Inventors: |
Kudo; Chie; (Tokyo, JP)
; Kawakami; Yutaka; (Tokyo, JP) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Assignee: |
Keio University
|
Family ID: |
40387133 |
Appl. No.: |
12/674881 |
Filed: |
August 22, 2008 |
PCT Filed: |
August 22, 2008 |
PCT NO: |
PCT/JP2008/064987 |
371 Date: |
April 20, 2010 |
Current U.S.
Class: |
435/371 ;
530/350; 530/389.2 |
Current CPC
Class: |
A61P 37/08 20180101;
C07K 16/24 20130101; A61P 35/02 20180101; A61P 37/06 20180101; C12N
15/113 20130101; A61P 43/00 20180101; C07K 16/30 20130101; C07K
16/2866 20130101; C12N 2501/148 20130101; A61K 2039/505 20130101;
A61K 38/1709 20130101; A61P 37/04 20180101; C07K 16/18 20130101;
A61P 37/02 20180101; A61K 31/00 20130101; C07K 2317/73 20130101;
C07K 14/4702 20130101; A61P 35/04 20180101; A61K 38/195 20130101;
C12N 2510/00 20130101; A61P 35/00 20180101; C12N 5/0693 20130101;
C12N 2502/1157 20130101; A61K 38/1793 20130101; C12N 2310/14
20130101; C12N 2502/30 20130101; C12N 2502/1114 20130101 |
Class at
Publication: |
435/371 ;
530/350; 530/389.2 |
International
Class: |
C12N 5/071 20100101
C12N005/071; C07K 14/47 20060101 C07K014/47; C07K 16/26 20060101
C07K016/26 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 24, 2007 |
JP |
2007-218977 |
Claims
1. A gene expression enhancer for enhancing expression of FoxP3
gene in a cell, wherein the enhancer activates MCP1 signaling in
the cell.
2. A gene expression enhancer for enhancing expression of FoxP3
gene in a cell, comprising a cell expressing a Snail protein, an
MCP1 protein, an FSTL1 protein, a membrane IL-13Ra2 protein or a
secretory IL-13Ra2 protein.
3. A gene expression enhancer for enhancing expression of FoxP3
gene in a cell, comprising an MCP1 protein, an FSTL1 protein or a
secretory IL-13Ra2 protein.
4. The gene expression enhancer according to claim 3, comprising a
culture supernatant of a cell expressing a Snail protein or a cell
secreting an MCP1 protein, an FSTL1 protein or a secretory IL-13Ra2
protein.
5. The gene expression enhancer according to claim 2 or 4, wherein
the cell is a tumor cell.
6. A cell differentiation inducer for inducing differentiation of a
cell into a regulatory T cell, wherein the inducer activates MCP1
signaling in the cell.
7. A cell differentiation inducer for inducing differentiation of a
cell into a regulatory T cell, comprising a cell expressing a Snail
protein, an MCP1 protein, an FSTL1 protein, a membrane IL-13Ra2
protein or a secretory IL-13Ra2 protein.
8. A cell differentiation inducer for inducing differentiation of a
cell into a regulatory T cell, comprising an MCP1 protein, an FSTL1
protein or a secretory IL-13Ra2 protein.
9. An immunosuppressor, wherein the immunosuppressor activates MCP1
signaling.
10. An immunosuppressor comprising a cell expressing a Snail
protein or a cell expressing an MCP1 protein, an FSTL1 protein, a
membrane IL-13Ra2 protein or a secretory IL-13Ra2 protein.
11. An immunosuppressor comprising an MCP1 protein, an FSTL1
protein or a secretory IL-13Ra2 protein.
12. An agent for treating hyperimmune disease, wherein the agent
activates MCP1 signaling.
13. An agent for treating hyperimmune disease, comprising a cell
expressing a Snail protein, an MCP1 protein, an FSTL1 protein, a
membrane IL-13Ra2 protein or a secretory IL-13Ra2 protein.
14. An agent for treating hyperimmune disease, comprising an MCP1
protein, an FSTL1 protein or a secretory IL-13Ra2 protein.
15. An inhibitor of enhancement of gene expression for inhibiting
enhancement of expression of FoxP3 gene in a cell, wherein the
inhibitor suppresses MCP1 signaling in the cell.
16. An inhibitor of enhancement of gene expression for inhibiting
enhancement of expression of FoxP3 gene in a cell, wherein the
inhibitor suppresses function of an MCP1 protein, an FSTL1 protein,
a membrane IL-13Ra2 protein or a secretory IL-13Ra2 protein.
17. The inhibitor of enhancement of gene expression according to
claim 15 or 16, comprising an anti-MCP1 antibody with MCP1
inhibiting activity.
18. The inhibitor of enhancement of gene expression according to
claim 16, comprising an anti-FSTL1 antibody with FSTL1 protein
inhibiting activity, an anti-membrane IL-13Ra2 antibody with
membrane IL-13Ra2 protein inhibiting activity or an anti-secretory
IL-13Ra2 antibody with secretory IL-13Ra2 protein inhibiting
activity.
19. An inhibitor of induction of cell differentiation for
inhibiting induction of differentiation of a cell into a regulatory
T cell, wherein the inhibitor suppresses MCP1 signaling in the
cell.
20. An inhibitor of induction of cell differentiation for
inhibiting induction of differentiation of a cell into a regulatory
T cell, wherein the inhibitor suppresses function of an MCP1
protein, an FSTL1 protein, a membrane IL-13Ra2 protein or a
secretory IL-13Ra2 protein.
21. The inhibitor of induction of cell differentiation according to
claim 19 or 20, comprising an anti-MCP1 antibody with MCP1
inhibiting activity.
22. The inhibitor of induction of cell differentiation according to
claim 20, comprising an anti-FSTL1 antibody with FSTL1 protein
inhibiting activity, an anti-membrane IL-13Ra2 antibody with
membrane IL-13Ra2 protein inhibiting activity or an anti-secretory
IL-13Ra2 antibody with secretory IL-13Ra2 protein inhibiting
activity.
23. A reducer of immunosuppression suppressing MCP1 signaling.
24. A reducer of immunosuppression suppressing function of an MCP1
protein, an FSTL1 protein, a membrane IL-13Ra2 protein or a
secretory IL-13Ra2 protein.
25. A reducer of immunosuppression suppressing function of an IL-13
protein, a CCR2 protein or an IL-10 protein.
26. A stimulator of tumor immunity suppressing MCP1 signaling.
27. A stimulator of tumor immunity, wherein the stimulator
suppresses function of an MCP1 protein, an FSTL1 protein, a
membrane IL-13Ra2 protein or a secretory IL-13Ra2 protein.
28. A stimulator of tumor immunity, wherein the stimulator
suppresses function of an IL-13 protein, a CCR2 protein or an IL-10
protein.
29. An antitumor agent suppressing MCP1 signaling.
30. An antitumor agent, wherein the agent suppresses function of an
MCP1 protein, an FSTL1 protein, a membrane IL-13Ra2 protein or a
secretory IL-13Ra2 protein.
31. An antitumor agent, wherein the agent suppresses function of an
IL-13 protein, a CCR2 protein or an IL-10 protein.
32. A suppressor of tumor growth, wherein the suppressor suppresses
MCP1 signaling or function of a TSP1 protein.
33. A suppressor of tumor cell infiltration, wherein the suppressor
suppresses MCP1 signaling or function of an FSTL1 protein.
34. A suppressor of tumor metastasis, wherein the suppressor
suppresses MCP1 signaling or function of an FSTL1 protein.
35. A gene expression enhancer of FoxP3 gene, 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 cell, comprising a substance enhancing Snail
activity.
36. The gene expression enhancer according to claim 35, wherein the
substance enhancing Snail activity is an expression vector of a
snail gene.
37. The gene expression enhancer according to claim 35, wherein the
cell is a Panc-1 cell.
38. An anticancer agent for blood cancer, comprising an inhibitory
substance inhibiting function of a Snail protein.
39. The anticancer agent according to claim 38, wherein the
inhibitory substance inhibits expression of the snail gene.
40. The anticancer agent according to claim 38 or 39, wherein the
blood cancer is leukemia.
Description
TECHNICAL FIELD
[0001] The present invention is related to reducers of
immunosuppression by tumor cells and antitumor agents using the
same.
BACKGROUND ART
[0002] 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).
[0003] For example, 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).
[0004] 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).
[0005] 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).
[0006] 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).
[0007] 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).
CITATION LIST
[Non Patent Literature]
[0008] [NPL 1] Miyara and Sakaguchi Trends Mol. Med. 13, 108-116,
2007 [0009] [NPL 2] Fontenot et al. Nat. Immunol. 4, 330-336, 2003
[0010] [NPL 3] Fontenot et al. Immunity 22, 329-341, 2005 [0011]
[NPL 4] Zheng et al. J. Immunol. 172, 5213-5221, 2004 [0012] [NPL
5] Bisikirska et al. J. Clin. Invest. 115, 2904-2913, 2005 [0013]
[NPL 6] Hori et al. Science 299, 1057-1061, 2003 [0014] [NPL 7]
Jiang and Chess J. Clin. Invest. 114, 1198-1208, 2004 [0015] [NPL
8] Atabani et al. Eur. J. Immunol. 35, 2157-2162, 2005 [0016] [NPL
9] Ichihara et al. Clin. Cancer Res. 9, 4404-4408, 2003 [0017] [NPL
10] Wolf et al. Clin. Cancer Res. 9, 606-612, 2003 [0018] [NPL 11]
Hicky et al. Semin. Immunol. 11, 125-137, 1999 [0019] [NPL 12]
Liyanage et al. J. Immunol. 169, 2756-2761, 2002 [0020] [NPL 13]
Sasada et al. Cancer 98, 1098-1099, 2003 [0021] [NPL 14] Woo et al.
Cancer Res. 61, 4766-4772, 2001 [0022] [NPL 15] Sakaguchi et al.
Immunol. Rev. 182, 18-32, 2001 [0023] [NPL 16] Turk et al. Immunol.
Rev. 188, 122-135, 2002 [0024] [NPL 17] Andaloussi and Lesniak
Neuro-Oncology 8, 234-243, 2006
SUMMARY OF INVENTION
Technical Problem
[0025] 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.
[0026] Accordingly, 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.
Solution to Problem
[0027] Snail, a Zinc-finger transcription factor, 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).
[0028] As a reason for this fact, it is considered that by
suppressing expression of intercellular adhesion molecules such as
E-cadherin, Snail regulates the epithelial-mesenchymal transition
(EMT) which occurs during, for example, the processes of
gastrulation and tissue/organ development in an ontogenesis, the
repairing process when a normal tissue or cells are lost, and the
metastatic process when cancer cells metastasize (Nature Rev Cancer
7, 415-428, 2007).
[0029] Thus, 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.
[0030] Accordingly, the embodiments of the present invention are as
follows. [0031] (1) A gene expression enhancer for enhancing
expression of FoxP3 gene in a cell, wherein the enhancer activates
MCP1 signaling in the cell. [0032] (2) A gene expression enhancer
for enhancing expression of FoxP3 gene in a cell, containing a cell
expressing a Snail protein, an MCP1 protein, an FSTL1 protein, a
membrane IL-13Ra2 protein or a secretory IL-13Ra2 protein. [0033]
(3) A gene expression enhancer for enhancing expression of FoxP3
gene in a cell, comprising an MCP1 protein, an FSTL1 protein or a
secretory IL-13Ra2 protein. [0034] (4) The gene expression enhancer
according to (3), comprising a culture supernatant of a cell
expressing a Snail protein or a cell secreting an MCP1 protein, an
FSTL1 protein or a secretory IL-13Ra2 protein. [0035] (5) The gene
expression enhancer according to (2) or (4), wherein the cell is a
tumor cell. [0036] (6) A cell differentiation inducer for inducing
differentiation of a cell into a regulatory T cell, wherein the
inducer activates MCP1 signaling in the cell. [0037] (7) A cell
differentiation inducer for inducing differentiation of a cell into
a regulatory T cell, containing a cell expressing a Snail protein,
an MCP1 protein, an FSTL1 protein, a membrane IL-13Ra2 protein or a
secretory IL-13Ra2 protein. [0038] (8) A cell differentiation
inducer for inducing differentiation of a cell into a regulatory T
cell, comprising an MCP1 protein, an FSTL1 protein or a secretory
IL-13Ra2 protein. [0039] (9) An immunosuppressor, wherein the
immunosuppressor activates MCP1 signaling. [0040] (10) An
immunosuppressor comprising a cell expressing a Snail protein or a
cell expressing an MCP1 protein, an FSTL1 protein, a membrane
IL-13Ra2 protein or a secretory IL-13Ra2 protein. [0041] (11) An
immunosuppressor comprising an MCP1 protein, an FSTL1 protein or a
secretory IL-13Ra2 protein. [0042] (12) An agent for treating
hyperimmune disease, wherein the agent activates MCP1 signaling.
[0043] (13) An agent for treating hyperimmune disease comprising a
cell expressing a Snail protein, an MCP1 protein, an FSTL1 protein,
a membrane IL-13Ra2 protein or a secretory IL-13Ra2 protein. [0044]
(14) An agent for treating hyperimmune disease comprising an MCP1
protein, an FSTL1 protein or a secretory IL-13Ra2 protein. [0045]
(15) An inhibitor of enhancement of gene expression for inhibiting
enhancement of expression of FoxP3 gene in a cell, wherein the
inhibitor suppresses MCP1 signaling in the cell. [0046] (16) An
inhibitor of enhancement of gene expression for inhibiting
enhancement of expression of FoxP3 gene in a cell, wherein the
inhibitor suppresses function of an MCP1 protein, an FSTL1 protein,
a membrane IL-13Ra2 protein or a secretory IL-13Ra2 protein. [0047]
(17) The inhibitor of enhancement of gene expression according to
(15) or (16), comprising an anti-MCP1 antibody with MCP1 inhibiting
activity. [0048] (18) The inhibitor of enhancement of gene
expression according to (16), comprising an anti-FSTL1 antibody
with FSTL1 protein inhibiting activity, an anti-membrane IL-13Ra2
antibody with membrane IL-13Ra2 protein inhibiting activity or an
anti-secretory IL-13Ra2 antibody with secretory IL-13Ra2 protein
inhibiting activity. [0049] (19) An inhibitor of induction of cell
differentiation for inhibiting induction of differentiation of a
cell into a regulatory T cell, wherein the inhibitor suppresses
MCP1 signaling in the cell. [0050] (20) An inhibitor of induction
of cell differentiation for inhibiting induction of differentiation
of a cell into a regulatory T cell, wherein the inhibitor
suppresses function of an MCP1 protein, an FSTL1 protein, a
membrane IL-13Ra2 protein or a secretory IL-13Ra2 protein. [0051]
(21) The inhibitor of induction of cell differentiation according
to (19) or (20), comprising an anti-MCP1 antibody with MCP1
inhibiting activity. [0052] (22) The inhibitor of induction of cell
differentiation according to (20), comprising an anti-FSTL1
antibody with FSTL1 protein inhibiting activity, an anti-membrane
IL-13Ra2 antibody with membrane IL-13Ra2 protein inhibiting
activity or an anti-secretory IL-13Ra2 antibody with secretory
IL-13Ra2 protein inhibiting activity. [0053] (23) A reducer of
immunosuppression suppressing MCP1 signaling. [0054] (24) A reducer
of immunosuppression suppressing function of an MCP1 protein, an
FSTL1 protein, a membrane IL-13Ra2 protein or a secretory IL-13Ra2
protein. [0055] (25) A stimulator of tumor immunity suppressing
MCP1 signaling. [0056] (26) A stimulator of tumor immunity
suppressing function of an MCP1 protein, an FSTL1 protein, a
membrane IL-13Ra2 protein or a secretory IL-13Ra2 protein. [0057]
(27) An antitumor agent suppressing MCP1 signaling. [0058] (28) An
antitumor agent, wherein the agent suppresses function of an MCP1
protein, an FSTL1 protein, a membrane IL-13Ra2 protein or a
secretory IL-13Ra2 protein. [0059] (29) A suppressor of tumor
growth, wherein the suppressor suppresses MCP1 signaling or
function of a TSP1 protein. [0060] (30) A suppressor of tumor cell
infiltration, wherein the suppressor suppresses MCP1 signaling or
function of an FSTL1 protein. [0061] (31) A suppressor of tumor
metastasis, wherein the suppressor suppresses MCP1 signaling or
function of an FSTL1 protein. [0062] (32) A gene expression
enhancer of FoxP3 gene, 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 cell,
comprising a substance enhancing Snail activity. [0063] (33) The
gene expression enhancer according to (32), wherein the substance
enhancing Snail protein activity is an expression vector of a snail
gene. [0064] (34) The gene expression enhancer according to (32),
wherein the cell is a Panc-1 cell. [0065] (35) An anticancer agent
for blood cancer, comprising an inhibitory substance inhibiting
function of a Snail protein. [0066] (36) The anticancer agent
according to (35), wherein the inhibitory substance inhibits
expression of the snail gene. [0067] (37) The anticancer agent
according to (35) or (36), wherein the blood cancer is leukemia.
[0068] (38) A gene expression enhancing method for enhancing
expression of FoxP3 gene in a cell, comprising the step of
activating MCP1 signaling in the cell. [0069] (39) A gene
expression enhancing method for enhancing expression of FoxP3 gene
in a cell, comprising the step of allowing the cell to contact with
a cell expressing one or more proteins selected from the group
consisting of a Snail protein, an MCP1 protein, an FSTL1 protein, a
membrane IL-13Ra2 protein and a secretory IL-13Ra2 protein. [0070]
(40) The gene expression enhancing method according to (39),
wherein the cell is a tumor cell. [0071] (41) A gene expression
enhancing method for enhancing expression of FoxP3 gene in a cell,
comprising the step of allowing the cell to contact with one or
more proteins selected from the group consisting of a Snail
protein, an MCP1 protein, an FSTL1 protein, a membrane IL-13Ra2
protein and a secretory IL-13Ra2 protein. [0072] (42) The gene
expression enhancing method for enhancing expression of FoxP3 gene
in a cell according to (40), comprising the step of allowing the
cell to contact with a culture supernatant of a cell expressing one
or more proteins selected from the group consisting of a Snail
protein, an MCP1 protein, an FSTL1 protein, a membrane IL-13Ra2
protein and a secretory IL-13Ra2 protein. [0073] (43) The gene
expression enhancing method according to (42), wherein the cell is
a tumor cell. [0074] (44) A cell differentiation inducing method
for inducing differentiation of a cell into a regulatory T cell,
comprising the step of activating MCP1 signaling in the cell.
[0075] (45) A cell differentiation inducing method for inducing
differentiation of a cell into a regulatory T cell, comprising the
step of allowing the cell to contact with a cell expressing a Snail
protein, an MCP1 protein, an FSTL1 protein, a membrane IL-13Ra2
protein or a secretory IL-13Ra2 protein. [0076] (46) A cell
differentiation inducing method for inducing differentiation of a
cell into a regulatory T cell, comprising the step of allowing the
cell to contact with an MCP1 protein, an FSTL1 protein or a
secretory IL-13Ra2 protein. [0077] (47) A method for treating a
patient with hyperimmunity, comprising the step of administering to
the patient an activator to activate MCP1 signaling. [0078] (48) A
method for treating a patient with hyperimmunity, comprising the
step of administering to the patient a cell expressing a Snail
protein or a cell expressing an MCP1 protein, an FSTL1 protein, a
membrane IL-13Ra2 protein or a secretory IL-13Ra2 protein. [0079]
(49) A method for treating a patient with hyperimmunity, comprising
the step of administering to the patient an MCP1 protein, an FSTL1
protein or a secretory IL-13Ra2 protein. [0080] (50) An agent for
treating hyperimmune disease activating MCP1 signaling. [0081] (51)
A gene expression enhancement-inhibiting method for inhibiting
enhancement of FoxP3 gene expression in a cell, comprising the step
of suppressing MCP1 signaling in the cell. [0082] (52) A gene
expression enhancement-inhibiting method for inhibiting enhancement
of FoxP3 gene expression in a cell, comprising the step of
suppressing function of an MCP1 protein, an FSTL1 protein, a
membrane IL-13Ra2 protein or a secretory IL-13Ra2 protein in the
cell. [0083] (53) The gene expression enhancement-inhibiting method
according to (51), wherein the cell is allowed to contact with an
anti-MCP1 antibody with MCP1 inhibiting activity. [0084] (54) The
gene expression enhancement-inhibiting method according to (52),
wherein the cell is allowed to contact with an anti-FSTL1 antibody
with FSTL1 protein inhibiting activity, an anti-membrane IL-13Ra2
antibody with membrane IL-13Ra2 protein inhibiting activity or an
anti-secretory IL-13Ra2 antibody with secretory IL-13Ra2 protein
inhibiting activity. [0085] (55) A cell differentiation
induction-inhibiting method for inhibiting induction of
differentiation of a cell into a regulatory T cell, comprising the
step of suppressing MCP1 signaling in the cell. [0086] (56) A cell
differentiation induction-inhibiting method for inhibiting
induction of differentiation of a cell into a regulatory T cell,
comprising the step of suppressing function of an MCP1 protein, an
FSTL1 protein, a membrane IL-13Ra2 protein or a secretory IL-13Ra2
protein in the cell. [0087] (57) The cell differentiation
induction-inhibiting method according to (55), wherein the cell is
allowed to contact with an anti-MCP1 antibody with MCP1 inhibiting
activity. [0088] (58) The cell differentiation induction-inhibiting
method according to (56), wherein the cell is allowed to contact
with an anti-FSTL1 antibody with FSTL1 protein inhibiting activity,
an anti-membrane IL-13Ra2 antibody with membrane IL-13Ra2 protein
inhibiting activity or an anti-secretory IL-13Ra2 antibody with
secretory IL-13Ra2 protein inhibiting activity. [0089] (59) An
immunosuppression reducing method for reducing immunosuppression of
a patient with suppressed immunity, comprising the step of
suppressing MCP1 signaling in the patient. [0090] (60) An
immunosuppression reducing method for reducing immunosuppression of
a patient with suppressed immunity, comprising the step of
suppressing function of an MCP1 protein, an FSTL1 protein, a
membrane IL-13Ra2 protein or a secretory IL-13Ra2 protein in the
patient. [0091] (61) A method for stimulating tumor immunity in a
patient with suppressed tumor immunity, comprising the step of
suppressing MCP1 signaling in the patient. [0092] (62) A method for
stimulating tumor immunity in a patient with suppressed tumor
immunity, comprising the step of suppressing function of an MCP1
protein, an FSTL1 protein, a membrane IL-13Ra2 protein or a
secretory IL-13Ra2 protein. [0093] (63) A method for treating a
tumor patient, including the step of suppressing MCP1 signaling in
the patient. [0094] (64) A method for treating a tumor patient,
including suppressing function of an MCP1 protein, an FSTL1
protein, a membrane IL-13Ra2 protein or a secretory IL-13Ra2
protein in the patient. [0095] (65) A method for treating a tumor
patient, comprising suppressing function of a TSP1 protein in the
patient. [0096] (66) A gene expression enhancing method for FoxP3
gene, 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 cell,
comprising the step of allowing the cell to contact with a
substance for enhancing Snail protein activity. [0097] (67) The
gene expression enhancing method according to (66), wherein the
substance for enhancing Snail protein activity is a snail gene
expression vector. [0098] (68) The gene expression enhancing method
according to (66), wherein the cell is a Panc-1 cell. [0099] (69) A
method for treating a patient with blood cancer, comprising the
step of administering to the patient a substance for inhibiting
function of a Snail protein. [0100] (70) The treating method
according to (69), wherein the substance inhibits expression of
snail gene. [0101] (71) The treating method according to (69),
wherein the blood caner is leukemia.
BRIEF DESCRIPTION OF DRAWINGS
[FIG. 1]
[0102] 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]
[0103] 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]
[0104] 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]
[0105] 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]
[0106] 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]
[0107] 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]
[0108] 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]
[0109] 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]
[0110] 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]
[0111] 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]
[0112] 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]
[0113] 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]
[0114] 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]
[0115] 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]
[0116] 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]
[0117] 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]
[0118] 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]
[0119] 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]
[0120] 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]
[0121] 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]
[0122] FIG. 17B shows suppression of the infiltration of leukemia
cells using snail gene-specific siRNAs in one example of the
present invention.
[FIG. 18]
[0123] 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]
[0124] 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]
[0125] 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).
DESCRIPTION OF EMBODIMENTS
[0126] Hereinafter the embodiments of the present invention thus
accomplished based on the abovementioned findings are described in
detail by giving examples. Where no explanation is given in the
embodiments or examples, methods described in standard protocols
such as J. Sambrook, E. F. Fritsch & T. Maniatis (Ed.),
Molecular cloning, a laboratory manual (3rd edition), Cold Spring
Harbor Press, Cold Spring Harbor, N.Y. (2001); and F. M. Ausubel,
R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith,
K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley
& Sons Ltd., as well as their modifications/variations, are
employed. Where a commercially available reagent kit or a measuring
instrument is used, the protocol attached thereto will be
followed.
[0127] It is to be understood that the object, characteristics, and
advantages of the present invention as well as the ideas thereof
will be apparent to those skilled in the art from the descriptions
given herein, and the present invention can be easily reproduced by
those skilled in the art based on the descriptions given herein.
The embodiments and specific examples of the invention described
herein are to be taken as preferred embodiments of the present
invention, and these descriptions are presented only for
illustrative and/or explanatory purposes. Therefore, the present
invention is not limited to these examples, and all the embodiments
those skilled in the art can assume from the examples are also
encompassed by the present invention. It is further apparent to
those skilled in the art that various changes and modifications may
be made based on the descriptions given herein within the intent
and scope of the present invention disclosed herein, and such
variations and modifications are also encompassed by the present
invention.
==Gene Expression Enhancer I. Genes of MCP1, TSP1, FSTL1 and
IL-13Ra2==
[0128] 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.
[0129] 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.
[0130] 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.
[0131] While 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.
==Gene Expression Enhancer II. FoxP3 Gene==
[0132] 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.
[0133] 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.
[0134] In a method for using the gene expression enhancer, 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. In an example, 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.
[0135] 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 and CD8+ T cells.
==Gene Expression Enhancer III. FoxP3 Gene==
[0136] 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.
[0137] 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.
[0138] 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.
[0139] Specific examples 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.
[0140] The gene expression enhancer may contain one or more than
one of the abovementioned substances.
[0141] In a method for using the gene expression enhancer, the
enhancer may be, for example, directly administered to the cells to
be treated. In this case, 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. Alternatively, 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.
[0142] 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.
==Cell Differentiation Inducer, Immunosuppressor==
[0143] 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. Indeed,
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.
[0144] 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 cell differentiation
inducer and the immunosuppressor may be used either in vivo or in
vitro. The immunosuppressor may suppress hyperimmunity and/or
normal immunity.
==Agent for Treating Hyperimmune Disease==
[0145] Hypofunction of regulatory T cells causes hyperimmune
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.
[0146] While the method for using the agent for treating
hyperimmune diseases may be chosen appropriately, the agent is
preferably administered to patients by either a systemic
administration or a topical administration to the site of
hyperimmunity.
==Inhibitor of Enhancement of FoxP3 Gene Expression==
[0147] 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.
[0148] 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.
[0149] 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.
[0150] 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.
[0151] In a method for using the inhibitor of enhancement of gene
expression, the inhibitor may be administered in vivo or in vitro
to the cells to be treated or its vicinity. In the case where 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.
[0152] 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.
==Inhibitor of Induction of Cell Differentiation, Reducer of
Immunosuppression==
[0153] As mentioned above, 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. Thus, 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.
[0154] In a method for using the inhibitor of induction of cell
differentiation or the 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.
In the case where the inhibitor of induction of cell
differentiation or the reducer of immunosuppression 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.
[0155] While 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.
==Stimulator of Tumor Immunity, Antitumor Agent==
[0156] The inventors of the present invention discovered that 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. Namely, it was discovered that 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. Based on this fact, 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. Examples of 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.
[0157] Indeed, 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. However, the inhibitory substance for inhibiting
the action of the mediating proteins can suppress the inhibition of
phagocytosis by the Snail-expressing tumor cells.
[0158] While 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.
==Suppressor of Tumor Growth==
[0159] 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.
[0160] While 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.
==Suppressor of Tumor Cell Infiltration, Suppressor of Tumor
Metastasis==
[0161] 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.
[0162] 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.
[0163] 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).
[0164] While 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.
[0165] With regard to any of the abovementioned agents according to
the present invention, 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.
==Anticancer Agent for Blood Cancer==
[0166] The anticancer agent for blood cancers according to the
present invention 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.
[0167] It should be noted that the relation of Snail with the blood
cancer has never been known beforehand. Even if the involvement of
the expression of Snail in the metastasis of cancers might have
been imagined, it was due to the regulation of intercellular
adhesion molecules such as E-cadherin found in the solid cancer,
and the involvement of Snail in cancers of blood cells in a
suspension state has never been obvious by those skilled in the
art.
[0168] While the blood cancers are not particularly limited, the
examples include leukemia, malignant lymphoma and multiple
myeloma.
Examples
{1} Forced Expression of Snail Gene in Panc-1 Cell
<Purpose>
[0169] 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.
<Experimental Methods>
(1) Construction of Expression Vector for Snail Gene and
Introduction of the Vector
[0170] 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.
(2) Analysis of Gene Expression in snail Gene-Introduced Cell Line
by RT-PCR
[0171] RNAs were extracted from the human tumor cell lines by using
RNeasy (QIAGEN) and reverse-transcribed by using AMV (incubated at
42.degree. C. for 50 min and at 70.degree. 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.
TABLE-US-00001 Forward 5'-CAGATGAGGACAGTGGGAAAGG-3' (SEQ ID NO: 1)
Reverse 5'-ACTCTTGGTGCTTGTGGAGCAG-3' (SEQ ID NO: 2)
(3) Analysis of Protein Expression in snail Gene-Introduced Cell
Line by Immunohistochemical Staining
[0172] In order to examine the expression levels of proteins in the
tumor cells, the tumor cells (5.times.10.sup.4 cells) were cultured
in slide chambers overnight (37.degree. C., 5% CO.sub.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.degree. C., 20 min), then stained with various antibodies (e.g.
an anti-Snail antibody (SANTA CRUZ) plus an Alexa488-labelded
anti-goat IgG (Molecular Probes), or an anti-E-cadherin antibody
(BD Bioscience) plus an Alexa568-labelded anti-mouse IgG (Molecular
Probes)), for 1 hour at 4.degree. C. Afterwards, cells were mounted
with using Vecter Shield (Vector Laboratories), and observed under
a fluorescence microscope (LSM 5 PASCAL, Carl Zeiss).
<Results>
[0173] FIG. 1A summarizes the phenotypes of the cell lines
introduced with snail gene.
[0174] While intrinsic expression of Snail protein was observed in
the Panc-1 cell as the parent cell line, the expression level of
Snail protein was further increased by the introduction of the
expression vector for snail gene in the clones listed in the table.
As the results, in each of the clones, the cellular shape was
changed from round shape to spindle/spreading shape, the cellular
growth capability ("Proliferation") was reduced both in vitro and
in vivo, the protein level of E-cadherin as well as the cellular
adhesion capability ("Adhesion") were reduced, and the cellular
mobility ("Migration") as well as the cellular infiltration
capability ("Invasion") were increased. In particular, a prominent
effect was observed in Clone F3 with regard to the
epithelial-mesenchymal transition (EMT), and thus F3 was used in
the following Examples.
{2} Induction of expression of FoxP3 in CD4+ Cells by Coculturing
with Hs294T Cells
<Purpose>
[0175] By coculturing human melanoma Hs294T cells treated by
TGF-beta with PBMCs, it will be shown that an enhancement of
expression of FoxP3 protein occurs in CD4+ cells among PBMCs and
that the expression of FoxP3 protein disappears by a knockdown of
snail gene.
<Experimental Methods>
(1) TGF-Beta Treatment of Tumor Cells
[0176] The following 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
.mu.g/3.times.10.sup.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.
Sequences of Snail Gene-Specific siRNAs #1:
TABLE-US-00002 Forward 5'- GCGAGCUGCAGGACUCUAA-3' (SEQID NO: 3)
Reverse 5'- UUAGAGUCCUGCAGCUCGC-3' (SEQID NO: 4)
Sequences of Snail Gene-Specific siRNAs #2:
TABLE-US-00003 Forward 5'- CCCACUCAGAUGUCAAGAA-3' (SEQ ID NO: 5)
Reverse 5'- UUCUUGACAUCUGAGUGGG-3' (SEQ ID NO: 6)
Sequences of siRNA Control:
TABLE-US-00004 Forward 5'-GCGCGUCAGGACUCGAUAA-3' (SEQ ID NO: 7)
Reverse 5'-UUAUCGAGUCCUGACGCGC-3' (SEQ ID NO: 8)
(2) Analysis of Gene Expression by RT-PCR in Tumor Cell
[0177] 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.
TABLE-US-00005 Forward 5'-GTCAACGGATTTGGTCGTATT-3' (SEQ ID NO: 9)
Reverse 5'-ATCACTGCCACCCAGAAGACT-3' (SEQ ID NO: 10)
(3) Isolation of Human Peripheral Blood Cell (PBMC)
[0178] 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".
(4) Coculture of PBMC with Tumor Cell (Hs294T Cell)
[0179] The tumor cells obtained as above were first inactivated by
either a treatment with MMC (100 .mu.g/mL, 2 hours at 37.degree.
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.times.10.sup.5 of PBMCs and 1.times.10.sup.4 tumor
cells in a 96-well plate, or 5.times.10.sup.5 of PBMCs and
5.times.10.sup.4 of tumor cells in a 24-well plate) at 37.degree.
C. in 5% CO.sub.2 for 3 to 5 days, and then the PBMCs were
recovered.
(5) Expression Analysis of FoxP3 Protein
[0180] 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.degree. C., 20 min), then incubated with
an anti-FoxP3 antibody (eBioscience) at 4.degree. 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.
<Results>
[0181] FIG. 2A shows the results of the analysis for the expression
of snail gene and the expression of FoxP3. While the expression of
snail gene was increased in the human melanoma Hs294T cells by
treating with TGF-beta ("Control" in the figure), the expression
was suppressed by the snail gene-specific siRNAs. The content of
the cells expressing FoxP3 in the CD4+ cell fraction was increased
(31%) by the treatment with TGF-beta in comparison to the cells not
treated (25%), but the contents were decreased by suppressing the
expression of snail gene with using the snail gene-specific siRNAs,
regardless of the TGF-beta treatment (no treatment: from 25% to
17%, treated: from 31% to 20%). This indicates that the increase in
the expression of FoxP3 in the CD4+ cells is due to the increased
expression of snail gene in the Hs294T cells.
{3} Induction of FoxP3 Expression and Acquisition of Suppressive
Activity of T Cell Proliferation in CD4+ Cell by Coculturing of
Panc-1 Cell with F3 Cell or D10 Cell
<Purpose>
[0182] By coculturing PBMCs with either F3 cells or D10 cells, it
will be shown that an enhancement of expression of FoxP3 protein
occurs in CD4+ cells among the PBMCs and that the CD4+ cells
acquire an activity to suppress the proliferation of T cells.
<Experimental Method>
[0183] (1) Coculture of PBMCs with Tumor Cells (Panc-1 Cell or F3
Cell)
[0184] Panc-1 cells or F3 cells were coculturd with PBMCs in the
same method as in {2} above.
(2) Coculture of CD4+ Cells with Fresh T Cells
[0185] 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.degree. C. for 30 minutes; and
CD4+ cells were isolated by using a MACS automated cell sorter
(Miltenyi Biotec).
[0186] In the meantime, for observing proliferation responses of T
cells, fresh PBMCs were obtained in the same method as in {2} above
from the same healthy individual, and 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).
These fresh T cells (2.times.10.sup.5) were added with an anti-CD3
antibody (final concentration of 1 .mu.g/mL) along with the CD4+
cells (2.times.10.sup.5) which were isolated from the PBMCs having
been cultured with the tumor cells, and cultured in a 96-well plate
for 4 days. After an addition of 1/10 volume of Premix WST-1
Solution (Takara Bio), the cells were further cultured for 24
hours. Then 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.
(3) Expression Analysis of FoxP3 Protein
[0187] The expression of FoxP3 protein was analyzed in the same
method as in {2} above.
<Results>
(1) Expression Analysis of FoxP3 Protein
[0188] FIG. 2B shows the results of the FACS analyses.
[0189] Upper panels ("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. Lower panels ("FoxP3+ in CD4+ fraction") show the
expression levels of FoxP3+ protein in the CD4+ cell fractions (the
expression level of FoxP3+ protein increases as the x-axis advances
in each graph), and the numbers in the graphs indicate the contents
(%) of FoxP3+ cells among the fraction of CD4+ cells present within
the range where the expression was judged to be positive in
comparison to the isotype control (rat IgG2a) for the anti-FoxP3
antibody used.
[0190] An enhancement of expression of FoxP3 was observed in the
CD4+ cells by coculturing with the Panc-1 cells in which snail gene
was not forced to be expressed ("Parent" in the figure: the
expression level of FoxP3 was 30.83) in comparison to the cells
without the coculturing with tumor cells ("No tumor" in the figure:
14.56). In contrast, further enhancement of the expression of FoxP3
was achieved by coculturing with the F3 cells where snail gene was
forced to be expressed ("F3" in the figure: 43.44). These results
indicate that there is a correlation between the expression level
of snail gene and the expression enhancing capability for
FoxP3.
[0191] Thus, 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.
[0192] It should be noted that because of the fact that the
expression enhancing capability for FoxP3 was reduced when the
expression of snail gene was suppressed by introducing the siRNAs
specific for snail gene into the F3 cells, the expression enhancing
capability for FoxP3 has been confirmed not to be an artifact due
to variations among clones or the like, but indeed the consequence
of the forced expression of snail gene.
(2) Coculture of CD4+Cells with T Cells
[0193] 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. As the
positive controls, 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).
[0194] While the proliferation of T cells was strongly suppressed
by coculturing with Panc-1 cells (1 to 1.5 for "Prt" or "Mock" in
the figure), the proliferation was even further suppressed when the
T cells were cocultured with D10 cells or F3 cells, showing their
significant suppressive effect (P<0.001).
[0195] Thus, 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.
{4} Induction of Expression of FoxP3 in CD4+ Cells by Coculturing
with HCT116 Cells
<Purpose>
[0196] An experiment according to {1} and {2} above was conducted
with using HCT116 cells, a human intestinal cancer cell line, in
place of the Panc-1 cells. The experimental methods were the same
as those in {1} and {2} above.
<Results>
[0197] FIG. 3 shows the results of the FACS analyses.
[0198] 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). In contrast, 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). Thus,
a correlation between the expression level of snail gene and the
expression enhancing capability for FoxP3 was observed in multiple
cancer types.
{5} Induction of Expression of FoxP3 in CD4+ Cells by Cell Culture
Supernatant from Clone with Forced Expression of Snail Gene
<Purpose>
[0199] It will be shown that not only the F3 cell by itself but
also a cell culture supernatant from the F3 cell enhances the
expression of FoxP3 protein in CD4+ cells.
<Methods>
(1) Method for Preparing Culture Supernatant
[0200] 1.times.10.sup.5 of tumor cells were cultured in a 25
cm.sup.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.degree. C.), from which a supernatant was taken as the
culture supernatant, and stored at 4.degree. C. until used in the
following experiment.
(2) Method for Treatment by Culture Supernatant
[0201] A suspension of 5.times.10.sup.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.degree. C. and 5% CO.sub.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.
(3) Others
[0202] The expression analysis of FoxP3 protein was conducted
according to {2} above.
<Results>
[0203] FIG. 4 shows the results of the FACS analyses.
[0204] The culture supernatant from the parent cell line ("Prt-sup"
in panel A, "Parent" in panel B) as well as the Snail-introduced
cells ("D6-snail+", "D10-snail+", "F3-snail+", and "F5-snail+" in
panel A, "Snail-Tr" in panel B) enhanced the expression of FoxP3
protein in each of CD4+ cells (panel A, top row), CD4+CD25- cells
(panel A, bottom row), and CD4+CD25+ cells (Panel B), in comparison
to the cases where only the culture medium was used ("Medium" in
panel A, "No stimulant" in panel B). This indicates that the
enhancing action for the expression of FoxP3 protein by the
expression of Snail protein is mediated by a humoral factor.
[0205] Thus, the culture supernatant from the cells expressing
Snail protein can be also used as an enhancer for the expression of
FoxP3 protein.
[0206] It should be noted that since the 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).
{6} Genes Whose Expression Increases by Forced Expression of Snail
Protein in Panc-1, HCT116 and Hs294T Cell Lines
<Purpose>
[0207] It will be shown that the expression of MCP1, TSP1, FSTL1
and secretory IL-13Ra2 increases by the forced expression of Snail
protein in Panc-1, HCT116 and Hs294T.
<Methods>
(1) Method for Measuring Expression Level of MCP1, TSP1, FSTL1 or
IL-13Ra2
[0208] Commercially available 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. As for the negative control, a similar
experiment was conducted with using a medium which was not used for
a culture.
[0209] Meanwhile, the gene expression of FSTL1 was measured by the
RT-PCR method as described in {1} above. The following
oligonucleotides were used as the primers. As for the negative
control (NC), a similar experiment was conducted without addition
of an mRNA.
TABLE-US-00006 Forward2 5'-GCACAGGCAACTGTGAGAAA-3' (SEQ ID NO: 11)
Reverse2 5'-CATAGTGTCCAAGGGCTGGT-3' (SEQ ID NO: 12)
(2) Method for Detecting Intracellular Localization of IL-13Ra2
Protein
[0210] The expression of the membrane-bound form of IL-13Ra2 as
well as those present in cytoplasm/nuclei of tumor cells were
detected similarly to the method for the expression of Snail
protein, by the immunostaining as described in {1}(3) above.
<Results>
[0211] The results are shown in FIG. 5. (A) 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.
[0212] In each of Snail-expressing clones (D6, D10, F3 and F5), the
expression of the abovementioned cytokines were increased in
comparison to the parent cell line (Prt). The increase in the
expression of IL-13Ra2 was observed not only for its secretory form
(FIG. 5A) but also those on the membrane as well as in the
cytoplasm and nuclei of each clone as shown in FIG. 5C.
[0213] Thus, an enhancement of the activity of Snail protein can
enhance the expression of MCP1, TSP1, IL-13Ra2 and FSTL-1.
{7} Suppression of Enhancement of FoxP3 Protein Expression by F3
Clone Using Antibodies to Each of MCP1, TSP1, FSTL1 and IL13Ra2
Proteins.
<Purpose>
[0214] It will be shown that antibodies to each of MCP1, TSP1,
FSTL1 and IL13Ra2 proteins inhibit the action of F3 clone to
enhance the expression of FoxP3 protein.
<Methods>
[0215] (1) Antibodies Used in this Example
[0216] Commercially available products of 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.
(2) Recovery of Culture Supernatants Added with Antibodies
[0217] 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 .mu.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.
(3) Others
[0218] Preparation of the culture supernatant from tumor cells and
the expression analysis of FoxP3 protein were conducted according
to {5} above.
<Results>
[0219] 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.
[0220] 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. For example, 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. 7) in CD4+ cells, from 38.39 to 28.86 in CD4+CD25+
cells, and from 35.66 to 28.41 in CD4+CD25- cells. Similarly, the
anti-IL-10 antibody ("Anti-IL-10" in the figures) reduced the
expression levels of FoxP3 protein to 11.42 (FIG. 6) or to 30.88
(FIG. 7) (in CD4+ cells), to 29.89 (in CD4+ CD25+ cells) and to
28.05 (in CD4+ CD25- cells).
[0221] Meanwhile, the administration of the anti-MCP1 antibody
("Anti-MCP1" in the figures) 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). Thus, 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.
[0222] When the anti-MCP1 antibody, the anti-TSP1 antibody, the
anti-FSTL1 antibody and the anti-IL13Ra2 antibody were used
together, a maximum effect of suppression of expression enhancing
capability for Fox3 protein was observed in CD4+ cells (reduction
from 35.34 to 8.54) (FIG. 6), indicating that each of these
cytokines redundantly contributes to the enhancing action for the
expression of FoxP3 protein by the culture supernatant of F3
clone.
[0223] Therefore, 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.
{8} Suppression for Enhancement of Expression of FoxP3 Protein
Using Anti-IL-13Ra2 Antibody
<Purpose>
[0224] It will be shown that anti-IL-13Ra2 antibody inhibits the
activity of B11 clone to enhance the expression of FoxP3
protein.
<Methods>
[0225] An experiment according to {7} above was conducted except
that the anti-MCP1 antibody and the anti-IL-13Ra2 antibody were
used as the antibodies and that B11 clone was used as the tumor
cells. As for the positive control to inhibit the activity to
enhance the expression of FoxP3, an anti-IL-13 antibody was used in
this case.
<Results>
[0226] FIG. 8 shows the results with using the anti-MCP1 antibody
and the anti-IL-13Ra2 antibody.
[0227] 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). Thus, 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.
[0228] Thus, 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.
{9} Suppression of Enhancement of Expression of Foxp3 Protein by
Mouse Melanoma B16-F10 Using Anti-MCP1 Antibody And Anti-IL-13Ra2
Antibody
<Purpose>
[0229] It will be shown that the anti-MCP1 antibody and the
anti-IL-13Ra2 antibody inhibit the activity for enhancement of the
FoxP3 protein expression in mouse melanoma B16-F10.
<Methods>
(1) Method for Preparing Mouse Spleen Cell
[0230] 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.
(2) Others
[0231] 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.
<Results>
[0232] FIG. 9 (upper panels) 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.
[0233] 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.
[0234] Thus, the effect of suppression of the enhancement of FoxP3
protein expression can be observed not only in human tumor cells
but also in mouse tumor cells.
{10} Enhancing Action of Cytokines MCP1, TSP1, FSTL1 and Secretory
IL-13Ra2 on the FoxP3 Protein Expression
<Purpose>
[0235] It will be shown that each of the cytokines MCP1, TSP1,
FSTL1 and secretory IL-13Ra2 enhances the expression of FoxP3
protein in CD4+ cells.
<Methods>
[0236] An experiment according to {5} above was conducted except
that a medium supplemented with either of the cytokines at 1 ng/mL
was used in place of the culture supernatant. As the positive
control, culture supernatants from Panc-1 cell and F3 clone as well
as the cytokines TGF-b and IL-10 were used. As for the each
cytokine, the following commercially available products were used:
MCP1: R&D, #279-MC; TSP1: R&D #3074-TH; FSTL1: R&D
#669-FO; secretory IL-13Ra2: Abcam #ab46112; TGF-b: R&D #100-B;
and IL-10: eBioscience #34-8109.
<Results>
[0237] FIG. 10 shows the results of the measurements for the
expression of FoxP3 protein.
[0238] In comparison to the case where the medium without the
addition of a cytokine was used (designated as "Medium" in the
figure; the expression level was 2.55), enhancing actions on the
expression of FoxP3 protein, similar to that of the culture
supernatant from F3 clone (the expression level was 17.08), were
observed by the addition of each of MCP1 protein (the expression
level was 17.35), TSP1 protein (the expression level was 18.40),
FSTL1 protein (the expression level was 16.00) and secretory
IL-13Ra2 protein (the expression level was 20.21).
[0239] Thus, each of the cytokines MCP1, TSP1, FSTL1 and secretory
IL-13Ra2 is useful as an agent for enhancing the expression of
FoxP3 protein.
{11} Direct and Indirect Actions of Snail-Expressing Cell on
Enhancement of the FoxP3 Protein Expression
<Purpose>
[0240] It will be shown that there are direct and indirect actions
of Snail-expressing cells on enhancement of the FoxP3 protein
expression, and that the indirect action involves at least a
dendritic cell.
<Methods>
[0241] By using isolated CD4+ cells as the immune cell in place of
the bulk PBMCs, 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.
[0242] The CD4+ cells and CD11c+ cells (dendritic 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.
<Results>
[0243] 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.
[0244] The addition of Bulk PBMCs instead of CD4+ cells only to F3
cells decreased the expression level of FoxP3 protein (from 35.34
in "Bulk+F3 cells" to 10.93 in "CD4+F3 cells"). When DCs or others
were further added, the expression level of FoxP3 protein was
increased (to 25.72 in "CD4+DC+F3 cells" or 16.15 in "CD4+others+F3
cells"), indicating that the increase in the expression level of
FoxP3 protein in CD4+ cells among the bulk PBMCs involves cells
other than the CD4+ cells (DCs or cells other than DCs).
[0245] Meanwhile, when F3 cells were added to CD4+ cells only, the
expression level of FoxP3 protein was increased (to 10.93 in "CD4+
F3") in comparison to the case where the bulk PBMCs was not
stimulated (2.55 in "Bulk (no stim)"), indicating that there is
also a mechanism where the F3 cell by itself or a humoral factor in
its culture supernatant acts directly on the CD4+ cell.
[0246] In all the cases where the cells were added, the expression
levels of FoxP3 protein were about twice as high as the cases where
the corresponding cell culture supernatants were added. For
example, "CD4+F3 cells" yielded 10.93, whereas "CD4+F3sup" yielded
5.48; "CD4+DC+F3 cells" yielded 25.72, whereas "CD4+sup (Tu+DC)"
yielded 14.9; and "CD4+others+F3 cells" yielded 16.15, whereas
"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.
{12} Induction of Expression of FoxP3 in CD8+ Cell by Culture
Supernatant of Cell Clone with Forced Expression of Snail Gene
<Purpose>
[0247] It will be shown that similarly to the case where CD4+ cells
are used, a cell culture supernatant of Clone F3 in which snail
gene is forced to be expressed also induces the expression of FoxP3
protein in CD8+ cells.
<Methods>
[0248] An experiment was conducted in the same method as in {5}
above except that CD8+ cells were used as the cells to be treated
in place of the CD4+ cells.
<Results>
[0249] FIG. 12 shows the results of the measurements for the
expression of FoxP3 protein.
[0250] Similarly to the results obtained by using CD4+ cells, 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).
[0251] Thus, a culture supernatant of Snail-expressing cells can
also enhance the expression of FoxP3 protein in CD8+ cells.
{13} Suppression of Enhancement of FoxP3 Protein Expression in CD8+
Cell Using Anti-TSP1 Antibody and Anti-IL-13Ra2 Antibody
<Purpose>
[0252] It will be shown that similarly to the case where CD4+ cells
are used, the anti-TSP1 antibody and the anti-IL-13Ra2 antibody
also suppress the enhancement of expression of FoxP3 protein in
CD8+ cells.
<Methods>
[0253] An experiment was conducted in the same method as in {7}
above except that CD8+ cells were used as the cells to be treated
in place of the CD4+ cells. The anti-MCP1 antibody or the anti-TSP1
antibody was used for F3 cells, and the anti-IL-13Ra2 antibody was
used for B11 cells. As for the negative control, the same
experiment was conducted with using the mouse IgG antibody.
<Results>
[0254] 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, and FIG. 14 shows the
results where the anti-IL-13Ra2 antibody was used for B11
cells.
[0255] 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. 10.21 by the
anti-IL-13Ra2 antibody (Anti-IL13Ra2)). Therefore, it is indicated
that the major contributors to the enhancing action of the cell
culture supernatant on the FoxP3 protein expression are at least
MCP1, TSP1 and IL-13Ra2.
[0256] Thus, at least MCP1, TSP1 and IL-13Ra2 also have the
enhancing action on the FoxP3 protein expression in CD8+ cells.
{14} Suppression of Proliferation of Snail-Expressing Tumor Cell
Using Anti-MCP1 Antibody And Anti-TSP1 Antibody
<Purpose>
[0257] It will be shown that an addition of the anti-MCP1 antibody
or the anti-TSP1 antibody to the tumor cells expressing snail gene
suppresses the proliferation of the tumor cells.
<Methods>
(1) Method for Measuring Cell Proliferation
[0258] The anti-MCP1 antibody, the anti-TSP1 antibody or the
anti-TGF-b antibody as a control was added at 2 .mu.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%.
<Results>
[0259] 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%. Thus, the proliferation capability of
tumor cells can be reduced by inhibiting function of MCP1 or
TSP1.
{15} Suppression for Infiltration of Snail-Expressing Tumor Cell
Using Anti-MCP1 Antibody and Anti-FSTL1 Antibody
<Purpose>
[0260] It will be shown that an addition of the anti-MCP1 antibody
or the anti-FSTL1 antibody to tumor cells expressing snail gene
suppresses the infiltration of the tumor cells.
<Methods>
[0261] 5.times.10.sup.4 of F3 cells placed in the upper chamber of
a Transwell Chamber (pore size 8 .mu.m, BD Bioscience) with its
membrane coated by matrigel were cultured overnight (37.degree. C.,
5% CO.sub.2). The anti-MCP1 antibody or the anti-FSTL1 antibody was
added into both the upper chamber and the lower chamber at 1
.mu.g/ml, during the culture. After complete removal of the cells
above the membrane, the cells infiltrate into the lower chamber
were fixed and stained using crystal violet solution, and counted
under a microscope to evaluate the cellular infiltration
capability. The content of the infiltrated cells were also
calculated by bringing the result obtained with using the negative
control (mIgG) to 100%. For the positive control, the anti-IL-10
antibody and the anti-TGF-b antibody were used.
<Results>
[0262] FIG. 16 shows the results of the measurements for the
cellular infiltration capability.
[0263] The cellular infiltration capability of F3 cells (designated
as "mIgG" in the figure) was enhanced in comparison to the parent
cell line of Panc-1 cells (designated as "parent" in the figure).
When 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. Thus, the
anti-MCP1 antibody and the anti-FSTL1 antibody have the action to
reduce the cellular infiltration capability of Snail-expressing
tumor cells.
{16} Suppression of Infiltration by Suppressing Expression of Snail
in Leukemia Cell
<Purpose>
[0264] It will be shown that an infiltration capability of leukemia
cells can be reduced by suppressing the expression of Snail in the
leukemia cells.
<Methods>
[0265] First, the expressions of Snail, MCP-1 and FSTL-1 in
leukemia cell lines (Molt-3, Molt-4, UF1, KT1, K562 and MC3) were
examined by RT-PCR. The same method as in {1}, {2} and {6} above
was employed except that primers of the following sequences were
used for MCP-1.
TABLE-US-00007 Forward 5'-GTGTTTGACATCTTTGAACTC-3' (SEQ ID NO: 13)
Reverse 5'-CCAAAGACAAACCTCACATTC-3' (SEQ ID NO: 14)
[0266] Next, 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 .mu.g/1.times.10.sup.6 cells/2 mL, Invitrogen) were cultured in
a 6-well plate.
[0267] After 2 days of culturing, the cells were placed in the
matrigel-coated Transwell Chamber (pore size 8 .mu.m, BD
bioscience) and cultured for 4 hours. Then the number of cells
infiltrated through the filter was counted.
<Results>
[0268] As shown in the results of the RT-PCR in FIG. 17A, strong
expression of Snail was detected in all the leukemia cell lines
tested.
[0269] FIG. 17B shows the number of infiltrated cells counted for
each of the leukemia cell lines. In all 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).
[0270] Thus, 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.
{17} Reduction of Tumor Immunity Suppression by Tumor Cell that
Inhibits the Action of Mediating Protein Produced by
Snail-Expressing Tumor Cell
<Purpose>
[0271] It will be shown that 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.
<Antibodies Used>
[0272] 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.
<Methods>
[0273] 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 .mu.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.degree. 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.degree. C.) for 2 hours.
<Results>
[0274] About 25% of PBMCs treated with the HCT116 cells not
expressing snail gene ("Mock-treated PBMCs" in FIG. 18) were
labeled with both fluorescence. In contrast, only about 10% of
PBMCs treated by the B11 clone ("B11-treated PBMCs" in FIG. 18)
were labeled with both fluorescence. Thus, a Snail-expressing tumor
cell is capable of suppressing the action of phagocytotic cells
among PBMCs.
[0275] However, when each of the antibodies was added while PBMCs
were allowed to contact with the B11 clone, the content of cells
labeled with both fluorescence was increased to 15%.about.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. Thus, by inhibiting function of the mediating
protein for suppression of tumor immunity, the suppression of tumor
immunity by tumor cells can be reduced, thereby stimulating the
tumor immunity.
{18} Experiment of In Vivo Treatment Using siRNA Specific for Snail
Gene or MCP1 Gene
<Purpose>
[0276] It will be shown that suppression of tumor immunity by tumor
cells can be reduced by suppressing function of Snail protein or
MCP1 signaling in vivo.
<Methods>
[0277] The cells in which snail gene was forced to be expressed
(H6-snail+) 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.times.10.sup.6 cells and an
intravenous injection with 2.times.10.sup.5 cells were both applied
to one individual simultaneously). After 7 days, the siRNAs
specific for Snail or MCP1 gene or the control siRNAs (5
.mu.g/mouse, Invitrogen) formed in lipid complexes by using PEI
(Polyplus Transfection) were injected into the subcutaneously
transplanted tumor.
The Sequences of the Mouse Snail Gene-Specific siRNAs:
TABLE-US-00008 Sense 5'-GGAAGAUCUUCAACUGCAA-3' (SEQ ID NO: 15)
Antisense 5'-UUGCAGUUGAAGAUCUUCC-3' (SEQ ID NO: 16)
The Sequences of the Mouse MCP1 Gene-Specific siRNAs:
TABLE-US-00009 Sense 5'-CCAGCAAGAUGAUCCCAAU-3' (SEQ ID NO: 17)
Antisense 5'-AUUGGGAUCAUCUUGCUGG-3' (SEQ ID NO: 18)
The Sequences of the Control for Mouse siRNA:
TABLE-US-00010 Sense 5'-CCAGAAGUACUACCGCAAU-3' (SEQ ID NO: 19)
Antisense 5'-AUUGCGGUAGUACUUCUGG-3' (SEQ ID NO: 20)
[0278] After 1 week, the tumor and a lung were removed from the
transplanted mouse, the volume of the tumor and the number of
metastatic lung nodules were measured, and 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.degree. C. for 1
hour, and subjected to the analysis.
<Results>
[0279] 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).
[0280] As shown in FIG. 19A and FIG. 19B, in the mouse transplanted
with B16-F10 ("Mock" in the figure), the growth of the tumor was
significant, whereas the metastasis into the lung was little. In
contrast, in the mouse transplanted with H6-snail+("Cont" in the
figure), the growth of tumor was not as significant as Mock,
whereas the metastasis into the lung was significantly promoted.
However, when the siRNAs specific for snail gene or MCP1 gene were
injected ("Snail" and "MCP1" in the figure, respectively), both the
proliferation of cells and the metastasis into the lung were
suppressed.
[0281] As shown in FIG. 19C, while the number of cells infiltrated
into the tumor was significantly decreased in the "H6-snail+" where
snail gene was forced to be expressed in comparison to the case of
B16-F10 ("Mock" in the figure), the number of cells infiltrated
into the tumor was increased to a level over the case of B16-F10 by
injecting the siRNAs specific for snail gene or MCP1 gene.
[0282] Thus, 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.
{19} Experiment of In Vivo Treatment Using Anti-TSP1 Antibody
<Purpose>
[0283] It will be shown that the suppression of tumor immunity by
tumor cells can be reduced by suppressing function of TSP1 protein
in vivo.
<Methods>
[0284] 1.times.10.sup.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 .mu.g/mouse, Calbiochem)
was injected into the subcutaneously transplanted tumor, and the
following assays were conducted after 1 week.
[0285] (1) The tumor volume was measured.
[0286] (2) In order to detect micrometastasis of tumor cells in
various tissues (LG: lung; SP: spleen; PB: peripheral blood; BM:
bone marrow), the gene expression of gp100, a cancer antigen
specifically expressed by B16-F10 cells, was analyzed by RT-PCR.
The same method for RT-PCR as in {1} above was employed except that
the following primers were used.
Sequences of Primers Specific for gp100:
TABLE-US-00011 Forward 5'-ACAGCCAGTGTATCCACAGG-3' (SEQ ID NO: 22)
Reverse 5'-ACTTCCATTGTGTGTGTGCC-3' (SEQ ID NO: 23)
Sequences of Primers Specific for GAPDH as the Control:
TABLE-US-00012 [0287] Forward 5'-TTGACCTCAACTACATGGTCTA-3' (SEQ ID
NO: 24) Reverse 5'-ACCAGTAGACTCCACGACATAC-3' (SEQ ID NO: 25)
[0288] (3) Intratumoral infiltrated cells were analyzed by using
the FACScan flow cytometer in the same method as in {18} above.
<Results>
[0289] 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).
[0290] (1) As shown in FIG. 20A, in both cases where B16-F10 was
transplanted ("Mock" in the figure) and where H6-snail+ was
transplanted ("H6-snail+" in the figure), the administeration of
the anti-TSP-1 antibody ("Anti-TSP1+" in the figure) significantly
reduced the tumor volumes.
[0291] (2) As shown in FIG. 20B, in the cases where H6-snail+ was
transplanted and a mouse IgG was administered as the control of the
antibody ("H6+Control mIgG" in the figure), a higher expression
level of gp100 was detected in each of the tissues in comparison to
the cases where B16-F10 was transplanted ("Mock" in the figure),
indicating the promotion of tumor metastasis. By administering the
anti-TSP-1 antibody ("H6+Anti-TSP1" in the figure), the expression
level of gp100 was decreased in all the tissues, indicating that
the metastasis into the tissues was significantly suppressed.
[0292] (3) As shown in FIG. 20C, while the number of cells
infiltrated into the tumor was significantly decreased in the
H6-snail+ where snail gene was forced to be expressed ("H6-snail+;
mIgG" in the figure) in comparison to the case of B16-F10 ("Mock;
mIgG" in the figure), the number of cells infiltrated into the
tumor was increased to a level over the case of B16-F10 ("Mock;
mIgG" in the figure) by administering the anti-TSP-1 antibody
("Anti-TSP1+" in the figure).
[0293] Thus, 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.
INDUSTRIAL APPLICABILITY
[0294] 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.
Sequence CWU 1
1
25122DNAArtificial SequencePrimer 1cagatgagga cagtgggaaa gg
22222DNAArtificial SequencePrimer 2actcttggtg cttgtggagc ag
22319RNAArtificial SequencesiRNA 3gcgagcugca ggacucuaa
19419RNAArtificial SequencesiRNA 4uuagaguccu gcagcucgc
19519RNAArtificial SequencesiRNA 5cccacucaga ugucaagaa
19619RNAArtificial SequencesiRNA 6uucuugacau cugaguggg
19719RNAArtificial SequencesiRNA 7gcgcgucagg acucgauaa
19819RNAArtificial SequencesiRNA 8uuaucgaguc cugacgcgc
19921DNAArtificial SequencePrimer 9gtcaacggat ttggtcgtat t
211021DNAArtificial SequencePrimer 10atcactgcca cccagaagac t
211120DNAArtificial SequencePrimer 11gcacaggcaa ctgtgagaaa
201220DNAArtificial SequencePrimer 12catagtgtcc aagggctggt
201321DNAArtificial SequencePrimer 13gtgtttgaca tctttgaact c
211421DNAArtificial SequencePrimer 14ccaaagacaa acctcacatt c
211519RNAArtificial Sequencesi RNA 15ggaagaucuu caacugcaa
191619RNAArtificial SequencesiRNA 16uugcaguuga agaucuucc
191719RNAArtificial SequencesiRNA 17ccagcaagau gaucccaau
191819RNAArtificial SequencesiRNA 18auugggauca ucuugcugg
191919RNAArtificial SequencesiRNA 19ccagaaguac uaccgcaau
192019RNAArtificial SequencesiRNA 20auugcgguag uacuucugg
19218PRTArtificial SequenceSynthetic protein 21Lys Ser Pro Trp Phe
Thr Thr Leu1 52220DNAArtificial SequencePrimer 22acagccagtg
tatccacagg 202320DNAArtificial SequencePrimer 23acttccattg
tgtgtgtgcc 202422DNAArtificial SequencePrimer 24ttgacctcaa
ctacatggtc ta 222522DNAArtificial SequencePrimer 25accagtagac
tccacgacat ac 22
* * * * *