U.S. patent application number 10/379537 was filed with the patent office on 2003-08-21 for method of screening apoptosis inducing substances.
This patent application is currently assigned to CHUGAI SEIYAKU KABUSHIKI KAISHA. Invention is credited to Fukushima, Naoshi.
Application Number | 20030157577 10/379537 |
Document ID | / |
Family ID | 13654837 |
Filed Date | 2003-08-21 |
United States Patent
Application |
20030157577 |
Kind Code |
A1 |
Fukushima, Naoshi |
August 21, 2003 |
Method of screening apoptosis inducing substances
Abstract
The present invention provides a method of screening substances
having property of causing apoptosis, and relates to a method of
screening substances having property of causing apoptosis
characterized by using cells which are expressing IAP (Integrin
Associated Protein), and the relates to above screening method,
wherein the cells used are myeloid cells, and relates to
pharmaceutical compositions containing as the active ingredient the
substances obtained by the above method, and the present invention
makes it possible to differentiate, identify and screen readily and
highly efficiently the substances, such as antibodies and the like,
that have property of causing apoptosis on myeloid cells by using
cells which are expressing IAP while using specific binding
reactions of the substances, and the above specific substances thus
obtained can be used by virtue of their characteristics as the
active ingredient of pharmaceutical compositions such as anticancer
agents and medicines for myelocytic leukemia and the like.
Inventors: |
Fukushima, Naoshi;
(Gotemba-shi, JP) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
CHUGAI SEIYAKU KABUSHIKI
KAISHA
5-1, Ukima 5-chome, Kita-ku
Tokyo
JP
115
|
Family ID: |
13654837 |
Appl. No.: |
10/379537 |
Filed: |
March 6, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10379537 |
Mar 6, 2003 |
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09147004 |
Sep 8, 1998 |
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6579692 |
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09147004 |
Sep 8, 1998 |
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PCT/JP97/00702 |
Mar 6, 1997 |
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Current U.S.
Class: |
435/7.23 ;
424/155.1 |
Current CPC
Class: |
C07K 14/70596 20130101;
C07K 14/4747 20130101; A61K 38/00 20130101 |
Class at
Publication: |
435/7.23 ;
424/155.1 |
International
Class: |
G01N 033/574; A61K
039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 6, 1996 |
JP |
8/78182 |
Claims
What is claimed is:
1. A method of screening substances having property of causing
apoptosis characterized by using cells which are expressing IAP
(Integrin Associated Protein) and screening the substances
thereby.
2. The method of screening according to claim 1, wherein the cells
used are myeloid cells.
3. The substances having property of causing apoptosis obtained by
the method of screening according to claim 1 or claim 2.
4. The substances having property of causing apoptosis according to
claim 3, wherein the substances are antibodies.
5. Pharmaceutical compositions containing as an active ingredient
the substances according to claim 3 and/or claim 4.
6. The pharmaceutical compositions according to claim 5, wherein
the pharmaceutical compositions are anticancer agents.
7. The pharmaceutical compositions according to claim 5, wherein
the pharmaceutical compositions are medicines for myelocytic
leukemia.
8. Substances having property of causing apoptosis which have a
binding potency to IAP.
9. Pharmaceutical compositions containing as an active ingredient
the substances according to claim 8.
10. The pharmaceutical compositions according to claim 9, wherein
the pharmaceutical compositions are anticancer agents.
11. The pharmaceutical compositions according to claim 10, wherein
the pharmaceutical compositions are medicines for myelocytic
leukemia.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method of screening
substances having property of causing apoptosis and the like, in
particular, relates to a novel screening method which makes it
possible to screen readily and highly efficiently the substances,
such as monoclonal antibodies and the like, that have property of
causing apoptosis on myeloid cells by using cells which are
expressing IAP (Integrin Associated Protein), and relates to the
substances having property of causing apoptosis obtained by the
above screening method, pharmaceutical compositions containing as
the active ingredient the above substances, and relates to
substances having property of causing apoptosis which have a
specific binding activity to IAP, pharmaceutical compositions
containing as the active ingredient the above substances.
BACKGROUND ART
[0002] Granulocyte colony-stimulating factors, for example,
recombinant granulocyte colony-stimulating factors (rG-CSF), have
been known primarily as humoral factors to stimulate the
differentiation and proliferation of granulocyte cells, and it has
been reported in an experiment upon mice in vivo that the
administration of rG-CSF enhances the hematopoiesis of the bone
marrow and in addition causes remarkable extramedullary
hematopoiesis in the spleen to proliferate hematopoietic stem cells
and all hematopoietic precursor cells in the spleen. And it has
been thought as extramedullary hematopoietic mechanism in the
spleen that hematopoiesis occurs due to a splenic hematopoietic
microenvironment modifications according to the stimulation of
rG-CSF to enhance hematopoietic potential.
[0003] Hence, the present inventors have noted splenic stromal
cells administered rG-CSF with a view to clarifying the
hematopoietic potential in the spleen, and established a
hematopoietic stromal cell line (CF-1 cells) from the spleen of a
mouse administered rG-CSF with a view to attempting the analysis of
the enhancement of-the hematopoietic potential by stromal cells
with rG-CSF, and examined the potential effect on hematopoiesis
using the hematopoietic stromal cells, and as a result,
colony-stimulating activities in vitro and potency supportive of
hematopoietic stem cells in vivo have been recognized [Blood, 80,
1914 (1992)].
[0004] However, though some of splenic stromal cells have been
established as a cell line (CF-1 cells), and cytological
characteristics thereof have been examined, a specific antibody
recognizing surface antigens thereof has been hardly prepared so
far, and characteristics thereof have been scarcely known.
[0005] Hence, the present inventors have engaged in assiduous
studies with a view to developing specific antibodies capable of
recognizing splenic stromal cells on the basis of the above
information upon splenic stromal cells and the results of the
studies, and prepared monoclonal antibodies using the splenic
stromal cell lines as antigens for immunization, and as a result,
novel monoclonal antibodies unreported so far have been
obtained.
[0006] And as a result of examining the properties of the obtained
monoclonal antibodies, the inventors found that they had the
property of causing apoptosis on myeloid cells, and had reported
this result previously, and further have found that the antigens
recognized by the monoclonal antibodies are identical to IAP
(Integrin Associated Protein) and the IAP have functions relating
to apoptosis, and that it is possible to differentiate, identify
and screen the substances, such as antibodies and the like, that
have property of causing apoptosis by using cells which are
expressing the IAP, and further have engaged in assiduous studies,
which have led to the completion of the present, invention.
SUMMARY OF INVENTION
[0007] It is the objective of the present invention to provide a
method of screening substances having property of causing apoptosis
and the like.
[0008] This invention relates to a method of screening substances
having property of causing apoptosis characterized by using cells
which are expressing IAP (Integrin Associated Protein) and
screening the substances. The invention relates to the screening
method, wherein the cells used are myeloid cells, and relates to
pharmaceutical compositions containing as the ingredient the
substances obtained by the above screening method. The invention
makes it possible to differentiate, identify and screen readily and
highly efficiently the substances, such as antibodies and the like,
that have property of causing apoptosis on myeloid cells by using
cells which are expressing IAP while using specific binding
reactions of the substances. The above specific substances thus
obtained by the screening method of the invention can be used by
virtue of their characteristics as the active ingredient of
pharmaceutical compositions such as anticancer agents and medicines
for myelocytic leukemia and the like which are useful in the field
of remedies for myelocytic leukemia and the like.
DISCLOSURE OF INVENTION
[0009] It is the objective and purpose of the present invention to
provide a method of screening substances having property of causing
apoptosis by using cells which are expressing IAP, and further to
provide novel substances having property of causing apoptosis on
cells obtained by the above screening method and pharmaceutical
compositions as the active ingredient the above substances.
[0010] The above monoclonal antibody is remarkably useful as an
antibody recognizing antigens causing the apoptosis [it is also
called self-destruction of cells, phenomenon that a nuclear
chromatin DNA is digested at a nucleosome unit (so-called ladder
formation) to result in the death of cells ] of myeloid cells and
having a function of identifying them or a function of causing
apoptosis on myeloid cells. Incidentally, myeloid cells include
cells other than lymphoid cells, such as neutrophils,
megakaryocytes, myeloblasts, myelocytes, mast cells, macrophages,
monocytes and erythroblasts, and the myeloid cells according to the
present invention also mean the same as mentioned above. No
monoclonal antibody having the property of causing apoptosis on
myeloid cells has been known so far other than the above monoclonal
antibody, and hence the above monoclonal antibodies are defined to
include all monoclonal antibodies having the property of causing
apoptosis on myeloid cells.
[0011] The monoclonal antibody may be prepared basically as stated
below.
[0012] Namely, the above monoclonal antibody may be prepared, for
example, by using splenic stromal cells derived from an animal
administered rG-CSF as antigens, immunizing them according to an
ordinary immunization method, cell-fusing the immunized cells
according to an ordinary cell fusion method, and cloning the fused
cells according to an ordinary cloning method.
[0013] As a method of preparing the above monoclonal antibody can
be preferably exemplified a method comprising using CF-1 cells,
splenic stromal cells of an animal administered rG-CSF established
as culture cell line by the present inventors, as the antigen
[Blood, Vol. 80, 1914 (1992)] , fusing plasma cells (immunocyte) of
a mammal immunized with the antigen with myeloma cells of a mammal
such as a mouse, cloning the obtained fused cells (hybridomas),
selecting clones producing the above antibody recognizing the above
cell line among them, and culturing them to recover objective
antibody. However, the method is only an example, and in this case,
for example, not only the above CF-1 cells but also cell lines
derived from human splenic stromal cells obtained according to the
case of CF-1 cells may be used as the antigens properly to prepare
antibodies binding to objective human myeloid cells in the same
manner as in the case of the above CF-1 cells.
[0014] In the method of preparing such monoclonal antibodies,
mammals to be immunized with the above antigen are not particularly
restricted; it is preferable to make selection taking into account
suitability with myeloma cells to be used in cell fusion, and
preferably a mouse, a rat and a hamster and the like are used.
[0015] Immunization is performed according to an ordinary method,
for example, by administering splenic stromal cells such as the
above CF-1 cells and the like into abdominal cavity of a mammal by
injection. More specifically, it is preferable to administer one
diluted with or suspended in a proper amount of PBS or isotonic
sodium chloride solution to an animal several times every month. It
is preferable to use splenic cells removed after the final
administration of the above cells as immunocytes.
[0016] As a myeloma cell of a mammal as the other parent cell fused
with the above immunocytes can be used preferably known various
cells including P3(P3X63Ag8.653) [J. Immunol., 123, 1548 (1978)],
p3-U1 [Current Topics in Micro-biology and Immunology, 81, 1-7
(1978) ], NS-1 [Eur. J. Immunol., 6, 511-519 (1976)], MPC-11 [Cell,
8, 405-415 (1976)], Sp2/0-Ag14 [Nature, 276, 269-270 (1978)], FO
[J. Immunol. Meth., 35, 1-21 (1980)], S194 [J. Exp. Med., 148,
313-323 (1978)] and R210 [Nature, 277, 131-133 (1979)].
[0017] The cell fusion of the above immunocyte and a myeloma cell
performed basically according to an ordinary method, for example, a
method by Milstein et al. [Methods Enzymol., 73, 3-46 (1981)] and
the like.
[0018] More specifically, the above cell fusion may be performed,
for example, in an ordinary nutrition medium in the presence of a
fusion-accelerating agent. As a fusion-accelerating agent,
polyethylene glycol (PEG) and Sendai virus (HVJ) and the like, and
furthermore, adjuvants such as dimethyl sulfoxide and the like may
be added properly if required in order to enhance the fusing
effect. Regarding the ratios of immunocytes and myeloma cells, the
former is preferably used in an amount 1-10 times that of the
latter. Examples of a medium used in the above cell fusion include
a RPMI-1640 medium and a MEM medium suitable for the proliferation
of the above myeloma cell and other mediums ordinarily used for the
culture of this kind of cell, and in addition, supplementary serum
such as fetal bovine serum (FBS) may be used together.
[0019] Cell fusion is performed by mixing prescribed amounts of the
above immunocytes and myeloma cells in the above medium, adding a
PEG solution preheated to about 37.degree. C., for example, PEG
with an average molecular weight of the order of 1,000-6,000 to the
medium, ordinarily, at a concentration of about 30-60% (W/V), and
mixing them. Subsequently, by repeating the operations of adding
proper mediums one after another, centrifuging the reaction mixture
and removing the supernatants can be formed objective
hybridomas.
[0020] Said hybridomas are selected by culturing in an ordinary
selective medium, for example, a HAT medium (medium supplemented
with hypoxanthine, aminopterin and thymidine). The culture in the
HAT medium is continued for a period sufficient for cells other
than objective hybridomas (non-fused cells) to die out, ordinarily
for several days to several weeks. Subsequently, the screening and
monocloning of the hybridomas producing the objective antibodies
are performed according to ordinary limiting dilution analysis.
[0021] The prepared hybridomas producing the above monoclonal
antibodies may be subcultured in an ordinary medium and stored in
liquid nitrogen for a long time.
[0022] In order to collect the above monoclonal antibodies from the
hybridomas may be employed a method comprising culturing the
hybridomas according to an ordinary method, and obtaining them from
the supernatants, or a method comprising administering a hybridoma
into a appropriate mammal to proliferate, and obtaining them from
its ascites. The former is suitable for obtaining antibodies with a
high purity and the latter is suitable for the mass production of
antibodies.
[0023] Furthermore, the antibodies obtained according to the above
methods may be purified to a high degree employing an ordinary
purification means such as a salting-out technique, gel filtration
and affinity chromatography and the like.
[0024] As the monoclonal antibody, BMAP-1 is used in Example
described later, the monoclonal antibody to be used is not limited
to the same, and the monoclonal antibody may be any one so far as
it has a specific property to be described specifically in Example
later, namely, a property of causing apoptosis on myeloid cells,
and those having the property are included in the scope of the
present invention, irrespective of the kind of the same. Next, as
cells which are expressing IAP, myeloid cells and the like are
exemplified, and for example, transfectant Jurkat cell with murine
IAP gene (Genbank, Accession number Z 25524) [The Journal of Cell
Biology, 123, 485-496 (1993)] transfected by the usual way and the
like are used preferably. The IAP gene is not limited to the murine
IAP gene, and it is possible to use another IAP gene, such as human
IAP gene and the like. Further, cells which are expressing IAP (for
example, human leukemia cell and the like) are used in the same
way.
[0025] Then, the screening method of the present invention is
characterized by using cells which are expressing IAP, and a method
of screening substances having property of causing apoptosis by
using the cells comprises differentiating, identifying and
screening by the usual way the substances, such as monoclonal
antibody which recognizes the IAP specifically as an antigen, and
fragments thereof, while using the same cells which are not
expressing IAP as a blank, and further a definite method of the
same is not limited to the specified method.
[0026] The monoclonal antibody thus obtained by the screening
method of the present invention, fragments thereof, substances
having property of causing apoptosis which have a binding ability
to IAP and the like can be used by virtue of their characteristics
as the active ingredient of pharmaceutical compositions, such as
anticancer agents, medicines for myelocytic leukemia and the like
which are useful in the field of remedies for myelocytic leukemia
and the like.
[0027] Needless to say, the establishment of a specific system for
identifying and recognizing antigens causing apoptosis on myeloid
cells according to utilizing the monoclonal antibody, or for using
it as medicine for myelocytic leukemia according to utilizing the
specific property thereof, and modification and application thereof
are put into practice according to an ordinary method obvious to
those skilled in the art.
BRIEF DESCRIPTION OF DRAWINGS
[0028] FIG. 1 shows an analysis (a control in the absence of an
antibody, CF-1 cell) according to immunofluorescence.
[0029] FIG. 2 shows an analysis of the binding properties of the
GSPST-1 antibody to CF-1 cells according to immunofluorescence.
[0030] FIG. 3 shows an analysis of the binding properties of the
BMAP-1 antibody to CF-1 cells according to immunofluorescence.
[0031] FIG. 4 shows an analysis (a control in the absence of an
antibody, bone marrow cell) according to immunofluorescence.
[0032] FIG. 5 shows an analysis of the binding properties of the
GSPST-1 antibody to bone marrow cells according to
immunofluorescence.
[0033] FIG. 6 shows an analysis of the binding properties of the
BMAP-1 antibody to bone marrow cells according to
immunofluorescence.
[0034] FIG. 7 shows an analysis (a control in the absence of an
antibody, NFS-60) according to immunofluorescence.
[0035] FIG. 8 shows the binding properties of the GSPST-1 antibody
to NFS-60 cells according to immunofluorescence.
[0036] FIG. 9 shows an analysis (a control according to rat IgG1,
NFS-60) according to immunofluorescence.
[0037] FIG. 10 shows the binding properties of the BMAP-1 antibody
to NFS-60 cells according to immunofluorescence.
[0038] FIG. 11 shows an assay for the monoclonal antibody (BMAP-1)
to inhibit NFS-60 cell proliferation.
[0039] FIG. 12 shows an assay for the monoclonal antibody (GSPST-1)
to inhibit the bone marrow transplantation.
[0040] FIG. 13 shows an assay for the monoclonal antibody (BMAP-1)
to inhibit the bone marrow transplantation.
[0041] FIG. 14 is an explanatory view [microphotograph (stained
with H. E.) of bone marrow samples (.times.4000)] showing dead bone
marrow cells (2) on 6 days after the administration of the
monoclonal antibody BMAP-1 of the present invention, and the
control (1) in the absence of the antibody.
[0042] FIG. 15 is an explanatory view (migration-photo according to
electrophoretic chromatography) showing the ladder formation of the
DNA of bone marrow cells observed when the monoclonal antibody
BMAP-1 of the present invention was administered.
[0043] FIG. 16 shows a cytotoxicity assay by TNF FIG. 17 shows a
cytotoxicity assay by the monoclonal antibody (BMAP-1).
[0044] FIG. 18 shows an analysis (a control according to rat IgG2a,
BWV1) according to immunofluorescence.
[0045] FIG. 19 shows the binding properties of the antimouse MHC
class I antibody to BWV1 cells according to immunofluorescence.
[0046] FIG. 20 shows an analysis (a control according to rat IgG1,
BWV1) according to immunofluorescence.
[0047] FIG. 21 shows the binding properties of the BMAP-1 antibody
to BWV1 cells according to immunofluorescence.
[0048] FIG. 22 shows proliferation inhibitory action to BMAP-1
cells (Jurkat cells transfected with murine IAP gene)
[0049] FIG. 23 shows an analysis of apoptosis--an action to Jurkat
cells transfected with expression vector (IgG1 1 .mu.g/ml, A:
Apoptosis ratio, 6.2%)
[0050] FIG. 24 shows an analysis of apoptosis--an action of BMAP-1
to Jurkat cells transfected with expression vector (BMAP-1 1
.mu.g/ml, A: Apoptosis ratio, 3.5%)
[0051] FIG. 25 shows an analysis of apoptosis an action to Jurkat
cells transfected with murine IAP gene (IgG 1 1 .mu.g/ml, A:
Apoptosis ratio, 3.2%)
[0052] FIG. 26 shows an analysis of apoptosis--an action of BMAP-1
to Jurkat cells transfected with murine IAP gene (BMAP-1 1 g g/ml,
A: Apoptosis ratio, 25.6%)
EXPLANATION OF SYMBOLS
[0053] a: DNA of the thymus of a mouse administered BMAP-1 (24
hours)
[0054] b: DNA of the bone marrow of a mouse administered BMAP-1 (24
hours)
[0055] c: DNA of the bone marrow of a mouse administered BMAP-1 (8
hours)
[0056] d: DNA of the bone marrow of a mouse administered BMAP-1 (4
hours)
[0057] e: DNA of the bone marrow of a non-treated mouse (bone
marrow cells)
[0058] f: Molecular weight marker
[0059] Best Mode for Carrying Out the Invention
[0060] Next, the present invention will be described further in
detail according to Reference Example and Example, but the present
invention is not restricted to the Example.
REFERENCE EXAMPLE
[0061] Establishment of Splenic Stromal Cells and Their
Characteristics Thereof
[0062] 1) Establishment of Splenic Stromal Cells
[0063] A splenic stromal cell line was established from the primary
culture of the splenic cells of a C57BL/6J mouse administered
rG-CSF 100 .mu.g/kg for 5 days according to serial administration
of rG-CSF. Namely, this spleen was removed after the administration
of rGCSF under germ-free conditions, cultured in a 25-cm.sup.2
plastic flask (Corning Co.) for 6 weeks and in an Isocove's
modified Dulbecco's medium (IMDM) (Boehringer-Mannheim Co.) with
10% heat-inactivated fetal bovine serum (FBS) (Sanko Junyaku,
Tokyo), 100 U/ml penicillin and 100 .mu.g/ml streptomycin in an
incubator under the condition of 37.degree. C. and 5% CO.sub.2 and
the medium was exchanged for a fresh growth medium twice a
week.
[0064] In the confluent culture, the adherent cell populations
(stromal cells) were harvested from the flask by using 0.05%
trypsin plus 0.02% EDTA (Sigma Chemical Co.) in Ca-,Mg-free PBS,
and were transferred into new flasks. These passages were repeated
approximately once or twice a week. In the early passages (1st
through 10th passages), the split ratio of the cells was 1/4 to
1/8, and subsequently the ratio was 1/16 to 1/32. The stromal cells
became homogeneous and fibroblastoid after approximately the 10th
passage.
[0065] At the 20th passage, these stromal cells were harvested as
described above and forwarded to cell cloning by using a limiting
dilution technique; cell cloning was repeated twice to establish a
stromal cell line (CF-1 cell line).
[0066] Subsequently, these cells were maintained in 5 ml of IMDM
supplemented with 10% heat-inactivated FBS in a 25-cm.sup.2 flask
(Corning Co.), and subcultured once every 5 days at the split ratio
of 1/32. Splenic stromal cell lines can be established from other
animals than mouse; for example, human splenic stromal cell lines
can be established using the same method as described above by
transforming the cells with an SV-40 adenovirus vector [J. Cell.
Physiol., 148, 245 (1991) ]
[0067] 2) Characteristics of CF-1 Cells
[0068] CF-1 cells established as a cell line as described above
were examined for alkaline phosphatase, acid phosphatase,
.beta.-glucuronidase, .alpha.-naphthyl acetate esterase and oil red
O using standard cytochemical techniques. CF-1 cells were also
characterized by immunoenzymatic histochemistry using the following
monoclonal and polyclonal antibodies: macI (Sero Tec.); factor
VIII-related antigen (Dakopatts); and collagen type I, collagen
type III and fibronectin (Chemicon International Inc.).
Phagocytosis was tested by latex bead uptake (particle diameter:
1.09 .mu.m; Sigma), and the potency of CF-1 cells to convert to
adipocytes was tested by exposure to 10.sup.-6 mol/1 hydrocortisone
phosphate (Sigma) in a 25-cm.sup.2 flask for 4 weeks after the
confluent culture.
[0069] As a result, the CF-1 cells were negative for alkaline
phosphatase, factor VIII-related antigen, mac I and phagocytosis,
whereas they were positive for collagen type I, collagen type III
and fibronectin. CF-1 cells were not converted to adipocytes during
4 weeks in a confluent culture with 10.sup.-6 mol/1 hydrocortisone,
although CF-1 cells had only traces of lipid. From these data, CF-1
cells do not have the characteristics of preadipocytes, macrophages
and endothelial cells, and therefore it has become obvious that
they are derived from stromal cells different from them.
[0070] 3) Maintenance of Hematopoietic Stem Cells by CF-1 Cells
[0071] To examine whether hematopoietic stem cells are maintained
by CF-1 cells or not, CFU-S assays (assays of spleen
colonies-forming cells) were performed by the technique of Till and
McCulloch. Ten mice per group were irradiated with 900 cGy
(MBR-1520R; Hitachi, Tokyo) and injected intravenously with bone
marrow mononuclear cells (BM cells) (1.0.times.10.sup.5/head,
5.0.times.10.sup.4/head, or 2.5.times.10.sup.4/head) and CF-1 cells
(1.0.times.10.sup.5/head), and colonies in the spleen were counted
on the 12th day as CFU-S clones (spleen colonies).
[0072] As a result, when bone marrow mononuclear cells (BM cells)
and CF-1 cells were transplanted into irradiated mice, the number
of spleen colonies of every group of BM cells increased
significantly (between 1.4-1.8 times) as compared to the mice
without CF-1 cells, and, on the 12th day after the transplantation,
the survival ratios of the mice transplanted with BM cells and CF-1
cells were higher than those with only BM cells, showing a low
death rate; hence it has become apparent that hematopoietic stem
cells are maintained by CF-1 cells.
[0073] The embodiment of the invention will be described in detail
hereinafter.
EXAMPLE
[0074] Establishment of Monoclonal Antibodies
[0075] 1) Antigens and Immunization
[0076] Immunization was performed by using CF-1 cells obtained in
the above Reference Example as antigens. The cells were cultured in
an incubator under the condition of 5% CO.sub.2 and 37.degree. C.,
using an Isocove's modified Dulbecco's medium (IMDM)
(Boehringer-Mannheim Co.) supplemented with 10% fetal bovine serum
(FBS; Sanko Junyaku) as a medium.
[0077] The cells were treated with 1 mM EDTA/PBS, and removed from
a culture flask according to pipetting. The cells were suspended
into 1 mM EDTA/PBS at the cell number of about 1.times.10.sup.7/ml,
and administered to a Wistar Imamich rat (7-week-old, female;
Animal Breeding Research Laboratory). One ml of cells of about
1.times.10.sup.7/ml were injected into the abdominal cavity of the
rat at the initial immunization, and 1 ml of cells of about
1.times.10.sup.7/ml were administered additionally one month later.
Further, 1 ml of cells of about 1.times.10.sup.7/ml were
administered additionally several times at an interval of a month,
and after the reactivity between the immunized rat antibody and
CF-1 cells was recognized, 1 ml of cells of 1.times.10.sup.8/ml
were administered as the final immunization. Three days after the
final immunization, the rat was killed to remove spleen.
[0078] 2) Cell Fusion
[0079] After the spleen removed from the rat was minced, splenic
cells isolated were centrifuged, suspended in an IMDM medium
(Boehringer-Mannheim Co.), and washed intensively. On the other
hand, the cells obtained by cultured mouse myeloma cell line
Sp2/0-Ag14 [Nature, 276, 269-270 (1978)] in an IMDM
(Boehringer-Mannheim Co.) supplemented with 10% fetal bovine serum
(FBS; Sanko Junyaku) were washed in the above IMDM medium in the
same manner, and 1.times.10.sup.8 thereof and 2.times.10.sup.8 of
the above splenic cells were put into a centrifuge tube and mixed
to perform cell fusion by polyethylene glycol 4000 (Nakarai Kagaku)
according to an ordinary procedure [Clin. Exp. Immunol., 42 ,
458-462 (1980)]
[0080] Subsequently, the obtained fused cells were dispensed into a
96-well plate with an IMDM medium supplemented with 20% FBS, and
cultured in an incubator under the condition of 37.degree. C. and
5% CO.sub.2. They were replaced into a HAT selective medium
gradually from the following day, and continued to be cultured.
[0081] After the start of the culture, the supernatants were
replaced into a new HAT medium twice a week to continue the culture
and maintain the proliferation.
[0082] Next, the obtained fused cells were cloned according to an
ordinary procedure using limiting dilution analysis. Namely, only
clones having strong binding properties to antigens were cloned
according to an ordinary procedure employing limiting dilution
analysis by examining binding properties thereof to the antigens,
utilizing antibodies in the supernatants of the above fused
cells.
[0083] 3) Screening
[0084] The screening of fused cells (hybridomas) was performed
according to indirect fluorescent antibody technique using flow
cytometry.
[0085] The screening of clones producing objective antibodies was
performed using CF-1 cells as target cells. Namely, cells suspended
in a reaction buffer (PBS supplemented with 2% FBS and 0.02%
NaN.sub.3 ) were centrifuged and recovered as pellets, then
suspended in 100 .mu.l of the hybridoma culture supernatants (about
1.times.10.sup.6/100 .mu.l ) and reacted at 4.degree. C. for 1
hour. After they were washed with the above buffer once, an
FITC-labelled goat anti-rat IgG (FC) antibody (Chemicon) was added
thereto and incubated for 1 hour. After they were washed once, they
were analyzed according to flow cytometry (FACScan, Becton
Dickinson).
[0086] 4) Purification of Antibodies
[0087] The fused cells screened in the manner of the above 3) were
cultured according to an ordinary procedure, and antibodies
produced in the supernatants were separated according to an
ordinary procedure, and purified.
[0088] Namely, hybridomas were recovered from wells with high
antibody titers to the antigens, spread in a tissue culture plastic
dish (Corning Co.), cultured under the condition of 5% CO.sub.2 and
37.degree. C., proliferated, and purified according to an ordinary
procedure to obtain monoclonal antibodies GSPST-1 and BMAP-1.
[0089] Regarding GSPST-1, obtained cells were injected into the
abdominal cavity of a BALB/cAJc1-nu nude mouse (8-week-old, male,
Nippon Kurea). Produced ascites was recovered after 10-14 days,
salted out with 33% ammonium sulfate, and dialyzed with PBS.
Regarding the BMAP-1 antibody, it was cultured in a large scale in
an Iscove's modified MEM medium supplemented with 10% FBS, and the
supernatants were concentrated, salted out with 33% ammonium
sulfate, dialyzed with PBS, purified again by means of a protein A
column kit (Amersham), and dialyzed with PBS. Incidentally, in the
above Example was described the case in which the CF-1 cells were
used as antigens for immunization; however, it is possible to
establish a monoclonal antibody in the same manner also in case of
using other stromal cells having potency supportive of
hematopoietic stem cells.
[0090] A hybridoma producing the above monoclonal antibody BMAP-1
is a novel fused cell prepared from a Wistar Imamich rat splenic
cell and a mouse myeloma cell line SP2/0-Ag14 as parent cells, and
was deposited on Aug. 9, 1993, under the name of BMAP-1 (rat mouse
hybridoma) with the accession number of FERM BP-4382, at National
Institute of Bioscience and Human Technology, Agency of Industrial
Science and Technology in Japan [address: 1-3, Higashi 1-chome,
Tsukuba-shi, Ibaraki 305, Japan], international depositary
authority according to Budapest Treaty on the international
recognition of the deposit of microorganisms for the purpose of
patent procedures.
[0091] 5) Properties of Antibodies
[0092] (i) Reactivity of Antibodies
[0093] Reactivity to CF-1 Cells
[0094] The results of examining the reactivity of the obtained
monoclonal antibodies GSPST-1 and BMAP-1 to CF-1 cells according to
immunofluorescence analysis are shown in FIG. 1 through FIG. 3.
Here, FIG. 1 shows the results of analysis of the control in the
absence of an antibody, FIG. 2 the results of analysis of the
binding properties of GSPST-1 to CF-1 cells, and FIG. 3 the results
of analysis of the binding properties of BMAP-1 to CF-1 cells. In
the drawings, vertical axes show relative number of cells and
transverse axes fluorescence intensity.
[0095] As is apparent from FIG. 1 through FIG. 3, it has been
revealed that monoclonal antibodies GSPST-1 and BMAP-1 have
properties binding to CF-1 cells and recognize surface antigens of
CF-1 cells.
[0096] Reactivity to Bone Marrow Cells
[0097] Next, the results of analysis of the reactivity of GSPST-1
and BMAP-1 to normal bone marrow cells according to flow cytometry
(FACScan, Becton Dickinson) are shown in FIG. 4 through FIG. 6.
Here, FIG. 4 shows the results of analysis of the control in the
absence of an antibody, FIG. 5 the results of analysis of the
binding properties of GSPST-1 to bone marrow cells, and FIG. 6 the
results of analysis of the binding properties of BMAP-1 to bone
marrow cells. In the drawings, vertical axes show relative number
of cells and transverse axes fluorescence intensity.
[0098] As is shown in FIG. 4 through FIG. 6, it has been revealed
that GSPST-1 has not a property binding to bone marrow cells at
all, and that BMAP-1 has a property binding to all bone marrow
cells.
[0099] Reactivity to Myelocytic Leukemic Cell Line (NFS-60)
[0100] The results of analysis of the the reactivity of GSPST-1 and
BMAP-1 to NFS-60 cells [Proc. Natl. Acad. Sci. USA, 82, 6687-6691
(1985)] according to flow cytometry (FACScan, Becton Dickinson) are
shown in FIG. 7 through FIG. 10. Here, FIG. 7 shows the results of
analysis of the control in the absence of an antibody, FIG. 8 shows
the results of analysis of the binding properties of GSPST-1 to
NFS-60 cells, FIG. 9 shows the results of analysis of the control
using rat IgG1 on the market (Zymed) and FIG. 10 shows the results
of analysis of the binding properties of BMAP-1 to NFS-60 cells. In
the drawings, vertical axes show relative numbers of cells and
transverse axes fluorescence intensity.
[0101] As is shown in FIG. 7 through FIG. 10, it has been revealed
that GSPST-1 does not react with NFS-60 cells, and that BMAP-1 has
a property binding to NFS-60 cells.
[0102] Assay for BMAP-1 to Inhibit Proliferation of NFS-60
Cells
[0103] The results of examining the action of BMAP-1 to NFS-60
cells in the presence of G-CSF 100 ng/ml and cycloheximide
10.sup.-9 M according to the MTT assay method are shown in FIG. 11.
Using culture plates with 96 wells, 10 .mu.l/well of BMAP-1
solution were added at concentrations of 0, 1, 10, 100 ng/ml, and
1, 10 .mu.g/ml to 4.times.10.sup.3/well/100 .mu.l of NFS-60 cells,
and two days after the numbers of living cells were measured
according to the MTT method. It has been revealed as shown in FIG.
11 that the proliferation of NFS-60 cells is inhibited remarkably
by BMAP-1.
[0104] Next, as a result of typing the subclass of IgG of the
obtained monoclonal antibodies [using a rat Mono Ab-ID-Sp kit
(Zymed) and a biotin-labelled mouse anti-rat IgG1 antibody
(Zymed)], it has become apparent that GSPST-1 is IgG2a, and that
BMAP-1 is IgG1.
[0105] (iii) Potency Inhibiting Bone Marrow Transplantation
[0106] Next, a test upon the inhibition of bone marrow
transplantation was performed using these antibodies to examine
characteristics thereof. The results are shown in FIG. 12 and FIG.
13. As is shown in FIG. 12 and FIG. 13, while BMAP-1 has the effect
inhibiting the bone marrow transplantation, the effect has not been
found in GSPST-1. Namely, the above results were obtained by
administering 1.0.times.10.sup.5/head of bone marrow cells and
monoclonal antibodies to C57BL/6J mice, irradiated at a fatal dose
of radiation (900 cGy), through the veins of tails, and counting
the number of spleen colonies. Incidentally, "Non-treated" in FIG.
13 shows the case with no administration of bone marrow cells.
[0107] As is shown in FIG. 13, it has been confirmed that it is
because BMAP-1 reacts with bone marrow cells to cause apoptosis
that the monoclonal antibody inhibits transplantation completely in
the test upon the inhibition of bone marrow transplantation.
Namely, when a hybridoma producing BMAP-1 was administered into the
abdominal cavity of a nude mouse, it died at the time when its
ascites was stored in a small amount. In addition it has been
revealed that all bone marrow cells died out according to the
intravenous administration of 50 .mu.g/head BMAP-1 to a normal
C57BL/6J mouse, and in FIG. 14 is shown a microphotograph backing
up the fact that bone marrow cells on 6 days after the intravenous
administration of BMAP-1 died out. As is apparent from the
microphotograph, it has been observed that not only lymphoid cells
but also neutrophils, megakaryocytes, myeloblasts, myelocytes, mast
cells, macrophages, monocytes and erythroblasts (so-called myeloid
cells) died out. In addition, as a result of investigating the DNAs
of the bone marrow cells of a mouse administered 30 .mu.g/head
BMAP-1, apparently ladder formation has been observed as is shown
in FIG. 15, and it has been revealed that the above reaction of
BMAP-1 to bone marrow cells is due to apoptosis.
[0108] The Fc region of the IgG of the BMAP-1 antibody was digested
with pepsin (Sigma) and purified by means of a GPC column as
F(ab.sup.-)2, and 33.5 .mu.g/head (corresponding to 50 .mu.g/head
of the whole IgG) were administered to a C57BL/6J mouse
intravenously; as a result of it, it was observed that bone marrow
cells died out in the bone marrow. It has become apparent according
to the above fact that neither antibody-dependent cell cytotoxicity
nor complement-dependent cell cytotoxicity participates in the cell
death of bone marrow cells by BMAP-1.
[0109] As an antigen causing apoptosis has been reported the Fas
antigen of cell surface protein; regarding the Fas antigen, the
expressions of mRNAs of it are recognized in the thymus, heart,
liver, lungs and ovary, but its mRNAs are scarcely detected in the
bone marrow [J. Immunol., 148, 1274-1279 (1992)], and hence it is
apparent that antigens recognized by BMAP-1 are different from the
conventionally known Fas antigen.
[0110] Furthermore, in order to make it clear whether an antigen
recognized by BMAP-1 would be a TNF receptor or not, the function
of BMAP-1 was investigated using L-929 cells reacting with TNF to
cause cell death. The final concentrations of a mouse TNF.alpha.
(Genzyme) were 0, 1, 10, 100 pg/ml, 1, 10, 100 ng/ml, and 1
.mu.g/ml, and those of BMAP-1 were 0, 10, 100 pg/ml, 1, 10, 100
ng/ml, and 1, 10 .mu.g/ml, and the numbers of living cells of L-929
cells were measured according to the MTT method on the second day
after the addition of the TNF.alpha. and BMAP-1. As a result of it,
as shown in FIG. 16, FIG. 17, while L-929 cells were reduced by
TNF.alpha. remarkably, BMAP-1 had no effect upon L-929 cells.
Hence, it has become apparent that an antigen recognized by BMAP-1
is not a TNF receptor.
[0111] The results of investigating whether antigens recognized by
BMAP-1 would be MHC class I antigens or not according to flow
cytometry (FACScan, Becton Dickinson) are shown in FIG. 18 through
FIG. 21. Here, FIG. 18 shows the results of analysis of the control
using rat IgG1 (Zymed) FIG. 19 shows the results of analysis of the
binding properties of the anti-mouse MHC class I antibody (rat
IgG2a, BMA) to BWV1 cells (mouse lymphoma derived from BW5147
cells), FIG. 20 shows the results of analysis of the control using
rat IgG1 (Zymed) and FIG. 21 shows the results of analysis of the
binding properties of BMAP-1 to BWV1 cells. In the drawings,
vertical axes show relative numbers of cells and transverse axes
fluorescence intensity. As a result, BMAP-1 did not recognize BWV1
cells but the MHC class I antibody reacted with BWV1 cells.
[0112] As described above, it has been confirmed experimentally
that BMAP-1 has the function of causing apoptosis on myeloid cells;
according to the present inventor's knowledge, no monoclonal
antibody having the property of causing apoptosis on myeloid cells
has been reported so far as described above, and hence monoclonal
antibodies having such a function are ones found by the present
inventors.
[0113] It became clear by direct expression cloning that the
antigen recognized by BMAP-1 is murine IAP. The action of BMAP-1
was examined employing recombinant cells transfected with murine
IAP genes. That is, the action of BMAP-1 on IAP expressing cells
was examined by the MTS method and the analysis of DNA
fragmentation by a flow cytometry employing recombinant Jurkat
cells expressing murine IAP obtainable by inducing IAP genes into
Jurkat cells expressing no murine IAP by an ordinary procedure. The
result are shown in FIGS. 22 to 26.
[0114] The MTS method is an assay method (Promega) measuring the
number of living cells, and the action of BMAP-1 on recombinant
Jurkat cells was examined by this method. That is, the number of
living cells was measured by the MTS method, employing a culture
plate with 96 wells, by adding 1, 10, 100 ng/ml and 1, 10 .mu.g/ml
of BMAP-1 at the final concentration and 10 .mu.g/ml of IgG1 as a
control to 1.times.10.sup.4/well/100 .mu.l of recombinant Jurkat
cells in the presence of G418 (final concentration: 1 mg/ml)
(produced by Gibco BRL) and culturing them for two days. It became
clear as a result of it that, as shown in FIG. 22, the recombinant
Jurkat cells are prohibited from proliferating remarkably by
BMAP-1.
[0115] The analysis of the DNA fragmentation of the recombinant
Jurkat cells by BMAP-1 was performed by a flow cytometry (EPICS
(registered trademark) XL-MCL, produced by Coulter). That is,
employing a culture plate with 6 wells, IgG1 and BMAP-1 were added
at the final concentration of 1 .mu.g/ml to
1.5.times.10.sup.5/well/3 ml of recombinant Jurkat cells in the
presence of G418 (final concentration: 1 mg/ml) (produced by Gibco
BRL), and the mixture was cultured for two days and then subjected
to measurement. The cells were recovered from the culture plate,
and cell pellets were centrifuged at 200.times.g and fixed in 2 ml
of cold 70% ethanol at 4.degree. C. for 60 minutes. Subsequently,
the cells were centrifuged and resuspended in 1 ml of PBS. To 0.5
ml of the cell sample were added 0.5 ml of RNAse (Type I-A, Sigma,
St. Louis, Mo., USA, 1 mg/ml in PBS) and then the mixture was mixed
into 1 ml of a propidium iodide (PI, Sigma, 100 .mu.g/ml in PBS)
solution. The mixed cells were incubated at room temperature in a
dark place for 15 minutes, then kept at 4.degree. C. in a dark
place and subjected to measurement by a flow cytometry.
[0116] It became clear as a result of it that, as shown in FIG. 26,
in the cells transfected with murine IAP genes, apoptosis is caused
by BMAP-1.
[0117] On the other hand, the above action of BMAP-1 was not
observed in Jurkat cells with only an expression vector and
expressing no murine IAP (FIG. 24). It became clear from these
matters that the antigen recognized by the BMAP-1 antibody is
identical with IAP, and that IAP has a function related to
apoptosis.
[0118] According to information at present, as functions of IAP are
reported an action of being bound to a 6 chain of .alpha..sub..nu.
.beta..sub.3 of integrin and supporting the bond between
.alpha..sub..nu. .beta..sub.3 and vitronectin as a ligand thereof
(J. Cell Biol., 123, 485-496 (1993)), an action of causing the flow
of Ca.sup.2+ to the endothelium in adhesion between neutrophils and
the endothelium (J. Biol. Chem., 268, 19931-19934 (1993)) and an
action of supporting neutrophils to pass through the endothelium
(Proc. Natl. Acad. Sci. USA, 92, 3978-3982 (1995)); however, no
function related to apoptosis has been reported.
[0119] Since BMAP-1 is one of monoclonal antibodies being bound to
myeloid cells and causing apoptosis in myeloid cells, as described
above, it becomes possible to discriminate, identify and screen
substances causing apoptosis in myeloid cells by utilizing cells
expressing IAP as an antigen recognized by said BMAP-1 antibody
specifically.
[0120] As described above, the present inventor has made it clear
that the antigen recognized by the BMAP-1 antibody is identical
with IAP, and that IAP has a function related to apoptosis, and has
established, according to the above information, a method of
screening substances causing apoptosis in myeloid cells simply and
efficiently by employing cells transfected with IAP genes or cells
expressing IAP.
[0121] By utilizing the apoptosis action of the substances such as
monoclonal antibody obtained by the method according to the present
invention, fragments thereof and substances causing apoptosis
possessing binding potency to IAP to myeloid cells, since it can be
thought that the substances like said monoclonal antibody and the
like can kill myelocytic leukaemia cells which are thought to
express antigens thereof remarkably, the substances such as said
monoclonal antibody causing apoptosis in myeloid cells and
fragments thereof are useful as the effective components of medical
compositions such as anticancer agents and medicines for myelocytic
leukemia and the like.
[0122] The method according to the present invention has been
described specifically according to Examples; though as substances
causing apoptosis according to the present invention are mentioned
those exemplified as specific examples and typical ones, they are
not always restricted thereto; it goes without saying that all the
substances possessing the same characteristics and functions
prepared in the same manner are included.
[0123] Industrial Applicability
[0124] The present invention makes it possible to discriminate,
identify and screen substances such as antibodies and the like
causing apoptosis in myeloid cells simply and efficiently by
employing cells expressing IAP and utilizing the specific binding
reaction thereof. The substances possessing property of causing
apoptosis in myeloid cells obtained by the method according to the
present invention can be used by virtue of their characteristics as
the effective components of anticancer agents and medical
compositions such as medicines for myelocytic leukemia and the like
useful in the field of the remedies of myelocytic leukemia and the
like.
[0125] Indications relating to a Deposited Microorganism
[0126] Name and Address of Depositary Institution: National
Institute of Bioscience and Human Technology, Agency of industrial
Science and Technology, Ministry of Trade and Industry in Japan
(address: 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki 305, Japan
(Postal code 350))
[0127] Date of Deposite: Aug. 8, 1993
[0128] Accession Number: FERM BP-4382
[0129] Indication of microorganism: BMAP-1 (rat mouse
hybridoma)
* * * * *