U.S. patent application number 10/376628 was filed with the patent office on 2003-08-07 for monoclonal antibodies having property of causing apoptosis.
This patent application is currently assigned to CHUGAI SEIYAKU KABUSHIKI KAISHA. Invention is credited to Fukushima, Naoshi.
Application Number | 20030147894 10/376628 |
Document ID | / |
Family ID | 17084452 |
Filed Date | 2003-08-07 |
United States Patent
Application |
20030147894 |
Kind Code |
A1 |
Fukushima, Naoshi |
August 7, 2003 |
Monoclonal antibodies having property of causing apoptosis
Abstract
It is the objective and purpose of the present invention to
provide a monoclonal antibody having the property of causing
apoptosis on myeloid cells. This invention relates to a monoclonal
antibody having the property of causing apoptosis on myeloid cells,
and fragments thereof, and furthermore relates to a hybridoma
producing the monoclonal antibody. Since the monoclonal antibodies
of the present invention are useful as antibodies recognizing and
identifying antigens causing apoptosis on myeloid cells
specifically and besides have the property of causing apoptosis on
myeloid cells, they may be used as medicine useful in the field of
remedies for myelocytic leukemia utilizing the property.
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
Tokyo
JP
|
Family ID: |
17084452 |
Appl. No.: |
10/376628 |
Filed: |
March 3, 2003 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10376628 |
Mar 3, 2003 |
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09736146 |
Dec 15, 2000 |
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09736146 |
Dec 15, 2000 |
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09154498 |
Sep 17, 1998 |
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6267959 |
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09154498 |
Sep 17, 1998 |
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08605059 |
Apr 15, 1996 |
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5840344 |
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08605059 |
Apr 15, 1996 |
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PCT/JP94/01453 |
Sep 2, 1994 |
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Current U.S.
Class: |
424/155.1 ;
435/344; 530/388.8 |
Current CPC
Class: |
C07K 16/28 20130101;
A61P 35/02 20180101; A61K 38/00 20130101 |
Class at
Publication: |
424/155.1 ;
530/388.8; 435/344 |
International
Class: |
A61K 039/395; C12N
005/06; C07K 016/30 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 3, 1993 |
JP |
5-242110 |
Claims
What is claimed is:
1. A monoclonal antibody having the property of causing apoptosis
on myeloid cells through binding to the myeloid cells.
2. Fragments of a monoclonal antibody having the property of
causing apoptosis on myeloid cells through binding to the myeloid
cells.
3. A hybridoma producing the monoclonal antibody according to claim
1.
Description
TECHNICAL FIELD
[0001] The present invention relates to a novel monoclonal antibody
having the property of causing apoptosis on myeloid cells and being
useful as medicine for myelocytic leukemia, and fragments thereof,
and furthermore, relates to a hybridoma producing the monoclonal
antibody.
[0002] Since the monoclonal antibodies of the present invention are
useful as antibodies recognizing and identifying antigens causing
apoptosis on myeloid cells specifically and besides have the
property of causing apoptosis on myeloid cells, they may be used as
medicine useful in the field of remedies for myelocytic leukemia
utilizing the property.
BACKGROUND ART
[0003] 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.
[0004] 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
hematopqietic 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)].
[0005] However, though some of splenic stromal cells have been
established as a cell line (CF-1 cells), and cytological
characteristics thereof have been examined, no specific antibody
recognizing surface antigens thereof has been prepared so far, and
characteristics thereof have been scarcely known yet.
[0006] 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.
[0007] And as a result of examining the properties of the obtained
monoclonal antibodies, the inventors have found surprisingly that
they have the property of causing apoptosis on myeloid cells, which
has led to the completion of the present invention.
[0008] Disclosure of Invention
[0009] It is the objective and purpose of the present invention to
provide a novel monoclonal antibody having the property of causing
apoptosis on myeloid cells and being useful as medicine for
myelccytic leukemia, and fragments thereof, and in addition a
hybridoma producing the monoclonal antibody.
[0010] The monoclonal antibody of the present invention 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, and hence the
monoclonal antibodies of the present invention are defined to
include all monoclonal antibodies having the property of causing
apoptosis on myeloid cells.
[0011] The monoclonal antibody of the present invention may be
prepared basically as stated below.
[0012] Namely, the monoclonal antibody of the present invention 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 monoclonal antibody of the
present invention 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 antibody according to the
present invention 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 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 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(P3.times.63Ag8.653) [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
may be performed basically according to an ordinary method, for
example, a method by Milstein et al. [Methods Enzymol., 73, 3-46
(1981)].
[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 furthermore,
adjuvants such as dimethyl sulfoxide 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 m ure
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 prepaLed hybridomas producing the monoclonal antibodies
of the present invention may be subcultured in an ordinary medium
and stored in liquid nitrogen for a long time.
[0022] In order to collect the monoclonal antibodies of the present
invention 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 ascite. 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.
[0024] The monoclonal antibody of the present invention 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 antigens; the monoclonal antibody of the present invention
may be used as useful medicine for myelocytic leukemia according to
utilizing the property.
[0025] 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 of the present
invention, or for using it as medicine for myelocytic leukemia
according to utilizing the specific property thereof, and
modification and application thereof are included within the scope
of the present invention so far as they are put into practice
according to an ordinary method obvious to those skilled in the
art.
BRIEF DESCRIPTION OF DRAWINGS
[0026] FIG. 1 shows an analysis (a control in the absence of an
antibody, CF-1 cell) according to immunofluorescence.
[0027] FIG. 2 shows an analysis of the binding properties of the
GSPST-1 antibody to CF-1 cells according to immunofluorescence.
[0028] FIG. 3 shows an analysis of the binding properties of the
BMAP-1 antibody to CF-1 cells according to immunofluorescence.
[0029] FIG. 4 shows an analysis (a control in the absence of an
antibody, bone marrow cell) according to immunofluorescence.
[0030] FIG. 5 shows an analysis of the binding properties of the
GSPST-1 antibody to bone marrow cells according to
immunofluorescence.
[0031] FIG. 6 shows an analysis of the binding properties of the
BMAP-1 antibody to bone marrow cells according to
immunofluorescence.
[0032] FIG. 7 shows an analysis (a control in the absence of an
antibody, NFS-60) according to immunofluorescence.
[0033] FIG. 8 shows the binding properties of the GSPST-1 antibody
to NFS-60 cells according to immunofluorescence.
[0034] FIG. 9 shows an analysis (a control according to rat IgG1,
NFS-60) according to immunofluorescence.
[0035] FIG. 10 shows the binding properties of the BMAP-1 antibody
to NFS-60 cells according to immunofluorescence.
[0036] FIG. 11 shows an assay for the monoclonal antibody (BMAP-1)
to inhibit NFS-60 cell proliferation.
[0037] FIG. 12 shows an assay for the monoclonal antibody (GSPST-1)
to inhibit the bone marrow transplantation.
[0038] FIG. 13 shows an assay for the monoclonal antibody (BMAP-1)
to inhibit the bone marrow transplantation.
[0039] FIG. 14 is an explanatory view [microphotograph (stained
with H. E.) of bone marrow samples (.times.400)] 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.
[0040] 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.
[0041] FIG. 16 shows a cytotoxicity assay using L929 cells by
TNF.alpha.
[0042] FIG. 17 shows a cytotoxicity assay by the monoclonal
antibody (BMAP-1).
[0043] FIG. 18 shows an analysis (a control according to rat IgG2a,
BWV1) according to immunofluorescence.
[0044] FIG. 19 shows the binding properties of the antimouse MHC
class I antibody to BWV1 cells according to immunofluorescence.
[0045] FIG. 20 shows an analysis (a control according to rat IgG1,
BWV1) according to immunofluorescence.
[0046] FIG. 21 shows the binding properties of the BMAP-1 antibody
to BWV1 cells according to immunofluorescence.
EXPLANATION OF SYMBOLS
[0047] a: DNA of the thymus of a mouse administered BMAP-1 (24
hours)
[0048] b: DNA of the bone marrow of a mouse administered BMAP-1 (24
hours)
[0049] c: DNA of the bone marrow of a mouse administered BMAP-1 (8
hours)
[0050] d: DNA of the bone marrow of a mouse administered BMAP-1 (4
hours)
[0051] e: DNA of the bone marrow of a non-treated mouse (bone
marrow cells)
[0052] f: Molecular weight marker
[0053] 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.
[0054] Reference Example
[0055] Establishment of Splenic Stromal Cells and Their
Characteristics Thereof
[0056] 1) Establishment of Splenic Stromal Cells
[0057] 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. Namely, this spleen was removed
after the administration of rG-CSF 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.
[0058] 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 {fraction (1/16)} to {fraction
(1/32)}. The stromal cells became homogeneous and fibroblastoid
after approximately the 10th passage.
[0059] 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).
[0060] 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 {fraction (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)].
[0061] 2) Characteristics of CF-1 Cells
[0062] 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.
[0063] 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.
[0064] 3) Maintenance of Hematopoietic Stem Cells by CF-1 Cells
[0065] 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).
[0066] 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.
[0067] Best Mode for Carrying Out the Invention
[0068] The embodiment of the invention will be described in detail
hereinafter.
EXAMPLE
[0069] Establishment of Monoclonal Antibodies
[0070] 1) Antigens and Immunization
[0071] 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.
[0072] 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.
[0073] 2) Cell Fusion
[0074] After the spleen remove 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)].
[0075] 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.
[0076] 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.
[0077] 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.
[0078] 3) Screening
[0079] The screening of fused cells (hybridomas) was performed
according to indirect fluorescent antibody technique using flow
cytometry.
[0080] The screening of clones producing objective antibodies was
performed using CF-1 cells as target cells. Namely, cells suspended
in a reaction buffer (PBS suppplemented 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).
[0081] 4) Purification of Antibodies
[0082] 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.
[0083] 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.
[0084] 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 ascite 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, and the present invention is not
restricted to the above monoclonal antibodies but includes all
monoclonal antibodies having the same characteristics and all
hybridomas producing the monoclonal antibodies.
[0085] A hybridoma producing the monoclonal antibody BMAP-1 of the
present invention 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.
[0086] 5) Properties of Antibodies
[0087] (i) Reactivity of Antibodies
[0088] (Reactivity to CF-1 Cells)
[0089] 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.
[0090] 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.
[0091] (Reactivity to Bone Marrow Cells)
[0092] 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.
[0093] 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.
[0094] (Reactivity to Myelocytic Leukemic Cell Line (NFS-60 ))
[0095] 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.
[0096] 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.
[0097] (Assay for BMAP-1 to Inhibit Proliferation of NFS-60
Cells)
[0098] The results of examining the action of BMAP-1 to NFS-60
cells in the presence of G-CSF 100 ng/ml and cyclohexyimide
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, 10, 100 ng/ml, and 1,
10, 100 .mu.g/ml to .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.
[0099] (ii) Typing of Antibodies
[0100] 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.
[0101] (iii) Potency Inhibiting Bone Marrow Transplantation
[0102] 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.
[0103] 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
ascite 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.
[0104] 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.
[0105] 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 few mRNAs of it are 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.
[0106] 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.
[0107] 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.
[0108] As described above, it has been confirmed experimentally
that BMAP-1 has the function of causing apoptosis on myeloid cells;
according to the present inventors 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 novel ones found by the
present inventors. And, since it is thought that the monoclonal
antibodies of the present invention represented by BMAP-1 can cause
death of myelocytic leukemic cells considered to be high in the
expression of antigens thereof by utilizing the function of the
apoptosis of the monoclonal antibody on bone marrow cells, the
monoclonal antibody of the present invention having the property of
causing apoptosis on myeloid cells is useful as medicine for
myelocytic leukemia.
[0109] The monoclonal antibodies of the present invention have been
described specifically according to the Example as above; as the
monoclonal antibodies having the property of causing apoptosis on
myeloid cells according to the present invention may be exemplified
those mentioned as specific examples above, but they are not always
restricted to them but include all monoclonal antibodies having the
same characteristic and function prepared in the same manner,
irrespective of the kind of antigens.
[0110] Industrial Applicability
[0111] Since the monoclonal antibodies of the present invention are
useful as antibodies recognizing and identifying antigens causing
apoptosis on myeloid cells specifically and besides have the
property of causing apoptosis on myeloid cells, they may be used as
medicine useful in the field of remedies for myelocytic leukemia
utilizing the property.
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