U.S. patent application number 12/339129 was filed with the patent office on 2009-07-16 for method for detecting regulatory t cells using expression of folate receptor 4 as indicator, method for treating diseases using the detection method, pharmaceutical composition for immunostimulation, and method for treating diseases using the compostion.
This patent application is currently assigned to KYOTO UNIVERSITY. Invention is credited to Keiji Hirota, Shimon SAKAGUCHI, Tomoyuki Yamaguchi.
Application Number | 20090181012 12/339129 |
Document ID | / |
Family ID | 37234691 |
Filed Date | 2009-07-16 |
United States Patent
Application |
20090181012 |
Kind Code |
A1 |
SAKAGUCHI; Shimon ; et
al. |
July 16, 2009 |
METHOD FOR DETECTING REGULATORY T CELLS USING EXPRESSION OF FOLATE
RECEPTOR 4 AS INDICATOR, METHOD FOR TREATING DISEASES USING THE
DETECTION METHOD, PHARMACEUTICAL COMPOSITION FOR IMMUNOSTIMULATION,
AND METHOD FOR TREATING DISEASES USING THE COMPOSTION
Abstract
An object of the present invention is to provide a technique to
distinguish between T.sub.reg cells and activated T cells in a live
state. Another object of the present invention is to provide a
pharmaceutical composition for immunostimulation that can reduce
the number of T.sub.reg cells in vivo and effectively express the
immune response of activated T cells. In the method of the present
invention, T.sub.reg cells are detected from test cells containing
(i) regulatory T cells and (ii) at least one type of cell selected
from the group consisting of naive T cells and activated T cells,
wherein expressions of folate receptor 4 on the surfaces of cells
are measured and T.sub.reg cells are detected using the expressions
as an indicator. The present invention uses anti-folate receptor 4
antibody or folate receptor 4-binding fragment as an active
ingredient contained in a pharmaceutical composition for
immunostimulation.
Inventors: |
SAKAGUCHI; Shimon;
(Kyoto-shi, JP) ; Hirota; Keiji; (Kyoto-shi,
JP) ; Yamaguchi; Tomoyuki; (Kyoto-shi, JP) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W., SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
KYOTO UNIVERSITY
Kyoto-shi
JP
RIKEN
Wako-shi
JP
|
Family ID: |
37234691 |
Appl. No.: |
12/339129 |
Filed: |
December 19, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11240361 |
Oct 3, 2005 |
7488474 |
|
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12339129 |
|
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Current U.S.
Class: |
424/130.1 ;
424/93.71; 435/372.3; 435/7.24 |
Current CPC
Class: |
A61P 37/06 20180101;
A61P 37/04 20180101; C07K 16/28 20130101; G01N 33/56972 20130101;
A61K 2039/505 20130101; A61P 35/00 20180101 |
Class at
Publication: |
424/130.1 ;
435/7.24; 435/372.3; 424/93.71 |
International
Class: |
A61K 39/395 20060101
A61K039/395; G01N 33/53 20060101 G01N033/53; C12N 5/08 20060101
C12N005/08; A61K 45/00 20060101 A61K045/00; A61P 37/06 20060101
A61P037/06 |
Foreign Application Data
Date |
Code |
Application Number |
May 2, 2005 |
JP |
2005-134279 |
Claims
1. A method for detecting regulatory T cells from test cells
containing (i) regulatory T cells and (ii) at least one type of
cell selected from the group consisting of naive T cells and
activated T cells, the method comprising the steps of measuring
expressions of folate receptor 4 on the surfaces of the test cells
and detecting regulatory T cells using the expressions as an
indicator.
2. A detection method according to claim 1, wherein the expressions
of folate receptor 4 are measured using anti-folate receptor 4
antibody or folate receptor 4-binding fragment.
3. A method for isolating regulatory T cells comprising the step of
isolating cells contained in a cell group having the most intensive
folate receptor 4 expressions from test cells containing (i)
regulatory T cells, and (ii) at least one type of cell selected
from the group consisting of naive T cells and activated T
cells.
4. An isolation method according to claim 3, wherein the test cells
are those comprising T cells isolated from a mammal or comprising
cells obtained by subjecting naive T cells to antigenic
stimulus.
5. (canceled)
6. A method for suppressing immunity after organ transplant
comprising: step (a) collecting cells containing T cells from a
patient received an organ transplant; step (b) obtaining regulatory
T cells by isolating cells contained in a cell group having the
most intensive folate receptor 4 expressions from the cells
obtained in the step (a) or cells in which regulatory T cells were
induced by subjecting the cells obtained in the step (a) to
antigenic stimulus; and step (c) administering regulatory T cells
obtained in the step (b) to the patient.
7. A method for treating autoimmune diseases comprising: step (a)
collecting cells containing T cells from a patient suffering from
an autoimmune disease; step (b) obtaining regulatory T cells by
isolating cells contained in a cell group having the most intensive
folate receptor 4 expressions from the cells obtained in the step
(a) or cells in which regulatory T cells were induced by subjecting
the cells obtained in the step (a) to antigenic stimulus; and step
(c) administering regulatory T cells obtained in the step (b) to
the patient.
8. A reagent for detecting regulatory T cells comprising
anti-folate receptor 4 antibody or folate receptor 4-binding
fragment.
9. A pharmaceutical composition for immunostimulation comprising
anti-folate receptor 4 antibody or folate receptor 4-binding
fragment.
10. An pharmaceutical composition for treating tumors comprising
anti-folate receptor 4 antibody or folate receptor 4-binding
fragment.
11. An immunostimulation method comprising the step of
administering an amount of anti-folate receptor 4 antibody or
folate receptor 4-binding fragment effective for activating
immunity to a mammal including human.
12. A method for treating a patient suffering from a malignant
tumor or infectious disease comprising the step of administering an
amount of anti-folate receptor 4 antibody or folate receptor
4-binding fragment effective for treating malignant tumor to the
patient.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for detecting
regulatory T cells, a method for isolating regulatory T cells, a
method for treating diseases using these detection and isolation
methods, and a reagent for detecting regulatory T cells.
[0002] The present invention also relates to a pharmaceutical
composition for immunostimulation that can effectively express the
immune response of activated T cells by reducing the number of
regulatory T cells, and an immunostimulation method using the
composition.
BACKGROUND ART
[0003] CD4.sup.+CD25.sup.+ T cells are called regulatory T cells
(hereunder referred to as "T.sub.reg cells"), and it is known that
such T cells have an immunosuppressive activity and play an
important role in maintaining immunological tolerance (for example,
see Sakaguchi, S., et al., 1995, "Immunologic self-tolerance
maintained by activated T cells expressing IL-2 receptor
alpha-chains (CD25). Breakdown of a single mechanism of
self-tolerance causes various autoimmune diseases." J. Immunol.
155, 1151-1164). Because such T.sub.reg cells give an immune
response adverse to that of activated T cells, it is reported that
immunoactivation or immunosuppression can be more effectively
achieved by distinguishing between T.sub.reg cells and activated T
cells and applying them clinically (see, for example, Shimizu, J.,
et al., 1999, "Induction of tumor immunity by removing
CD25.sup.+CD4.sup.+ T cells: a common basis between tumor immunity
and autoimmunity.", J. Immunol. 163, 5211-5218). For example, it
has been reported that CD25.sup.+T cells derived from normal mice
having very low expressions of activated T cells are useful in
maintaining immunologic tolerance after organ transplantation
(Nishimura, E., Sakihama, T., Setoguchi, R., Tanaka. K., and
Sakaguchi, S.: Induction of antigen-specific immunologic tolerance
by in vivo and in vitro antigen-specific expansion of naturally
arising CD25+CD4+ regulatory T cells. Int. Immunol. 16: 1189-1201,
2004). Similar results have been reported in humans. (Yagi, H.,
Nomura, T., Nakamura, K., Kitawaki, T., Hori, S., Maeda, M.,
Onodera, M., Uchiyama, T., Fujii, S., and Sakaguchi, S.: Crucial
role of FOXP3 in the development and function of human CD25+CD4+
regulatory T cells. Int. Immunol. 16: 1643-1656, 2004). Therefore,
in order to clinically apply T.sub.reg cells, it is necessary to
establish a technique that distinguishes between T.sub.reg cells
and activated T cells.
[0004] Several methods for distinguishing between T.sub.reg cells
and activated T cells have been reported using substances expressed
on T.sub.reg cells as indicators. For example, CD25 is expressed on
T.sub.reg cells at extremely high levels, and therefore it has been
reported that T.sub.reg cells can be collected by isolating T cells
having extremely high CD25 expressions (Hoffmann P, Eder R,
Kunz-Schughart L A, Andreesen R, Edinger M. Large-scale in vitro
expansion of polyclonal human CD4(+)CD25 high regulatory T cells.
Blood. 104:895-903, 2004). Furthermore, CD25 and GITR have been
reported to be expressed on T.sub.reg cells at high levels (see
Shimizu, J., et al., 2002, "Stimulation of CD25(+)CD4(+) regulatory
T cells through GITR breaks immunological self-tolerance", Nat.
Immunol. 3, 135-142). However, because CD25 and GITR are expressed
not only on T.sub.reg cells but also on activated T cells at high
levels, it is known that CD25 and GITR cannot be used with high
accuracy as indicators for distinguishing between T.sub.reg cells
and activated T cells. There is another report stating that Foxp3
is specifically expressed in T.sub.reg cells (see Hori, S., et al.,
2003, "Control regulatory T cell development by the transcription
factor Foxp3.", Science 299, 1057-1061). However, since Foxp3 is a
transcription factor, which is not expressed on the surface of
cells, it is impossible to clinically use a T cell population from
which T.sub.reg cells have been removed or isolated T.sub.reg cells
in a live state by employing a method wherein T.sub.reg cells are
isolated or removed using Foxp3 as an indicator. As described
above, it is impossible to isolate or remove T.sub.reg cells by
detecting and distinguishing the T.sub.reg cells from activated T
cells in a live state using prior art techniques.
[0005] Furthermore, there is no report to date regarding not only a
technique for isolating or depleting live T.sub.reg cells but also
a technique by which the number of T.sub.reg cells is selectively
reduced in vivo.
DISCLOSURE OF THE INVENTION
[0006] An object of the present invention is to provide a technique
for distinguishing between T.sub.reg cells and activated T cells in
a live state. Another object of the present invention is to provide
a method for treating malignant tumors or infectious diseases or
for suppressing immunity after organ transplantation, using
above-mentioned technique.
[0007] Still another object of the present invention is to provide
a pharmaceutical composition that effectively expresses the immune
response of activated T cells by reducing the number of T.sub.reg
cells in vivo, and a method for activating immunity and for
treating disease using the pharmaceutical composition.
[0008] The present inventors conducted extensive research for
achieving the above objects and found that T.sub.reg cells are
expressed on the surface of folate receptor 4 (folate receptor
.delta., folate binding protein 3) at higher levels than activated
T cells and naive T cells, and therefore T.sub.reg cells can be
distinguished from activated T cells and naive T cells using the
expression of folate receptor 4 on the surface of the cell as an
indicator. Furthermore, an anti-folate receptor 4 antibody or a
folate receptor 4-binding fragment, which is used for specifically
detecting T.sub.reg cells, has an effect for reducing T.sub.reg
cells in vivo, stimulating immunity activity in vivo, and in
particular an effect for treating tumors or infectious diseases.
The present invention was accomplished based on these findings and
by adding improvements thereto.
[0009] In other words, with respect to a technique that detects or
isolates regulatory T cells, the present invention provides the
following inventions:
[0010] Item 1. A method for detecting regulatory T cells from test
cells containing (i) regulatory T cells and (ii) at least one type
of cell selected from the group consisting of naive T cells and
activated T cells,
[0011] the method comprising the steps of measuring expressions of
folate receptor 4 on the surfaces of the test cells and detecting
regulatory T cells using the expressions as an indicator.
[0012] Item 2. A detection method according to Item 1, wherein the
expressions of folate receptor 4 are measured using anti-folate
receptor 4 antibody or folate receptor 4-binding fragment.
[0013] Item 3. A method for isolating regulatory T cells comprising
the step of isolating cells contained in a cell group having the
most intensive folate receptor 4 expressions from test cells
containing (i) regulatory T cells, and (ii) at least one type of
cell selected from the group consisting of naive T cells and
activated T cells.
[0014] Item 4. An isolation method according to Item 3, wherein the
test cells are those comprising T cells isolated from a mammal or
comprising cells obtained by subjecting naive T cells to antigenic
stimulus.
[0015] Item 5. A method for treating malignant tumors or infectious
disease comprising:
[0016] step (a) collecting cells containing T cells from a patient
suffering from malignant tumors or infectious diseases;
[0017] step (b) obtaining cells containing T cells from which
regulatory T cells have been removed by depleting cells contained
in a cell group having the most intensive folate receptor 4
expressions from the cells obtained in the step (a); and
[0018] step (c) administering the cells obtained in the step (b) to
the patient.
[0019] Item 6. A method for suppressing immunity after organ
transplant comprising:
[0020] step (a) collecting cells containing T cells from a patient
received an organ transplant;
[0021] step (b) obtaining regulatory T cells by isolating cells
contained in a cell group having the most intensive folate receptor
4 expressions from the cells obtained in the step (a) or cells in
which regulatory T cells were induced by subjecting the cells
obtained in the step (a) to antigenic stimulus; and
[0022] step (c) administering regulatory T cells obtained in the
step (b) to the patient.
[0023] Item 7. A method for treating autoimmune diseases
comprising:
[0024] step (a) collecting cells containing T cells from a patient
suffering from an autoimmune disease;
[0025] step (b) obtaining regulatory T cells by isolating cells
contained in a cell group having the most intensive folate receptor
4 expressions from the cells obtained in the step (a) or cells in
which regulatory T cells were induced by subjecting the cells
obtained in the step (a) to antigenic stimulus; and
[0026] step (c) administering regulatory T cells obtained in the
step (b) to the patient.
[0027] Item 8. A reagent for detecting regulatory T cells
comprising anti-folate receptor 4 antibody or folate receptor
4-binding fragment.
[0028] The present invention also provides the following inventions
with respect to the technique that selectively reduces the number
of regulatory T cells in vivo:
[0029] Item 9. A pharmaceutical composition for immunostimulation
comprising anti-folate receptor 4 antibody or folate receptor
4-binding fragment.
[0030] Item 10. An pharmaceutical composition for treating tumors
comprising anti-folate receptor 4 antibody or folate receptor
4-binding fragment.
[0031] Item 11. An immunostimulation method comprising the step of
administering an amount of anti-folate receptor 4 antibody or
folate receptor 4-binding fragment effective for activating
immunity to a mammal including human.
[0032] Item 12. A method for treating a patient suffering from a
malignant tumor or infectious disease comprising the step of
administering an amount of anti-folate receptor 4 antibody or
folate receptor 4-binding fragment effective for treating malignant
tumor to the patient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 shows the results of Reference Example 3, i.e., the
TH6 antibody reaction specificity. In FIG. 1, the left chart shows
the results obtained using TH6 antibody, and the right chart shows
the results obtained using IgG2b (control). In FIG. 1, folate
receptor 4 (hereunder, folate receptor 4 may be referred to as
FLR4)-vector transfected cells (referred to as FLR4 in the figure)
are shown by a solid line, and cells introduced to only empty
vectors (referred to as Mock in the figure) are shown as shade. In
both the left and right charts in FIG. 1, the vertical axis
indicates PE fluorescence intensities per cell and the horizontal
axis indicates the numbers of cells.
[0034] FIG. 2 shows the results of Reference Example 4 in which
expressions of folate receptor 4 in thymocytes were measured.
Specifically, in FIG. 2, APC fluorescence intensities in the cells
of CD8.sup.+CD4.sup.- (upper left chart), CD8.sup.+CD4.sup.+ (upper
right chart), CD8.sup.-CD4.sup.- (lower left chart), and
CD8.sup.-CD4.sup.+ (lower right chart) using TH6 antibody are shown
by solid lines and those using IgG2b (control) are shown as shade.
In FIG. 2, the horizontal axis indicates the numbers of cells and
the vertical axis indicates APC fluorescence intensities per
cell.
[0035] FIG. 3 shows the results of Reference Example 4 in which
expressions of folate receptor 4 in lymph-node cells were measured.
Specifically, in FIG. 3, APC fluorescence intensities in the cells
of CD4.sup.+ (upper left chart), CD8.sup.+ (lower left chart), and
B220.sup.+ (upper right chart) using TH6 antibody are shown by
solid lines and those using IgG2b (control) are shown as shade. In
FIG. 3, the horizontal axis indicates the numbers of cells and the
vertical axis indicates APC fluorescence intensities per cell.
[0036] FIG. 4 shows the result of Example 1-1 in which expressions
of CD25 and folate receptor 4 in CD4.sup.+ cells derived from lymph
nodes or spleens of BALB/c mice by a panning method before and
after antigenic stimulus were measured. In FIG. 4, the left chart
shows the results when the CD4.sup.+ cells before antigenic
stimulus were used as a test sample and the right chart shows the
results when the CD4.sup.+ cells after antigenic stimulus were used
as a test sample. In both the left and right charts of FIG. 4, the
horizontal axis indicates Alexa Fluor488 fluorescence intensities
and the vertical axis indicates PE fluorescence intensities.
[0037] FIG. 5 shows the results of Example 1-2 in which expressions
of CD25 and folate receptor 4 per cell of cells mixture of
CD25.sup.+CD4.sup.+ cells and CD25.sup.-CD4.sup.+ cells before and
after antigenic stimulus were measured. The left chart of FIG. 5
shows the results when the cells mixture before stimulation was
used as a test sample, and the right chart shows the results when
the cell mixture population after stimulation was used as a test
sample. In both the right and left charts of FIG. 5, Thy1.2.sup.+
cells are shown as black dots, Thy1.2.sup.- cells (Thy1.1.sup.+
cells) are shown as gray dots, the horizontal axis indicates PE
fluorescence intensities, and the vertical axis indicates Alexa
Fluor488 fluorescence intensities.
[0038] FIG. 6 shows the results of Example 2 in which expressions
of CD25 and folate receptor 4 in CD4.sup.+ cells derived from
spleens or lymph nodes of BALB/c mice were measured. In FIG. 6, the
left chart shows the expressions of CD25 and folate receptor 4 in
CD4.sup.+ cells derived from spleens or lymph nodes of BALB/c mice
measured without antigenic stimulus and the right chart shows those
after antigenic stimulus. In both the right and left charts of FIG.
6, the horizontal axis indicates PE fluorescence intensities, and
the vertical axis indicates Alexa Fluor488 fluorescence
intensities.
[0039] FIG. 7 shows the results of Example 2 in which expressions
of foxp3 gene in the cell groups a to e of FIG. 6 were measured. In
FIG. 7, the vertical axis indicates the relative expressions of
foxp3 gene with the expression of foxp3 gene in the cell group a
being defined as 100.
[0040] FIG. 8 shows the results of Example 2 in which
incorporations of .sup.3H thymidine (1 .mu.M Ci/well) into the cell
groups a to e of FIG. 6 for 6 hours until completion of cell
culturing were measured after restimulating with alloantigen. In
FIG. 8, the vertical axis indicates the amount of radioactive
substance in Cpm (counts per minute).
[0041] FIG. 9 shows the results of Example 2 in which
immunoreaction suppression activities of the cell groups a and C of
FIG. 6 were measured. In FIG. 9, the vertical axis indicates the
amount of radioactive substance in Cpm (counts per minute).
[0042] FIG. 10 shows the results of Example 2 in which the effects
of the cell groups a, c and d of FIG. 6 for prolonging graft
survival of allo skin grafts were measured. In FIG. 10, the
horizontal axis indicates the days after initiation of the
experiment and the vertical axis indicates the proportion of mice
in which a skin graft is favorably adhered (Graft Survival, %).
[0043] FIG. 11 shows the results of Experiment 1 in Example 3 in
which expressions of CD25 and CD4 were measured in lymph-node cells
collected from mice to which Fab fragments of TH6 antibody (left
chart), PBS (middle figure), or TH6 antibody (right chart) were
intravenously administered.
[0044] FIG. 12 shows the results of Experiment 2 in Example 3
wherein percentages of CD25.sup.+CD4.sup.+ cell fractionations
(upper chart) and percentages of CD4.sup.+ cell fractionations
(lower chart) were measured with respect to peripheral blood
collected from mice to which TH6 antibody or rat IgG were
intravenously administered.
[0045] FIG. 13 shows the results of Experiment 3 in Example 3 in
which anti-gastric parietal cell autoantibody titers were measured
in the sera of BALB/c mice that had been administered TH6 antibody
or rat IgG. In FIG. 13, the vertical axis indicates OD405 nm
values; each .largecircle. indicates a mouse serum OD405 nm value;
and the line in the figure indicates the mean value of the OD405 nm
of the normal BALB/c mouse sera.
[0046] FIG. 14 shows micrographs taken while observing using a
microscope of HE stained stomachs extracted from BALB/c mice that
had been administered TH6 antibody or rat IgG in Experiment 3 of
Example 3. The upper micrograph shows the stomach of a mouse that
had been administered TH6 antibody, and the lower micrograph shows
a stomach of a mouse that had been administered rat IgG.
[0047] FIG. 15 shows the results of Example 4 in which the effect
of anti-folate receptor 4 antibody (TH6) for treating tumors was
measured. In FIG. 15, A shows the results obtained by administering
fibroblastoma Meth A cells and TH6 antibody or rat IgG on the same
day; B shows the results obtained by administering TH6 antibody or
rat IgG to mice having tumors of at least 4 mm of axial length
after inoculation with fibroblastoma Meth A cells; and C shows the
results obtained by administering TH6 antibody or rat IgG to mice
having tumors of at least 3 mm of axial length after administering
colon-carcinoma Colon 26 cells. In FIG. 15, top and middle figures
show change in average tumor diameter (the vertical axes indicate
average tumor axial lengths, the horizontal axes indicate the
numbers of days after administration; the top figures show the
results when TH6 antibody was administered and the middle figures
show the results when rat IgG was administered), and the bottom
figures show the survival rate of mice after tumor inoculation.
[0048] FIG. 16 shows the result of Experiment I in Example 5 in
which various kinds of surface markers on cells that had been
subjected to various cell treatments were measured. In FIG. 16,
figures show CD25 and folate receptor 4 expressions in cells, from
the left to right, T cells without TH6 antibody (whole), CD4.sup.+
cells, CD8.sup.+ cells, and TH6 highly depleted cells (TH6.sup.hi
depleted). In FIG. 16, the horizontal axes indicate PE fluorescence
intensities and the vertical axes indicate FITC fluorescence
intensities.
[0049] FIG. 17 shows the results of Experiment II of Example 5 in
which a T cell group without TH6 antibody (whole) was added or a T
cell group that had been treated using TH6 antibody or complement
thereof (TH6.sup.hi depleted) was administered to mice together
with fibroblastoma Meth A cells. In FIG. 17, the left charts show
the changes in average tumor axial lengths and the right chart
shows the survival rate after tumor inoculation.
[0050] FIG. 18 shows the results of Experiment III of Example 5 in
which anti-gastric parietal cell autoantibody titers in sera of
BALB/c. nude mice to which T cells without adding TH6 antibody
(whole) or T cells that had been treated using TH6 antibody and
complement thereof (TH6.sup.hi depleted) had been administered were
measured. In FIG. 18, the vertical axis indicates measured values
of OD405 nm; each .largecircle. indicates an OD405 nm value
measured in mouse serum; the line in the figure indicates the mean
OD405 nm value of normal BALB/c mice sera.
[0051] FIG. 19 shows the results of Example 6 in which expressions
of folate receptor 4 and HPRT in human CD4.sup.- cells,
CD25.sup.-CD4.sup.+ cells, and CD25.sup.+CD4.sup.+ cells were
measured by PCR.
(I) DETECTION AND ISOLATION OF T.sub.reg CELLS
[0052] The method for detecting T.sub.reg cells of the present
invention is such that T.sub.reg cells are detected from cells
(hereinafter referred to as "test cells") comprising (i) T.sub.reg
cells, and (ii) naive T cells and/or activated T cells. The
detection method comprises the steps of measuring expressions of
folate receptor 4 on the surfaces of T cells in the test cells, and
detecting T.sub.reg cells using the measured expressions of folate
receptor 4 as an indicator.
[0053] The method for detecting T.sub.reg cells of the present
invention is such that T.sub.reg cells are specifically detected in
the test cells by distinguishing (i) T.sub.reg cells from (ii)
naive T cells and/or activated T cells. The test cells in this
detection method are not limited as long as they comprise (i)
T.sub.reg cells and (ii) naive T cells and/or activated T cells.
Specific examples of test cells to be used are those comprising T
cells isolated from mammals, and those comprising cells obtained by
subjecting naive T cells isolated from mammals to antigenic
stimulus.
[0054] In the present invention, the test cells may include
hematopoietic cells, lymph-node cells, etc., in addition to T
cells. Since cells other than T cells have low expressions of
folate receptor 4, even if cells other than T cells are included in
the test cells, such cells will not adversely affect the accuracy
of T.sub.reg cell detection.
[0055] In the method for detecting T.sub.reg cells of the present
invention, expression of folate receptor 4 on the surfaces of T
cells can be measured by a known method. Examples of such methods
are those using antigen antibody reactions; ligands binding to
folate receptor 4; and techniques for in situ hybridization.
[0056] The above-mentioned method for measuring expressions of
folate receptor 4 on the surfaces of T cells using an antigen
antibody reaction can be specifically conducted by using
anti-folate receptor 4 antibody, and subjecting the anti-folate
receptor 4 antibody to antigen antibody reaction with folate
receptor 4 on T cells in the test cells, and detecting the antibody
bound to the folate receptor 4.
[0057] Here, the anti-folate receptor 4 antibody used may be either
of monoclonal antibody or polyclonal antibody; however, monoclonal
antibody is preferable. Such anti-folate receptor 4 polyclonal
antibody can be obtained by immunizing a mouse, hamster, rabbit or
like mammal using folate receptor 4 or T.sub.reg cells as
immunogen, and collecting anti-folate receptor 4 polyclonal
antibody from the immunized animal by a conventional method.
Anti-folate receptor 4 monoclonal antibody can be obtained by
culturing hybridoma cells that produce anti-folate receptor 4
monoclonal antibody. Hybridoma cells that produce anti-folate
receptor 4 monoclonal antibody can be prepared by collecting cells
that produce anti-folate receptor 4 monoclonal antibody from an
animal immunized with folate receptor 4, folate receptor
4-expressing cells, or T.sub.reg cells, and fusing such cells with
myeloma cells using a cell fusion method. This is an established
technique in the art. Note that TH6 cell strain (FERM BP-10382) is
deposited with the International Patent Organism Depositary,
National Institute of Advanced Industrial Science and Technology as
a mouse-rat hybridoma that can produce antibody against a folate
receptor 4 of mouse origin.
[0058] It is also possible to use an anti-folate receptor 4-binding
fragment such as Fab fragment and F(ab').sub.2 fragment of
anti-folate receptor 4 antibody instead of anti-folate receptor 4
antibody. Such folate receptor 4-binding fragment can be prepared
by known methods.
[0059] For detection purposes, the above-described antibody or
fragment thereof may be directly labeled with standard marker(s) or
indirectly labeled through a second antibody to which a marker is
bound.
[0060] Markers used for labeling the antibody are not limited, and,
for example, .sup.125I, .sup.3H, .sup.14C and like radioactive
isotopes; alkaline phosphatase, peroxidase and like enzymes;
phycoerythrin (PE), fluorescein isothiocyanate (FITC),
tetramethylrhodamine isothiocyanate (RITC) and like fluorescent
materials; biotin, etc., can be used. Labeling using such markers
can be conducted by known methods.
[0061] In the above-mentioned method using an antigen antibody
reaction, the conditions for the antigen antibody reaction can be
suitably selected from typically-employed conditions, and the
method for detecting antibody binding to folate receptor 4 can also
be suitably selected depending on the types of the markers labeling
the antibody.
[0062] Measurements of expressions of folate receptor 4 on the
surface of T cells using ligands binding to folate receptor 4 may
be conducted using ligands binding to folate receptor 4 instead of
antibody in the method using antigen antibody reactions. The
ligands binding to folate receptor 4 are not limited and known
ligands and ligands obtained by known methods may be used. The
conditions employed in such methods can be suitably selected
depending on the types of ligands used and the types of
markers.
[0063] Measurement of expressions of folate receptor 4 on the
surface of T cells using a technique of in situ hybridization can
be conducted by a standard method. As a specific example, the
expressions of folate receptor 4 can be measured by using a labeled
probe for mRNA of folate receptor 4, hybridizing the labeled probe
with mRNA of folate receptor 4 in the test cells, and detecting the
labeled probe which hybridized with the mRNA. Labeled probes used
in such in situ hybridization have a sequence specifically
complementary to mRNA of folate receptor 4, and have a marker bound
to ease detection. It is preferable that such probes have base
sequences that have a low cross-reactivity with other RNAs; such
probes can be prepared by cutting known folate receptor 4 cDNAs.
Markers for labeling the probes are not limited and, for example,
.sup.125I, .sup.3H, .sup.14C, and like radioactive isotopes; FITC,
RITC, and like fluorescent materials; digoxigenin; biotin, etc.,
can be used. Such markers can be bound to probes by methods known
in the art. In in situ hybridization, conditions for pretreatment
(immobilization) of the test cells, hybridization conditions,
conditions for detecting markers, etc., can be suitably selected in
accordance with methods known in the art.
[0064] Among the above-described methods for measuring the
expressions of folate receptor 4, preferable are those using
antigen antibody reactions and ligands binding to folate receptor
4. By employing such methods, it is possible to measure expressions
of folate receptor 4 in the test cells in a live state. Among these
methods, methods using antigen antibody reactions are the most
preferable.
[0065] Using the thus-measured expressions of folate receptor 4 on
T cells as an indicator enables T.sub.reg cells to be detected
separately from naive T cells and activated T cells. In other
words, based on the expressions of folate receptor 4, it is
possible to detect in the test cells two or three separated cell
groups comprising (1) T.sub.reg cells, and (2) activated T cells
and/or naive T cells; or two separated cell groups comprising (1)
T.sub.reg cells, and (2) mixture of activated T cells and naive T
cells. T.sub.reg cells, activated T cells, and naive T cells
exhibit degrees of expression of folate receptor 4 from greatest to
least in this order. Among the above-mentioned cell groups to be
detected, T.sub.reg cells are contained in the cell group that
exhibits the greatest expression of folate receptor 4.
[0066] Among the test cells, T.sub.reg cells, activated T cells,
and naive T cells tend to have CD25 expressions from greatest to
least in that order, and therefore in the detection method of the
present invention, differentiation of T.sub.reg cells, activated T
cells, and naive T cells can be conducted more accurately by using
expressions of CD25 in addition to those of folate receptor 4 as an
indicator. In other words, by using expressions of both folate
receptor 4 and CD25 as indicators, it is possible to separate the
test cells into three cell groups, i.e., a cell group comprising
T.sub.reg cells, a cell group comprising activated T cells, and a
cell group comprising naive T cells. Note that CD25 expressions can
be measured by known methods such as using anti-CD25 antibody,
etc.
[0067] Isolation of T.sub.reg cells from test cells can be
conducted by known methods. A preferable example of such an
isolation method is that T.sub.reg cells are isolated from the test
cells based on the expression levels of folate receptor 4 using a
flow cytometer with a sorting function. Another example of an
isolation method is such that T.sub.reg cells are isolated from the
test cells using magnetic beads.
[0068] Since the method of the present invention can isolate
T.sub.reg cells and distinguish between T.sub.reg cells and
activated T cells, it is possible to clinically apply T.sub.reg
cells and activated T cells with these being separated.
[0069] Malignant tumors, infectious disease, etc., can be treated
by effectively expressing the action of activated T cells in the
body of the patient by, for example, following steps:
[0070] collecting cells containing T cells from a patient suffering
from malignant tumors, infectious disease, etc.;
[0071] subjecting the collected cells to, if necessary, antigenic
stimulus to induce activated T cells; and
[0072] administering the cells from which T.sub.reg cells has been
removed using the method of the present invention to the
patient.
[0073] In other words, the present invention provides a method for
treating malignant tumors or for treating infectious diseases
comprising steps (a) to (c) as described below:
[0074] step (a) collecting cells containing T cells from a patient
suffering from a malignant tumor or infectious disease;
[0075] step (b) obtaining cells containing T cells from which
regulatory T cells have been removed by removing the cells of the
group having the greatest folate receptor 4 expression from the
cells obtained in the step (a); and
[0076] step (c) administering the cells obtained in the step (b) to
the patient.
[0077] In the above-described treatment method, collection of cells
containing T cells from a patient suffering from malignant tumors
or infectious diseases in step (a) and administering of the cells
from which T.sub.reg cells have been removed to the patient in step
(c) may be conducted by methods and/or conditions known in the
art.
[0078] It is also possible to enhance the immunosuppressive effect
for healing autoimmune diseases by making T.sub.reg cells
effectively exert their ability in the patient's body by following
steps:
[0079] collecting cells containing T cells from a patient who has
received an organ transplant or a patient suffering from an
autoimmune disease;
[0080] inducing the T.sub.reg cells, if necessary, by subjecting
the thus-collected cells to antigenic stimulus; and
[0081] administering the T.sub.reg cells isolated using the method
of the present invention to the patient.
[0082] In other words, the present invention provides an
immunosuppressive method conducted after organ transplantation
comprising the steps of:
step (a) collecting cells containing T cells from a patient who has
received an organ transplant; step (b) obtaining T.sub.reg cells by
isolating cells of the cell group having the greatest folate
receptor 4 expression from the cells obtained in the step (a) or
cells in which T.sub.reg cells were induced by subjecting the cells
obtained in the step (a) to antigenic stimulus; and step (c)
administering T.sub.reg cells obtained in the step (b) to the
patient.
[0083] Furthermore, the present invention provides a method for
treating autoimmune diseases comprising the steps of:
step (a) collecting cells containing T cells from a patient
suffering from an autoimmune disease; step (b) obtaining T.sub.reg
cells by isolating of the cell group having the greatest folate
receptor 4 expression from the cells obtained in the step (a) or
cells in which T.sub.reg cells were induced by subjecting the cells
obtained in the step (a) to antigenic stimulus; and step (c)
administering T.sub.reg cells obtained in the step (b) to the
patient.
[0084] In the above-described immunosuppressive method and method
for treating autoimmune diseases, the collection of cells
containing T cells from patients in step (a) and administering the
T.sub.reg cells to the patient in step (c) may be conducted by
methods and/or conditions known in the art.
[0085] Furthermore, as described above, by using anti-folate
receptor 4 antibody or folate receptor 4-binding fragment, it is
possible to specifically detect T.sub.reg cells with those being
distinguished from activated T cells. Therefore, the present
invention also provides a reagent for detecting T.sub.reg cells
that contains anti-folate receptor 4 antibody or folate receptor
4-binding fragment and, if necessary, carrier.
(II) PHARMACEUTICAL COMPOSITION FOR IMMUNOSTIMULATION
[0086] The above-described anti-folate receptor 4 antibodies or
folate receptor 4-binding fragments used as reagents for detecting
T.sub.reg cells can bind to T.sub.reg cells in vivo to selectively
deplete T.sub.reg cells, and thereby the effects of activated T
cells can be exerted effectively in vivo. Therefore, the present
invention also provides a pharmaceutical composition for
immunostimulation containing anti-folate receptor 4 antibody or
folate receptor 4-binding fragment as an active ingredient.
[0087] When the pharmaceutical composition is applied to a human,
it is preferable that the anti-folate receptor 4 antibody or folate
receptor 4-binding fragment contained therein be genetically
modified antibody (e.g., chimera antibody, humanized antibody) that
have been artificially modified to reduce heterogenetic antigens
against humans. Such modified antibody can be prepared using known
methods. A chimeric antibody is an antibody comprising a variable
region of an antibody derived from a mammal other than a human and
a constant region of human antibody origin. A humanized antibody is
an antibody comprising a complementarity determining region of an
antibody derived from a mammal other than a human and a framework
region and C region of human antibody origin.
[0088] The pharmaceutical composition is prepared by mixing
anti-folate receptor 4 antibody or folate receptor 4-binding
fragment with pharmaceutically acceptable base material(s) and/or
carrier(s). There is no limitation on the form of the
pharmaceutical composition, but injection is an example of a
preferable form.
[0089] A dose of the pharmaceutical composition is an amount
effective for activating immunity in vivo and is suitably selected
depending on the age and sex of the patient, administration method,
type of disease, etc. An example of the dose of the pharmaceutical
composition for an adult per day is such that anti-folate receptor
4 antibody or folate receptor 4-binding fragments is contained in
an amount of about 1-5000 mg, and preferably about 3-3000 mg.
[0090] Examples of administration routes of the pharmaceutical
composition are subdermal, intramuscular, intraperitoneal,
intra-abdominal, intrapleural, intravenous, etc.
[0091] The pharmaceutical composition for immunostimulation is
effective for treating malignant tumors, infectious diseases, etc.,
and useful as an agent for treating malignant tumors or for
treating infectious diseases. In particular, the pharmaceutical
composition for immunostimulation is effective for treating
malignant tumors, and useful as an agent for treating malignant
tumors.
[0092] Furthermore, the present invention provides a method for
activating immunity or treating malignant tumors or infectious
diseases using anti-folate receptor 4 antibody or folate receptor
4-binding fragment.
[0093] In other words, the present invention provides an
immunostimulation method of the following embodiment:
[0094] An immunostimulation method comprising a step of
administering anti-folate receptor 4 antibody or folate receptor
4-binding fragment in an amount effective for activating immunity
in a mammal, including a human.
[0095] Furthermore, the present invention provides a method for
treating malignant tumors or infectious diseases of the following
embodiment:
[0096] A method for treating malignant tumors or infectious
diseases comprising a step of administering an effective amount of
anti-folate receptor 4 antibody or folate receptor 4-binding
fragment to a patient suffering from a malignant tumor or
infectious disease.
EXAMPLES
[0097] The present invention is explained in detail with reference
to Reference Examples, Examples, etc., although the present
invention is not limited to these.
Reference Example 1
Preparation of Mouse Hybridoma Cells that Produce Anti-Folate
Receptor 4 Antibody
[0098] First, CD25.sup.+CD4.sup.+ T cell strains for serving as an
immunogen were prepared by the following method. Spleen/lymph-node
cells derived from normal BALB/c mice were placed in an RPMI
culture medium containing culture supernatant (a 8-fold dilution)
of hybridoma J11d (anti-24CD antibody-producing cell, purchased
from the American Type Culture Collection) and culture supernatant
(a 10-fold dilution) of hybridoma 3.155 (anit-CD8
antibody-producing cell, purchased from the American Type Culture
Collection), and allowed to stand on ice for 30 minutes. The
spleen/lymph-node cells in the RPMI culture medium were put into 10
cm dishes (2 dishes per mouse) coated with anti-rat IgG caprine
antibody (product of ICN Pharmaceuticals, 5 ml/dish of anti-rat IgG
caprine antibody diluted to 5 .mu.g/ml), and incubated at 4.degree.
C. for 30 minutes. The suspension cells were then collected as
cells rich in CD4.sup.+ cells by a panning method. The collected
cells were reacted on ice with biotin-labeled anti-CD25 antibody
(clone name: 7D4, product of PharMingen Company, a 200-fold
dilution), PE-labeled streptavidin (product of PharMingen Company,
a 400-fold dilution), and beads-labeled anti-PE antibody (product
of Miltenyi Biotec GmbH, a 10-fold dilution) in that order for 30
minutes each, and CD25.sup.+CD4.sup.+ cells were then collected as
a positive fraction by passing through a magnetic beads column
twice. The thus-obtained cells were stimulated by being subjected
to repeated co-culture with anit-CD3 antibody (10 vol % of culture
supernatant of hybridoma 2C11, hybridoma 2C11 was purchased from
the American Type Culture Collection), interleukin 2 (IL-2,
provided by Shionogi & Co. Ltd., 200 U/ml), and splenic cells
irradiated with 15 Gy radiation, obtaining CD25.sup.+CD4.sup.+ T
cell strains.
[0099] Rats (Wister rat; product of Clea Japan, Inc.) were
immunized against the thus-obtained CD25.sup.+CD4.sup.+ T cell
strains (5.times.10.sup.6) by intraperitoneally injecting the
CD25.sup.+CD4.sup.+ T cell strains to the mice every two weeks
(total 3 times). The spleen cells of each rat were collected three
days after the final injection and subjected to cell fusion using
P3U1 mouse-myeloma cells (provided by Juntendo University) and
Polyethyleneglycol 4000 (product of Merck & Co., Inc., 1 g/ml).
Screening of hybridomas was conducted using FACS by determining
whether or not the culture supernatant can stain
CD25.sup.+CD4.sup.+ cells more intensely than CD25.sup.-CD4.sup.+
cells. Specifically, CD4.sup.+ lymphocytes collected from a BALB/c
mouse by a panning method were reacted with each culture
supernatant, reacted with FITC-labeled anti-rat IgG mouse antibody
F(ab').sup.2 fragments (product of Jackson ImmunoResearch, a
1000-fold dilution), blocked with rat serum (a 50-fold dilution),
reacted with PE-labeled anti-CD25 antibody (clone name: PC61,
product of PharMingen Company), and then analyzed using a FACS
Calibur.
[0100] Mouse hybridoma TH6 cell strain (FERM BP-10382) was thus
obtained.
Reference Example 2
Preparation of Anti-Folate Receptor 4 Antibody (TH6 Antibody)
[0101] The mouse hybridoma TH6 cells (5.times.10.sup.6) obtained in
Reference Example 1 were administered to the abdominal cavities of
SCID mice (product of Clea Japan, Inc.), which are immunodeficient
mice, and abdominal dropsy was extracted on day 10 and after from
the injection. The extracted abdominal dropsy was subjected to
centrifugation, cell components and the like were removed therefrom
by being passed through a 0.45 .mu.m filter (product of Millipore),
and then the monoclonal antibody (TH6 antibody) was purified using
Protein G column (product of Amersham Biosciences KK). Elution of
monoclonal antibody from the column was conducted using 0.1 M
glycine-HCl at pH 2.7. Thus-obtained solution of monoclonal
antibody was subjected to dialysis with PBS using a dialysis
membrane (Spectrum Laboratories, MWCO 12-14000) and filtration
using 0.2 .mu.m filter (product of Millipore), obtaining purified
antibody.
Reference Example 3
Reaction Specificity of Anti-Folate Receptor 4 Antibody (TH6
Antibody)
[0102] A PMXS-IG vector (provided by the Institute of Medical
Science, the University of Tokyo) obtained by incorporating
full-length cDNAs of mouse folate receptor 4 (FLR4) into Plat-E
cells that are subclone of human HEK293T cells (provided by the
Institute of Medical Science, the University of Tokyo) or an empty
pMXS-IG vector was subjected to transfection using Fugene 6 (Roche
Molecular Biochemicals). Biotin-labeled TH6 antibody (labeled using
an Amersham biotinization kit) or biotin-labeled IgG2b (product of
PharMingen Company) was added to the cells (1.times.10.sup.8
cells/ml) in such a manner that the content of the biotin-labeled
TH6 antibody or biotin-labeled IgG2b became 1 .mu.g/ml, and reacted
on ice for 30 minutes. After washing, PE-labeled streptavidin
(product of PharMingen Company) was added thereto in such a manner
that the amount of the PE-labeled streptavidin became 0.5 .mu.g/ml,
reacted on ice for 30 minutes, and FACS analysis was then
conducted.
[0103] FIG. 1 shows the results. In FIG. 1, the left chart shows
the results when TH6 antibody was used, and the right chart shows
the results when IgG2b (control) was used. In FIG. 1, FLR4-vector
transfected cells (indicated as FLR4 in the figure) are shown by a
solid line, and cells introduced into only empty vectors (indicated
as Mock in the figure) are shown as shade. As shown in FIG. 1, the
TH6 antibody obtained in Reference Example 2 bind specifically to
FLR4-vector transfected cells, and therefore it was confirmed from
these results that TH6 antibody specifically bind to FLR4.
Reference Example 4
Verification of Expression of Folate Receptor 4 in Thymocytes and
Lymph-Node Cells
<Verification of Expression of Folate Receptor 4 in
Thymocytes>
[0104] Biotin-labeled TH6 antibody (labeled using an Amersham
biotinization kit) or biotin-labeled IgG2b (product of PharMingen
Company) were added to thymocytes (1.times.10.sup.9 cells/ml)
collected from BALB/c mice in such a manner that the content of the
biotin-labeled TH6 antibody or biotin-labeled IgG2b became 1
.mu.g/ml, and reacted on ice for 30 minutes. Subsequently,
FITC-labeled anti-CD4 antibody (product of PharMingen Company),
PE-labeled anti-CD8 antibodies (product of PharMingen Company) and
APC-labeled Streptavidin (product of PharMingen Company) were added
thereto in such a manner that the content of each became 0.4
.mu.g/ml, reacted on ice for 30 minutes, and FACS analysis was then
conducted.
[0105] FIG. 2 shows the results. In FIG. 2, APC fluorescence
intensities in the cells of CD8.sup.+CD4.sup.- (upper left),
CD8.sup.+CD4.sup.+ (upper right), CD8.sup.-CD4.sup.- (lower left),
and CD8.sup.-CD4.sup.+ (lower right) using TH6 antibody are shown
by solid lines and those using IgG2b (control) are shown as shade.
From the results, it became clear that in thymocytes, cells having
folate receptor 4 expressions exist in CD8.sup.-CD4.sup.+ cell
fractionations at high levels.
<Verification of Expression of Folate Receptor 4 in Lymph-Node
Cells>
[0106] Biotin-labeled TH6 antibody (labeled using an Amersham
biotinization kit) or biotin-labeled IgG2b (product of PharMingen
Company) were added to lymph-node cells (1.times.10.sup.8 cells/ml)
collected from BALB/c mice in such a manner that the content of the
biotin-labeled TH6 antibody or biotin-labeled IgG2b became 1
.mu.g/ml, and reacted on ice for 30 minutes. Subsequently,
FITC-labeled anti-CD4 antibody (product of PharMingen Company),
FITC-labeled anti-CD8 antibody (product of PharMingen Company) or
FITC-labeled anti-B220 antibody (product of PharMingen Company)
were added thereto in such a manner that the content thereof became
0.4 .mu.g/ml, APC-labeled Streptavidin (product of PharMingen
Company) was also added thereto in such a manner that its content
thereof became 0.4 .mu.g/ml, then reacted on ice for 30 minutes,
and FACS analysis was then conducted.
[0107] FIG. 3 shows the results. In FIG. 3, APC fluorescence
intensities in the cells of CD4.sup.+ (upper left), CD8.sup.+
(lower left), and B220.sup.+ (upper right) using TH6 antibody are
shown by solid lines and those using IgG2b (control) are shown as
shade. From the results, it became clear that, in lymph-node cells,
folate receptor 4 is expressed in CD4.sup.+ cells at higher levels
than in CD8.sup.+ cells and B cells.
Example 1-1
Detection of T.sub.reg Cells
[0108] CD4.sup.+ cells were collected from lymph-nodes and spleens
of BALB/c mice by a panning method. The thus-obtained CD4.sup.+
cells (1.times.10.sup.6 to 2.5.times.10.sup.6 cells/ml) were
stimulated by the addition of an equivalent amount of C57Bl/6
mouse-derived spleen cells irradiated with 15 Gy radiation as
antigen presenting cells (APC), and also IL-2 (final concentration
of 50 U/ml), and then cultured at 37.degree. C. for 9 days.
[0109] Using CD4.sup.+ cells before and after stimulation as test
samples, detection of T.sub.reg cells was conducted by the
following method.
[0110] Alexa Fluor488-labeled TH6 antibody (product of Molecular
Probes, labeled using an Alexa Fluor 488 Monoclonal antibody
labeling kit), PE-labeled anti-CD25 antibody (product of PharMingen
Company), and CyCrome-labeled anti-CD4 antibody (product of
PharMingen Company) were added to a CD4.sup.+ cell-containing
solution (2.times.10.sup.8 cells/ml) in such a manner that their
contents became 1 .mu.g/ml, 2 .mu.g/ml and 0.4 .mu.g/ml
respectively, and, after being reacted on ice for 30 minutes, FACS
analysis was then conducted.
[0111] FIG. 4 shows the results. In FIG. 4, the left chart shows
the results when the CD4.sup.+ cells before antigenic stimulus were
used as a test sample and the right chart shows the results when
the CD4.sup.+ cells after antigenic stimulus were used as a test
sample. In both the left and right charts of FIG. 4, the horizontal
axis indicates Alexa Fluor488 fluorescence intensities and the
vertical axis indicates PE fluorescence intensities. As is clear
from FIG. 4, CD25.sup.+CD4.sup.+ cells, which are T.sub.reg cells,
expressed folate receptor 4 at higher levels than
CD25.sup.-CD4.sup.+ cells.
[0112] From these results, it was confirmed that the CD4.sup.+
cells before antigenic stimulus can be divided into two fractions,
one being a cell group having high expressions of folate receptor 4
and CD25, and the other being a cell group having low to medium
folate receptor 4 expression and low CD25 expression. The CD4.sup.+
cells after antigenic stimulus can be divided into three fractions,
i.e., a cell group having high expressions of folate receptor 4 and
CD25, a cell group having medium folate receptor 4 expression and
medium to high CD25 expression, and a cell group having low
expressions of folate receptor 4 and CD25.
Example 1-2
Detection of T.sub.reg Cells
[0113] Using CD25.sup.+CD4.sup.+ cells collected from a
Thy1.2+BALB/c mouse by MACS using magnetic beads and
CD25.sup.-CD4.sup.+ cells collected from a Thy1.1+ BALB/c mouse by
MACS using magnetic beads, the following Experiment was conducted.
Using a mixture of CD25.sup.+CD4.sup.+ cells and
CD25.sup.-CD4.sup.+ cells as test cells, antigenic stimulus was
conducted by the same method as described in Example 1-1.
Biotin-labeled ani-Thy1.2 antibody (product of PharMingen Company)
were added to a sample of the cells mixture (5.times.10.sup.7
cells/ml) before stimulation (FIG. 5, the left chart) and 9 days
after stimulation (FIG. 5, the right chart) in such a manner that
the content of the biotin-labeled ani-Thy1.2 antibody became 0.2
.mu.g/ml, and then reacted on ice for 30 minutes. Subsequently,
Alexa Fluor488-labeled TH6 antibody (product of Molecular Probes,
labeled using an Alexa Fluor 488 Monoclonal antibody labeling kit),
PE-labeled anti-CD25 antibody (product of PharMingen Company)
APC-labeled anti-CD4 antibody (product of PharMingen Company) and
PerCpCy5.5-labeled streptavidin (product of PharMingen Company)
were added thereto in such a manner that their contents became 1
.mu.g/ml, 2 .mu.g/ml, 0.25 .mu.g/ml and 0.4 .mu.g/ml respectively,
reacted on ice for 30 minutes, and FACS analysis was then
conducted.
[0114] FIG. 5 shows the results. In FIG. 5, the left chart shows
the results when the cells mixture of CD25.sup.+CD4.sup.+ cells and
CD25.sup.-CD4.sup.+ cells before stimulation was used as a test
sample, and the right chart shows the results when the cells
mixture of CD25.sup.+CD4.sup.+ cells and CD25.sup.-CD4.sup.+ cells
after stimulation was used as a test sample. In both the left and
right charts of FIG. 5, Thy1.2+ cells are expressed as black dots,
Thy1.2 negative (Thy1.1+) cells are expressed as gray dots, the
horizontal axis indicates PE fluorescence intensities, and the
vertical axis indicates Alexa Fluor488 fluorescence intensities.
From these results, it became clear that the CD25.sup.+-derived
cells could exhibit high expressions of folate receptor 4 and CD25
after being stimulated.
Example 2
Isolation of T.sub.reg Cells
<Cell Isolation Based on the Folate Receptor 4
Expression>
[0115] CD4.sup.+ cells were collected from spleens and lymph nodes
of BALB/c mice by a panning method. To the CD4.sup.+ cells
(2.times.10.sup.8 cells/ml), Alexa Fluor488-labeled TH6 antibody
(product of Molecular Probes, labeled using an Alexa Fluor 488
Monoclonal antibody labeling kit), PE-labeled anti-CD25 antibody
(product of PharMingen Company), and CyChrome-labeled anti-CD4
antibody (product of PharMingen Company) were added in such a
manner that the their contents became 1 .mu.g/ml, 2 .mu.g/ml and
0.4 .mu.g/ml respectively, reacted on ice for 30 minutes, and FACS
analysis was then conducted (see the left chart of FIG. 6).
[0116] To CD4.sup.+ cells (2.times.10.sup.6 cells/ml) collected
from spleens and lymph nodes of BALB/c mice by a panning method,
were added APC (spleen cells irradiated with 15 Gy X-rays) of
C57Bl/6 mice in such a manner that the APC content became
2.times.10.sup.6 cells/ml. IL-2 was subsequently added thereto in
such a manner that the content of the IL-2 became 50 U/ml, and then
cultured at 37.degree. C. for 9 days. Cells obtained by depleting
dead cells from the above-obtained cells by a density
centrifugation method using Lympholyte-M (product of Cedarlane) was
reacted with anti-Fc receptor antibody (culture supernatant of
hybridoma2.4G2 cells; the hybridoma cells were purchased from the
American Type Culture Collection) on ice for 30 minutes, and the
expressions of folate receptor 4 and CD25 were analyzed using FACS
(see right chart in FIG. 6) in the same manner as described
above.
[0117] CD4.sup.+ cells whose folate receptor 4 and CD25 expressions
had been measured in the above-described manner were classified
into a to e cell groups as shown in FIG. 6, and each cell group was
isolated using a Moflow (Dako Cytomation) to purity of 95% or
greater.
<Measurement of foxp3 Gene Expressions in a to e Cell
Groups>
[0118] Expressions of Foxp3 gene in cells contained in the
above-obtained a to e cell groups were determined by real-time PCR
assay, and the ratio of HPRT gene, which was used as an internal
standard, relative to mRNA was calculated.
[0119] Quantitative determination was conducted by extracting RNAs
using Isogen (product of Nippon Gene Co., Ltd.) from about
5.times.10.sup.5 isolated cells, and subjecting the RNAs to reverse
transcription using Superscript II reverse-transcriptase and
oligo(dT).sub.12-18 primer (Invitrogen), obtaining cDNAs. Real-time
PCR assay was conducted using an ABI/PRISM770 sequence detection
system (PE Applied Biosystems). Primers and probes were as follows:
Foxp3 primers: 5'-CCC AGG AAA GAC AGC AAC CTT-3' and 5'-TTC TCA CAA
CCA GGC CAC TTG-3'; Foxp3 probe: 5'-FAM-ATC CTA CCC ACT GCT GGC AAA
TGG AGT C-3'; HPRT primers: 5'-TGA AGA GCT ACT GTA ATG ATC AGT CAA
C-3' and 5'-AGC AAG CTT GCA ACC TTA ACC A-3'; HPRT probe:
5'-VIC-TGC TTT CCC TGG TTA AGC AGT ACA GCC C-3', and were designed
at intron/exon boundaries. Using a QuantiTect Probe PCR kit
(product of Qiagen), each triplicate sample was subjected to 40
cycles each comprising 10 minutes at 95.degree. C., 15 seconds at
95.degree. C. and 60 seconds at 60.degree. C. with the
concentrations of primer and TaqMan probe being 0.4 .mu.M and 0.2
.mu.M respectively. Average amounts of mRNAs in Foxp3 and HPRT of
the triplicate samples were calculated and determined as relative
amounts thereof. The amount of mRNA in Foxp3 was divided by the
amount of mRNA in HPRT mRNA (ratio of Foxp3/HPRT), and relative
Foxp3/HPRT ratios for each fraction were determined with the
Foxp3/HPRT ratio in cell group a being set as 100.
[0120] FIG. 7 shows the results. As is clear from the results, the
expression of foxp3, which is a specific marker for T.sub.reg
cells, in cell group C is essentially as high as in cell group a.
It is therefore clear that the cell group C mainly contains
T.sub.reg cells. This result also confirmed that T.sub.reg cells
can be specifically detected and isolated using folate receptor 4
expression as an indicator.
<Restimulus to Cell Groups a to e Using Alloantigen>
[0121] Cells (1.times.10.sup.4) from each of the thus-obtained cell
groups a to e were cultured in U-bottomed 96-well plates with APC
(1.times.10.sup.5 spleen cells irradiated with 15 Gy X-rays)
derived from c57/Bl/6 mice for 5 and 7 days at 37.degree. C.
Incorporation of .sup.3H thymidine (1 .mu.M Ci/well) during the
last 6 hours of culturing was measured. The mean values of
duplicates are shown with standard deviations.
[0122] FIG. 8 shows the results. As is clear from the results,
cells with high folate receptor 4 expression (cell groups a and a)
did not react to the restimulus by alloantigen, but cells with
medium folate receptor 4 expression and medium CD25 expression
(cell group d) after restimulus exhibited high proliferation
reaction against the restimulus from the early stage of reaction.
Furthermore, cells with low CD25 expression (cell groups b and e)
tended to exhibit a low proliferation reaction at the early stage
of reaction but a relatively high proliferation reaction at the
later stage of the reaction. From these results, it was confirmed
that cell groups a and a mainly contain T.sub.reg cells, cell group
d mainly contains activated T cells, and cell groups b and e mainly
contain naive T cells.
<Measurement of Immunoreaction Suppression Activity>
[0123] Cell group a or c was added to cell group b
(5.times.10.sup.4 cells/ml) in such a manner that the content of
cell group a or a became 1.times.10.sup.4 cells/ml (in FIG. 9,
indicated as 1/5) or 2.5.times.10.sup.3 cells/ml (in FIG. 9,
indicated as 1/20), and cultured in C57Bl/6 mouse-derived APC
(1.times.10.sup.5 cells/ml) for 7 days at 37.degree. C.
Incorporation of .sup.3H thymidine (1 .mu.M Ci/well) during the
last 6 hours of culturing was measured. The mean values of
duplicates are shown together with standard deviations. For
comparison, incorporations of .sup.3H thymidine in cells without
addition of cell group a or a (in FIG. 9, shown as b, second from
the right), and those without addition of any of cell groups a to c
(in FIG. 9, shown as--at the right) were also measured.
[0124] FIG. 9 shows the results. From these results, it is clear
that cell group a had more intense immunoreaction suppression
activity than cell group a (i.e., normal mouse T.sub.reg
cells).
<Measurement of the Effect for Prolonging the Skin Graft>
[0125] Skins of C57Bl/6 mice were transplanted to BALB/c nude mice
lacking T cells, and, two weeks or more after healing of the wound,
1.times.10.sup.5 cells of cell group a, a or d together with
2.times.10.sup.5 BALB/c T cells (cell population obtained by
depleting J11d positive cells from spleen/lymph-node cells by a
panning method; unstimulated cells (Fresh T cells)) were
administered through intravenous injection. FIG. 10 shows the
number of days until the skin grafts were rejected, counting the
transplantation day as zero. For comparison, the same experiment
was conducted except that the above-descried BALB/c T cells
(unstimulated T cells; Fresh T cells) alone were administered
through intravenous injection.
[0126] FIG. 10 shows the results. Based on these results, it was
confirmed that use of cell groups a and C leads to prolongation of
allo skin graft survival.
Example 3
Affect of Anti-Folate Receptor 4 Antibody (TH6 Antibody) on
T.sub.reg Cells
<Experiment 1>
[0127] Monoclonal antibody (TH6 antibody) prepared in Reference
Example 2 or Fab fragments thereof (30 .mu.g) were diluted with 300
.mu.l of PBS (pH7.2) and intravenously administered to BALB/c mice.
Four days after administration, lymph-node cells were extracted
from the mice. After blocking the obtained lymph-node cells with
anti-Fc receptor antibody, the lymph-node cells were reacted with
FITC-labeled anti-CD4 antibody and PE-labeled anti-CD25 antibody,
and FACS analysis was then conducted. For comparison, the same
experiment was conducted except that only PBS was administered to
the BALB/c mice. Note that the Fab fragments of TH6 antibody used
in this experiment were obtained by using an ImmunoPure Fab
Preparation Kit (product of Pierce) and dialyzing the prepared Fab
fragments with PBS.
[0128] FIG. 11 shows the results. From these results, it is clear
that CD25.sup.+CD4.sup.+ cells (i.e., T.sub.reg cells) can be
depleted by administering folate receptor 4 antibody or Fab
fragment thereof.
<Experiment 2>
[0129] Monoclonal antibody (TH6 antibody) prepared in Reference
Example 2 or rat IgG (product of Sigma Chemical Company) in an
amount of 1 to 100 .mu.g was intravenously administered to BALB/c
mice. Peripheral blood was collected from the mice four days after
administration, the red blood corpuscles of which were subjected to
hemolyzation, CD25 and CD4 were stained in the same manner as in
Experiment 1, and FACS analysis was then conducted.
[0130] FIG. 12 shows the results. FIG. 12 shows, together with the
standard deviations, the mean values of proportions of
CD25.sup.+CD4.sup.+ cell fractions (upper chart) and of CD4.sup.+
cell fractionations (lower chart) in lymphocytes fractions, which
can be sorted by forward scatter light (FSC) and side scatter light
(SSC). As shown in FIG. 12, by the administration of TH6 antibody,
the number of CD25.sup.+CD4.sup.+ cells was dose-dependently
reduced to one fifth. The number of CD4.sup.+ cells was also
dose-dependently reduced almost by half, but the reduction was
slighter than for CD25.sup.+CD4.sup.+ cells. Based on the results
of this Experiment, it was confirmed that TH6 antibody can
selectively deplete CD25.sup.+CD4.sup.+ cells in vivo.
<Experiment 3>
[0131] To BALB/c mice, 100 .mu.g of monoclonal antibody (TH6
antibody) prepared in Reference Example 2 or rat IgG (product of
Sigma Chemical Company) was intra-abdominally administered on the
10.sup.th day and 20.sup.th day from birth. Three months after the
final administration, blood sera and the stomachs were extracted
from the mice. Titer of anti-gastric parietal cell autoantibody of
the sera collected from the mice were measured by ELISA as follows.
First, wells of a ELISA plate with 96 flat wells (product of ICN,
Limbro/Titertek plate) were coated with gastric mucosa extract of
BALB/c mice diluted with PBS over a night, washed with 0.05%
Tween-20/PBS, blocked with 1% BSA/PBS at room temperature for 1
hour. And then, the sera collected from the mice were added into
the wells that had been subjected to the above-treatment and
allowed to incubate at room temperature for 1 hour. Subsequently,
thus treated wells were washed with 0.05% Tween-20/PBS, and
subjected reaction with ALP-labeled anti-mouse IgG (product of
Sigma Chemical Company, 1/1000 dilution) at room temperature for 1
hour. Thereafter, the wells were washed with 0.05% Tween-20/PBS,
ALP substrate (product of Sigma Chemical Company, 1 mg/ml)
dissolved with 10 wt. % diethanol amine solution (pH 9.8) was added
in to the wells and allowed to react for 30 minutes, and OD405 nm
values were then measured.
[0132] FIG. 13 shows the results. In FIG. 13, each .largecircle.
indicates a measured OD405 nm value for each mouse serum; and the
line in the figure indicates the mean OD405 nm value of normal
BALB/c mouse sera. From these results, it is clear that in all the
mice administered with TH6 antibody, anti-gastric parietal cell
autoantibody were produced at high levels.
[0133] The stomachs extracted from the mice were fixed using 10%
formalin, subjected to thin sectioning, stained with HE
(hematoxylin and eosin), and observed using a microscope (FIG. 14).
In the mouse administered with TH6 antibody, thickening of stomach
walls and elimination of gastric parietal cells (cells in the
fundus which dyed deep red), and infiltration of lymphocytes were
observed. In the mouse administered with TH6 antibody, autoimmune
gastritis was induced.
<Overall Review>
[0134] By administrating TH6 antibody, T.sub.reg cells could be
depleted in vivo and develop autoimmune diseases. Since even Fab
fragment of TH6 lack Fc portions, which bind to complements, etc.,
T.sub.reg cells were depleted the same as complete antibody; it is
therefore clear that folate receptor 4 is necessary for T.sub.reg
cells to survive, and there is a high possibility that TH6 antibody
blocks the function of the folate receptor 4. In other words,
T.sub.reg cells may be depleted by inhibiting the function of
folate receptor 4.
Example 4
Effect of Anti-Folate Receptor 4 Antibody (TH6) for Treating
Tumor-1
<Experiment A>
[0135] Fibroblastoma Meth A cells (provided by Okayama University,
2.times.10.sup.5) were subdermally administered to BALB/c mice, and
100 .mu.g of monoclonal antibody (TH6 antibody) prepared in
Reference Example 2 or rat IgG (product of Sigma Chemical Company)
were intravenously administered to the mice on the same date of
administration of the fibroblastoma Meth A cells. After
administration, the longer and shorter axes of tumors were measured
about every four days.
[0136] FIG. 15 A shows the changes in average tumor axial lengths
in the mice (top and middle charts), and survival rates after
inoculation of tumor (bottom chart). Note that, in evaluating mouse
survival, a mouse was considered to be dead if the average tumor
axial length exceeded 15 mm. As is clear from FIG. 15 A, in the
IgG-administered group, the tumors did not persist in three mice
out of 12; however, in the TH6 antibody-administered group, the
tumors were rejected in all 12 mice.
<Experiment B>
[0137] Fibroblastoma Meth A cells (provided by Okayama University,
2.times.10.sup.5) were subdermally administered to BALB/c mice on
day zero, and 10 .mu.g of monoclonal antibody (TH6 antibody)
prepared in Reference Example 2 or rat IgG (product of Sigma
Chemical Company) were administered three times, i.e., on days 8,
12, and 16, to those mice having a tumor at least 4 mm in axial
length as of day 8.
[0138] FIG. 15 B shows the changes in average tumor axial length in
each mouse (top and middle charts), and the survival rate after
inoculation of tumor (bottom chart). All 16 mice except one
administered with IgG died because of the tumor; however, in 9 mice
out of 16 administered with TH6 antibody, tumors were rejected.
Based on the results, it was confirmed that administration of TH6
antibody is effective even for palpable Meth A tumors.
<Experiment C>
[0139] Colon-carcinoma cells Colon 26 (2.times.10.sup.5, provided
by the Institute of Development, Aging and Cancer, Tohoku
University) were subdermally administered to BALB/c mice on day
zero of the experiment, and 10 .mu.g of monoclonal antibody (TH6
antibody) prepared in Reference Example 2 or rat IgG (product of
Sigma Chemical Company) were administered three times, i.e., on
days 8, 12, and 16, to those mice having a tumor at least 3 mm of
axial length as of day 8.
[0140] FIG. 15 C shows changes in average tumor axial length (top
and middle chart) in each mouse, and survival rate after
inoculation of tumor (bottom chart). All eight IgG-administered
mice except one died because of the tumor, but tumors were rejected
in five out of eight TH6 antibody-administered mice.
<Overall Review>
[0141] It is known that a method for depleting T.sub.reg cells by
administering anti-CD25 antibody is effective only if the
administration of anti-CD25 antibody is conducted before tumor
inoculation, and it is ineffective after tumor inoculation. It is
assumed that this is because CD25 is highly expressed not only in
T.sub.reg cells but also in activated T cells.
[0142] In contrast, by administering TH6 antibody, more than half
of palpable tumors could be cured. It is assumed that this is
because the effect of TH6 antibody could deplete just T.sub.reg
cells without depleting activated T cells.
Example 5
Effect of Anti-Folate Receptor 4 Antibody (TH6) for Treating
Tumor-2
<Experiment I>
[0143] After inoculating fibroblastoma Meth A cells
(2.times.10.sup.5 provided by Okayama University) to the backs of
BALB/c mice, 30 .mu.g of anti-GITR antibody (DTA-1antibody;
prepared by using mouse hybridoma DTA-1 cells; the mouse hybridoma
DTA-1 cells are stored at the Institute for Frontier Medical
Sciences, Kyoto University) were intravenously administered to the
mice in order to increase immune response. Inguinal and
under-foreleg lymph nodes and splenic cells were collected from the
mice. The thus-collected cells were co-cultured with Meth A cells
(the concentration of Meth A cells was 1/25.sup.th to 1/5.sup.th of
lymphocytes) of which cell division was terminated by treating with
mitomycin C, IL-2 was added in such a manner that the content of
the IL-2 became 50 U/ml from the 6.sup.th day of the culturing, and
living cells were collected by the density centrifugation method
using Lympholyte-M on the 9.sup.th day of culturing. The
thus-obtained cells (about 5.times.10.sup.7 cells) were added to
either 0.5 ml of culture medium containing TH6 antibody (1
.mu.g/ml) prepared in Reference Example 2 or only 0.5 ml of culture
medium, incubated on ice for 30 minutes, 5 ml of rabbit origin
complement-containing liquid (product of Cedarlane, prepared by
dissolving 1 vial of rabbit origin complement-containing liquid in
1 ml of sterile water, and diluting to 1/10 using a RPMI solution
containing 2% FCS) was added, and incubated at 37.degree. C. for 30
minutes. Cells to which TH6 antibody was added before being treated
with complement thereof was defined as cells treated with TH6
antibody and complement thereof (TH6.sup.hi depleted) and cells to
which TH6 antibody was not added was defined as a whole. After
washing, about 5.times.10.sup.5 cells were reincubated using 50
.mu.l of culture medium containing TH6 antibody (5 .mu.g/ml), and
FITC-labeled anti-rat antibody (product of Jackson ImmunoResarch)
were added thereto in such a manner that the content of the
FITC-labeled anti-rat antibody became 1 .mu.g/ml, and reacted on
ice for 30 minutes. After blocking with rat serum on ice for 20
minutes, PE-labeled anti-CD25 antibody (product of PharMingen
Company), APC-labeled anti-CD4 antibody (product of PharMingen
Company) or biotin-labeled anti-CD8 antibody (product of PharMingen
Company) were added in such a manner that the content became 0.25
.mu.g/ml, APC-labeled streptavidin was added in such a manner that
the content thereof became 0.4 .mu.g/ml and reacted on ice for 30
minutes, and FACS analysis was then conducted.
[0144] FIG. 16 shows the results. In FIG. 16, the charts show, from
the left, the analytical results for the cells without TH6 antibody
treatment (whole), CD4.sup.+ cells without TH6 antibody treatment,
CD8.sup.+ cells without TH6 antibody treatment, and cells treated
with TH6 antibody and complement thereof (TH6.sup.hi depleted)
after TH6 antibody treatment, respectively. In FIG. 16, the
horizontal axes indicate PE fluorescence intensities (corresponding
to expression of CD25), and the vertical axes indicate fluorescence
intensities of FITC (corresponding to expression of folate receptor
4).
[0145] From these results, it was confirmed that, as the same as
the cells stimulated with alloantigen, cells stimulated with tumor
cells could be divided into three cell groups, i.e., having high
expressions of both folate receptor 4 and CD25; moderate folate
receptor 4 expression and moderate to high CD25 expression; and low
expressions of both folate receptor 4 and CD25. The cells having
high expressions of folate receptor 4 and CD25 existed in CD4.sup.+
cells at high levels and was substantially not observed in
CD8.sup.+ cells. It was also confirmed that the cells having high
expressions of folate receptor 4 and CD25 could be depleted by
treating with TH6 antibody and complements thereof.
<Experiment II>
[0146] The cells cultured in Experiment I were divided into two
groups, one without being treated with TH6 antibody (whole) and the
other being treated with TH6 antibody and complements thereof
(TH6.sup.hi depleted). Subsequently, 2.times.10.sup.6 cells of each
group were administered to different BALB/c nude mice, which had no
T cells. After the administration (day zero), fibroblastoma Meth A
cells (2.times.10.sup.5 cells, provided by Okayama University) were
subdermally administered to the mice, and changes in diameter of
the tumor were measured.
[0147] FIG. 17 shows the results, wherein changes in average tumor
axial length of each mouse (left charts) and the survival rate
after inoculation of tumor (right chart) are shown. Note that, in
evaluating mouse survival, a mouse was considered to be dead when
the average tumor axial length exceeded 15 mm or its body weight
was significantly reduced, even if the mouse was actually not
dead.
<Experiment III>
[0148] In the above-described Experiment II, either 90 days after
tumor inoculation or at the time a mouse was considered to be dead,
sera of the mice were collected and titers of anti-gastric parietal
cell autoantibody of the sera collected from the mice were measured
by the same method as in Example 3 of Experiment 3.
[0149] FIG. 18 shows the results. The line in FIG. 18 indicates the
mean OD405 nm value measured using sera collected from normal
BALB/c mice. From these results, it became clear that mice
administered the cells treated with TH6 antibody and complement
(TH6.sup.hi depleted) had a higher antibody titer in the serum
compared to the mice administered the cells that had not been
treated with TH6 antibody (whole).
<Overall Review>
[0150] In mice administered with the cells from which cells with
folate receptor 4 high expression were depleted (TH6.sup.hi
depleted), proliferation of tumors was modest and the tumors were
rejected in 5 mice out of 12. In contrast, when the entire cells
after culturing (whole) was administered, proliferation reduction
of tumors was observed in 2 or 3 out of 12 mice, but tumors grew as
rapidly as in mice to which non-stimulated lymphocytes were
administered in other mice, i.e., 9 or 10 out of the 12 mice. From
these results, it is assumed that an intensive tumor immune
response was induced by proliferating T cells having anti-tumor
activity induced by stimulation in vitro, and depleting the
T.sub.reg cells from the proliferated T cells while retaining
activated T cells. In all mice administered with the cells from
which cells having high folate receptor 4 expression had been
removed (TH6.sup.hi depleted), anti-gastric parietal cell
antibodies were observed, and the antibody titers thereof tended to
be high. Therefore, it is assumed that in such mice, severe
autoimmune diseases can be easily induced.
[0151] From the above-described results, it was confirmed that
tumor immunity activity can be increased by inducing T cells which
respond to a tumor in vitro, depleting the cells having high folate
receptor 4 expressions from the T cells, and administering the thus
obtained remaining cells in vivo.
Example 6
Assay of Human CD25.sup.+CD4.sup.+
[0152] Peripheral blood mononuclear cells (PBMCs) were collected
from a healthy donor and purified by gradient centrifugation using
Ficoll-Paque.TM. plus (Amersham Biosciences). PBMCs were stained
with magnetic beads conjugated anti-CD4 antibody (Miltenyi Biotec,
human CD4 multisort kit) and CD4.sup.+ cells were separated from
CD4.sup.- cells with magnetic column. After dissociating the
magnetic beads from the antibody, CD4.sup.+ cells were stained with
magnetic beads-conjugated anti-CD25 antibody. CD25.sup.+CD4.sup.+
cells were separated from CD25.sup.-CD4.sup.+ cells with magnetic
column. RNAs were eluted from the sorted cells using an RNeasy mini
kit (Qiagen). Reverse transcription of RNAs was performed with a
SuperScript reverse transcriptase (Invitrogen). PCR for human
folate receptor 4 fragment comprises a denaturation step at
94.degree. C. for 2.5 minutes; followed by 5 cycles at 94.degree.
C. for 30 seconds, at 72.degree. C. for 15 seconds; 5 cycles at
94.degree. C. for 30 seconds, at 70.degree. C. for 15 seconds, at
72.degree. C. for 15 seconds; and 35 cycles at 94.degree. C. for 30
seconds, at 68.degree. C. for 15 seconds, at 72.degree. C. for 15
seconds, using an advantage cDNA PCR kit (Clontech) with primers
having the following sequences; ccccactctacaacttcagcctgtttcac and
actcgctctccctgcccactcgg. PCR for human HPRT fragment comprises a
denaturation step at 94.degree. C. for 2.5 minutes, followed by 30
cycles at 94.degree. C. for 30 seconds, at 60.degree. C. for 30
seconds, and at 72.degree. C. for 45 seconds, with primers having
the following sequences; tatggacaggactgaacgtcttgc and
gacacaaacatgattcaaatccctga. DNA fragments with about 170 and 500
base pairs are shown for FLR4 and HPRT, respectively (see FIG.
19).
[0153] About human counterpart of the mouse FLR4, a gene sequence
is predicted from a genomic sequence using a gene prediction method
(Reference; O. Spiegelstein, J. D. Eudy, R. H. Funnell, Gene 258,
117-25 (Nov. 27, 2000)). This gene is also called as FR.delta. or
"predicted gene: similar to folate receptor 3". We found that human
CD25.sup.+CD4.sup.+ cells in peripheral blood express mRNAs for
this predicted gene.
INDUSTRIAL APPLICABILITY
[0154] In the T.sub.reg cell detection and isolation methods of the
present invention, by using the expressions of folate receptor 4 on
the surface of cells as an indicator, T.sub.reg cells can be
detected and isolated separately from activated T cells and/or
naive T cells. Therefore, by applying the method of the present
invention clinically, it becomes possible to selectively remove
T.sub.reg cells from a patient, and selectively collect T.sub.reg
cells and administer the collected T.sub.reg cells to a patient.
Thus, the methods of the present invention are useful in
immunosuppression after organ transplantation or cell therapy for
treating malignant tumors.
[0155] The pharmaceutical composition for immunostimulation of the
present invention can effectively exhibit the action of activated T
cells by binding to T.sub.reg cells in vivo, and selectively
reducing the number of T.sub.reg cells. The pharmaceutical
composition of the present invention is particularly effective for
treating malignant tumors, infectious diseases, etc., and is usable
as an agent for treating these diseases.
* * * * *