U.S. patent application number 11/513178 was filed with the patent office on 2008-08-07 for antibodies to ocif-binding molecules.
Invention is credited to Masaaki Goto, Kanji Higashio, Masahiko Kinosaki, Fumie Kobayashi, Tomonori Morinaga, Nobuaki Nakagawa, Nobuyuki Shima, Ken Takahashi, Akihiro Tomoyasu, Eisuke Tsuda, Naohiro Washida, Kyoji Yamaguchi, Kazuki Yano, Hisataka Yasuda.
Application Number | 20080187540 11/513178 |
Document ID | / |
Family ID | 27525882 |
Filed Date | 2008-08-07 |
United States Patent
Application |
20080187540 |
Kind Code |
A9 |
Yamaguchi; Kyoji ; et
al. |
August 7, 2008 |
Antibodies to OCIF-binding molecules
Abstract
A novel protein which binds to Osteoclastogenesis Inhibitory
Factor (OCIF-binding molecule; OBM), a process for preparing the
same, DNA encoding said protein, a protein having an amino acid
sequence encoded by this DNA, a method for producing said protein
by genetic engineering technique, and a pharmaceutical composition
containing said protein. Screening methods for a substance for
controlling expression of said protein using said protein and the
DNA, a substance which inhibits or modulates the biological
activity of said protein, or a receptor which transmits the action
of said protein through binding to said protein, the substance
obtained by the screening methods, and a pharmaceutical composition
which contains this substance. An antibody for said protein, a
process for preparing the same, a measuring method of said protein
using the antibody, and a medicine comprising this antibody.
Inventors: |
Yamaguchi; Kyoji;
(Oomiya-shi, JP) ; Yasuda; Hisataka; (Kawachi-gun,
JP) ; Nakagawa; Nobuaki; (Shimotsuga-gun, JP)
; Shima; Nobuyuki; (Kawachi-gun, JP) ; Kinosaki;
Masahiko; (Kawachi-gun, JP) ; Tsuda; Eisuke;
(Kawachi-gun, JP) ; Goto; Masaaki;
(Shimotsuga-gun, JP) ; Yano; Kazuki;
(Shimotsuga-gun, JP) ; Tomoyasu; Akihiro;
(Shimotsuga-gun, JP) ; Kobayashi; Fumie;
(Kawachi-gun, JP) ; Washida; Naohiro;
(Shimotsuga-gun, JP) ; Takahashi; Ken;
(Shimotsuga-gun, JP) ; Morinaga; Tomonori;
(Shimotsuga-gun, JP) ; Higashio; Kanji;
(Kawagoe-shi, JP) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
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Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20070009520 A1 |
January 11, 2007 |
|
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Family ID: |
27525882 |
Appl. No.: |
11/513178 |
Filed: |
August 31, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10854300 |
May 27, 2004 |
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11513178 |
Aug 31, 2006 |
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10167182 |
Jun 11, 2002 |
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10854300 |
May 27, 2004 |
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09202455 |
Dec 15, 1998 |
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PCT/JP98/01728 |
Apr 15, 1998 |
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10167182 |
Jun 11, 2002 |
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Current U.S.
Class: |
424/144.1 ;
435/320.1; 435/325; 435/334; 435/6.14; 435/69.1; 435/7.2; 530/350;
530/388.22; 536/23.5 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 19/10 20180101; C07K 14/70578 20130101; C07K 2317/92 20130101;
C07K 16/2875 20130101; C07K 14/705 20130101; C07K 2319/00 20130101;
G01N 33/566 20130101; G01N 2333/525 20130101; C07K 16/28 20130101;
A61K 38/00 20130101; C07K 2317/76 20130101; A61K 2039/505 20130101;
G01N 2333/70578 20130101; G01N 2800/108 20130101; A61P 19/00
20180101; C07K 16/2878 20130101; C07K 14/525 20130101; A61P 29/00
20180101; A61P 19/02 20180101; A61P 3/14 20180101; A61P 19/04
20180101; A61P 19/08 20180101 |
Class at
Publication: |
424/144.1 ;
530/350; 435/006; 435/007.2; 435/069.1; 435/320.1; 435/325;
536/023.5; 530/388.22; 435/334 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/567 20060101 G01N033/567; C07H 21/04 20060101
C07H021/04; C12P 21/06 20060101 C12P021/06; A61K 39/395 20060101
A61K039/395; C07K 14/705 20060101 C07K014/705; C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 15, 1997 |
JP |
97808/1997 |
Jun 9, 1997 |
JP |
151434/1997 |
Aug 12, 1997 |
JP |
217897/1997 |
Aug 21, 1997 |
JP |
224803/1997 |
Dec 2, 1997 |
JP |
332241/1997 |
Claims
1-64. (canceled)
65. A method of reducing bone resorption in a patient in need
thereof, comprising administering to said patient a therapeutic
amount of a human monoclonal antibody that binds osteoclastogenesis
inhibitory factor-binding molecule (OBM), wherein said therapeutic
amount is effective to reduce bone resorption and said OBM consists
of the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:17.
66. The method of claim 65, wherein said OBM does not consist of
SEQ ID NO:16.
67. The method of claim 66, wherein said OBM does not consist of
SEQ ID NO:1.
68. The method of claim 65, wherein said monoclonal antibody
exhibits a dissociation constant in an order of 1.times.10.sup.-9
(M) for said OBM.
69. The method of claim 65, wherein said monoclonal antibody
exhibits a high affinity for said OBM.
70. The method of claim 65, wherein said monoclonal antibody
exhibits an affinity for said OBM equal to the affinity osteoclast
inhibitory factor (OCIF) exhibits for said OBM.
71. The method of claim 65, wherein said monoclonal antibody
exhibits a dissociation constant for said OBM of 1.times.10.sup.-9
(M) or less.
72. The method of claim 65, wherein said monoclonal antibody
exhibits a dissociation constant for said OBM between an order of
1.times.10.sup.-11 (M) and an order of 1.times.10.sup.-10 (M).
73. The method of claim 65, wherein said monoclonal antibody
exhibits a dissociation constant for said OBM between an order of
1.times.10.sup.-11 (M) and an order of 1.times.10.sup.-8 (M).
74. The method of claim 65, wherein said monoclonal antibody
exhibits a dissociation constant for said OBM between an order of
1.times.10.sup.-9 (M) and an order of 1.times.10.sup.-8 (M).
75. A method of inhibiting osteoclast formation in a patient in
need thereof, comprising administering to said patient a
therapeutic amount of an antibody that binds osteoclastogenesis
inhibitory factor-binding molecule (OBM), wherein said therapeutic
amount is effective to inhibit osteoclast formation, and wherein
said OBM consists of the amino acid sequence of SEQ ID NO:11 or SEQ
ID NO:17 and said antibody exhibits a dissociation constant for
said OBM between 1.times.10.sup.-11 (M) and 1.times.10.sup.-8
(M).
76. The method of claim 75, wherein said dissociation constant for
said OBM is between 1.times.10.sup.-9 (M) and 1.times.10.sup.-8
(M).
77. The method of claim 75, wherein said antibody has a high
affinity for said OBM.
78. A method for treating a disease accompanying abnormal bone
metabolism comprising administering a therapeutic amount of an
antibody that binds osteoclastogenesis inhibitory factor-binding
molecule (OBM), wherein said therapeutic amount is effective to
reduce bone resorption or inhibit osteoclast formation, and wherein
said OBM consists of the amino acid sequence of SEQ ID NO:11 or SEQ
ID NO:17 and said antibody does not bind a protein consisting of
the amino acid sequence of SEQ ID NO:16.
79. The method of claim 78, wherein said disease accompanying
abnormal bone metabolism is a disease selected from the group
consisting of osteoporosis, hypercalcemia, Paget's disease, renal
osteodystrophy, rheumatoid arthritis, and osteoarthritis.
80. The method of claim 78, wherein said disease accompanying
abnormal bone metabolism is osteoporosis.
81. The method of claim 78, wherein said disease accompanying
abnormal bone metabolism is hypercalcemia.
82. The method of claim 78, wherein said disease accompanying
abnormal bone metabolism is Paget's disease.
83. The method of claim 78, wherein said disease accompanying
abnormal bone metabolism is renal osteodystrophy.
84. The method of claim 78, wherein said disease accompanying
abnormal bone metabolism is rheumatoid arthritis.
85. The method of claim 78, wherein said disease accompanying
abnormal bone metabolism is osteoarthritis.
86. A method for treating a disease accompanying abnormal bone
metabolism comprising administering a therapeutic amount of a human
monoclonal antibody that binds osteoclastogenesis inhibitory
factor-binding molecule (OBM), wherein said therapeutic amount is
effective to reduce bone resorption or inhibit osteoclast
formation, and wherein said OBM consists of the amino acid sequence
of SEQ ID NO:11 or SEQ ID NO:17.
87. The method of claim 86, wherein said disease accompanying
abnormal bone metabolism is osteoporosis.
88. The method of claim 86, wherein said disease accompanying
abnormal bone metabolism is hypercalcemia.
89. The method of claim 86, wherein said disease accompanying
abnormal bone metabolism is Paget's disease.
90. The method of claim 86, wherein said disease accompanying
abnormal bone metabolism is renal osteodystrophy.
91. The method of claim 86, wherein said disease accompanying
abnormal bone metabolism is rheumatoid arthritis.
92. The method of claim 86, wherein said disease accompanying
abnormal bone metabolism is osteoarthritis.
93. A human monoclonal antibody which specifically binds to
osteoclastogenesis inhibitory factor binding molecule (OBM),
wherein said OBM consists of the amino acid sequence of SEQ ID
NO:11 or SEQ ID NO:17 and said antibody inhibits bone resorption or
inhibits the formation of osteoclasts.
94. The human monoclonal antibody of claim 93, wherein said anibody
exhibits a dissociation constant for said OBM of 1.times.10.sup.-9
(M) or less.
95. The human monoclonal antibody of claim 93, wherein said
antibody does not bind a protein consisting of the amino acid
sequence of SEQ ID NO:16.
96. The human monoclonal antibody of claim 93, wherein said
monoclonal antibody exhibits a dissociation constant for said OBM
between an order of 1.times.10.sup.-11 (M) and an order of
1.times.10.sup.-10 (M).
97. The human monoclonal antibody of claim 93, wherein said human
monoclonal antibody exhibits an affinity for said OBM equal to the
affinity osteoclast inhibitory factor (OCIF) exhibits for said
OBM.
98. A monoclonal antibody which specifically binds to
osteoclastogenesis inhibitory factor binding molecule (OBM),
wherein said OBM consists of the amino acid sequence of SEQ ID
NO:11 or SEQ ID NO:17 and said antibody inhibits bone resorption or
inhibits the formation of osteoclasts and exhibits a dissociation
constant for said OBM between an order of 1.times.10.sup.-9 (M) and
an order of 1.times.10.sup.-8 (M).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/854,300 filed May 27, 2004, which is a continuation of U.S.
application Ser. No. 10/167,182, filed Jun. 11, 2002, which is a
divisional of U.S. application Ser. No. 09/202,455, filed Dec. 15,
1998 (now abandoned), which is a national phase application under
35 U.S.C. .sctn. 371 of International Application No.
PCT/JP98/01728, filed Apr. 15, 1998, which claims priority to
Japanese Patent Application No. 097808/1997, filed Apr. 15, 1997,
Japanese Patent Application No. 151434/1997, filed Jun. 9, 1997,
Japanese Patent Application No. 217897/1997, filed Aug. 12, 1997,
Japanese Patent Application No. 224803/1997, filed Aug. 21, 1997
and Japanese Patent Application No. 332241/1997, filed Dec. 2,
1997.
FIELD OF TECHNOLOGY
[0002] The present invention relates to a novel protein
(OCIF-binding molecule, the protein may be hereinafter called OBM)
which binds to osteoclastogenesis inhibitory factor (hereinafter it
may be called OCIF) and a method to produce this protein.
[0003] The present invention also relates to DNA encoding this
protein, proteins containing the amino acid sequence encoded by
this DNA, a method for the preparation of this protein utilizing
genetic engineering techniques, and pharmaceutical compositions
comprising this protein.
[0004] The present invention further relates to methods for
screening, using this protein and the DNA, substances to control
the expression of this protein, substances inhibiting or regulating
the biological activity of this protein, or receptors transducing
the signal of the protein by interacting with this protein, to
substances obtained by the screening, and to pharmaceutical
compositions which comprise the resulting substances.
[0005] The present invention further relates to antibodies against
this protein, methods for preparing the antibodies, and
pharmaceutical compositions comprising these antibodies.
BACKGROUND OF THE INVENTION
[0006] Bone metabolism is dependent on the overall activity of
osteoblasts which control bone formation and osteoclasts which
control bone resorption. Abnormality of bone metabolism is
considered to be caused by an imbalance of the bone formation and
the bone resorption. Osteoporosis, hypercalcemia, Paget's disease,
renal osteodystrophy, chronic rheumarthritis, osteoarthristis, and
the like are known as diseases accompanying abnormality of bone
metabolism. Osteoporosis is a typical disease caused by such
abnormality of bone metabolism. This disease is generated when bone
resorption by osteoclasts exceeds bone formation by osteoblasts.
The disease is characterized by a decrease in both the bone
calcified material and the bone matrix. Although the mechanism of
this disease is not completely elucidated, the disease causes aches
in bones, makes them fragile, and may result in fracturing. This
disease is becoming a social problem because it increases the
number of bedridden aged persons as the aged population becomes
larger. Development of therapeutic agent for this disease is
urgently desired. Disease due to a decrease in bone mass is
expected to be cured by suppressing bone resorption, accelerating
bone formation, or improving the balance between bone resorption
and formation. Bone formation is expected to increase by
accelerating proliferation, differentiation, or activation of
osteoblasts which form bone, or by suppressing proliferation,
differentiation, or activation of osteoclasts which resorb bone. In
recent years, strong interest has been directed to hormones, low
molecular weight substances, or physiologically active proteins
exhibiting such activities, and energetic basic research and
development is underway on these subjects.
[0007] Drugs such as a calcitonin agents, active-form vitamin
D.sub.3 agents, hormone agents containing estradiol, ipriflavon,
vitamin K.sub.2, and bisphosphonate compounds have already been
known as drugs to treat and shorten the treatment period of
diseases related to bone. Clinical tests are in progress on
active-form vitamin D.sub.3 derivatives, estradiol derivatives, and
bisphosphonate compounds of the second and the third generation to
develop therapeutic agents with excellent efficacy and minimal side
effects.
[0008] However, therapies using these agents were found not
necessarily satisfactory in terms of efficacy and therapeutic
results. Development of novel therapeutic agents which are safer
and with higher efficacy is urgently desired. Some agents used for
the treatment of diseases related to bone metabolism are used only
limitedly due to their side effects. Furthermore, treatments using
two or more agents in combination are currently the mainstream in
the treatment of diseases related to bone metabolism such as
osteoporosis. From such a point of view, development of drugs
having action mechanisms different from those of conventional
drugs, and exhibiting a higher efficacy and minimal side effects is
desired.
[0009] As mentioned above, the cells controlling bone metabolism
are osteoblasts and osteoclasts. These cells are known to have
close mutual interactions called "coupling". Specifically,
cytokines such as Interleukins 1 (IL-1), 3 (IL-3), 6 (IL-6), and 11
(IL-11), granulocytic macrophage-colony stimulating factor
(GM-CSF), macrophage-colony stimulating factor (M-CSF),
Interferon-.gamma. (IFN-.gamma.), tumor necrosis factor .alpha.
(TNF-.alpha.), and transforming growth factor-.beta. (TGF-.beta.),
secreted by osteoblastic stromal cells are known to accelerate or
suppress differentiation or maturation of osteoclasts (Raisz:
Disorders of Bone and Mineral Metabolism, 287-311, 1992; Suda et
al.: Principles of Bone Biology, 87-102, 1996; Suda et al.:
Endocrine Reviews, 4, 266-270, 1995, Lacey et al.: Endocrinology,
186, 2369-2376, 1995). It has been reported that osteoblastic
stromal cells play an important role in the differentiation and
maturation of osteoclasts, as well as in osteoclast functions such
as bone resorption by mature osteoclasts, through cell-to-cell
contact with immature osteoclast precursors or mature
osteoclasts.
[0010] A factor called osteoclast differentiation factor (ODF, Suda
et al.: Endocrine Rev. 13:66-80, 1992; Suda et al.: Bone 17,
87S-91S, 1995) is thought to be expressed on the membrane of
osteoblastic stromal cells and involved in the formation of
osteoclasts through cell-to-cell contact. According to this
hypothesis, an ODF receptor is present in the precursor cells of
osteoclasts. However, so far neither the ODF nor the receptor has
been purified or identified. There are also no reports relating to
their characteristics, action mechanism, or structure. Thus, the
mechanism involved in differentiation and maturation of osteoclasts
has not yet been sufficiently elucidated. Clarification of this
mechanism will greatly contribute not only to the basic medicine,
but also to the development of novel drugs for the treatment of
diseases associated with abnormality of bone metabolism.
[0011] The present inventors have conducted extensive studies in
view of this situation and discovered an osteoclastogenesis
inhibitory factor (OCIF) in a culture broth of human embryonic lung
fibroblast, IMR-90 (ATCC Deposition No. CCL186) (WO 96/26217).
[0012] The present inventors have been successful in cloning DNA
encoding OCIF, production of recombinant OCIF in animal cells, and
confirmation of in vivo pharmaceutical effects (improving effect on
bone metabolism, etc.) of the recombinant OCIF. OCIF is expected to
be used as an agent for the prevention or treatment of diseases
related to abnormality of bone metabolism, with higher efficacy
than conventional drugs and less side effects.
DISCLOSURE OF THE INVENTION
[0013] The present inventors have searched for a protein which
binds to osteoclastogenesis inhibitory factor (OCIF) and discovered
that an OCIF-binding protein is specifically expressed on the
osteoblastic stromal cells cultured in the presence of a bone
resorption factor such as active-form vitamin D.sub.3 and
parathyroid hormone (PTH). In addition, the present inventors have
investigated the characteristics and physiological functions of
this OCIF-binding protein and found that the protein exhibits
biological activity of a factor which supports or promotes the
osteoclast differentiation and maturation from immature precursors
of osteoclasts. These findings have led to the completion of the
present invention. Further investigation into the protein of the
present invention has proven that this is an important protein
controlling the differentiation and maturation of osteoclasts from
immature precursors of osteoclasts in a co-culture system of the
osteoblastic stromal cells and spleen cells. The success in
identification and isolation of the protein which functions as a
factor supporting or promoting differentiation and maturation of
osteoclasts in the present invention has enabled screening for a
novel medicine useful for abnormality of bone metabolism based on
mechanism of bone metabolism utilizing the protein of the present
invention.
[0014] Accordingly, an object of the present invention is to
provide a novel protein (OCIF-binding molecule or OBM) which binds
to osteoclastogenesis inhibitory factor (OCIF), and a method to
produce this protein.
[0015] Another object of the present invention is to provide DNA
encoding this protein, proteins containing an amino acid sequence
encoded by this DNA, a method for producing this protein utilizing
genetic engineering techniques, and pharmaceutical compositions
comprising this protein.
[0016] A further object of the present invention is to provide
methods for screening substances which control expression of this
protein using this protein and the DNA, substances inhibiting or
regulating the biological activity of this protein, receptors
transducing the action of the protein by binding to the protein,
substances obtained by the screening, and pharmaceutical
compositions which comprises these substances.
[0017] A still further object of the present invention is to
provide antibodies against this protein, methods for preparing the
antibodies, and pharmaceutical compositions comprising these
antibodies.
[0018] The protein of the present invention has the following
physicochemical properties and biological activity. [0019] (a)
Affinity: specifically binds to the osteoclastogenesis inhibitory
factor (OCIF) and exhibits high affinity to OCIF (dissociation
constant on cell membrane: Kd=10.sup.-9 M or less); [0020] (b)
Molecular weight: has a molecular weight of approximately
30,000-40,000 when determined by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) under non-reducing conditions and an
apparent molecular weight of approximately 90,000-110,000 when
cross-linked to a monomer form OCIF; and [0021] (c) Biological
activity: exhibits activity supporting or promoting osteoclast
differentiation and maturation in a co-culture system of the mouse
osteoblastic stromal cells and mouse spleen cells in the presence
of bone resorption factors such as active-form vitamin D.sub.3 and
parathyroid hormone (PTH).
[0022] A co-culture system of ST2, a mouse osteoblastic stromal
cell line, and mouse spleen cells in the presence of active-form
vitamin D.sub.3 or PTH is well known as a typical in vitro culture
system for osteoclast formation. The cells expressing the protein
of the present invention can be determined by testing the binding
of OCIF to mouse osteoblastic stromal cells or mouse spleen cells
cultured in the presence or absence of active-form vitamin D.sub.3.
The protein of the present invention is specified as the protein
which is induced specifically on the osteoblastic stromal cells
cultured in the presence of an osteotropic factor such as
active-form vitamin D.sub.3 or PTH. In addition, the protein of the
invention can be specified as a protein exhibiting biological
activity supporting or promoting differentiation and maturation of
osteoclasts from the following results. That is, the osteoclast
formation is inhibited dose dependently by the addition of 1 to 40
ng/ml of OCIF to the above-mentioned co-culture system in the
presence of the active-form vitamin D.sub.3, the time course of
expression of the protein of the present invention on ST2 cells in
the presence of active-form vitamin D.sub.3 well correlates with
the time course of osteoclast formation in the co-culture. In
addition, the amount of protein of the present invention expressed
on ST2 cells correlates with the capability of the cells to support
the osteoclast formation, and the binding of OCIF to the protein of
the present invention on the ST2 cells completely suppresses
osteoclasts formation.
[0023] The affinity of the protein of the present invention to OCIF
can be evaluated by labeling OCIF and examining the binding of the
labeled OCIF to the surface of animal cell membrane. OCIF can be
labeled by a conventional protein-labeling method such as
radioisotope or fluorescent labeling. Labeling of tyrosine residues
with .sup.125I can be given as a specific example of labeling of
the OCIF with an radioisotope. Labeling methods such as iodogen
method, chloramine T method, and enzymatic method can be utilized.
The binding of the labeled OCIF to the surface membrane of animal
cell can be tested by a conventional method. The addition of
unlabeled OCIF to the medium used for the binding assay to a
concentration, 100 to 400 times the concentration of labeled OCIF,
ensures measurement of non-specific binding. The amount of specific
binding of OCIF can be calculated by subtracting the amount of
non-specific binding from the total amount of binding of the
labeled OCIF. The affinity of the protein of the present invention
expressed on the cell membrane to OCIF can be evaluated by changing
the amount of labeled OCIF and analyzing the specific binding by
Scatchard plot.
[0024] The determined affinity of the protein of the present
invention to OCIF is approximately 100-500 pM. The protein of the
present invention is specified by a high affinity (dissociation
constant on cell membrane: Kd=10.sup.-9 M or less) to
osteoclastogenesis inhibitory factor (OCIF). The molecular weight
of OBM can be accessed by gel filtration chromatography, SDS-PAGE,
or the like. SDS-PAGE is preferred in order to accurately determine
the molecular weight. The OBM is specified as a protein having a
molecular weight of approximately 40,000 (40,000.+-.4,000) under
reducing conditions.
[0025] The protein of the present invention can be obtained from
mouse osteoblastic stromal cell line, ST2, mouse preadipocyte cell
line, PA6, human osteoblastic cell lines, or other osteoblastic
cells selected from mammalians such as humans, mice, or rats. As
the substances to induce expression of the protein of the present
invention, osteotropic factors such as active-form vitamin D.sub.3
(calcitriol), parathyroid hormone (PTH), interleukin (IL)-1, IL-6,
IL-11, Oncostatin M, and leukemia inhibitory factor (LIF) can be
given. These substances can be added in the concentration of
10.sup.-8 M (active-form vitamin D.sub.3 and PTH), 10 ng/ml
(IL-11), or 1 ng/ml (Oncostatin M). IL-6 is preferably used at a
concentration of 20 ng/ml with 500 ng/ml soluble IL-6 receptor.
Preferably, confluent cells of mouse osteoblastic stromal cell
line, ST2, cultured in .alpha.-MEM medium to which 10.sup.-8 M of
active-form vitamin D.sub.3, 10.sup.-7 M of dexamethasone, and 10%
fetal bovine serum were added can be used. The cultured cells may
be collected by scraping with a cell scraper. The collected cells
may be stored at -80.degree. C. until use.
[0026] The protein of the present invention can be purified
efficiently from the membrane fractions of the collected cells. The
membrane fractions can be prepared by a conventional method which
is used to prepare intracellular organella. Various types of
protease inhibitors may be added to the buffer solution used for
the preparation of the membrane fractions. Examples of the protease
inhibitors include serine protease inhibitors, thiol protease
inhibitors, and metaprotease inhibitors. PMSF, APMSF, EDTA,
o-phenanthroline, leupeptine, pepstatin A, aprotinin, soybean
trypsin inhibitor are givens as specific examples. A Daunce
homogenizer, a polytron homogenizer, or a ultrasonic processor can
be used to homogenize the cells. The cell homogenate is suspended
in a buffer solution containing 0.5 M of sucrose and centrifuged
for 10 minutes at 600.times.g, to separate the nucleus and
undisrupted cells as precipitate. The supernatant is centrifuged
for 90 minutes at 150,000.times.g to obtain a membrane fractions as
precipitate. The obtained membrane fraction is treated by various
types of detergents to efficiently solubilize and extract the
protein of the present invention from the cell membrane. Detergents
which are commonly used to solubilize cell membrane proteins, such
as CHAPS
(3-[(3-cholamidopropyl)-dimethylamonio]-1-propanesulfonate), Triton
X-100, Nikkol, and n-octyl glycoside, can be used. Preferably, 0.5%
CHAPS is added to the membrane fraction and the mixture is stirred
for 2 hours at 4.degree. C. to solubilize the protein of the
present invention. The sample thus prepared is centrifuged at
150,000.times.g for 60 minutes to obtain the solubilized membrane
fraction as a supernatant.
[0027] The protein of the present invention can be purified from
the solubilized membrane fraction with a column, gel, or resin
coupled with OCIF. The immobilized OCIF may be that isolated from a
culture broth of human embryonic lung fibroblasts, IMR-90,
described in WO 96/26217 or rOCIF prepared using genetic
engineering techniques. rOCIF can be prepared by introducing human
cDNA, mouse cDNA, or rat cDNA into an expression vector according
to a conventional method, transducing the constructed vector in
animal cells such as CHO cells, BHK cells, or Namalwa cells, or in
insect cells to produce ROCIF, and purifying rOCIF. Obtained OCIF
has a molecular weight of approximately 60 kDa (monomer-form) or
120 kDa (dimer-form). The dimer-form OCIF is preferable for
immobilization. Given as examples of the gels and resins to which
OCIF is immobilized are ECH Sepharose 4B, EAH Sepharose 4B,
Thiopropyl Sepharose 6B, CNBr-activated Sepharose 4B, activated CH
Sepharose 4B, Epoxy activated Sepharose 6B, activated thiol
Sepharose 4B (these are manufactured by Pharmacia Co.), TSKgel
AF-Epoxy Toyopal 650, TSKgel AF-Amino Toyopal 650, TSKgel AF-Formyl
Toyopal 650, TSKgel AF-Carboxy Toyopal 650, TSKgel AF-Tresyl
Toyopal 650 (these are manufactured by Tosoh Corporation),
Amino-Cellulofine, Carboxy-Cellulofine, FMP activated Cellulofine,
Formyl-Cellulofine (these are manufactured by Seikagaku Kogyo Co.),
Affigel 10, Affigel 15, and Affiprep 10 (these are manufactured by
BioRad Co.). As columns to which OCIF is immobilized, HiTrap
NHS-activated column (Pharmacia Co.), TSKgel Tresyl-5PW (Tosoh
Corporation), etc. can be given. As a specific example of the
method for immobilizing OCIF to a HiTrap NHS-activated column (1
ml, Pharmacia Co.), the following method can be given.
Specifically, 1 ml of 0.2M NaHCO.sub.3/0.5 M NaCl solution (pH 8.3)
containing 13.0 mg of OCIF is injected to the column to perform
coupling reaction at room temperature for 30 minutes. 0.5 M
ethanolamine/0.5 M NaCl (pH 8.3) and 0.1 M acetic acid/0.5 M NaCl
(pH 4.0) are sequentially applied to the column. Then, the column
is again washed with 0.5 M ethanolamine/0.5 M NaCl (pH 8.3) and the
column is allowed to stand for one hour at room temperature to
block excess active groups. The column is sequentially washed twice
with 0.5 M ethanolamine/0.5 M NaCl (pH 8.3) and 0.1 M acetic
acid/0.5 M NaCl (pH 4.0), and then washed with 50 mM Tris/1M
NaCl/0.1% CHAPS solution (pH 7.5), thereby obtaining a
OCIF-immobilized column. The protein of the present invention can
be efficiently purified by a OCIF-immobilized column prepared in
this manner, or an OCIF-immobilized gel or resin.
[0028] It is desirable to add the various above-mentioned protease
inhibitors to the buffer solutions used for the purification of the
protein to suppress degradation of the protein of the present
invention. The protein of the present invention can be purified by
loading the above-mentioned solubilized membrane fraction on the
OCIF-immobilized column or by mixing with the OCIF-immobilized gel
or resin, and eluting the protein from the column, gel, or resin
with acid, various protein denaturing agents, cacodylate buffer,
and the like. It is desirable to use an acid for elution and to
neutralize immediately after elution to minimize denaturation of
the protein of the present invention. As the acidic solution used
for elution, 0.1 M glycine-hydrochloric acid solution (pH 3.0), 0.1
M glycine-hydrochloric acid solution (pH 2.0), 0.1 M sodium citrate
solution (pH 2.0), and the like can be given.
[0029] The protein of the present invention can be fuirther
purified by conventional purification methods used for purification
of various proteins from biological materials and by various
purification methods utilizing the physicochemical properties of
this protein. To concentrate solutions containing the protein of
the present invention, conventional techniques used in the
purification process for proteins such as ultra filtration, freeze
drying, and salting-out, can be used. Ultra filtration with
Centricon-10 (BioRad Co. ), for example, is preferably used. As a
means for the purification, various techniques conventionally
utilized for the purification of proteins, such as ion exchange
chromatography, gel filtration chromatography, hydrophobic
chromatography, reverse phase chromatography, and preparative
electrophoresis, are used in combination. More specifically, it is
possible to purify the protein of the present invention by a
combination of gel filtration chromatography with Superose 12
column (Pharmacia Co.) and reverse phase chromatography. To detect
the protein of the present invention in the purification process,
the binding activity of the protein of the present invention to the
immobilized OCIF is examined or the material bound to the
immobilized OCIF is immuno precipitated with an anti-OCIF antibody
and analyzed by SDS-polyacrylamide gel electrophoresis
(SDS-PAGE).
[0030] The obtained protein of the present invention is useful as
an agent for treating diseases caused by abnormality of bone
metabolism such as osteopetrosis or as a reagent for research and
diagnosis of these diseases.
[0031] The present invention further provides DNA encoding a novel
protein (OCIF-binding molecule or OBM) which binds to
osteoclastogenesis inhibitory factor, proteins containing the amino
acid sequence encoded by this DNA, a method for the preparation of
this protein by the genetic engineering technique, and
pharmaceutical compositions comprising this protein. Furthermore,
the present invention provides methods for screening substances to
regulate expression of OBM, a method for screening substances
inhibiting or modifying the biological activity of OBM, or a method
for screening receptors transducing the action of OBM by binding to
OBM, and pharmaceutical compositions which comprises substances
obtained as a result of the screening.
[0032] The novel protein OBM which is encoded by the DNA of the
present invention has the following physicochemical properties and
biological activity. [0033] (a) binds specifically to
osteoclastogenesis inhibitory factor (OCIF), [0034] (b) has a
molecular weight of approximately 40,000 (.+-.4,000) when
determined by SDS-PAGE under reducing conditions and an apparent
molecular weight of approximately 90,000-110,000 when crosslinked
to monomer-form OCIF, and [0035] (c) exhibits activity supporting
or promoting differentiation and maturation of osteoclasts.
[0036] Human osteoclastogenesis inhibitory factor (OCIF) which is
used as a probe to identify the DNA encoding OBM, the OCIF-binding
molecule of the present invention, and to evaluate properties of
OBM can be isolated from a culture broth of a human embryonic lung
fibroblast cell line, IMR-90, according to WO No. 96/26217.
Recombinant human OCIF, recombinant mouse OCIF, recombinant rat
OCIF, and the like can also be used for the isolation and
identification of the DNA coding OBM. These recombinant OCIF
proteins can be produced by inserting DNA fragments encoding these
proteins into expression vectors according to conventional methods,
expressing in animal cells such as CHO cells, BHK cells, or Namalwa
cells, or in insect cells, and purifying them.
[0037] As a method for isolating cDNA encoding a target protein
(cDNA cloning), the method comprising determination of a partial
amino acid sequence of the protein and isolation of the target cDNA
by hybridization utilizing the nucleotide sequence corresponding to
the amino acid sequence can be employed.
[0038] Another available method, even in the case where the amino
acid sequence of the protein is not known, comprises constructing a
cDNA library in a expression vector, introducing the cDNA into
cells, and screening for the expression of the target protein to
isolate the objective cDNA (expression cloning method, D'Andrea et
al.: Cell 57, 277-285, 1989; Fukunaga et al.: Cell 61, 341-350,
1990). In the expression cloning method, suitable host cells such
as bacteria, yeast, animal cells, and the like are selected
depending on the objective. In many cases, animal cells are
selected as the host cells for cloning cDNA encoding a protein such
as the protein of the present invention which is considered to be
present in animal cell membrane surface. Normally, host cells
showing high efficiency for DNA transfection and achieving
expression of the introduced DNA at high levels are selected.
[0039] One of such animal cells is the monkey kidney cells, COS-7,
used in the present invention. Because SV40 large T antigen is
expressed in the COS-7 cell, a plasmid which has a replicator of
SV40 can be present as an episome of multiple copies in the cell,
so that a high level of expression is expected. In addition,
because expression of a target protein by COS-7 cells reaches a
maximum within a few days after introduction of DNA, the cell is
suitable for rapid screening. A combination of this host cell with
a plasmid capable of high expression ensures gene expression of an
extremely high level. The factor exhibiting the greatest influence
on the expression of a gene on a plasmid is a promoter. Promoters
such as SRa promoter and cytomegalovirus-derived promoters are used
as high expression promoters. To screen for the cDNA encoding a
membrane protein by the expression cloning strategy, screening
procedures such as binding method, panning method, or film emulsion
method are used.
[0040] The present invention relates to DNA encoding the protein
(OBM) which specifically binds to OCIF, isolated by the combination
of the expression cloning strategy and the screening by the binding
method, to the expressed protein, and to screening of
physiologically active substances using the DNA or the expressed
protein. OBM encoded by the DNA of the present invention can be
detected by labeling OCIF and testing the binding of the labeled
OCIF to membrane surface of an animal cell. OCIF can be labeled by
a conventional labeling method such as radioisotope labeling method
or fluorescent labeling method which is used for labeling common
proteins.
[0041] Labeling tyrosine residues by .sup.125I can be given as a
specific example of labeling OCIF with a radioisotope. Labeling
methods such as the iodogene method, chloramine T method, and
enzymatic method can be utilized. The binding of labeled OCIF to
the animal cell membrane surface can be tested by conventional
methods. The addition of unlabeled OCIF to the medium used for the
test to a concentration, 100 to 400 times the concentration of
labeled OCIF, enables quantification of the amount of non-specific
binding. The amount of specific binding of OCIF can be calculated
by subtracting the amount of non-specific binding from the total
amount of binding of the labeled OCIF.
[0042] The present inventors assumed that there is interaction
between the factor involved in differentiation of osteoclasts and
OCIF. Based on this assumption, to isolate the protein to which
recombinant OCIF binds, the inventors screened the expression
library prepared from mRNA of mouse osteoblastic stromal cell line,
ST2, according to the following method. Specifically, DNA
synthesized using ST2 mRNA was inserted into an expression vector
for animal cells and the vector with the insert was introduced into
monkey kidney COS-7 cells. The objective protein expressed on the
COS-7 cells was screened using OCIF labeled with .sup.125I as a
probe. As a result, DNA encoding the protein which binds
specifically to OCIF was isolated. The nucleotide sequence of the
DNA encoding this OCIF-binding molecule (OCIF-binding molecule;
OBM) was then determined. Moreover, OBM encoded by this DNA was
found to bind specifically and strongly to OCIF, on the cell
membrane.
[0043] Comparatively mild conditions for hybridization of DNA in
the present invention are the conditions, for example, wherein DNA
is transferred to a nylon membrane and immobilized thereto
according to conventional methods and hybridized in a buffer
solution for hybridization with a probe DNA labeled with an isotope
at a temperature of 40-70.degree. C. for about 2 hours to
overnight, followed by washing in 0.5.times.SSC (0.075 M sodium
chloride and 0.0075 M sodium citrate) at 45.degree. C. for 10
minutes. Specifically, Highbond N (Amersham Co.) is used as the
nylon membrane to transfer and immobilize DNA thereon. DNA is then
hybridized with a probe DNA labeled with .sup.32P in a rapid
hybridization buffer (Amersham Co.) at 65.degree. C. for 2 hours,
followed by washing with 0.5.times.SSC (0.075 M sodium chloride and
0.0075 M sodium citrate) at 45.degree. C. for 10 minutes.
[0044] A co-culture system of mouse osteoblastic stromal cells,
ST2, and mouse spleen cells in the presence of active-form vitamin
D.sub.3 or PTH is well known as a typical in vitro culture system
for osteoclast-formation. The protein of the present invention is
specified as the protein which is induced specifically on the
osteoblastic stromal cells cultured in the presence of an agent
which accelerates bone resorption such as active-form vitamin
D.sub.3 or PTH. In addition, because of the fact that formation of
osteoclasts is stimulated by the addition of the protein encoded by
the DNA of the present invention to mouse spleen cells cultured
even in the absence of active-form vitamin D.sub.3 or PTH, OBM
which is encoded by the DNA of the present invention is considered
to be involved in the differentiation and maturation of
osteoclasts.
[0045] Recombinant OBM can be produced by inserting the DNA of the
present invention into an expression vector to construct a plasmid
and introducing the plasmid into various cells or microorganisms to
express recombinant OBM. As a host in which recombinant OBM is
expressed, mammalianian cells such as COS-7, CHO, Namalwa, or
bacteria such as Escherichia coli can be used. OBM may be expressed
as a membrane-bound-form protein using the full length DNA or as a
secretion-form or a soluble-form protein by removing the portion
encoding the transmembrane domain. The produced recombinant OBM can
be efficiently purified using a suitable combination of
conventional purification methods used for common proteins, such as
affinity chromatography using OCIF-immobilized columns, ion
exchange chromatography, and gel filtration chromatography. The
obtained protein of the present invention is useful as an agent for
treating diseases caused by abnormality of bone metabolism such as
osteopetrosis or as a reagent for research and diagnosis of such
diseases.
[0046] The following screening operations can be carried out using
the protein OBM encoded by the DNA of the present invention: (1)
screening of substances which regulate expression of OBM, (2)
screening of substances which specifically bind to OBM and inhibit
the biological activity of OBM, and (3) screening of proteins which
are present in osteoclast precursor cells and transduce the
biological activity of OBM (OBM receptor). It is also possible to
develop antagonists and agonists using this OBM receptor. In the
combinatorial chemistry using the above-mentioned OBM or OBM
receptor, a peptide library used for the screening of the
antagonists or agonists can be prepared by the following method.
Specifically, one of the methods is split method (Lam et al.;
Nature 354, 82-84, 1991). According to this method, synthetic
carriers (beads) each comprising a specific amino acid (unit) bound
thereto are prepared separately for all units. The synthesized
carriers are mixed altogether and divided into portions equal to
the number of the units. Then, the next units are bound. This
procedure is repeated "n" times to produce a library containing
carriers to which "n" units are bound. According to this synthetic
method, each carrier pool has one type of sequence. Therefore, it
is possible to identify a peptide specifically binding to the
protein of the present invention by selecting the pool which gives
a signal positive in this screening method using the protein of the
present invention, and determining the amino acid sequence of the
peptide bound on the pool. Another method is phage display method
which utilizes phage carrying synthetic DNA which encode peptides
with random amino acid sequences. The method has the advantage of
increasing the number of molecules in the library as compared with
the above-mentioned synthetic peptide library method, but has the
disadvantage of less variety for a given number of molecules
because there can be particular sequences which are missing in the
library if the phages are unable to express those sequences. In the
phage display method, the screening system using the protein of the
present invention can also be applied to determine the nucleotide
sequence encoding the peptide. That is, the phage specifically
binding to the protein of the present invention is concentrated by
panning, the selected phage is amplified in Escherichia coli, and
the nucleotide sequence encoding the peptide is determined. In
addition, a peptide exhibiting high specificity and high affinity
to OBM or OBM receptor can be screened from a peptide library using
the screening systems mentioned above in (2) and (3) by screening
in the presence of OBM or OCIF while increasing the concentration
of OBM or OCIF. Only positive carrier pools or phages are selected
in this manner. For example, low molecular weight peptide agonists
exhibiting an EPO (erythropoietin)-like activity were screened from
a peptide library using a receptor of erythropoietin (EPO) which is
a hematopoietic hormone, the tertiary structure of this substance
was analyzed, and based on this tertiary structure, low molecular
weight substances (antagonist) exhibiting the EPO-like activity
were synthesized (Nicholas et al.: Science, 273, 458-463,
1996).
[0047] The present inventors have previously discovered using the
osteoclastogenesis inhibitory factor, OCIF, that an OCIF-binding
protein is specifically expressed on osteoblastic stromal cell
line, ST2, cultured in the presence of a osteotropic factor such as
active-form vitamin D.sub.3 or parathyroid hormone (PTH). The
inventors further found that this protein exhibits a biological
activity to support or stimulate differentiation or maturation of
osteoclasts from immature osteoclast precursor cells, and clarified
various physicochemical properties and the biological activity of
this protein by purification thereof. In order to compare the
recombinant OBM expressed by the DNA of the present invention and
the above-mentioned purified natural type protein which
specifically binds to OCIF, the present inventors investigated the
physicochemical properties and biological activities of the two
proteins. As a result, the two proteins were confirmed .rarw.to be
both membrane-bound proteins which specifically bind to OCIF,
.uparw. to have molecular weights of approximately 40,000
determined by SDS-PAGE, and .fwdarw.to have apparent molecular
weights of about 90,000-110,000 when cross-linked to a monomer form
OCIF. Not only are these physicochemical properties identical, but
both proteins exhibit a biological activity to support or stimulate
differentiation or maturation of osteoclasts, suggesting the
possibility that these are the same protein. In addition, a rabbit
anti-OBM polyclonal antibody produced using the purified protein
prepared by expressing the DNA of the present invention by a
genetic engineering technique (recombinant OBM) was confirmed to
cross react with the above-described purified natural type protein,
to inhibit specific binding of this purified natural type protein
and OCIF in the same manner as the antibody inhibits specific
binding of OBM and OCIF. Based on these results, it is clear that
the recombinant OBM expressed by the DNA of the present invention
is identical to the natural type protein which specifically binds
to OCIF.
[0048] To isolate a gene (cDNA) encoding human OCIF-binding protein
(hereinafter called human OBM) which specifically binds to OCIF and
exhibits the activity to support and stimulate differentiation and
maturation of osteoclasts from mouse spleen cells in the same
manner as the natural type or recombinant mouse OBM dose, a cDNA
library prepared from mRNA derived from human lymph nodes was
screened using a human OBM cDNA fragment as a probe. The human OBM
cDNA fragment was obtained by polymerase chain reaction (PCR) in
accordance with the method mentioned above using both cDNA prepared
from human lymph node as a template and the primer which was
prepared from mouse OBM cDNA. As a result, cDNA encoding the human
protein which specifically binds to OCIF was isolated and the
nucleotide sequence of the cDNA encoding this human OCIF-binding
protein molecule (i.e. the cDNA encoding human OBM) was determined.
Similar to mouse OBM, this human OBM encoded by the cDNA has
characteristics to bind to OCIF strongly and specifically on the
cell membrane and exhibits the activity to support and promote
differentiation and maturation of osteoclasts from mouse spleen
cells. Specifically, the present invention provides DNA encoding
novel human OBM protein which binds to osteoclastogenesis
inhibitory factor (OCIF), a protein which possesses the amino acid
sequence encoded by the DNA, a method for producing the protein
exhibiting characteristics of specifically binding to OCIF and the
activity to support and promote differentiation and maturation of
osteoclasts from mouse spleen cells by genetic engineering
techniques, pharmaceutical compositions comprising this protein for
the treatment of diseases caused by abnormality of bone metabolism,
a method for screening substances regulating expression of human
OBM, a method for screening substances which inhibit or modulate
the activity of human OBM by binding to it, a method for screening
receptors which bind to human OBM and transmit the action of OBM,
and a pharmaceutical compositions comprising the substances
obtained by these screenings.
[0049] The present invention further provides DNA encoding novel
human OBM protein which specifically binds to OCIF and exhibits the
biological activity to support and promote differentiation and
maturation of osteoclasts, a protein which possesses the amino acid
sequence encoded by the DNA, a method for producing the protein
exhibiting characteristics of specifically binding to OCIF and the
activity to support and promote differentiation and maturation of
osteoclasts by genetic engineering techniques, and pharmaceutical
compositions comprising this protein for the treatment of diseases
causing abnormality of bone metabolism. Furthermore, the present
invention provides a method for screening substances regulating
expression of human OBM, a method for screening substances which
inhibit or modulate the activity of human OBM by binding to it, a
method for screening receptors binding to human OBM and
transmitting the action of OBM, antibodies against human OCIF
binding protein, and pharmaceutical compositions comprising these
antibodies for the prevention or treatment of diseases causing
abnormality of bone metabolism.
[0050] The novel, human OCIF-binding protein molecule (OBM) which
is encoded by the DNA of the present invention has the following
physicochemical properties and biological activity. [0051] (a)
binds specifically to osteoclastogenesis inhibitory factor (OCIF)
(WO 96/26217), [0052] (b) has a molecular weight of approximately
40,000 (.+-.5,000) when determined by SDS-PAGE under reducing
conditions and an apparent molecular weight of approximately
90,000-110,000 when crosslinked with a monomer form OCIF, and
[0053] (c) exhibits activity to support and stimulate
differentiation and maturation of osteoclasts.
[0054] Mouse OBM cDNA which encodes mouse OCIF-binding protein and
used as a probe to isolate and identify the cDNA encoding human OBM
of the present invention, can be isolated according to the
above-mentioned method from a cDNA library of mouse osteoblastic
stromal cell line, ST2. Human osteoclastogenesis inhibitory factor
(OCIF) which is necessary to evaluate the properties and the
biological activity of the protein obtained by expression of human
OBM cDNA, can be prepared according to the method described in WO
96/26217 by isolating from a culture broth of human fibroblast cell
line, IMR-90, or by genetic engineering techniques using the DNA
encoding OCIF. Recombinant human OCIF, recombinant mouse OCIF,
recombinant rat OCIF, or the like can be used for the assessment of
the properties and biological activity of human OBM. These
recombinant OCIF can be obtained according to conventional methods
by inserting cDNA into an expression vector, expressing the cDNA in
animal cells such as CHO cells, BHK cells, or Namalwa cells, or in
insect cells, and purifying the expressed proteins.
[0055] The following methods can be used to isolate human cDNA
encoding the target protein (cDNA cloning). .rarw. A method
comprising purifying the protein, determining the partial amino
acid sequence of the protein, and isolating the target cDNA by
hybridization using the DNA fragment comprising nucleotide sequence
corresponding to the amino acid sequence as a probe, .uparw. a
method applied even in the case where the amino acid sequence of
the protein is not known, which comprises constructing a cDNA
library in a expression vector, introducing the cDNA library into
cells, and screening for the expression of the target protein to
isolate the objective cDNA (expression cloning method), and
.fwdarw.a method of isolating cDNA encoding the target human
protein from the cDNA library constructed using human cells or
tissues by hybridization or by the use of polymerase chain reaction
(PCR) using the cDNA encoding the protein of mammalian origin
(other than human) which possesses the same characteristics and
biological activity as the target protein of human origin as a
probe, assuming that the cDNA probe has high homology with the
human-origin cDNA which to be cloned. Based on the assumption that
human OBM cDNA has a high homology with mouse OBM cDNA, it is
possible to determine which cells or tissues produce human OBM by
Northern hybridization method using the mouse OBM cDNA as a probe.
Human OBM cDNA can be obtained by the following method using the
mouse OBM primer prepared from the mouse OBM cDNA. Human OBM cDNA
fragments can be prepared by the PCR method using cDNA prepared
from human OBM-producing tissues such as human lymph nodes as a
template. These human OBM cDNA fragments are used as probes for
screening the cDNA library of human OBM-producing cells or tissues
which were identified according to the method mentioned above. The
present invention relates to the DNA encoding human OBM which has
characteristics of specific binding to OCIF and exhibits activity
to support and promote differentiation and maturation of
osteoclasts. Because the OBM which is encoded by the DNA of the
present invention is a membrane-bound type protein which comprises
a transmembrane domain, this protein can be detected by labeling
OCIF and by examining the binding of the labeled OCIF to the
surface of animal cells in which the cDNA of the present invention
was expressed. The above-described labeling method using
radioisotope or fluoresceine conventionally applied to labeling
proteins can be used for labeling OCIF.
[0056] The molecular weight of the protein expressed by the human
OBM cDNA of the present invention can be accessed by gel filtration
chromatography, SDS-PAGE, or the like. In order to accurately
determine the molecular weight, it is desirable to use the SDS-PAGE
method, by which human OBM was specified as a protein having a
molecular weight of approximately 40,000 (40,000.+-.5,000) under
reducing conditions.
[0057] Comparatively mild conditions for hybridization of DNA in
the present invention are the conditions, for example, wherein DNA
is transferred to a nylon membrane and immobilized thereto
according to a conventional method and hybridized with a probe DNA
labeled with an isotope in a buffer solution for hybridization at a
temperature of 40-70.degree. C. for about 2 hours to overnight,
followed by washing in 0.5.times.SSC (0.075 M sodium chloride and
0.0075 M sodium citrate) at 45.degree. C. for 10 minutes.
Specifically, Highbond N (Amersham Co.) is used as the nylon
membrane to transfer and immobilize DNA thereon. The DNA is then
hybridized with a probe DNA labeled with .sup.32P in a rapid
hybridization buffer (Amersham Co.) at 65.degree. C. for 2 hours,
followed by washing with 0.5.times.SSC at 45.degree. C. for 10
minutes.
[0058] A co-culture system of mouse osteoblastic stromal cells,
ST2, and mouse spleen cells in the presence of active-form vitamin
D.sub.3 or PTH is well known as a typical in vitro culture system
for osteoclast-formation. Interaction by adhesion of osteoblastic
stromal cells and spleen cells and presence of an osteotropic
factor such as active-form vitamin D.sub.3 or PTH are indispensable
for the osteoclasts formation in this in vitro culture system. In
this in vitro culture system, COS cells, monkey kidney cells having
no osteoclast formation-supporting capability, acquire capability
to support osteoclasts formation from spleen cells in the absence
of an osteotropic factor when the cDNA of the present invention was
expressed as osteoblastic stromal cell line ST2 did. Based on the
fact that the cDNA of the present invention encodes a protein
comprising a transmembrane domain form, this cDNA can be expressed
as a secretion form or soluble-form by removing the part which
encodes this transmembrane domain. It was confirmed that
osteoclasts can be formed by the addition of the secretion form
human OBM to the above-mentioned in vitro culture system in the
absence of osteotropic factors. Based on these results, the human
OBM which is encoded by the cDNA of the present invention is
specified as the factor involved in the differentiation and
maturation of osteoclasts.
[0059] A recombinant human OBM can be prepared by inserting the
cDNA of the present invention into an expression vector, preparing
a human OBM expression plasmid, introducing the plasmid into
various cell strains and expressing OBM in the cells. Mammalianian
cells such as COS-7, CHO, Namalwa cells, or bacteria such as
Escherichia coli can be used as a host for expressing OBM. In this
case, OBM may be expressed as a membrane-bound-form protein, using
the full length DNA, or as a secretion-form or soluble-form protein
by removing the part encoding the transmembrane domain. The
recombinant OBM thus produced can be efficiently purified using a
suitable combination of conventional purification methods used for
common proteins such as affinity chromatography using OCIF
immobilized columns, ion exchange chromatography, and gel
filtration chromatography. Human OBM of the present invention thus
obtained is useful as an agent for treating diseases caused by
abnormality of bone metabolism such as osteopetrosis or as a
reagent for research and diagnosis of such diseases.
[0060] The following screening operations can be carried out using
the protein OBM encoded by the DNA of the present invention: (1)
screening of substances which can regulate expression of human OBM,
(2) screening of substances which specifically bind to human OBM
and inhibit or modify the biological activity of OBM, and (3)
screening of human proteins which are present in osteoclast
precursor cells and transmit the biological activity of human OBM
(human OBM receptor). It is also possible to develop antagonists
and agonists using this human OBM receptor. In the combinatorial
chemistry using the human OBM or human OBM receptor, peptide
libraries required for the screening of antagonists or agonists can
be produced by the same method as used for the screening using
mouse OBM. A peptide with extremely high specificity and affinity
can be obtained by screening peptide libraries using human OBM
instead of mouse OBM.
[0061] Although this OBM is very useful as mentioned above and
antibodies specifically recognizing OBM and enzyme immunoassay
using these antibodies are indispensable in determination of OBM
concentration, no antibodies useful for the access of OBM
concentration have been so far available. In addition, an anti-OBM
antibody or anti-sOBM antibody which neutralizes the biological
activity of OBM or sOBM is supposed to suppress the activity of OBM
or sOBM, specifically the activity to induce osteoclasts formation.
These are expected to be useful as therapeutic agents to treat
abnormality of bone metabolism. However, no such antibodies have so
far been available.
[0062] In view of this situation, the present inventors have
conducted extensive studies. As a result, the present inventors
have found antibodies (anti-OBM/sOBM antibodies) which recognize
both OBM, a membrane-bound protein which specifically binds to
osteoclastogenesis inhibitory factor (OCIF), and soluble OBM (SOBM)
which lack a transmembrane domain. Accordingly, the present
invention provides antibodies (anti-OBM/sOBM antibodies) which
recognizes both OBM, a membrane-bound protein which specifically
binds to osteoclastogenesis inhibitory factor (OCIF), and sOBM
which lack a transmembrane domain; a method for the preparation
thereof; a method for determination of OBM and sOBM concentrations
using these antibodies; and agents for the prevention or treatment
of diseases resulting from abnormality of bone metabolism.
[0063] The present invention relates to antibodies (anti-OBM/sOBM
antibodies) which recognize both the OBM, a membrane-bound protein
which specifically binds to osteoclastogenesis inhibitory factor
(OCIF), and soluble OBM (sOBM) which lack a transmembrane domain; a
method for the preparation thereof; a method for quantifying OBM
and sOBM using these antibodies; and agents for the prevention or
treatment of diseases resulting from abnormality of bone
metabolism. The antibodies of the present invention exhibit
activity of neutralizing the osteoclastogenesis accelerating
activity which is the biological activity of OBM and sOBM and
comprises the antibodies having the following characteristics:
[0064] (a) polyclonal antibody which recognizes both mouse OBM and
mouse sOBM (anti-mouse OBM/sOBM polyclonal antibody), [0065] (b)
polyclonal antibody which recognizes both human OBM and human sOBM
(anti-human OBM/sOBM polyclonal antibody), [0066] (c) monoclonal
antibodies which recognizes both mouse OBM and mouse sOBM
(anti-mouse OBM/sOBM monoclonal antibodies), [0067] (d) monoclonal
antibodies which recognize both human OBM and human sOBM
(anti-human OBM/sOBM monoclonal antibodies), and [0068] (e)
anti-human OBM/sOBM monoclonal antibodies which crossreact to both
mouse OBM and mouse sOBM.
[0069] The polyclonal antibody which recognizes both mouse OBM and
mouse sOBM (hereinafter called anti-mouse OBM/sOBM polyclonal
antibody) and the polyclonal antibody which recognizes both human
OBM and human sOBM (hereinafter called anti-human OBM/sOBM
polyclonal antibody) were produced by the following method. The
purified mouse OBM used as an antigen for immunization can be
obtained according to the above-mentioned method. Especially, mouse
osteoblastic stromal cell line, ST2, was treated with active-form
vitamin D.sub.3, and OBM on the cell membrane was purified using an
OCIF-immobilized column and gel filtration chromatography, thereby
obtaining natural mouse OBM (native OBM). The above-mentioned mouse
OBM cDNA (SEQ ID NO:15) or human OBM cDNA (SEQ ID NO:12) was
inserted into an expression vector according to conventional
methods. Recombinant mouse OBM (SEQ ID NO:1) and recombinant human
OBM (SEQ ID NO:11) can be obtained by expressing cDNA in animal
cells such as CHO cells, BHK cells, Namalwa, or COS-7 cells, insect
cells or Escherichia coli, and purifying them using the same
purification methods as mentioned above. These may be used as
antigens for immunization. In this instance, purifying a large
amount and a high level of mouse OBM or human OBM, which are
membrane-bound proteins, is a task requiring a great deal of labor.
On the other hand, as mentioned above, OBM, which is a
membrane-bound protein, and a soluble OBM (sOBM), which is obtained
by deleting transmembrane domain of OBM, are known to be almost the
same in their osteoclast differentiation and maturation activities.
It is possible to use mouse sOBM and human sOBM which are
relatively easily expressed and purified to a high level, as
antigens for immunization.
[0070] Mouse sOBM (SEQ ID NO:16) and human sOBM (SEQ ID NO:17) can
be obtained by adding a nucleotide sequence encoding a known signal
sequence originating from the other secretion protein in the
upstream side of the 5' end of, respectively, mouse sOBM cDNA (SEQ
ID NO:18) and human sOBM cDNA (SEQ ID NO:19), inserting these into
an expression vector by the use of genetic engineering techniques,
causing these proteins to be expressed in host cells such as
various animal cells, insect cells, or Escherichia coli, and
purifying the resultant products. The antigens for immunization
thus obtained are dissolved in phosphate buffered saline (PBS),
mixed with the same volume of Freund's complete adjuvant to
emulsify the solution if required, and subcutaneously administered
to animals about once a week to immunize these animals several
times. A booster injection is given when the antibody titer reaches
a maximum. Exsanguination is performed 10 days after the booster
administration. The resulting antiserum is treated with ammonium
sulfate precipitation. IgG fraction is purified using an anion
exchange chromatography or purified by protein A- or protein
G-Sepharose column chromatography after diluting the antiserum
two-fold with Binding Buffer.TM. (BioRad Co.), to obtain the
anti-mouse or, anti-human OBM/sOBM polyclonal antibody.
[0071] The monoclonal antibodies of the present invention can be
obtained according to the following method. In the same manner as
in the case of the polyclonal antibodies, natural mouse OBM (native
OBM), recombinant mouse or human OBM, or recombinant mouse or human
sOBM can be used as immunogens to prepare monoclonal antibodies.
Hybridomas are produced according to conventional methods by
immunizing mammals with these antigens or by immunizing lymphocytes
in vitro and fusing the immunized cells with myeloma cells. By
analyzing the hybridoma culture supernatant thus obtained by a
solid phase ELISA method, antibody-producing hybridomas recognizing
the highly purified antigen are selected. The resulting hybridomas
are cloned and established as stable antibody-producing hybridoma
clones. These hybridomas are cultured to obtain the antibodies.
Small mammals such as mice or rats are commonly used to produce
hybridomas. Animals are immunized by intravenously or
intraperitoneally injecting the antigen diluted to a suitable
concentration using a suitable solvent such as physiological salt
solution. Optionally, Freund's complete adjuvant maybe used
together with antigen. These are usually injected 3-4 times, once a
week or every two weeks. The immunized animals are dissected three
days after final immunization. Splenocytes from the removed spleen
are used as immunized cells. As mouse myeloma to be fused with
immunized cells, p3/x63-Ag8, p3-U1, NS-1, MPC-11, SP-2/0, FO, P3x63
Ag8.653, and S194 can be given. A cell line such as R-210 is given
as the cell of rat origin. Human antibodies are produced by
immunizing human B lymphocytes in vitro and fusing the immunized
cells with human myeloma cells or a cell line transformed with EB
virus. The fusion of the immunized cells and myeloma cells can be
carried out according to a conventional method such as the method
of Koehler and Milstein (Koehler et al.: Nature 256, 495-497
(1975)). A method using electric pulse is also applicable.
Immunized lymphocytes and myeloma cells are mixed at a
conventionally accepted ratio and fused in an FCS-free (fetal
bovine serum-free) culture medium with an addition of polyethylene
glycol, and cultured in an FCS-containing HAT selection medium to
select fused cells (hybridomas). Next, the hybridomas which produce
antibodies were selected by using a conventional antibody detection
method such as an ELISA, a plaque technique, Ouchterlony method, or
aggregation method, to establish stable hybridomas. The hybridomas
established in this way can be subcultured by a conventional
culture method or can be stored by freezing as required. A
hybridoma can be cultured by a conventional method to collect the
culture supernatant or implanted in the abdominal cavity of mammals
to obtain the antibody from the ascitic fluid. The antibody in the
culture supernatant or ascitic fluid can be purified by a
conventional method such as salting out, ion exchange and gel
filtration chromatography, or protein A or protein G affinity
chromatography. Almost all monoclonal antibodies obtained using
sOBM as an antigen can specifically recognize not only sOBM but
also OBM (such antibodies are hereinafter called anti-OBM/sOBM
monoclonal antibodies). These antibodies can be used for the
quantification of OBM or sOBM. The amounts of OBM and sOBM can be
quantified by labeling these antibodies with a radioisotope or an
enzyme and by applying the labeled antibodies to a quantification
system known as a radioimmunoassay (RIA) or enzymeimmunoassay
(EIA). Using these quantification systems, the amount of sOBM in a
biological sample such as blood or urine can be determined with
ease at high sensitivity. In addition, the amount of OBM binding to
a tissue or surface of cells can be measured with ease at high
sensitivity utilizing a binding assay using these antibodies.
[0072] When an antibody is used as a medication for humans, it is
desirable to use a human-type anti-human OBM/sOBM antibody in view
of antigenicity. The human-type anti-human OBM/sOBM antibody can be
prepared according to the following methods .rarw., .uparw., or
.fwdarw.. In the method .rarw., human lymphocytes collected from
human peripheral blood or spleen are immunized with an antigen
human OBM or human sOBM in vitro in the presence of IL-4. The
resulting immunized human lymphocytes are fused with K6H6/B5 (ATCC
CRL1823) which is a hetero hybridoma of mouse and human, and
screened to obtain the objective antibody producing hybridoma. The
antibodies produced by the resulting antibody producing hybridomas
are human type anti-human OBM/sOBM monoclonal antibodies. The
antibodies neutralizing the activity of human OBM/sOBM are selected
from these antibodies. However, in general, it is difficult to
produce an antibody exhibiting high affinity to an antigen by the
method of immunizing human lymphocytes in vitro. Therefore, in
order to obtain monoclonal antibodies with high affinity to human
OBM and sOBM, it is necessary to increase the affinity of the
human-type anti-human OBM/sOBM monoclonal antibodies obtained by
the above method. This can be done according to the following
method. First, a random mutation is introduced into CDR region
(particularly CDR3 region) of a human-type anti-human OBM/sOBM
monoclonal antibody which neutralize OBM but have a low affinity,
and make the phage to express protein. Phages which can strongly
bind to human OBM/sOBM which are selected by a phage display method
using plates on which human OBM/sOBM antigens are immobilized. The
selected phages are grown in Escherichia coli. The amino acid
sequence of the CDR which exhibits high affinity is determined from
the nucleotide sequence of the DNA cloned in the phage. The
thus-obtained DNA encoding the human type anti-human OBM/sOBM
monoclonal antibodies is introduced into a commonly used expression
vector for mammalian cells to produce the human type anti-human
OBM/sOBM monoclonal antibodies. The target human type anti-human
OBM/sOBM monoclonal antibodies exhibiting high affinity and capable
of neutralizing the biological activity of human OBM/sOBM can be
selected from these monoclonal antibodies. In the method .uparw.,
mouse type anti-human OBM/sOBM monoclonal antibodies are produced
according to the same method as in the present invention using
BALB/c mouse (Koehler et al.: Nature 256, 495-49, 1975), and
monoclonal antibodies which can neutralize the biological activity
of human OBM/sOBM and exhibiting high affinity are selected. These
high affinity mouse anti-human OBM/sOBM monoclonal antibodies can
be converted into human-type using the CDR-grafting technique
(Winter and Milstein: Nature 349, 293-299, 1991) by implanting its
CDR regions (CDR-1, 2 and 3) into the CDR regions of human IgG. In
the method .fwdarw., human peripheral blood lymphocytes are
implanted into a severe combined immune deficiency (SCID) mouse.
Because the implanted SCID mouse can produce human antibodies
(Mosier D. E. et al.: Nature 335, 256-259, 1988; Duchosal M. A. et
al.: Nature 355, 258-262, 1992), lymphocytes which can produce the
human monoclonal antibodies having specificity to human OBM/sOBM
can be collected by screening SCID mouse immunized with human OBM
or sOBM. The resulting lymphocytes are fused with K6H6/B5 (ATCC
CRL1823) which is a heterohybridoma of mouse and human, according
to the procedure described above for the human antibodies in the
method .rarw.. The resulting hybridomas are screened to obtain
hybridomas which can produce the objective human monoclonal
antibodies. The thus-obtained hybridomas are cultured to produce
large amounts of the objective human monoclonal antibodies. The
antibodies can be purified by the above-mentioned purification
method. In addition, it is possible to produce recombinant human
monoclonal antibodies in large amounts by constructing a cDNA
library from the hybridoma which can produce the objective human
monoclonal antibodies to obtain a gene (cDNA) encoding the
objective human-type monoclonal antibodies by cloning, inserting
this gene into a suitable expression vector by using genetic
engineering techniques, and expressing the monoclonal antibodies in
host cells such as various animal cells, insect cells, or
Escherichia coli. A large amounts of purified human monoclonal
antibodies can be obtained by purifying from the resulting culture
supernatant by the purification methods mentioned above.
[0073] The antibodies which can neutralize the biological activity
of OBM/sOBM can be obtained from the anti-OBM/sOBM monoclonal
antibodies produced according to this method. The antibodies which
neutralize the biological activity of OBM/sOBM are expected to be
useful as agents for the treatment or prevention of bone metabolism
abnormality because of their capability of blocking in vivo
biological activity of OBM/sOBM, specifically the capability of
preventing the induction osteoclast formation. The activity of
anti-OBM/sOBM antibodies to neutralize the biological activity of
OBM or sOBM can be measured by determining the activity to suppress
osteoclast formation in the in vitro system. Specifically, the
following in vitro osteoclastogenesis culture system can be given:
.rarw. a co-culture system of mouse osteoblastic stromal cell
strain, ST2 cells, and mouse spleen cells in the presence of
active-form vitamin D.sub.3 and dexamethasone, .uparw. a co-culture
system comprising OBM expressing monkey kidney cell strain, COS-7,
immobilizing the OBM-expressing cells with formaldehyde, and
culturing mouse spleen cells on those cells in the presence of
M-CSF, and .fwdarw. a culture system of mouse spleen cells in the
presence of recombinant sOBM and M-CSF. The
osteoclastogenesis-inhibitory activity of the anti-OBM/sOBM
antibodies can be measured by adding the anti-OBM/sOBM antibodies
at various concentrations to these culture systems and
investigating their effects on osteoclast formation. The
osteoclastogenesis-inhibitory activity of the anti-OBM/sOBM
antibodies can also be evaluated by measuring their bone
resorption-inhibitory activity utilizing experimental animals in
vivo. Especially, ovariectomized animal model is given as an animal
model with progressive osteoclast formation. The
osteoclastogenesis-inhibitory activity of the anti-OBM/sOBM
antibodies can be determined by administering the anti-OBM/sOBM
antibodies to such experimental animals and evaluating the
suppression of bone resorption (a bone density increasing
activity).
[0074] The thus-obtained antibodies capable of neutralizing the
OBM/sOBM biological activity are useful in pharmaceutical
compositions, particularly pharmaceutical compositions to prevent
or treat bone metabolism abnormality or as antibodies for an
immunological diagnosis of such diseases. The preparations
comprising the antibodies of the present invention can be
administered either orally or non-orally. Such preparations can be
safely administered to humans or animals as pharmaceutical
compositions which contain the antibodies recognizing OBM and/or
sOBM as an active component. As the forms of pharmaceutical
composition, injection agents including intravenous drip,
suppository agents, sublingual agents, percutaneous absorption
agents, and the like are given. Because monoclonal antibodies are
macromolecule proteins, they not only readily adhere to a glass
container such as a vial or a syringe, but also are easily
denatured by physicochemical factors such as heat, pH, or humidity.
Therefore, the preparations should be stabilized by the addition of
stabilizers, pH adjusters, buffering agents, solubilizing agents,
or detergents. As the stabilizers, amino acids such as glycine and
alanine, saccharides such as dextran 40 and mannose, and sugar
alcohols such as sorbitol, mannitol, and xylytol can be given.
These stabilizers may be used either individually or in
combinations of two or more. The amount of stabilizers to be added
is preferably from 0.01 to 100 times, particularly preferably from
0.1 to 10 times, the amount of the antibody. The addition of these
stabilizers increases storage stability of liquid preparations or
lyophilized products thereof. Phosphate buffers and citrate buffers
are given as examples of the buffering agents. The buffering agents
not only adjust the pH of the liquid preparations or aqueous
solutions obtained by re-dissolving the lyophilized products
thereof, but also increase stability and solubility of the
antibody. It is desirable to add the buffering agent in an amount
to make from 1 mM to 10 mM concentration of the liquid preparation
or of the aqueous solution prepared from the lyophilized product.
Polysolbate 20, Pulluronic F-68, and polyethylene glycol are given
as examples of the detergent. A particularly preferred example is
Polysolbate 80. These detergents may be used either individually or
in combinations of two or more. Macromolecule proteins such as an
antibody is easily adhere to glass containers. Adherence to
containers of the antibody in a liquid preparation or in an aqueous
solution prepared by re-dissolving a lyophilized product can be
prevented by adding such detergents at a concentration from 0.001
to 1.0%. The preparations comprising the antibodies of the present
invention can be obtained by adding stabilizers, buffering agents,
or agents which prevent adsorption. When the preparations are used
as injection agents for medication or for animals, such injection
agents should preferably have an osmotic pressure ratio of 1 to 2.
The osmotic pressure ratio can be adjusted by increasing or
decreasing the amount of sodium chloride when making the
preparations. The amount of an antibody in a preparation can be
suitably adjusted depending on the disease, route of
administration, and the like. A dose of a human antibody to humans
may be changed depending on the affinity of the antibody to human
OBM/sOBM, especially, on the dissociation constant (Kd value) to
human OBM/sOBM. The higher the affinity (or the smaller the Kd
value), the less the dose to be administered to humans to obtain a
certain medicinal effect. Because a human-type antibody has a long
half-life in blood of about 20 days, it is sufficient to administer
it to humans at a dose of about 0.1-100 mg/kg once or more in a
1-30 day period.
BRIEF DESCRIPTION OF THE DRAWINGS
[0075] FIG. 1 shows the result of SDS-PAGE of mouse OBM protein of
the present invention obtained in Example 3.
21 Explanation of Symbols>
[0076] (A): Lane 1: Molecular weight markers [0077] Lane 2: A
partially purified sample (Gly-HCl (pH 2.0) elution fraction)
obtained from ST2 cells cultured in the presence of active-form
vitamin D.sub.3 and dexamethasone. [0078] Lane 3: A partially
purified sample (Gly-HCl (pH 2.0) elution fraction) obtained from
ST2 cells cultured in the absence of active-form vitamin D.sub.3
and dexamethasone. [0079] (B): Lane 1: Molecular weight markers
[0080] Lane 2: Mouse OBM protein of the present invention after
purification by reverse phase high performance liquid
chromatography (Example 3)
[0081] FIG. 2 shows the result of the binding assay of .sup.125I
labeled OCIF to osteoblastic stromal cells, ST2, in Example 4.
[0082] FIG. 3 shows the osteoclast formation capability of
osteoblastic stromal cells ST2 from different generations in
Example 5(1).
<Explanation of Symbols>
[0083] 1: Ability of ST2 cells from about a .sub.1 th subculture to
support osteoclast formation. [0084] 2: Ability of ST2 cells from
about a .sub.40th subculture to support osteoclast formation.
[0085] FIG. 4 shows change with the passage of time in expression
of the protein of the present invention on the cell membrane of
osteoblastic stromal cells cultured in the presence of active-form
vitamin D.sub.3 and dexamethasone in Example 5(2).
[0086] FIG. 5 shows change with the passage of time in osteoclast
formation in the co-culture system of Example 5 (2).
[0087] FIG. 6 shows the inhibitory effect on osteoclast formation
when treated with OCIF for different culture periods during the
co-culture period in Example 5(3).
[0088] FIG. 7 shows the results of a crosslinking test of
.sup.125I-labeled OCIF with the protein of the present invention in
Example 6.
<Explanation of Symbols>
[0089] Lane 1: .sup.125I-labeled OCIF-CDD1 [0090] Lane 2:
.sup.125I-labeled OCIF-CDD1 crosslinked with ST2 cells [0091] Lane
3: .sup.125I-labeled OCIF-CDDl crosslinked in the presence of
400-fold excess of unlabeled OCIF
[0092] FIG. 8 shows the result of SDS-PAGE in Example 9.
<Explanation of Symbols>
[0093] Lane 1: Proteins of pOBM291-transfected COS-7 cells
immonoprecipitated in the absence of OCIF [0094] Lane 2: Proteins
of pOBM291 -transfected COS-7 cells immunoprecipitated in the
presence of OCIF
[0095] FIG. 9 shows the results of analysis of binding capability
of .sup.125I-labeled OCIF to COS-7 cells transfected with pOBM291
in Example 10.
<Explanation of Symbols>
[0096] Lanes 1 and 2: The amount of the .sup.125I-labeled OCIF
binding to COS-7 cells transfected with pOBM291 [0097] Lanes 3 and
4: The amount of the .sup.125I-labeled OCIF binding to COS-7 cells
transfected with pOBM291 in the presence of 400-fold excess of
unlabeled OCIF
[0098] FIG. 10 shows the result of a crosslinking test using OCIF
labeled with .sup.125I in Example 11.
<Explanation of symbols>
[0099] Lane 1: .sup.125I-labeled OCIF [0100] Lane 2:
.sup.125I-labeled OCIF crosslinked with COS-7 cells transfected
with pOBM291 [0101] Lane 3: .sup.125I-labeled OCIF crosslinked with
COS-7 cells transfected with pOBM291 in the presence of 400-fold
excess of unlabeled OCIF
[0102] FIG. 11 shows the result of a Northern Blot in Example
12.
<Explanation of Symbols>
[0103] Lane 1: RNA originating from ST2 cells cultured without
addition of Vitamin D and dexamethasone [0104] Lane 2: RNA
originating from ST2 cells cultured with the addition of Vitamind D
and dexamethasone
[0105] FIG. 12 shows the OCIF-binding capability of the proteins in
the conditioned medium at various OCIF concentrations in Example
14(2).
<Explanation of Symbols>
[0106] .largecircle.:pCEP4 [0107] .circle-solid.:pCEP sOBM
[0108] FIG. 13 shows the OCIF-binding capability of the protein in
the conditioned medium at various proportions of the conditioned
medium in Example 14(2).
<Explanation of Symbols>
[0109] .largecircle.:PCEP4 [0110] .circle-solid.:PCEP sOBM
[0111] FIG. 14 shows the result of SDS-PAGE of a fusion protein
consisting of thioredoxin and mouse OBM expressed in Escherichia
coli in Example 15(2).
<Explanation of Symbols>
[0112] Lane 1: Molecular weight markers [0113] Lane 2: Soluble
protein fractions originating from GI724/pTrxFus [0114] Lane 3:
Soluble protein fractions originating from GI724/pTrxOBM25
[0115] FIG. 15 shows the OCIF-binding capability at various
proportions of soluble protein fractions in Example 15(3).
<Explanation of Symbols>
[0116] .quadrature.: GI724/pTrxFus [0117] .largecircle.:
GI724/pTrxOBM25
[0118] FIG. 16 shows the OCIF-binding capability of soluble protein
fractions (1%) at various concentrations of OCIF in Example
15(3).
<Explanation of Symbols>
[0119] .quadrature.: GI724/pTrxFus [0120] .largecircle.:
GI724/pTrxOBM25
[0121] FIG. 17 shows the results of inhibition of specific binding
to OCIF of mouse OBM obtained by expression of mouse OBM cDNA of
the present invention and purification or natural OCIF-binding
protein by a rabbit anti-mouse OBM antibody.
<Explanation of Symbols>
[0122] 1: Purified OBM prepared by expression of the cDNA in the
presence of the antibody, OBM+.sup.125I-OCIF [0123] 2: The natural
protein in the presence of the antibody+.sup.125I-OCIF [0124] 3:
Mouse OBM prepared by expression of the cDNA in the absence of the
antibody, mouse OBM+.sup.125I-OCIF [0125] 4: The natural protein in
the absence of the antibody+.sup.125I-OCIF [0126] 5: 3+ unlabeled
OCIF (400-fold more than .sup.125I-OCIF) [0127] 6: 4+ unlabeled
OCIF (400-fold more than .sup.125I-OCIF)
[0128] FIG. 18 shows the result of SDS-PAGE of human OBM protein
expressed by the cDNA of the present invention.
<Explanation of Symbols>
[0129] Lane 1: Molecular weight markers [0130] Lane 2: Proteins of
COS-7 cells transfected with phOBM (an expression vector containing
a cDNA of the present invention), immunoprecipitated with a rabbit
anti-OCIF polyclonal antibody in the absence of OCIF [0131] Lane 3:
Proteins of COS-7 cells transfected with phOBM (an expression
vector containing a cDNA of the present invention),
immunoprecipitated with a rabbit anti-OCIF polyclonal antibody in
the presence of OCIF
[0132] FIG. 19 shows the result of analysis of binding of OCIF to
COS-7 cells transfected with phOBM, an expression vector containing
a cDNA of the present invention.
<Explanation of Symbols>
[0133] Lane 1: COS-7 cells transfected with phOBM and the addition
of .sup.125I-OCIF. [0134] Lane 2: COS-7 cells transfected with
phOBM and the addition of .sup.125I-OCIF, in the presence of a
400-fold more unlabeled OCIF
[0135] FIG. 20 shows the result of crosslinking of human OBM, which
is a protein encoded by a cDNA of the present invention, with
.sup.125I-OCIF (monomer-type).
<Explanation of Symbols>
[0136] Lane 1: .sup.125I-OCIF [0137] Lane 2: The crosslinked
products of .sup.125I-OCIF with the proteins on the membrane of
COS-7 cells transfected with phOBM. [0138] Lane 3: The crosslinked
products of .sup.125I-OCIF with the proteins on the membrane of
COS-7 cells transfected with pHOBM, in the presence of a 400-fold
more unlabeled OCIF.
[0139] FIG. 21 shows the OCIF-binding capability of the protein
(secreted-form hOBM) in the conditioned medium at various OCIF
concentrations in Example 24(2).
<Explanation of Symbols>
[0140] .largecircle.: Conditioned medium of 293-EBNA cells
transfected with pCEP4, which does not contain cDNA encoding
secreted-form human OBM [0141] 574 : Conditioned medium of 293-EBNA
cells transfected with pCEPshOBM, which contains cDNA encoding
secreted-form human OBM
[0142] FIG. 22 shows the OCIF-binding capability of the protein
(secreted-form human OBM) in the conditioned medium at a specific
OCIF concentration while changing the amount of conditioned medium
added in Example 24(2). [0143] .largecircle.: Conditioned medium of
293-EBNA cells transfected with pCEP4, which does not contain cDNA
encoding secreted-form human OBM [0144] .circle-solid.: Conditioned
medium of 293-EBNA cells transfected with pCEPshOBM, which contains
cDNA encoding secreted-form human OBM
[0145] FIG. 23 shows the result of SDS-PAGE of a fusion protein
consisting of thioredoxin and human OBM expressed in Escherichia
coli.
<Explanation of Symbols>
[0146] Lane 1: Molecular weight markers [0147] Lane 2: Soluble
protein fractions originating from Escherichia coli GI724/pTrxFus
[0148] Lane 3: Soluble protein fractions originating from
Escherichia coli GI724/pTrxhOBM
[0149] FIG. 24 shows the OCIF-binding capability of the fusion
protein consisting of thioredoxin and human OBM to OCIF, when the
amount of the soluble protein fraction originating from Escherichia
coli including the fusion protein added was varied in Example
25(3).
<Explanation of Symbols>
[0150] .largecircle.: Soluble protein fractions originating from
Escherichia coli GI724/pTrxFus [0151] .circle-solid.: Soluble
protein fractions originating from Escherichia coli
GI724/pTrxshOBM
[0152] FIG. 25 shows the OCIF-binding capability of the fusion
protein of thioredoxin and human OBM in soluble protein fractions
originating from Escherichia coli to OCIF in various concentrations
in Example 25(3).
<Explanation of Symbols>
[0153] .largecircle.: Soluble protein fractions originating from
Escherichia coli GI724/pTrxFus [0154] .circle-solid.: Soluble
protein fractions originating from Escherichia coli
GI724/pTrxshOBM
[0155] FIG. 26 shows the result of quantifying human OBM and human
sOBM by the sandwich ELISA method using the rabbit anti-human
OBM/sOBM polyclonal antibody of the present invention.
<Explanation of Symbols>
.quadrature.: Human OBM
.circle-solid.: Human sOBM
[0156] FIG. 27 shows the result of quantifying human OBM and human
sOBM by the sandwich ELISA method using the anti-human OBM/sOBM
monoclonal antibodies of the present invention.
<Explanation of Symbols>
.quadrature.: Human OBM
.circle-solid.: Human sOBM
[0157] FIG. 28 shows the result of quantifying mouse OBM and sOBM
by the sandwich ELISA method using the anti-human OBM/sOBM
monoclonal antibodies of the present invention which cross react
mouse OBM and sOBM. A
<Explanation of Symbols>
.quadrature.: Mouse OBM
.circle-solid.: Mouse sOBM
[0158] FIG. 29 shows the activity of the fusion protein consisting
of thioredoxin and mouse OBM to stimulate human osteoclast-like
cell formation.
[0159] FIG. 30 shows the suppression of the anti-OBM/sOBM antibody
of the bone resorption activity stimulated by vitamin D.sub.3.
[0160] FIG. 31 shows the suppression of the anti-OBM/sOBM antibody
of the bone resorption activity stimulated by prostaglandin E.sub.2
(PGE.sub.2).
[0161] FIG. 32 shows the suppression by the anti-OBM/sOBM antibody
of the bone-resorbing activity stimulated by parathyroid hormone
(PTH).
[0162] FIG. 33 shows the suppression by the anti-OBM/sOBM antibody
of the bone-resorbing activity stimulated by interleukin 1 .alpha.
(IL-1).
BEST MODE FOR CARRYING OUT THE INVENTION
EXAMPLES
[0163] The present invention will be described in more detail by
way of examples which are given for the purpose of illustration of
the invention and are not limiting thereof in any way of the
remainder of the disclosure.
Example 1
Preparation of the protein of the present invention
[0164] (1) Large-scale cultivation of ST2 Cells
[0165] Mouse osteoblastic stromal cell line ST2 (RIKEN CELL BANK
RCB0224) was cultured using .alpha.-MEM containing 10% fetal bovine
serum. ST2 cells cultured to confluence in a 225 cm.sup.2 T flask
for adherent-cell culture were treated with trypsin and harvested
from the T flask. After washing, the cells were transferred to five
225 cm.sup.2 T flasks. After the addition of 60 ml of .alpha.-MEM
containing 10.sup.-8 M active-form vitamin D.sub.3 (Calcitriol),
10.sup.-7 M dexamethasone, and 10% fetal bovine serum, cells in
each flask were cultured for 7-10 days in a CO.sub.2 incubator. The
cultured ST2 cells were harvested using a cell scraper and stored
at -80.degree. C. until use.
[0166] (2) Preparation of membrane fraction and solubilization of
membrane-bound proteins
[0167] To the ST2 cells (volume, about 12 ml) described in Example
1 (1), cultured using eighty 225 cm.sup.2 T flasks, was added three
times the volume (36 ml) of 10 mM Tris-HCl buffer (pH 7.2)
containing protease inhibitors (2 mM APMSFP, 2 mM EDTA, 2 mM
o-phenanthroline, 1 mM leupeptin, 1 .mu.g/ml pepstatin A, and 100
unit/ml aprotinin). After vigorously agitating for 30 seconds using
a vortex mixer, the cells were allowed to stand for 10 minutes on
ice. The cells were homogenized using a homogenizer (DOUNCE TISSUE
GRINDER, A syringe, WHEATON SCIENTIFIC Co.). The same volume (48
ml) of 10 mM Tris-HCl buffer (pH 7.2) containing the
above-mentioned protease inhibitors, 0.5 M sucrose, 0.1 M potassium
chloride, 10 mM magnesium chloride, and 2 mM calcium chloride was
added to the homogenized cells. After stirring, the mixture was
centrifuged at 600.times.g for 10 minutes at 4.degree. C., thereby
separating nuclei and non-homogenized cells as precipitate. The
supernatant obtained by the centrifuge was centrifuged at
150,000.times.g for 90 minutes at 4.degree. C., to obtain the
membrane fraction of the ST2 cells as a precipitate. Eight ml of 10
mM Tris-HCl buffer (pH 7.2) containing the above-mentioned protease
inhibitors, 150 mM sodium chloride, and 0.1 M sucrose was added to
this membrane fraction. After the addition of 200 .mu.l of 20%
CHAPS (3-[(3-cholamidopropyl)-dimethylamonio]-1-propanesulfonate,
Sigma Co.), the mixture was stirred for 2 hours at 4.degree. C. The
mixture was then centrifuged at 150,000.times.g for 60 minutes at
4.degree. C., to obtain supernatant as a solubilized membrane
fraction.
Example 2
Purification of the protein of the present invention
[0168] (1) Preparation of OCIF-immobilized affinity column
[0169] After replacing iso-propanol in a HiTrap NHS-activated
column (1 ml, manufactured by Pharmacia Co.) with 1 mM hydrochloric
acid, 1 ml of 0.2 M NaHCO.sub.3/0.5 M NaCl solution (pH 8.3)
containing 13.0 mg of recombinant OCIF prepared by the method of WO
96/26217 was added to the column using a syringe (5 ml,
manufactured by Terumo Corp.), to effect coupling reaction at room
temperature for 30 minutes. The column was fed with 3 ml of 0.5 M
ethanolamine/0.5 M NaCl (pH 8.3) and 3 ml of 0.1 M acetic acid/0.5
M NaCl (pH 4.0) three times in turn to inactivate excess active
groups, then the solution was replaced with 0.5 M ethanolamine/0.5
M NaCl (pH 8.3). After allowing to stand at room temperature for 1
hour, the column was washed twice alternately with 0.5 M
ethanolamine/0.5 M NaCl (pH 8.3) and 0.1 M acetic acid/0.5M NaCl
(pH 4.0), and the solution was then replaced with 50 mM Tris/1 M
NaCl/0.1% CHAPS buffer (pH 7.5).
[0170] (2) Purification of the protein of the present invention
using OCIF-immobilized affinity column
[0171] The purification of the OCIF-binding protein was carried out
at 4.degree. C., unless otherwise indicated. The above-mentioned
OCIF-immobilized affinity column was equilibrated with 10 mM
Tris-hydrochloride buffer (pH 7.2) to which the protease inhibitors
described in Example 1(2), 0.15 M sodium chloride, and 0.5% CHAPS
were added. About 8 ml of the solubilized membrane fraction
described in Example 1(2) was applied to the column at a flow rate
of 0.01 ml/minute. Then, the column was washed with 10 mM
Tris-hydrochloride buffer (pH 7.2) to which the above-mentioned
protease inhibitors, 0.15 M sodium chloride, and 0.5% CHAPS was
added, for 100 minutes at a flow rate of 0.5 ml/minute. Next, the
proteins adsorbed to the column was eluted with 0.1 M
glycine-hydrochloride buffer (pH 3.3) containing the protease
inhibitors, 0.2 M sodium chloride, and 0.5% CHAPS for 50 minutes at
a flow rate of 0.1 ml/minute. In the same manner, the proteins
adsorbed to the column was eluted with 0.1 M sodium citrate buffer
(pH 2.0) containing the protease inhibitors, 0.2 M sodium chloride,
and 0.5% CHAPS for 50 minutes at a flow rate of 0.1 ml/minute. The
eluate was collected in 0.5 ml fractions. Each fraction was
immediately neutralized by the addition of 2M Tris solution. The
fractions derived from the elution with these buffers (each
fraction consisting of 1.0-5.0 ml of eluate) were concentrated to
50-100 .mu.l using Centricon-10 (manufactured by Amicon of U.S.A.).
OCIF was added to a portion of each concentrated fraction and
immunoprecipitated with anti-OCIF polyclonal antibody. The
precipitated fractions were treated with SDS and subjected to
SDS-PAGE. Fractions (Fr. No. 3-10) in which the band of the protein
with specific binding ability to OCIF appeared were regarded as the
protein fractions of the present invention.
[0172] (3) Purification of the protein of the present invention by
gel filtration
[0173] The concentrated OCIF-binding protein (the fractions obtain
by the elution with 0.1 M glycine-hydrochloride buffer (pH 3.3) and
0.1 M sodium citrate buffer (pH 2.0)) prepared in Example 2(2) was
applied to a Superose 12 HR10/30 column (1.0.times.30 cm,
manufactured by Pharmacia Co.) which was equilibrated with 10 mM
Tris-HCl, 0.5M NaCl, 0.5% CHAPS (pH 7.0) and developed with the
equilibration buffer as a mobile phase at a flow rate of 0.5
ml/min, and each 0.5 ml fraction was collected. The fractions
containing the protein of the present invention (Fr. Nos. 27-32)
were identified according to the same method as described above.
Each of the fractions was concentrated using Centricon-10 (a
product of Amicon).
[0174] (4) Purification by reverse phase high performance liquid
chromatography
[0175] The above-mentioned OCIF-binding protein purified by the gel
filtration was applied to a C.sub.4 column (2.1.times.250 mm,
Vydac, USA) which was equilibrated with 0.1% trifluoroacetic acid
(TFA) and 30% acetonitrile. The proteins bound to the column were
eluted with linear gradients of acetonitrile from 30% to 55% for
the first 50 minutes and from 55% to 80% during the next 10 minutes
at a flow rate of 0.2 ml/min. Peaks of eluted proteins were
detected by measuring optical density at 215 nm. Proteins in the
different peaks were analyzed to identify the fractions containing
the protein of the present invention, and a highly purified protein
of the present invention was obtained.
Example 3
[0176] SDS-PAGE of the purified protein of the present
invention
[0177] The solubilized membrane fraction prepared from ST2 cells
which were cultured in the presence or absence of active-form
vitamin D.sub.3 was subjected to purification with the
OCIF-immobilized affinity column. The purified preparations were
subjected to SDS-PAGE. As shown in FIG. 1(A), a major protein band
with MW of about 30,000-40,000 was detected only in the purified
preparation from ST2 cells which was cultured in the presence of
the active-form vitamin D.sub.3, proving that the protein which
specifically binds to OCIF (i.e. the protein of the present
invention) can be selectively purified by the OCIF-immobilized
affinity column. However, bands of several proteins (other than the
protein of the present invention) which non-specifically bind to
carriers or spacers of the OCIF-immobilized column were detected in
both of the purified preparations. The proteins other than the
protein of the present invention were removed according to the
above-described method by gel filtration and C4 reverse phase
chromatography. SDS-PAGE of the obtained highly purified protein of
the present invention is shown in FIG. 1(B). The highly purified
protein of the present invention was found to be
electrophoretically homogeneous and had a molecular weight of about
30,000-40,000.
Example 4
Binding test of OCIF to osteoblasts
[0178] (1) Preiparation of .sup.125I-labeled OCIF
[0179] OCIF was labeled with .sup.125I by the lodogen method.
Specifically, 20 .mu.l of 2.5 mg/ml Iodogen-chloroform solution was
transferred to a 1.5 ml Eppendorf tube and chloroform was
evaporated off at 40.degree. C., to obtain a tube coated with
lodogen. The tube was washed three times with 400 .mu.l of 0.5 M
sodium phosphate buffer (Na-Pi, pH 7.0). Five .mu.l of 0.5 M Na-Pi
(pH 7.0) was added to the tube. Immediately after the addition of
1.3 .mu.l (18.5 MBq) of Na-.sup.125I solution (NEZ-033H20,
manufactured by Amersham Co.), 10 .mu.l of 1 mg/ml rOCIF solution
(monomer type or dimer type) was added to the tube. After mixing
with a vortex mixer, the mixture was allowed to stand at room
temperature for 30 seconds. The solution was transferred to a tube
containing 80 .mu.l of a solution of 10 mg/ml potassium iodide in
0.5 M Na-Pi (pH 7.0) and 5 .mu.l of a phosphate buffered saline
containing 5% bovine serum albumin, and stirred. The mixture was
applied to a spin column (1 ml, G-25 fine, manufactured by
Pharmacia Co.) which was equilibrated with phosphate buffered
saline containing 0.25% bovine serum albumin and the column was
centrifuged for 5 minutes at 2,000 rpm. Four hundred .mu.l of a
phosphate buffered saline containing 0.25% bovine serum albumin was
added to the fraction eluted from the column and the mixture was
stirred. Two .mu.l of the aliquot was removed to measure the
radioactivity using a gamma counter. The radiochemical purity of
the .sup.125I-labled OCIF was determined by measuring the
radioactivity precipitated with 10% TCA. The biological activity of
the labeled .sup.125I-labeled OCIF was measured according to the
method described in WO 96/26217. The concentration of the
.sup.125I-labeled OCIF was measured by ELISA according to the
following procedure.
[0180] (2) Measurement of the concentration of .sup.125I-labeled
OCIF by ELISA
[0181] One hundred: 1 of 50 mM NaHCO.sub.3 (pH 9.6) in which the
anti-OCIF rabbit polyclonal antibody described in WO 96/26217 was
dissolved to a concentration of 2 .mu.g/ml was added to each well
of a 96-well immuno-plate (MaxiSorp.TM., a product of Nunc Co.).
The plate was allowed to stand overnight at 4.degree. C. After
removing the solution by suction, 300:1 of Block Ace.TM. (Snow
Brand Milk Products Co., Ltd.)/phosphate buffered saline (25/75)
solution was added to each well. The plate was then allowed to
stand for two hours at room temperature. After removing the
solution by suction, the wells were washed three times with
phosphate buffered saline containing 0.01% Polysorbate 80 (P-PBS).
Next, 300 .mu.l of Block Ace.TM./phosphate buffered saline (25/75)
solution to which .sup.125I-labeled OCIF or the standard OCIF
preparation was mixed, was added to each well. The plate was then
allowed to stand for two hours at room temperature. After removing
the solution by suction, each well was washed six times with 200
.mu.l of P-PBS.
[0182] One hundred pI of Block Ace.TM. (Snow Brand Milk Products
Co., Ltd.)/phosphate buffered saline (25/75) solution containing
peroxidase labeled rabbit anti-OCIF polyclonal antibody was added
to each well. The plate was allowed to stand for two hours at room
temperature. After removing the solution by suction, the wells were
washed six times with 200 .mu.l P-PBS. Then, 100 .mu.l of TMB
solution (TMB Soluble Reagent, High Sensitivity, Scytek Co.) was
added to each well. After incubating at room temperature for 2-3
minutes, 100 .mu.l of stopping solution (Stopping Reagent, Scytek
Co.) was added to each well. Absorbance of each well was measured
at 490 nm using a microplate reader. The concentration of
.sup.125I-labeled OCIF was determined from a calibration curve
prepared using the standard preparation of OCIF.
[0183] (3) Binding test of OCIF to osteoblasts or spleen cells
[0184] Mouse osteoblastic stromal cell line ST2 or spleen cells
were suspended in .alpha.-MEM containing 10% fetal bovine serum
(FBS), either with or without 10.sup.-8 M active-form vitamin
D.sub.3 (Calcitriol) and 10.sup.-7 M dexamethasone, to a
concentration of 4.times.10.sup.4 cells/ml (ST2 cells) or
2.times.10.sup.6 cells/ml (spleen cells), respectively. Each cell
suspension was innoculated into a 24-well micro plate. The cells
were cultured for 4 days in a CO.sub.2 incubator. After washing the
cells with .alpha.-MEM, 200 .mu.l of medium for the binding test
(.alpha.-MEM to which 0.2% bovine serum albumin, 20 mM Hepes
buffer, and 0.2% NaN.sub.3 were supplemented), containing 20 ng/ml
of above-described .sup.125I-labeled OCIF (monomer form or dimer
form), was added to each well. To the wells for the measurement of
non-specific binding, 200 .mu.l of the medium for the binding test
containing 8 .mu.g/ml of rOCIF (400 times concentration) in
addition to 20 ng/ml of .sup.125I-labeled OCIF was added. The cells
were cultured for one hour in a CO.sub.2 incubator and washed 3
times with 1 ml of a phosphate buffered saline. In this procedure,
spleen cells were washed by centrifuging the 24-well plate in each
washing step, because the spleen cells were non-adherent. After
washing, 500 .mu.l of 0.1 N NaOH solution was added to each well
and the plate was allowed to stand for 10 minutes at room
temperature to dissolve the cells. The amount of RI in each well
was measured by a gamma counter.
[0185] As shown in FIG. 2, .sup.125I-labeled OCIF did not bind to
the cultured spleen cells, but specifically bound only to the
osteoblastic stromal cells which were cultured in the presence of
active-form vitamin D.sub.3. The results indicated that the protein
of the present invention is a membrane bound protein induced by
active-form vitamin D.sub.3 and dexamethasone on osteoblastic
stromal cells.
Example 5
Biological activity of the protein of the present invention
[0186] (1) Osteoclasts-formation supported by osteoblastic stromal
cells
[0187] The osteoclasts formation-supporting capability of
osteoblastic stromal cells was evaluated by measuring tartaric acid
resistant acid phosphatase activity (TRAP activity) of the formed
osteoclasts. Specifically, spleen cells (2.times.10.sup.5 cells/100
.mu.l/well) from a ddy mouse (8-12 weeks old) and mouse
osteoblastic stromal cells ST2 (5.times.10.sup.3 cells/100
.mu.l/well) were suspended in .alpha.-MEM to which 10.sup.-8 M
active-form vitamin D.sub.3, 10.sup.-7 M dexamethasone, and 10%
fetal bovine serum were added. The cells were innoculated into
96-well plates and cultured for one week in a CO.sub.2 incubator.
After washing each well with phosphate buffered saline, 100 .mu.l
of ethanol/acetone (1:1) was added to each well, and the cells were
immobilized at room temperature for one minute. After
immobilization, 100:1 of 50 mM citrate buffer (pH 4.5) containing
5.5 mM p-nitrophenol phosphate and 10 mM sodium tartarate was added
to each well. After 15 minutes of reaction at room temperature, 0.1
N NaOH solution was added to each well and absorbance at 405 nm was
measured using a microplate reader. The results of
osteoclasts-formation by ST2 cells with a passage number of about
10 or 40 after purchasing the cells from RIKEN CELL BANK are shown
in FIG. 3. The results indicate that the ST2 cells with a higher
passage number exhibit more potent ability to support
osteoclasts-formation.
[0188] (2) Time course change of expression of the protein of the
present invention on membrane of osteoblastic stromal cells in a
culture system which include active-form vitamin D.sub.3 and
dexamethasone and time course change of osteoclasts-formation in
the co-culture system
[0189] In the same manner as in Example 4(3), osteoblastic stromal
cell ST2 was cultured for 7 days in the presence of active-form
vitamin D.sub.3 and dexamethasone. The OCIF-binding test was
carried out using .sup.125I-labeled OCIF (monomer type) as
described in the experiment in Example 4(1). Non-specific binding
was measured by competing .sup.125I-OCIF binding to ST2 cells with
400-fold concentration of unlabeled OCIF. As a result, it was
confirmed that the amount of specific binding of .sup.125I-labeled
OCIF increase in accordance with increase in culture period in the
presence of active-form vitamin D.sub.3 and dexamethasone.
Specifically, as shown in FIGS. 4 and 5, the protein of the present
invention was expressed on the surface of ST2 cells by active-form
vitamin D.sub.3 in accordance with increase in culture period and
the expression reached a maximum on the fourth day of culture. On
the other hand, osteoclast-like cells are formed by coculturing
mouse spleen cells and ST2 cells in the presence of active-form
vitamin D.sub.3 TRAP (a marker enzyme of osteoclasts)--positive
mononuclear pre-osteoclast-like cells are formed on the third or
fourth day of the culture. More differentiated and mature
TRAP-positive multinuclear cells are formed on the fifth to sixth
day of the culture. A good correlation between time-course of the
expression of the protein of the present invention and
osteoclasts-formation was thus demonstrated.
[0190] (3) Inhibition of osteoclasts formation by OCIF treatment
for different period during the co-culture
[0191] To make it clear that the protein of the present invention
is a factor involved in the osteoclasts-formation, the cells were
treated with 100 mg/ml OCIF for different culture periods during
the six day co-culture period described in the above-mentioned
Example 5(2) (two consecutive days in the six-day period, except
for the 5th day for which a one-day period was applied). As a
result, as shown in FIG. 6, OCIF treatment at 48-96 hours after
start of the culture at which expression of the protein of the
present invention on ST2 cells is maximal was found to be most
effective for inhibiting formation of osteoclasts. Specifically, it
was confirmed that OCIF controls osteoclast formation by binding to
ST2 cells via the protein of the present invention.
[0192] Based on the results of the above experiments, the protein
of the present invention was confirmed to be induced on cell
membrane of osteoblastic stromal cells by active-form vitamin
D.sub.3 and dexamethasone and to exhibit a biological activity to
support or accelerate differentiation or maturation of
osteoclasts.
Example 6
Crosslinking test for .sup.125I-labeled OCIF and the protein of the
present invention
[0193] To identify the protein of the present invention more
clearly, the protein of the present invention was crosslinked with
.sup.125I-labeled OCIF. Mouse osteoblastic stromal cell line ST2
was cultured for four days in the presence or absence of
active-form vitamin D.sub.3 and dexamethasone in the same manner as
described in Example 4(3). After washing the cells with 1 ml of
phosphate buffered saline, 200:1 of medium for binding test
(.alpha.-MEM to which 0.2% bovine serum albumin, 20 mM Hepes
buffer, 0.2% NaN.sub.3, and 100: g/ml heparin were added),
containing 25 ng/ml of .sup.125I-labeled OCIF (monomer type) or 40
ng/ml of .sup.125I-labeled OCIF-CDD1 which was obtained by
expressing the protein of Sequence ID No. 76 (WO 96/26217) in
animal cells, was added. The above-mentioned culture medium for the
binding test was further supplemented with 400-fold concentration
of OCIF and was added to the other wells to assess non-specific
binding. After culturing for one hour in a CO.sub.2 incubator, each
well was washed three times with 1 ml of phosphate buffered saline
containing 100 .mu.g/ml heparin. Five hundred .mu.l of phosphate
buffered saline containing 100 .mu.g/ml crosslinking agent, DSS
(Disuccinimidyl suberate, Pierce Co.), was added to each well and
the plate was kept for 10 minutes at 0.degree. C. The wells were
washed twice with 1 ml of phosphate buffered saline at 0.degree. C.
One hundred .mu.l of 20 mM Hepes buffer containing 1% Triton X-100,
10 .mu.M pepstatin, 10 .mu.M leupeptin, 2 mM PMSF
(phenylmethylsulfonyl fluoride), 10 .mu.M antipain, and 2 mM EDTA,
was then added to each well. The plate was allowed to stand for 30
minutes at room temperature to dissolve the cells. Fifteen: 1 of
these samples were treated with SDS under non-reducing conditions
according to conventional method and subjected to
SDS-polyacrylamide gel electrophoresis (4-20% polyacrylamide
gradient, manufactured by Daiichi Chemical Co., Ltd.). After
electrophoresis, the gels were dried and exposed to BioMax MS film
(manufactured by Kodak) for 24 hours at -80.degree. C. using BioMax
MS intensifying screens (manufactured by Kodak). After exposure,
the film was developed by conventional method. A band of
crosslinking product with a molecular weight of 90,000-110,000 was
detected when the .sup.125I-labeled OCIF (monomer type, 60 kDa) was
used. When the .sup.125I-labeled OCIF-CDD1 (31 kDa) was used, a
band of crosslinking product of about 70-80 kDa (average, 78 kDa)
was detected as shown in FIG. 7.
Example 7
Analysis of the protein of the present invention expressed on ST
cells by Scatchard Plot
[0194] The above-mentioned .sup.125I-labeled OCIF (monomer type)
was added to a concentration of 1,000 pM to the culture medium for
binding test (.alpha.-MEM containing 0.2% bovine serum albumin, 20
mM Hepes buffer, and 0.2% NaN.sub.3) and the culture medium was
serially diluted at a rate to 1/2 with the culture medium not
containing .sup.125I-labeled OCIF. Solutions for measuring
non-specific binding were prepared by further adding 400-fold
concentration of monomer-form OCIF to these solutions. Two hundred
.mu.l of the prepared solutions were added to the above-mentioned
wells with ST2 cells cultured for 4 days (passage number, about 10)
in the presence of 10.sup.-8 M active-form vitamin D.sub.3
(Calcitriol) and 10.sup.-7 M dexamethasone, to assess binding of
.sup.125I-labeled OCIF in the same method as described in Example
4(3). The results were subjected to Scatchard Plot analysis to
determine the dissociation constant of OCIF and OCIF-binding
protein and the number (site) of OCIF-binding protein per a ST2
cell. As a result, the dissociation constant of OCIF and the
protein of the present invention was found to be 280 pM, and the
number of the site of OCIF-binding protein per a ST2 cell was
approximately 33,000/cell. Based on the finding in Example 5(1)
that osteoclasts-formation supported by the ST2 cells with passage
number about 40 was more extensive than that with passage number
about 10, the number (the site) of the protein of the present
invention expressed on the ST2 cell with a passage number about 40
was assessed. The number (site) was 58,000/cell and was clearly
larger than the ST2 cells with passage number about 10, indicating
that the amount of the protein of the present invention expressed
on ST2 cells is related to their potency to support
osteoclasts-formation. The results indicated that the protein of
the present invention is a factor that supports or induces
differentiation or maturation of osteoclasts.
Example 8
Cloning of OBMcDNA
[0195] (1) Extraction of RNA from mouse ST2 cells
[0196] Mouse osteoblastic stromal cell line ST2 (RIKEN CELL BANK,
RCB0224) was cultured in .alpha.-MEM (Gibco BRL Co.) containing 10%
fetal bovine serum. ST2 cells cultured to confluent in a 225
cm.sup.2 T-flask for adherent cells were treated with trypsin to
harvest the cells from the T-flask. The cells were washed and
transferred to five 225 cm.sup.2 T-flasks. Sixty ml of .alpha.-MEM
containing 10.sup.-8 M active-form vitamin D.sub.3 (Calcitriol,
Wako Pure Chemicals Co., Ltd.), 10.sup.-7 M dexamethasone, and 10%
fetal bovine serum was added to each flask and the cells were
cultured for 5 days in a CO.sub.2 incubator. Total RNA was
extracted from the cultured ST2 cells using ISOGEN (Wako Pure
Chemicals Co., Ltd.). Poly A.sup.+ RNA was prepared from about 600:
g of the total RNA using an Oligo (dT)-cellulose column (5'-3'
Prime Co.). About 8 .mu.g of Poly A.sup.+ RNA was obtained.
[0197] (2) Construction of expression library
[0198] Double-stranded cDNA was synthesized from 2 .mu.g of
polyA.sup.+ RNA obtained in Example 8(1) using a Great Lengths cDNA
Synthesis kit (Clontech Co.) according to the instruction in the
manual. Specifically, 2 .mu.g of polyA.sup.+ RNA and Oligo
(dT).sub.25 (dN) primer were mixed and distilled water was added to
the mixture to make the final volume to 6.25 .mu.l. After
incubation for about 3 minutes at 70.degree. C., the mixture was
cooled on ice for 2 minutes. To the mixture were added 2.2 .mu.l of
distilled water, 2.5 .mu.l of 5X First-strand buffer, 0.25 .mu.l of
100 mM DTT (dithiothreitol), 0.5 .mu.l of PRIME RNase inhibitor
(1U/ml) (5'-3' Prime Co.), 0.5 .mu.l of [.alpha.-.sup.32P] dCTP
(Amersham Co., 3000 Ci/mmol) diluted 5-fold with distilled water to
make 2 .mu.Ci/:1, 0.65 .mu.l of dNTP (20 mM each), and 1.25 .mu.l
(250 unit) of MMLV (RNaseH.sup.-) reverse transcriptase. The
mixture was incubated for 90 minutes at 42.degree. C., followed by
the further addition of 62.25:1 of distilled water, 20 .mu.l of 5X
second-strand buffer, 0.75 .mu.l of dNTP (20 mM each), and 5 .mu.l
of Second-strand enzyme cocktail. The resulting mixture was
maintained at 16.degree. C. for two hours. Then, 7.5 units of T4DNA
polymerase was added to this reaction mixture. After incubation at
16.degree. C. for 30 minutes, the reaction was terminated by the
addition of 5 .mu.l of 0.2M EDTA. After a phenol-chloroform
treatment, the product was precipitated with ethanol. An
EcoRI-SalI-NotI linker (Clontech Co.) was attached to the ends of
the resultant double-stranded cDNA. Then, the ends were
phospholylated and the product was applied on a size fractionation
column to obtain cDNA with a length more than 500 bp. DNA was
precipitated with ethanol, dissolved in water and ligated to
pcDL-SR .alpha. 296 (Molecular and Cellular Biology, Vol. 8, pp
466-472, 1988) which had been cut with a restriction enzyme EcoRI
(Takara Shuzo Co.) and treated with CIAP (calf intestine alkaline
phophatase, Takara Shuzo Co.).
[0199] (3) Screening of expression library by means of binding to
OCIF
[0200] An escherichia coli strail, XL2 Blue MRF' (Toyobo Co.,
Ltd.), was transformed using the DNA produced in Example 8(2) and
cultured on L-Carbenisilin agar (1% trypton, 0.5% yeast extract, 1%
NaCl, 60 .mu.g/ml carbenisilin, 1.5% agar) prepared in a 24-well
plastic plates, to produce about 100 colonies per well.
Transformants in each well were suspended in 3 ml of Terrific Broth
ampicillin culture medium (1.2% trypton, 2.4% yeast extract, 0.4%
glycerol, 0.017 M KH.sub.2PO.sub.4, 0.072 M K.sub.2HPO.sub.4, 100
.mu.g/ml ampicillin) and cultured at 37.degree. C. overnight with
shaking. Cells were collected by centrifugation to prepare plasmid
DNA using a QIAwell kit (QIAGEN Co.). DNA concentration was
determined by measuring absorbance at 260 nm. DNA was concentrated
by precipitating with ethanol and dissolved in distilled water to a
concentration of 200 ng/.mu.l. Five hundred DNA pools, each of
which was obtained from about 100 colonies were prepared and were
used for transfection into COS-7 cells (RIKEN CELL BANK, RCB0539).
COS-7 cells were seeded into DMEM containing 10% fetal bovine serum
in each well of 24-well plates at a cell density of
8.times.10.sup.4 cells/well and cultured overnight at 37.degree. C.
in a CO.sub.2 incubator. Next day, the culture medium was removed
and the cells were washed with serum-free DMEM culture medium. The
above-described plasmid DNA which was previously diluted with an
OPTI-MEM culture medium (Gibco BRL Co.) and mixed with
Lipofectamine (a transfection reagent, manufactured by Gibco BRL
Co.) according to the protocol supplied with Lipofectamine. After
15 minutes, the mixture was added to the cells in each well. The
amount of Lipofectamine and DNA used were, respectively, 1 .mu.g
and 4 .mu.l per well. After 5 hours, the culture medium was removed
and 1 ml of DMEM culture medium (Gibco BRL Co.) containing 10%
fetal bovine serum was added to each well. The plates were
incubated for 2-3 days at 37.degree. C. in a CO.sub.2 incubator(5%
CO.sub.2). The COS-7 cells transfected and cultured for 2-3 days in
this manner were washed with a serum-free DMEM culture medium.
Then, 200 .mu.l of a culture medium for the binding assay
(serum-free DMEM culture medium containing 0.2% calf serum albumin,
20 mM Hepes buffer, 0.1 mg/ml heparin, and 0.02% NaN.sub.3) with 20
ng/ml of .sup.125I-labeled OCIF added thereto was added to each
well. After culturing for one hour at 37.degree. C. in a CO.sub.2
incubator (5% CO.sub.2), the cells were washed twice with 500 .mu.l
of a phosphate buffered saline containing 0.1 mg/ml heparin. After
washing, 500 .mu.l of 0.1 N NaOH solution was added to each well.
The plates were allowed to stand for 10 minutes at room temperature
to lyse the cells. The amount of .sup.125I in each well was
measured using a gamma counter (Packard Co.). One DNA pool
containing cDNA encoding the protein which specifically binds to
OCIF was found by screening a total of 500 pools. The DNA pool
containing the cDNA was further divided, and the above-described
transfection and screening operations were repeated to isolate the
cDNA which encodes the protein which binds to OCIF. The plasmid
containing this cDNA was named pOBM291. The Escherichia coli
containing this plasmid was deposited with The National Institute
of Bioscience and Human Technology, Agency of Industrial Science
and Technology, Biotechnology Laboratory, as pOBM291 on May 23,
1997 under the deposition No. FERM BP-5953.
[0201] The methods of labeling OCIF with .sup.125I and quantitative
analysis of the .sup.125I-labeled OCIF by ELISA are shown below.
Labeling of OCIF with .sup.125I was carried out according to the
lodogen method. Twenty .mu.l of 25 mg/ml lodogen-chloroform
solution was added to a 1.5 ml Eppendorf tube and chloroform was
evaporated by heating at 40.degree. C., to prepare an
Iodogen-coated tube. The tube was washed three times with 400 .mu.l
1 of 0.5 M sodium phosphate buffer (Na-Pi, pH 7.0), and 5 .mu.l of
0.5 M Na-Pi (pH 7.0) was added. Immediately after the addition of
1.3 .mu.l (18.5 MBq) of Na-.sup.125I solution (NEZ-033H20, Amersham
Co.), 10 .mu.l of 1 mg/ml rOCIF solution (monomer type or dimer
type) was added to the tube. After mixing the contents with a
vortex mixer, the tube was allowed to stand at room temperature for
30 seconds. The solution in the tube was transferred to a tube to
which 80 .mu.l of 10 mg/ml potassium iodide, 0.5 M Na-Pi (pH 7.0)
and 5 .mu.l of a phosphate buffered saline containing 5% bovine
serum albumin (BSA-PBS) were previously added. After stirring, the
mixture was applied to a spin column (1 ml, G-25 fine, manufactured
by Pharmacia Co.) equilibrated with BSA-PBS, and the column was
centrifuging for 5 minutes at 2000 rpm. Four hundred .mu.l of
BSA-PBS was added to the fraction eluted from the column. After
stirring, 2 .mu.l of an aliquot of this solution was sampled to
measure the radioactivity by a gamma counter. The radiochemical
purity of the .sup.125I-labeled OCIF solution thus prepared was
determined by measuring radioactivity precipitated by 10% TCA. The
biological activity of the .sup.125I-labeled OCIF was measured
according to the method of WO 96/26217. The concentration of the
.sup.125I-labeled OCIF was determined by the ELISA as follows.
Specifically, 100 .mu.l of 50 mM NaHCO.sub.3 (pH 9.6) in which the
anti-OCIF rabbit polyclonal antibody described in WO 96/26217 was
dissolved to a concentration of 2 .mu.g/ml was added to each well
of a 96-well immuno-plate (MaxiSorp.TM., a product of Nunc Co.).
The plate was allowed to stand overnight at 4.degree. C. After
removing the solution by suction, 300 .mu.l of Block Ace.TM. (Snow
Brand Milk Products Co., Ltd.)/phosphate buffered saline (25/75)
(B-PBS) was added to each well. The plate was then allowed to stand
for two hours at room temperature. After removing the solution by
suction, the wells were washed three times with phosphate buffered
saline containing 0.01% Polysorbate 80 (P-PBS). Next, 100 .mu.l of
B-PBS containing .sup.125I-labeled OCIF or standard OCIF was added
to each well. The plate was then allowed to stand for two hours at
room temperature. After removing the solution by suction, each well
was washed six times with 200 .mu.l of P-PBS. One hundred .mu.l of
peroxidase-labeled rabbit anti-OCIF polyclonal antibody diluted
with B-PBS was added to each well. The plate was allowed to stand
for two hours at room temperature. After removing the solution by
suction, the wells were washed six times with 200 .mu.l of P-PBS.
Then, 100 .mu.l of TMB solution (TMB Soluble Reagent, High
Sensitivity, Scytek Co.) was added to each well. After incubating
the plate at room temperature for 2-3 minutes, 100 .mu.l of
stopping solution (Stopping Reagent, Scytek Co.) was added to each
well. Absorbance at 450 nm of each well was measured using a
microplate reader. The concentration of .sup.125I-labeled OCIF was
determined based on a calibration curve drawn using the standard
preparation of OCIF.
[0202] (4) Determination of the nucleotide sequence of the cDNA
encoding the entire amino acid sequence of OBM
[0203] The nucleotide sequence of the OBM cDNA obtained in the
Example 8(3) was determined using a Taq DyeDeoxy Terminator Cycle
Sequencing kit (a product of Perkin Elmer Co.). Specifically, the
nucleotide sequence of the insert fragment was directly determined
using pOBM291 as a template. Fragments with a length of about 1.0
kb and 0.7 kb which were obtained by digesting pOBM291 with a
restriction enzyme EcoRI were inserted into the EcoRI site of
plasmid pUC19 (Takara Shuzo Co.). The nucleotide sequences of these
fragments were also determined. The following primers were used:
primer SRR2 which was used to determine nucleotide sequences of DNA
fragments inserted into pcDL-SR .alpha.296, M13PrimerM3 and
M13PrimerRV (both manufactured by Takara Shuzo Co.) which were used
to determine the nucleotide sequences of DNA fragments inserted
into plasmid pUC19, and synthesized primer OBM#8 designed based on
the nucleotide sequence of OBM cDNA. Sequences of these primers are
shown as the SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID
NO:6.
[0204] In addition, the nucleotide sequence of OBM cDNA is shown as
SEQ ID NO:2 and the amino acid sequence determined therefrom is
shown as the SEQ ID NO:1.
Example 9
[0205] Expression of the protein encoded by the cDNA of the present
invention
[0206] Plasmid pOBM291 was transfected into COS-7 cells in each
well of a 6-well plate using Lipofectamine and the transfected
COS-7 cells were cultured for two days in DMEM containing 10% fetal
bovine serum. The medium was replaced with a
cysteine-methionine-free DMEM (Dainippon Seiyaku Co. Ltd.) (800
.mu.l/well) containing 5% dialyzed fetal bovine serum. The cells
were cultured for 15 minutes, followed by the addition of 14 .mu.l
of Express Protein Labeling Mix (10 mCi/ml, manufactured by NEN
Co.). After culturing for four hours, 200 .mu.l of DMEM including
10% fetal bovine serum was added. After one hour culturing, the
cells were washed twice with PBS. Then, 0.5 ml of a TSA buffer (10
mM Tris-HCl (pH 8.0) containing 0.14 M NaCl, 0.025% NaN.sub.3),
containing 1% TritonX-100, 1% bovine hemoglobin, 10 .mu.g/ml
leupeptin, 0.2 TIU/ml aprotinin, 1 mM PMSF, was added and the
mixture was allowed to stand for one hour on ice. Cells were
disrupted by pipetting and centrifuged at 3000.times.g for 10
minutes at 4.degree. C. to obtain supernatant. 200 .mu.l of
dilution buffer (TSA buffer containing 0.1 % TritonX-100, 0.1%
bovine hemoglobin, 10 .mu.g/ml leupeptin, 0.2 TIU/ml aprotinin, 1
mM PMSF) was added to 100 .mu.l of this supernatant. The mixture
was shaken for one hour at 4.degree. C. with protein A Sepharose
(50 .mu.l). The resultant mixture was centrifuged at 1500.times.g
for one minute at 4.degree. C. to collect supernatant, and thereby
fraction(s) which is non-specifically adsorbed to Protein A
Sepharose was removed. OCIF (1 .mu.g) was added to this supernatant
and the mixture was shaken at 4.degree. C. for one hour to achieve
the binding of OCIF to OBM. Anti-OCIF polyclonal antibody (50
.mu.g) was added and the mixture was shaken for one hour at
4.degree. C. Then, Protein A Sepharose (10 .mu.l) was added and the
mixture was shaken for an additional hour at 4.degree. C., followed
by centrifuge at 1500.times.g for 1 minute at 4.degree. C. to
collect precipitate. The precipitate was washed twice with dilution
buffer, twice with a bovine hemoglobin-free dilution buffer, once
with TSA buffer, and once with 50 mM Tris-HCl (pH 6.5). After
washing, SDS buffer (0.125 M Tris-HCl, 4% sodium dodecylsulfate,
20% glycerol, 0.002% Bromophenol Blue, pH 6.8) containing 10%
.E-backward.-mercaptoethanol was added to the precipitate. The
mixture was heated for 5 minutes at 100.degree. C. and subjected to
SDS-PAGE (12.5% polyacrylamide gel, Daiichi Chemical Co., Ltd.).
The gel was fixed according to a conventional method. Isotope
signals were amplified using Amplify.TM. (Amersham Co.) and the
sample was exposed to Bio Max MR film (KODAK Co.) at -80.degree. C.
The results are shown in FIG. 8, which indicates that the protein
encoded by the cDNA of the present invention has a molecular weight
of about 40,000.
Example 10
Binding of the protein encoded by the cDNA of the present invention
to OCIF
[0207] Plasmid pOBM291 was transfected into COS cells in each well
of a 24-well plate using Lipofectamine. After culturing for 2-3
days, the cells were washed with serum-free DMEM culture medium.
200 .mu.l of culture medium for the binding assay (serum-free DMEM
culture medium containing 0.2% calf serum, albumin, 20 mM Hepes,
0.1 mg/ml heparin, and 0.2% NaN.sub.3), supplemented with 20 ng/ml
.sup.125I-labeled OCIF, was added to the wells. To the other wells,
200:1 of culture medium for the binding assay to which 8 .mu.g/ml
of unlabelled OCIF had been added, in addition to 20 ng/ml
.sup.125I-labeled OCIF, was added. After culturing for one hour at
37.degree. C. in a CO.sub.2 incubator (5% CO.sub.2), the cells were
washed twice with 500 .mu.l of phosphate buffered saline containing
0.1 mg/ml of heparin. Then, 500 .mu.l of 0.1 N NaOH solution was
added to each well and the plate was allowed to stand for 10
minutes at room temperature to dissolve the cells. The amount of
.sup.125I in each well was measured by a gamma counter. As a
result, as shown in FIG. 9, the .sup.125-labeled OCIF was found to
bind only to the cells in which plasmid pOBM291 was transfected. In
addition, the binding was confirmed to be conspicuously inhibited
by the addition of (unlabeled) OCIF at a 400-fold concentration.
These results have demonstrated that the protein OBM encoded by the
cDNA on plasmid pOBM291 specifically binds to OCIF on the surface
of the transfected COS-7 cells.
Example 11
Crosslinking of .sup.125I-labeled OCIF and the protein encoded by
the cDNA of the present invention
[0208] Crosslinking of .sup.125-labeled monomer type OCIF and the
protein encoded by the cDNA of the present invention was carried
out to investigate the characteristics of the protein encoded by
the cDNA of the present invention in further detail. After
transfection of plasmid pOBM291 into COS-7 cells according to the
method used in the Example 8(3), 200 .mu.l of the culture medium
for the binding assay, as described above, containing
.sup.125I-labeled OCIF (25 ng/ml) was added to the wells. The
culture medium for the binding assay to which unlabeled OCIF at a
400-fold concentration was added in addition to .sup.125I-labeled
OCIF was added to the other wells. After culturing for one hour at
37.degree. C. in a CO.sub.2 incubator (5% CO.sub.2), the cells were
washed twice with 500 .mu.l of phosphate buffered saline containing
0.1 mg/ml heparin. Five hundred .mu.l of phosphate buffered saline
containing 100 .mu.g/ml of a crosslinking agent, DSS
(disuccinimidyl suberate, manufactured by Pierce Co.) was added to
the cells, followed by a reaction for 10 minutes at 0.degree. C.
The cells in these wells were washed twice with 1 ml of cold
phosphate buffered saline (0.degree. C.). After the addition of 100
.mu.l of 20 mM Hepes buffer containing 1 % Triton X-100 (Wako Pure
Chemicals Co., Ltd.), 2 mM PMSF (Phenylmethylsulfonyl fluoride,
Sigma Co.), 10 .mu.M Pepstatin (Wako Pure Chemicals Co., Ltd.), 10
.mu.M Leupeptin (Wako Pure Chemicals Co., Ltd.), 10 .mu.M antipain
(Wako Pure Chemicals Co., Ltd.) and 2 mM EDTA (Wako Pure Chemicals
Co., Ltd.), the wells were allowed to stand for 30 minutes at room
temperature to dissolve the cells. Fifteen .mu.l aliquots of these
samples were heated in the presence of SDS under reducing
conditions according to a conventional method and subjected to
SDS-electrophoresis using 4-20% polyacrylamide gradient gel
(Daiichi Pure Chemical Co., Ltd.). After the electrophoresis, the
gel was dried and exposed for 24 hours at -80.degree. C. to a
BioMax MS film (Kodak Co.) using a BioMax MS sensitization screen
(Kodak Co.). The exposed film was developed according to a
conventional method. As a result, a band with a molecular weight of
a range of 90,000-110,000, shown in FIG. 10, was detected by
crosslinking the .sup.125I-labeled monomer type OCIF and the
protein encoded by the cDNA of the present invention.
Example 12
Northern blotting analysis
[0209] ST2 cells cultured to become confluent in a 25 cm.sup.2 T
flask for attached-cell cultures were treated with trypsin and
stripped from the T flask. After washing, the cells were seeded
into a 225 cm.sup.2 T flask and cultured for 4 days in a CO.sub.2
incubator with 60 ml of an .alpha.-MEM culture medium containing
10.sup.-8 M active-form vitamin D.sub.3, 10.sup.-7 M dexamethasone,
and 10% fetal bovine serum. Total RNA was extracted from the
cultured ST2 cells using ISOGEN (Wako Pure Chemicals Co., Ltd.).
The total RNA was also extracted in the same manner from ST2 cells
which were cultured in the absence of the active-form vitamin
D.sub.3 and dexamethasone. After the addition of 2.0 .mu.l of
5.times. gel electrophoresis buffer solution (0.2 M morpholino
propane sulfonic acid, pH 7.0, 50 mM sodium acetate, 5 mM EDTA) and
3.5 .mu.l of formaldehyde, and 10.0 .mu.l of formamide to 20 .mu.g
(4.5:1) of each of the total RNAs, the mixtures were incubated for
15 minutes at 55.degree. C and subjected to electrophoresis. The
gel for electrophoresis was prepared according to the formulation
of 1.0% agarose, 2.2 M deionized formaldehyde, 40 mM
morpholinopropane sulfonic acid (pH 7.0), 10 mM sodium acetate, and
1 mM EDTA. The electrophoresis was carried out in a buffer solution
of 40 mM morpholino propane sulfonic acid, pH 7.0, 10 mM sodium
acetate, and 1 mM EDTA. After the electrophoresis, RNA was
transferred onto nylon membrane. About 1.0 kb DNA fragment was
obtained by digesting pOBM29 1 with a restriction enzyme, EcoRI.
Hybridization was carried out using this DNA fragment, labeled with
a Megaprime DNA labeling kit (Amersham Co.) and
.alpha.-.sup.32.sub.p-dCTP (Amersham Co.), as a probe. As a result,
as shown in FIG. 11, it was confirmed that when ST2 cells were
cultured in the presence of active-form vitamin D.sub.3 and
dexamethasone, gene expression of the protein encoded by the cDNA
of the present invention (OBM) is induced strongly.
Example 13
Osteoclasts formation supporting capability of the protein encoded
by the cDNA of the present invention
[0210] pOBM291 was transfected into COS cells according to the same
method described in the Example 8(3). After three days, trypsinized
cells were washed once with phosphate buffered saline solution by
centrifugation, then fixed with PBS containing 1% paraformaldehyde
at room temperature for 5 minutes, followed by washing with PBS six
times by centriftigation. 700 .mu.l of 1.times.10.sup.6/ml mouse
spleen cells and 350 .mu.l of 4.times.10.sup.4/ml ST2 cells which
were suspended in .alpha.-MEM culture medium containing 10.sup.-8 M
active-form vitamin D.sub.3, 10-.sup.7 M dexamethasone, and 10%
fetal bovine serum, were added to a 24-well plate. TC insert (Nunc
Co.) was set in each well. The above-described fixed COS cells (350
pI) diluted to various concentrations with the above-mentioned
culture medium and OCIF solution (50 .mu.l) were added to the TC
insert and cultured for 6 days at 37.degree. C. As a result, it was
confirmed that the osteoclasts formation inhibitive activity of
OCIF can be inhibited by the protein encoded by the cDNA of the
present invention.
Example 14
Expression of secreted-form OBM
[0211] (1) Construction of a plasmid for the expression of
secreted- form OBM
[0212] A PCR reaction was carried out using OBM HF (SEQ ID NO:7)
and OBM XR (SEQ ID NO:8) as primers and pOBM291 as a template.
After purification by agarose gel electrophoresis, the product was
digested with restriction enzymes Hindlll and EcoRI, and further
purified by agarose gel electrophoresis. The purified fragment (0.6
kb), Hind III/EcoRI fragment (5.2 kb) of pSec TagA (Invitrogen
Co.), and EcoRI/Pmacl fragment (0.32 kb) of OBM cDNA were ligated
using a ligation kit ver. 2 (Takara Shuzo Co.). Escherichia coli
DH5 .alpha. was transformed using the reaction product. Plasmids
were purified by means of alkali SDS method from the resulting
ampicillin resistant strains and digested with restriction enzymes
to select a plasmid with fragments of a length of 0.6 Kb and 0.32
kb being inserted into pSec TagA. Selected plasmid was identified
as having a sequence encoding the secreted-form OBM (nucleotide
sequence: 338-1355 in SEQ ID NO:2, amino acid sequence: 72-316 in
the SEQ ID NO:1) by sequencing using a dyeterminator cycle
sequencing FS kit (Perkin Elmer Co.). This plasmid was digested
with restriction enzymes Nhel and Xhol to isolate a fragment (1.0
kb) containing the secreted-form OBM cDNA by agarose gel
electrophoresis. This fragment was inserted into the NheI/hoI
fragment (10.4 kb) of an expression vector, pCEP4 (Invitrogen Co.),
using a ligation kit and Escherichia coli DH5 a was transformed
using the reaction product thereof. Plasmids were purified by
alkali SDS method from the resulting ampicillin resistant strains
and digested with restriction enzymes to select an Escherichia coli
strain having the secreted-form OBM expression plasmid (PCEP sOBM)
with the correct structure. The Escherichia coli strain containing
the pCEP sOBM was cultured and pCEP sOBM was purified using QIA.TM.
filter plasmid midi kit (QIAGEN Co.).
[0213] (2) Expression of secreted-form OBM
[0214] 293-EBNA cells were suspended in IMDM containing 10% FCS
(IMDM-10% FCS) and seeded into a 24-well plate coated with collagen
(manufactured by Sumitomo Bakelite Co., Ltd.) in a cell density of
2.times.10.sup.5/2 ml/well and cultured overnight. The cells were
transfected with 1 .mu.g of pCEP sOBM or pCEP4 using 4 .mu.l of
Lipofectamine (Gibco Co.). After culturing for two days in 0.5 ml
of a serum-free IMDM or IMDM-10% FCS, the conditioned medium was
collected. Expression of the secreted-forrn OBM in the conditioned
medium was confirmed as follows. Sodium hydrogen carbonate was
added to the conditioned medium to a final concentration of 0.1 M
and the solution was added to a 96-well plate. The plate was
allowed to stand overnight at 4.degree. C., thereby immobilizing
OBM in the conditioned medium on the 96-well plate. The plate was
filled with a Block AceTM (Snow Brand Milk Products Co., Ltd.)
solution diluted four-fold with PBS (B-PBS) and allowed to stand
for two hours at room temperature to block residual binding sites
of the plate. After the addition to each well of 100 .mu.l of 3-100
ng/ml of OCIF which was diluted with B-PBS, the plate was allowed
to stand for two hours at 37.degree. C., followed by washing with
PBS containing 0.05% Tween 20 (PBS-T). Then, 100:1 of a
peroxidase-labeled rabbit anti-OCIF polyclonal antibody described
in WO 96/26217 which was diluted with B-PBS was added to each well.
After allowing to stand for two hours at 37.degree. C., the wells
were washed six times with PBS-T. Then, a TMB solution (TMB Soluble
Reagent, High Sensitivity, Scytek Co.) was added in the amount of
100 .mu.l per well and allowed to stand at room temperature for
about 10 minutes, whereupon the reaction was terminated by the
addition of 100 .mu.l of a termination solution (Stopping Reagent,
Scytek Co.) to each well. Absorbance at 450 nm of each well was
measured by a microplate reader. The results are shown in FIG. 12
which indicates that the absorbance at 450 nm increased according
to the concentration of the added OCIF in the plate in which the
conditioned medium of the cells transfected with pCEP sOBM was
immobilized. On the other hand, no increase in absorbance was seen
in the plate in which the conditioned medium of the cells
transfected with vector pCEP4 was immobilized. FIG. 13 shows the
results of an experiment wherein the proportion of the conditioned
medium which is used for immobilization was changed within a range
of 5-90% and a specific concentration of OCIF (50 ng/ml) was added.
It can be seen that the absorbance at 450 nm increased according to
the increase in the proportion of the conditioned medium in the
plate wherein the conditioned medium of the cells transfected with
pCEPsOBM was immobilized, whereas no such increase in absorbance
was seen in the plate wherein the conditioned medium of the cells
transfected with vector pCEP4 was immobilized. From these results,
it was confirmed that secreted-form OBM is produced into the
conditioned medium of the cells transfected with pCBP sOBM.
Example 15
Expression of thioredoxin-OBM fusion protein (Trx-OBM)
[0215] (1) Construction of a thioredoxin-OBM fusion protein
(Trx-OBM) expression vector
[0216] Ten .mu.l of 10.times. ExTaq buffer (Takara Shuzo Co.), 8
.mu.l of 10 mM dNTP (Takara Shuzo Co.), 77.5 .mu.l of sterilized
distilled water, 2 .mu.l of an aqueous solution of pOBM291 (10
ng/.mu.l ), 1 .mu.l of primer OBM3 (100 pmol/.mu.l, SEQ ID NO:9), 1
.mu.l of primer OBMSalR2 (100 pmol/.mu.l, SEQ ID NO:10), and 0.5
.mu.l of ExTaq (5 u/.mu.l ) (Takara Shuzo Co.) were mixed and
reacted (PCR reaction) in a micro centrifuge tube. After reacting
at 95.degree. C. for 5 minutes, at 50.degree. C. for one second, at
55.degree. C. for one minute, at 74.degree. C. for one second, and
at 72.degree. C. for 5 minutes, a cycle reaction consisting of a
reaction at 96.degree. C. for one minute, at 50.degree. C. for one
second, at 55.degree. C. for one minute, at 74.degree. C. for one
second, and at 72.degree. C. for 3 minutes, was repeated 25 times.
From the total reaction liquid DNA fragment of about 750 bp was
purified by 1% agarose gel electrophoresis using a QIAEX.TM. II gel
extraction kit (QIAGEN Co.). The whole amount of purified DNA
fragment was digested with restriction enzymes SaII and EcoRI
(Takara Shuzo Co.) and subjected to 1.5% agarose gel
electrophoresis to purify a DNA fragment of about 160 bp (Fragment
1), which was dissolved in 20 .mu.l of sterilized distilled water.
In the same manner, a DNA fragment of about 580 bp (Fragment 2)
obtained by digesting 4 .mu.g of pOBM291 with restriction enzymes
BamH1 and EcoRI (Takara Shuzo Co.) and a DNA fragment of about 3.6
kb (Fragment 3) obtained by digesting 2 .mu.g of pTrXFus
(Invitrogen Co.) with restriction enzymes BamHI and SaII (Takara
Shuzo Co.) were respectively purified and dissolved in 20 .mu.l of
sterilized distilled water. The QIAEXII gel extraction kit was used
for the purification of DNA fragments. Fragments 1-3 were ligated
by incubating at 16.degree. C. for 2.5 hours using DNA ligation kit
ver.2 (Takara Shuzo Co.). Using the ligation reaction liquid,
Escherichia coli strain GI724 (Invirogen Co.) was transformed
according to the method described in the Instruction Manual of
ThioFusion Expression System (Invirogen Co.). A microorganism
strain with plasmid in which the OBM cDNA fragment (nucleotide
sequence: 350-1111 in the (SEQ ID NO:2, amino acid sequence: 76-316
in the (SEQ ID NO:1) is fused in frame to a thioredoxin gene was
selected from the resulting ampicillin resistant transformants by
the analysis of restriction maps obtained by digestion with
restriction enzymes and DNA sequence determination. The
microorganism strain thus obtained was named GI724/pTrxOBM25.
[0217] (2) Expression of OBM in Escherichia coli
[0218] GI724/pTrxOBM25 and GI724 containing pTrxFus (GI724/pTrxFus)
were respectively cultured six hours with shaking at 30.degree. C.
in 2 ml of RMG-Amp culture medium (0.6% Na.sub.2HPO.sub.4, 0.3%
KH.sub.2PO.sub.4, 0.05% NaCl, 0.1% NH.sub.4Cl, 1.2% casamino acid
(Difco Co.), 1% glycerol, 1 mM MgCl.sub.2, and 100 .mu.g/ml
ampicillin (Sigma Co.), pH 7.4). The broth 0.5 ml of the broth was
added to 50 ml of Induction culture medium (0.6% Na.sub.2HPO.sub.4,
0.3% KH.sub.2PO.sub.4, 0.05% NaCl, 0.1% NH.sub.4Cl, 0.2% casamino
acid, 0.5% glucose, 1 mM MgCl.sub.2, 100 .mu.g/ml ampicillin, pH
7.4) and cultured with shaking at 30.degree. C. When OD.sub.600nm
reached about 0.5, L-tryptophan was added to a final concentration
of 0.1 mg/ml, followed by shaking the culture at 30.degree. C. for
an additional 6 hours. The culture broth was centrifuged at
3000.times.g to collect the cells, which were suspended in 12.5 ml
of PBS (10 mM phosphate buffer, 0.15 M NaCl, pH 7.4). The
suspension was subjected to an ultrasonic generator (Ultrasonics
Co.) to disrupt the cells. The disrupted cells were centrifuged at
7000.times.g for 30 minutes to obtain a supernatant liquid as a
soluble protein fraction. Ten .mu.l of this soluble protein
fraction was subjected to SDS polyacrylamide (10%) electrophoresis
under reducing conditions. As a result, a band with a molecular
weight of 40 kDa which was not detected in the soluble protein
fraction of GI724/pTrxFus was found in the soluble protein fraction
of GI724/pTrxOBM25 (FIG. 14). Accordingly, it was confirmed that a
fusion protein (Trx-OBM) of thioredoxin and OBM was expressed in
Escherichia coli.
[0219] (3) Binding capability of Trx-OBM to OCIF
[0220] Binding of the expressed Trx-OBM to OCIF was confirmed
according to the following experiment. Anti-thioredoxin antibody
(Invirogen Co.) which was diluted to 5000-fold with 10 mM sodium
hydrogen carbonate solution was added to a 96-well immunoplate
(Nunc Co.) in the amount of 100 .mu.l per well. After being allowed
to stand overnight at 4.degree. C., the liquid in the wells was
discarded. Two hundred .mu.l of a solution prepared by diluting
Block Ace.TM. (Snow Brand Milk Products Co., Ltd.) two-fold with
PBS (BA-PBS) was added to each well. After being allowed to stand
for one hour at room temperature, the solution was discarded and
soluble protein fractions originating from the above-described
GI724/pTrxOBM25 or GI724/pTrxFus, each diluted with BA-PBS in
various concentrations were added to each well in the amount of 100
.mu.l. After being allowed to stand for two hours at room
temperature, each well was washed three times with PBS-T and
charged with 100 .mu.l of OCIF (100 ng/ml) which was diluted with
BA-PBS. After being allowed to stand for two hours at room
temperature, each well was washed three times with PBS-T and
charged with 100 .mu.l of peroxidase-labeled rabbit anti- OCIF
polyclonal antibody (described in WO 96/26217) which was diluted
2,000-fold with BA-PBS. After being allowed to stand for two hours
at room temperature, each well was washed six times with PBS-T and
charged with 100 .mu.l of TMB solution (TMB Soluble Reagent, High
Sensitivity, Scytek Co.). After being allowed to stand for about 10
minutes at room temperature, each well was charged with 100 .mu.l
of termination solution (Stopping Reagent, Scytek Co.). Absorbance
of each well at 450 nm was measured by a microplate reader. The
results are shown in FIG. 15. There was no difference in absorbance
between the sample with the soluble protein fraction originating
from GI724/pTrxFus added thereto and the sample without the
addition of this soluble protein fraction. On the other hand, the
absorbance increased in the samples to which the soluble protein
fraction originating from GI724/pTrxOBM25 was added in proportion
to the concentration of the soluble protein fraction. The results
of the other experiment wherein the dilution rate of the soluble
protein fraction was maintained constant (1%) while adding OCIF
diluted with BA-PBS in different concentrations (0-100 ng/ml) are
shown in FIG. 16. It can be seen that the absorbance remained low
at any concentrations of OCIF in samples using a soluble protein
fraction originating from GI724/pTrxFus, whereas the absorbance
increased in proportion to the OCIF concentration in the samples to
which the soluble protein fraction originating from GI724/pTrxOBM25
was added. Based on these results, it was confirmed that Trx-OBM
which is produced from GI724/pTrxOBM25 has a capability of binding
to OCIF.
[0221] (4) Large-scale cultivation of Escherichia coli which
produces Trx-OBM
[0222] GI724/pTrxOBM25 cells were spread on RMG-Amp agar (0.6%
Na.sub.2 PO.sub.4, 0.3% KH.sub.2PO.sub.4, 0.05% NaCl, 0.1%
NH.sub.4Cl, 2% casamino acid, 1% glycerol, 1 mM MgCl.sub.2, 100:
g/ml ampicillin, 1.5% agar, pH 7.4) using a platinum transfer loop.
The cells were cultured overnight at 30.degree. C. The cultured
cells were suspended in 10 ml of Induction medium. The suspension
was added 5 ml for each to two 2 1 Erlenmeyer flasks containing 500
ml of Induction medium and cultured at 30.degree. C. with shaking.
When the OD.sub.600nm reached about 0.5, L-tryptophan was added to
a final concentration of 0.1 mg/ml. Culturing with shaking was
continued for six hours at 30.degree. C. The culture broth was
centrifuged for 20 minutes at 3000.times.g to collect the cells,
which were suspended in 160 ml of PBS. The suspension was subjected
to an ultrasonic generator (Ultrasonics Co.) to disrupt the cells.
The supernatant liquid was centrifuged for 30 minutes at
7000.times.g to obtain a soluble protein fraction.
[0223] (5) Preparation of OCIF-immobilized affinity column
[0224] Two g of TSKgel AF-Tolesyl Toyopal 650 (Tosoh Corp.) and 40
ml of 1.0 M potassium phosphate buffer (pH 7.5) containing 35.0 mg
of recombinant OCIF, which was prepared according to the method
described in WO 96/26217, were mixed. The mixture was gently shaken
overnight at 4.degree. C. to effect a coupling reaction. The
reaction mixture was centrifuged to remove the supernatant. To
inactivate excess active residues, 40 ml of 0.1 M Tris-HCl buffer
(pH 7.5) was added to the precipitated carrier and the mixture was
gently shaken at room temperature for one hour. The carrier in a
column was washed with 0.1 M glycine-HCl buffer (pH 3.3) containing
0.01% Polysorbate 80 and 0.2 M NaCl and 0.1 M sodium citrate buffer
(pH 2.0) containing 0.01% Polysorbate 80 and 0.2 M NaCl. The
carrier in the column was equilibrated by charging twice with 10 mM
sodium phosphate buffer (pH 7.4) containing 0.01% Polysorbate
80.
[0225] (6) Purification of Trx-OBM using OCIF-immobilized affinity
column
[0226] Unless otherwise indicated, purification of Trx-OBM was
carried out at 4.degree. C. The above-mentioned OCIF-immobilized
affinity carrier (10 ml) and the soluble protein fraction (120 ml)
prepared in Example 15(4) were mixed. The mixture was gently shaken
overnight at 4.degree. C. in four 50-ml centrifuge tubes using a
rotor. An Econo-column.TM. (internal diameter: 1.5 cm, length: 15
cm, manufactured by BioRad Co.) was filled with the carrier in the
mixture. The column was charged with 300 ml of PBS containing 0.01%
Polysorbate 80, 100 ml of 10 mM sodium phosphate buffer (pH 7.0)
containing 0.01% Polysorbate 80 and 2 M NaCl, and 100 ml of 0.1 M
glycine-HCl buffer (pH 3.3) containing 0.01% Polysorbate 80 and 0.2
M NaCl, in that order. Next, proteins adsorbed in the column were
eluted with 0.1 M sodium citrate buffer (pH 2.0) containing 0.01%
Polysorbate 80 and 0.2 M NaCl. The eluate was collected in 5 ml
portions. Each fraction thus collected was immediately neutralized
with addition 10% volume of 2 M Tris buffer (pH 8.0). Presence or
absence of Trx-OBM in the eluted fractions was determined according
to the method previously described in Example 15(3) (the binding
capability to OCIF). The fractions containing Trx-OBM were
collected and purified further.
[0227] (7) Purification of Trx-OBM by gel filtration
[0228] About 25 ml of Trx-OBM fractions obtained in Example 15 (6)
was concentrated to about 0.5 ml by centrifuge using Centriplus 10
and Centricon 10 (Amicon Co.). This sample was applied to a
Superose 12 HR 10/30 column (1.0.times.30 cm, Pharmacia Co.)
previously equilibrated with PBS containing 0.01% Polysorbate 80.
For the separation, PBS containing 0.01% Polysorbate 80 was used as
a mobile phase at a flow rate of 0.25 ml/min. The eluate from the
column was collected in 0.25 ml portions. The Trx-OBM in the
thus-collected fractions was detected by the same method as
previously described in Example 15(3) and by SDS-polyacrylamide
electrophoresis (10-15% polyacrylamide gel, Pharmacia Co.) using
Phast System (Pharmacia Co.) and silver staining. Fractions (Fr.
20-23) containing purified Trx-OBM were collected and the protein
concentration of Trx-OBM was determined. The measurement of the
protein concentration was carried out using bovine serum albumin as
a standard substance using DC-Protein assay kit (BioRad Co.).
Example 16
Osteoclast formation-inducing activity of OBM
[0229] pOBM291 and pcDL-SR .alpha. 296 were respectively
transfected into COS-7 cells using Lipofectamine (Gibco Co.). The
cells were cultured in DMEM containing 10% FCS for one day,
trypsinized, plated on cover slips (15 mm round shape, manufactured
by Matsunami Co.) in 24-well plates at 5.times.10.sup.4 cells per
well, and cultured for 2 days. The culture plate was washed once
with PBS. The cells were fixed with PBS containing 1%
paraformaldehyde at room temperature for 8 minutes. The plate on
which the fixed cells were attached was washed 6 times with PBS,
then 700 .mu.l of mouse spleen cells suspended at
1.times.10.sup.6/ml in .alpha.-MEM containing 10.sup.-8 M
active-form vitamin D.sub.3, 10.sup.-7 M dexamethasone, and 10%
fetal bovine serum were added to each well. Millicell PCF
(Millipore Co.) was set in each well and a suspension of ST2 cells
in the above-mentioned culture medium (4.times.10.sup.4/ml) were
added, 700 .mu.l per well, into the Millicell PCF followed by
incubation at 37.degree. C. for 6 days. After the culture, the
Millicell PCF was removed, the plate was washed once with PBS, and
the cells were fixed with acetone-ethanol solution (50:50) for one
minute. Then, cells exhibiting tartaric acid-resistant acid
phophatase activity (TRAP), which is a specific marker for
osteoclast, were selectively stained using LEUKOCYTE ACID
PHOSPHATASE kit (Sigma Co.). As a result of microscopic
observation, TRAP-positive cells were not detected in the wells in
which COS-7 cells transfected with pcDL-SR .alpha.296 were fixed.
In contrast, 45.+-.18 (average.+-.standard deviation, n=3) TRAP
positive cells were observed in the wells in which COS-7 cells
transfected with pOBM291 were fixed. Moreover, it was also
confirmed that calcitonin bound to these TRAP positive cells. Based
on these findings, it has been proven that OBM has osteoclast
formation-inducing activity.
Example 17
Osteoclast formation-inducing activity of Trx-OBM and secreted-form
OBM
[0230] Mouse spleen cells were suspended in .alpha.-MEM containing
10.sup.-8 M active-form vitamin D.sub.3, 10.sup.-7 M dexamethasone,
and 10% fetal bovine serum at a concentration of
2.times.10.sup.6/ml. The suspension was added to a 24 well plate in
the amount of 350 .mu.l per well. Each well was then charged with
350 .mu.l of a solution prepared by diluting purified Trx-OBM with
the above-mentioned culture medium (40 ng/ml), 350 .mu.l of
solution prepared by 10-fold diluting conditioned medium which was
produced by culturing 293-EBNA cells, in which pCEP sOBM or pCEP4
were transfected, in IMDM-10% FCS, with the above-mentioned culture
medium, or 350 .mu.l only of the above-mentioned culture medium.
The Millicell PCF (Mollipore Co.) was set on each well, to which
600 .mu.l of ST2 cells which was suspended in the above-mentioned
culture medium (4.times.10.sup.4/ml) were added. After cultured for
six days, Millicell PCF was removed. The plate was washed once with
PBS and the cells were fixed with acetone-ethanol solution (50:50)
for one minute. Then, the cells exhibiting the tartaric acid
resistant acidic phophatase activity (TRAP activity) were
selectively stained using LEUKOCYTE ACID PHOSPHATASE kit (Sigma
Co.). The result of microscopic observation revealed that no cells
exhibiting the TRAP activity were detected in the wells to which no
Trx-OBM was added, whereas 106.+-.21 (average.+-.standard
deviation, n=3) TRAP-positive cells were observed in the wells to
which Trx-OBM was added. Similarly, while no cells exhibiting TRAP
activity were detected in the wells to which conditioned medium of
293-EBNA transfected with pCEP4 had been added, 120.+-.31
(average.+-.standard deviation, n=3) TRAP positive cells were
observed in the wells to which conditioned medium of 293-EBNA
transfected with pCEPsOBM had been added. Moreover, it was also
confirmed that calcitonin binds to these TRAP positive cells. These
results have proven that Trx-OBM and secreted-form OBM exhibit
osteoclast formation-inducing activity.
Example 18
Identity of the protein OBM expressed by the cDNA of the present
invention and the natural type OCIF-binding protein of the present
invention
[0231] (1) preparation of rabbit anti-QBM polyclonal antibody
[0232] Three male Japanese white rabbits (weight: 2.5-3.0 kg,
supplied by Kitayama Labes Co.) were immunized with the purified
OBM (thioredoxin-OBM fusion protein) produced according to the
method in Examples 14(6) and 14(7) by subcutaneously injecting 1
ml/dose of emulsion prepared by mixing 200 .mu.g/ml of the purified
OBM with equal volume of Freund's complete adjuvant (DIFCO Co.),
six times, once a week. Ten days after the last immunization, the
rabbits were exsanguinated. Antibody was purified from the serum as
follows. Ammonium sulfate was added to the antiserum which was
diluted two-fold with PBS to a final concentration of 40% (w/v %).
After being allowed to stand for one hour at 4.degree. C., the
mixture was centrifuged for 20 minutes at 8000.times.g to obtain a
precipitate. The precipitate was dissolved in a small amount of
PBS, dialyzed against PBS at 4.degree. C., and loaded to a Protein
G-Sepharose column (manufactured by Pharmacia Co.). After washed
with PBS, the adsorbed immunoglobulin G was eluted with 0.1 M
glycine-HCl buffer solution (pH 3.0). The eluate was immediately
neutralized with 1.5 M Tris-HCl buffer (pH 8.7). After dialyzing
the eluted protein fractions against PBS, the absorbance at 280 nm
was measured to determine the protein concentration (E.sup.1%
13.5). Anti-OBM antibody labeled with horseradish peroxidase was
prepared using a maleimide-activated paroxidase kit (Pierce Co.) as
follows. 80 .mu.g of N-succinimide-S-acetyl thioacetic acid was
added to 1 mg of the purified antibody and reacted at room
temperature for 30 minutes. Five mg of hydroxylamine was added to
the resulting mixture to deacetylate the antibody. The modified
antibody was fractionated using a polyacrylamide desalting column.
The protein fractions were mixed with 1 mg of maleimide-activated
peroxidase and reacted for one hour at room temperature to obtain
enzyme-labeled antibody.
[0233] (2) Capability of rabbit anti-OBM polyclonal antibody to
inhibit specific binding of the protein (OBM) expressed by the cDNA
of the present invention or the natural type protein of the present
invention with OCIF
[0234] Purified OBM (thioredoxin-OBM fused protein) obtained
according to the method described in the Examples 15(6) and 15(7)
and the natural type purified OCIF-binding protein of the Example
2(4) were dissolved respectively in 0.1 M sodium carbonate buffer
to a concentration of 2 .mu.g/ml. An aliquot of each solution was
added 100 .mu.l per well respectively to a 96-well immunoplate
(manufactured by Nunc Co.). The plate was allowed to stand
overnight at 4.degree. C. 200 .mu.l of 50% Block Ace was added to
each well and the plate was allowed to stand at room temperature
for one hour. After washing each well three times with PBS
containing 0.1% Polysolbate 20 (P20-PBS), 100 .mu.l of rabbit
anti-OBM antibody solution which was dissolved in 25% Block Ace
prepared with P20-PBS to a concentration of 200 .mu.g/ml or 100
.mu.l of 25% Block Ace (containing no antibody) was added to each
well, followed by incubation at 37.degree. C. for one hour. Each
well was washed three times with P20-PBS and charged with 100
.mu.l/well of a binding test solution (P20-PBS containing 0.2% calf
serum albumin, 20 mM Hepes, and 0.1 mg/ml heparin) to which 20
ng/ml of .sup.125I-labeled OCIF described in the Example 8(3) was
added. Alternatively, each well was charged with 100 .mu.l/well of
another binding test solution containing 8: g/ml of unlabeled OCIF
in addition to 20 ng/ml .sup.125I-labeled OCIF. After incubating
these immunoplates at 37.degree. C. for one hour, the wells were
washed with P20-PBS six times. The amount of .sup.125I in each well
was measured by a gamma counter. The results are shown in FIG. 17.
As shown in the figure, both the purified OBM expressed using the
cDNA of the present invention and the protein that specifically
bind the natural type OCIF-specifically binding protein of the
present invention do not bind to the .sup.125I-labeled OCIF at all,
when they were treated with the rabbit anti-OBM polyclonal
antibody, whereas both proteins bound .sup.125I-labeled OCIF when
untreated with the antibody. The binding of both proteins to
.sup.125I-labeled OCIF was confirmed to be clearly specific,
because those bindings are significantly inhibited by the addition
of 400-fold concentration of unlabelled OCIF (8 .mu.g/ml). Based on
the results described above, the rabbit anti-OBM polyclonal
antibody recognizes both the OBM which is the protein expressed
using the cDNA of the present invention and the natural-type
OCIF-binding protein of the present invention, and it inhibits the
specific binding of these proteins with OCIF.
Example 19
Cloning of human OBM cDNA
[0235] (1) Preparation of mouse OBM primer
[0236] The mouse OBM primers prepared according to the method of
the Examples (OBM#3 and OBM#8) described above, were used for
screening of human OBM cDNA. The sequences are shown in SEQ ID NO:9
and SEQ ID NO:6, respectively.
[0237] (2) Isolation of human OBM cDNA fragment by PCR
[0238] Human OBM cDNA fragments were obtained by PCR using the
mouse OBM cDNA primers prepared in (1) above and Human Lymph Node
Marathon ready cDNA (Clontech Co.) as a template. The conditions
for PCR were shown as follows: TABLE-US-00001 10x EX Taq buffer
(Takara Shuzo Co.) 2:1 2.5 mM dNTP 1.6:1 cDNA solution 1:1 EX Taq
(Takara Shuzo Co.) 0.2:1 Distilled water 14.8:1 40:M primer OBM#3
0.2:1 40:M primer OBM#8 0.2:1
[0239] These solutions were mixed in a microfuge tube and
pre-incubated at 95.degree. C. for 2 minutes, followed by 40 cycles
of a three-stage reaction consisted of reactions at 95.degree. C.
for 30 seconds, at 57.degree. C. for 30 seconds, and at 72.degree.
C. for 2.5 minutes. After the reaction, the solution was incubated
for 5 minutes at 72.degree. C. and a portion of the solution was
subjected to electrophoresis on an agarose gel. A DNA fragment
(about 690 bp) amplified by the mouse OBM cDNA primers described
above was detected.
[0240] (3) Purification of the human OBM cDNA fragment amplified by
PCR and determination of the nucleotide sequence
[0241] The human OBM cDNA fragment obtained in Example 19 (2) was
separated by electrophoresis on an agarose gel and further purified
using a QIAEX gel extraction kit (Qiagen Co.). PCR was again
performed using the purified human OBM cDNA fragment as a template
and the mouse OBM cDNA primers described above, to produce a large
quantity of the human OBM cDNA fragment. The DNA fragment was
purified by a QIAEX gel extraction kit in the same manner as above.
The nucleotide sequence of the purified human OBM cDNA fragment was
determined using a Taq Dye Deoxy Terminator Cycle Sequencing FS kit
(Perkin Elmer Co.) using OBM#3 or OBM#8 SEQ ID NO:9 and SEQ ID
NO:6, respectively) as a primer. When compared with the sequence of
corresponding area of the mouse OBM cDNA, the nucleotide sequence
of the human OBM cDNA fragment showed 80.7% homology with that of
the mouse OBM cDNA.
[0242] (4) Screening of a full-length human OBM cDNA by
hybridization using the human OBM cDNA fragment (about 690 bp) as a
probe
[0243] A full-length human OBM cDNA was screened using the human
OBM cDNA fragment (about 690 bp) that was purified in the Example
19(3) and labeled with [.alpha..sup.32p] dCTP using a Megaprime DNA
Labeling kit (Amersham Co.). Human Lymph Node 5'-STRETCH PLUS cDNA
library (Clontech Co., the U.S.A) was screened using the DNA probe.
According to the manufacturer's protocol, Escherichia coli C600 Hfl
was infected with the recombinant phage for 15 minutes at
37.degree. C. The infected Escherichia coli was added to an LB agar
(1% trypton, 0.5% yeast extract, 1% NaCl, 0.7% agar) which was
heated at 45.degree. C. The LB agar was poured onto an LB agar
plate containing 1.5% agar. After overnight incubation at
37.degree. C., HyBond-N.TM. (Amersham Co.) was placed to the plate
on which plaques were produced and stored for about 3 minutes.
According to a conventional method, the filter was treated with
alkaline solution, neutralized, and dipped in 2.times.SSC solution.
DNA was then immobilized onto the filter using the UV CROSSLINKER
(Stratagene Co.). The resulting filter was dipped into Rapid-hyb
buffer (Amersham Co.). After pretreatment for 15 minutes at
65.degree. C., the filter was placed in Rapid-hyb buffer containing
the heat-denatured human OBM cDNA fragment (about 690 bp,
5.times.10.sup.5 cpm/ml) described above. After overnight
hybridization at 65.degree. C., the filter was washed with
2.times.SSC, 1.times.SSC, and 0.1.times.SSC, each containing 0.1%
SDS, in this order respectively for 15 minutes at 65.degree. C.
Several positive clones obtained were further purified by repeating
the screening twice. A clone possessing an insert (about 2.2 kb)
was selected from the purified clones and was used in the following
experiments. This purified phage was named .lamda. hOBM. About:g of
DNA was obtained from the purified .lamda. hOBM using a QIAGEN
Lambda kit (Qiagen Co.) according to the manufacturer's protocol.
The DNA was digested with restriction enzyme SaII and subjected to
electrophoresis on an agarose gel to separate the hOBM insert cDNA
(about 2.2 kb). This DNA fragment purified using the QIAEX gel
extraction kit (Qiagen Co.) was digested with restriction enzyme
Sail and inserted into plasmid pUC19 (MBI Co.) which was previously
digested with a restriction enzyme SaII and dephosphorylated, using
a DNA ligation kit ver. 2 (Takara Shuzo Co.). Escherichia coli DH 5
.alpha. (Gibco BRL Co.) was transformed with the pUC19 containing
the resulting DNA fragment. The resulting transformant was named
pUC19hOBM. The transformant was grown and pUC19hOBM in which the
human OBM cDNA (about 2.2 kb) was inserted and purified by a
conventional method.
[0244] (5) Determination of nucleotide sequence of cDNA encoding
the entire amino acid sequence of human OBM
[0245] The nucleotide sequence of the resulting human OBM cDNA
obtained in Example 19 (4) was determined using the Taq Dye Deoxy
Terminator Cycle Sequencing FS kit (Parkin Elmer Co.).
Specifically, the nucleotide sequence of the inserted fragment was
determined using pUC19hOBM as a template. As primers, primers for
the determination of the nucleotide sequence of the inserted
fragment DNA in pUC19hOBM, M13 Primer M3 and M13 Primer RV
(manufactured by Takara Shuzo Co.), and a synthetic primer, human
OBM#8, designed based on the nucleotide sequence of the human OBM
cDNA fragment (about 690 bp) were used.
[0246] The nucleotide sequence of the primers used, M13 Primer M3
and M13 Primer RV, are respectively shown as the Sequence ID No. 4
and No. 5. The amino acid sequence of human OBM deduced from the
nucleotide sequence of human OBM cDNA is shown in the Sequence
Table as Sequence ID No. 11. The nucleotide sequence of human OBM
cDNA is shown as Sequence ID No. 12.
[0247] The Escherichia coli which was transformed by the pUC19hOBM,
which is the plasmid containing the resulting human OBM cDNA, was
deposited in National Institute of Bioscience and Human Technology,
Agency of Industrial Science and Technology, on Aug. 13, 1997 as
deposition No. FERM BP-6058.
Example 20
[0248] Radioiodination of OCIF with .sup.125I and quantitative
analysis of .sup.125I-labled OCIF by ELISA
[0249] OCIF was labeled with .sup.125I using the IODO-GEN method.
Twenty .mu.l of 2.5 mg/ml IODO-GEN-chloroform solution were
transferred to a 1.5 ml Eppendorf tube and the chloroform was
evaporated at 40.degree. C., thereby providing a tube coated with
IODO-GEN. The tube was washed three times with 400 .mu.of 0.5 M
sodium phosphate buffer solution (Na-Pi, pH 7.0), followed by the
addition of 5 .mu.l 1 of 0.5 M Na-Pi (pH 7.0). To this tube was
added 1.3 .mu.l (18.5 MBq) of Na-.sup.125I solution (Amersham Co.,
NEZ-033H), immediately followed by the addition of 10 .mu.l of 1
mg/ml OCIF solution (monomer type or dimer type). The mixture was
mixed in a vortex mixer and allowed to stand at room temperature
for 30 seconds. This solution was transferred to a tube to which 80
.mu.l of 0.5 M Na-Pi (pH 7.0) solution containing 10 mg/ml
potassium iodine and 5 .mu.l of a phosphate buffered saline
solution containing 5% bovine serum albumin (BSA-PBS) were
previously added. The solution was mixed, applied to a spin column
(1 ml, G-25 Sephadex fine, manufactured by Pharmacia Co.) which was
equilibrated with BSA-PBS in advance, and centrifuged for 5 minutes
at 2,000 rpm. Four hundred .mu.l of BSA-PBS were added to the
fractions eluted from the column. After mixing, 2 .mu.l of the
solution were used to measure the radioactivity by a gamma counter.
The radiochemical purity of the .sup.125I-labeled OCIF solution
obtained above was measured by counting the radioactivity of
fractions precipitated by 10% trichloroacetic acid (TCA).
[0250] The biological activity of the .sup.125I-labeled OCIF was
measured according to the method described in WO 96/26217. The
concentration of the .sup.125I-labeled OCIF was measured using the
ELISA method as follows. Specifically, 50 mM NaHCO.sub.3 (pH 9.6)
in which rabbit anti-OCIF polyclonal antibody described in the WO
96/26217 was dissolved to a concentration of 2 .mu.g/ml was added
to each well of a 96-well immunoplate (MaxiSorp.TM., manufactured
by Nunc Co.) in the amount of 100 .mu.l per well. After these wells
were allowed to stand overnight at 4.degree. C., solution was
removed. Then the wells were charged with a mixed aqueous solution
of Block Ace.TM. (Snow Brand Milk Products Co., Ltd.) and a
phosphate buffered saline solution (25:75) (B-PBS) in the amount of
200 .mu.l/well. The plate was then allowed to stand for two hours
at room temperature. After the solution was removed, the wells were
washed three times with a phosphate buffered saline solution
containing 0.01% Polysolvate 80 (P-PBS). Next, B-PBS containing
.sup.125-labeled OCIF sample or the standard OCIF was added in the
amount of 100 .mu.l/well. The plate was then allowed to stand for
two hours at room temperature. After the solution was removed, each
well was washed six times with 200 .mu.l of P-PBS. A solution
prepared by diluting peroxidase-labeled rabbit anti-OCIF polyclonal
antibody with B-PBS was added in the amount of 100 .mu.l/well. The
plate was allowed to stand for two hours at room temperature. After
the solution was removed, the wells were washed six times with 200
.mu.l of P-PBS. Then, a TMB solution (TMB Soluble Reagent, High
Sensitivity, Scytek Co.) was added in the amount of 100 .mu.l/well.
After being allowed to stand at room temperature for 2-3 minutes,
100:1 of a termination solution (Stopping Reagent, Scytek Co.) was
added to each well. Absorbance of each well was measured at 450 nm
using a microplate reader. The concentration of .sup.125I-labeled
OCIF was determined with a calibration curve prepared using the
standard OCIF.
Example 21
Expression of the protein encoded by cDNA of the present
invention
[0251] (1) Construction of hOBM expression vector for animal cells
pUChOBM was digested with restriction enzyme Sail and a DNA
fragment (about 2.2 kb) were purified by electrophoresis on an 1%
agarose gel. The ends of the DNA fragments were blunted using a DNA
blunting kit (Takara Shuzo Co.) (blunted hOBMcDNA fragment).
Expression plasmid pcDL-SR .alpha. 296 (Molecular and Cellar
Biology, Vol. 8, pp 466-472 (1988)) was digested with restriction
enzyme EcoRI, blunted with blunting kit and ligated with the
blunted hoBM cDNA fragment using DNA ligation kit ver. 2.
Escherichia coli DH .alpha. was transformed with the ligation
reaction. A plasmid in the resulting ampicillin resistant
transformant was subjected to digestion with restriction enzyme to
analyze the DNA restriction map and determine the DNA sequence. As
a result, a strain having a plasmid in which hOBM cDNA is inserted
in the same direction of transcription as that of SR .alpha.
promotor was selected. The microorganism strain was named DH5
.alpha./phOBM.
[0252] (2) Expression of human OBM in COS-7 cells
[0253] Escherichia coli DH5 .alpha./phOBM was cultured and plasmid
phOBM was purified using Qiafilter Plasmid Midi kit (Qiagen Co.)
phOBM was transfected using Lipofectamine into COS-7 cells in the
wells of a 6-well plate and cultured for two days in DMEM
containing 10% fetal bovine serum. The culture medium was replaced
with cysteine-methionine-free DMEM (manufactured by Dainippon
Seiyaku Co., Ltd.) to which 5% dialysed fetal bovine serum was
added (88:1/well). The cells were incubated for 15 minutes,
followed by addition of 14:1 of Express Protein Labeling Mix (NEN
Co., 10 mCi/ml). After four hours incubation, 200:1 of DMEM
containing 10% fetal bovine serum was added to each well. The cells
were cultured for one hour and washed twice with PBS. Then, 0.5 ml
of a TSA buffer (10 mM Tris-HCl containing 0.14 M NaCl and 0.025%
NaN.sub.3, pH 8.0) containing 1% Triton X-100, 1% bovine
hemoglobin, 10 .mu.g/ml leupeptin, 0.2 TIU/ml aprotinin, and 1 mM
PMSF was added to each well and the mixtures were allowed to stand
for one hour on ice. The cells were mixed by pipetting and
centrifuged at 3,000.times.g, for 10 minutes at 4.degree. C., to
obtain supernatants. Two hundred .mu.l of a dilution buffer (TSA
buffer containing 0.1% Triton X-100, 0.1% bovine hemoglobin, 10
.mu.g/ml leupeptin, 0.2 TIU/ml aprotinin, and lmM PMSF) was added
to 100 .mu.l of the supernatant from each well. The resulting
mixtures were agitated at 4.degree. C. for one hour together with
Protein A Sepharose (50:1) and centrifuged at 1,500.times.g for one
minute at 4.degree. C., to collect supernatants, thereby removing
the protein which non-specifically adsorbed Protein A Sepharose.
OCIF (1 .mu.g) was added to the supernatants and the mixtures were
agitated for one hour at 4.degree. C. to bind human OBM and OCIF.
Then, rabbit anti-OCIF polyclonal antibody (50 .mu.g) was added,
followed by agitation at 4.degree. C. for one hour. Protein A
Sepharose (10 1.mu.l) was added to the resulting solution, followed
by agitation at 4.degree. C. for an additional hour. The mixtures
thus obtained were centrifuged for 1 minute at 1,500.times.g at
4.degree. C. to collect precipitates. The precipitates were washed
twice with a dilution buffer, twice with bovine hemoglobin-free
dilution buffer, once with TSA buffer, and once with 50 mM Tris-HCl
(pH 6.5). After addition of SDS buffer containing 10%
.beta.-mercaptoethanol (0.125 M Tris-HCl, 4% sodium dodecylsulfate,
20% glycerol, 0.002% Bromophenol Blue, pH 6.8), the mixture was
heated for 5 minutes at 100.degree. C. and subjected to SDS-PAGE
(12.5% polyacrylamide gel, Daiichi Pure Chemical Co.). The gel was
fixed and dried according to a conventional method. After isotope
signals were enhanced using Amplify.TM. (Amersham Co.), the dried
gel was subjected to autoradiography at -80.degree. C. using Bio
Max MR film (Kodak Co.). The results are shown in FIG. 18, which
shows that the molecular weight of the protein encoded by the cDNA
of the present invention is about 40,000.
Example 22
Binding of the protein encoded by cDNA of the present invention and
OCIF
[0254] PhOBM, which was purified in the same manner as in the
Example 21(2), was transfected into COS-7 cells in each well of a
24-well plate using Lipofectamine. After cultured for 2 to 3 days,
the cells were washed with serum-free DMEM. Two hundred .mu.l of a
culture medium for a binding test medium (serum-free DMEM to which
0.2% bovine serum albumin, 20 mM Hepes buffer solution, 0.1 mg/ml
heparin, and 0.2% NaN.sub.3 were added) containing 20 ng/ml of
.sup.125I-labeled OCIF was added to the wells. To the other wells,
200 .mu.l of culture medium for the binding test medium containing
8 .mu.g/ml of unlabeled OCIF in addition to 20 ng/ml of
.sup.125I-labeled OCIF, was added. After incubation for one hour at
37.degree. C. in a CO.sub.2 incubator (5% CO.sub.2), the cells were
washed twice with 500 .mu.l of a phosphate buffered saline solution
containing 0.1 mg/ml of heparin. Then, 500 .mu.l of 0.1 N NaOH
solution was added to each well and the plate was allowed to stand
for 10 minutes at room temperature to dissolve the cells. The
radioactivity of .sup.125I in the wells was measured by a gamma
counter. As a result, as shown in FIG. 19, it was confirmed that
the .sup.125I-labeled OCIF binds only to the cells transfected with
phOBM. Moreover, the binding was significantly inhibited by adding
400-fold excess unlabelled OCIF (8 .mu.g/ml). Based on the results
described above, the protein, human OBM encoded by the cDNA in the
phOBM was confirmed to specifically bind to OCIF on the surface of
COS-7 cells.
Example 23
Crosslinking of .sup.125I-labeled OCIF and the protein encoded by
the cDNA of the present invention
[0255] Crosslinking of .sup.125I-labeled monomer type OCIF and the
protein encoded by the cDNA of the present invention was carried
out to further investigate the characteristics of the protein
encoded by the cDNA of the present invention. After constructing
expression vector phOBM and transfecting into COS-7 cells according
to the method used in the Examples 21(1) and 21(2), 200:1 of
binding test medium containing .sup.125I-labeled OCIF (25 ng/ml)
described above was added. The binding test medium to which
unlabeled OCIF was added at a 400-fold concentration in addition to
.sup.125I-labeled OCIF was used for the other wells. After cultured
for one hour at 37.degree. C. in a CO.sub.2 incubator (5%
CO.sub.2), the cells were washed twice with 500 .mu.l of phosphate
buffered saline containing 0.1 mg/ml heparin. Five hundred il of
phosphate buffered saline in which 100 .mu.g/ml of a crosslinking
agent (DSS: disuccinimidyl suberate, manufactured by Pierce Co.)
was dissolved was added to the cells, followed by incubation for 10
minutes at 0.degree. C. The cells in these wells were washed twice
with 1 ml of ice-cold phosphate buffered saline. After an addition
of 100 .mu.l of 20 mM Hepes buffer solution containing 1% Triton
X-100 (Wako Pure Chemicals Co., Ltd.), 2 mM PMSF
(Phenylmethylsulfonyl fluoride, Sigma Co.), 10: M Pepstatin (Wako
Pure Chemicals Co., Ltd.), 10 .mu.M leupeptin (Wako Pure Chemicals
Co., Ltd.), 10: M antipain (Wako Pure Chemicals Co., Ltd.) and 2 mM
EDTA (Wako Pure Chemicals Co., Ltd.), the wells were allowed to
stand for 30 minutes at room temperature to dissolve the cells.
These samples (15 .mu.l aliquots) were treated with SDS under
reducing conditions according to a conventional method and
subjected to SDS-electrophoresis using 4-20% polyacrylamide
gradient gel (Daiichi Pure Chemical Co., Ltd.). After
electrophoresis, the gel was dried and subjected to autoradiography
for 24 hours at -80.degree. C. using BioMax MS film (Kodak Co.) and
BioMax MS sensitization screen (Kodak Co.). The film subjected to
autoradiography was developed according to a conventional method.
As a result, a band of a molecular weight in the range of
90,000-110,000, shown in FIG. 20, was detected by crosslinking of
.sup.125I-labeled monomer type OCIF and the protein encoded by the
cDNA of the present invention.
Example 24
Expression of secreted-form human OBM
[0256] (1) Construction of secreted-form human OBM expression
plasmid
[0257] A PCR was carried out using human OBM SF (SEQ ID NO:13) and
mouse OBM #8 (SEQ ID NO:6) as primers and pUC19hOBM as a template.
After purification by electrophoresis on an agarose gel, the
product was digested with restriction enzymes SpII and HindIII, and
further purified by electrophoresis on an agarose gel to obtain a
purified fragment (0.27 kb). Human OBM cDNA was partially digested
with restriction enzyme Dral and DNA fragments digested with Dral
at one site were purified by electrophoresis on an agarose gel. The
purified fragment was further digested with restriction enzyme
HindIII. The 0.53 kb Dral/HindIII fragment was purified by
electrophoresis on an agarose gel. The purified fragment was
ligated with the 0.27 kb SplI/HindIII fragment derived from the PCR
described above using ligation kit ver. 2 (Takara Shuzo Co.)
together with HindIII/EcoRI fragment (5.2 kb) of pSec TagA
(Invirogen Co.). Escherichia coli DH5 .alpha. was transformed using
the reaction product. Plasmids were purified by alkali SDS method
from the resulting ampicillin resistant transformants and digested
with restriction enzymes to select a plasmid containing 0.27 kb and
0.53 kb-fragments as inserts in pSec TagA. This plasmid was
confirmed to have a sequence encoding the secreted human OBM by
sequencing using a Tag dyedeoxyterminator cycle sequencing FS kit
(Perkin Elmer Co.). The plasmid was digested with restriction
enzymes Nhel and Xhol to prepare a fragment (0.8 kb) corresponding
to the secreted human OBM cDNA by electrophoresis on an agarose
gel. This fragment was inserted into the NheI and XhoI fragment
(10.4 kb) of an expression vector pCEP4 (Invirogen Co.) using
ligation kit and Escherichia coli DH5 .alpha. was transformed using
the reaction product. Plasmids were purified by alkali-SDS method
from the resulting ampicillin resistant transformants and digested
with restriction enzymes to select an Escherichia coli having the
expression plasmid for secreted-form human OBM (pCEPshOBM). The
Escherichia coli containing the pCEPshOBM was cultured and
pCEPshOBM was purified using a Qiafilter.TM. plasmid midi kit
(Qiagen Co.).
[0258] (2) Expression of secreted-form OBM
[0259] 293-EBNA cells were suspended in IMDM containing 10% FCS
(IMDM-10% FCS), added into a 24-well plate coated with collagen
(manufactured by Sumitomo Bakelite Co., Ltd.) in a cell density of
2.times.10.sup.5/2 ml/well and cultured overnight. The cells were
transfected with 1 .mu.g of pCEPshOBM or pCEP4 using 4 .mu.l of
Lipofectamine (Gibco Co.). After cultured for two days in 0.5 ml of
a serum-free IMDM or IMDM-10% FCS, the culture supernatants were
collected. Expression of the secreted human OBM in the culture
supernatant was detected as follows. Sodium bicarbonate was added
to the culture supernatants to a final concentration of 0.1 M and
the mixtures were added to a 96-well plate. The plate was allowed
to stand overnight at 4.degree. C., thereby immobilizing human OBM
in the culture supernatants on the 96-well plate. The plate was
blocked using Block Ace.TM. (Snow Brand Milk Products Co., Ltd.)
solution four-fold diluted with PBS (B-PBS) and allowed to stand
for two hours at room temperature. After adding 3-100 ng/ml of OCIF
which was diluted with B-PBS to each well, the plate was allowed to
stand for two hours at 37.degree. C., followed by wash with PBS
containing 0.05% Polysolvate 20 (P-PBS). Then, 100 .mu.l of a
peroxidase-labeled rabbit anti-OCIF polyclonal antibody described
in WO 96/26217 which was diluted with B-PBS was added to each well.
After allowing to stand for two hours at 37.degree. C., the wells
were washed six times with P-PBS. Then, TMB solution (TMB Soluble
Reagent, High Sensitivity, Scytek Co.) was added in the amount of
100:1 per well and the mixture was allowed to stand at room
temperature for about 10 minutes. The reaction was terminated by
the addition of 100 .mu.l of termination solution (Stopping
Reagent, Scytek Co.) to each well. Absorbance at 450 nm for each
well was measured by a microplate reader. The results are shown in
FIG. 21, which indicates that the absorbance at 450 nm increased
according to the concentration of the added OCIF in the plate in
which the conditioned medium of the cells transfected with
pCEPshOBM was immobilized. On the other hand, no increase in
absorbance was seen in the wells in which the conditioned medium of
the cells transfected with vector pCEP4 was immobilized. FIG. 22
shows the results of an experiment wherein the proportion of the
conditioned medium used for immobilization was changed within a
range of 5-90% in the presence of a constant concentration of OCIF
(50 ng/ml). The absorbance at 450 nm increased according to the
increase in the proportion of the conditioned medium in the plate
wherein the conditioned medium of the cells transfected with
pCEPshOBM was immobilized, whereas no such increase in absorbance
was seen in the plate wherein the conditioned medium of the cells
transfected with vector pCEP4 was immobilized. From these results,
it was confirmed that secreted-form human OBM is produced in the
conditioned medium of the cells transfected with pCBPshOBM.
Example 25
Expression of thioredoxin-human OBM fusion protein (Trx-hOBM)
[0260] (1) Construction of a thioredoxin-human OBM fusion protein
(Trx-hOBM) exression vector
[0261] Ten .mu.l of 10.times.ExTaq buffer (Takara Shuzo Co.), 8:1
of 10 mM dNTP (Takara Shuzo Co.), 77.5 .mu.l of sterilized
distilled water, 2:1 of an aqueous solution of pUC19hOBM (10
ng/.mu.l), 1 .mu.l of primer, mouse OBM #3 (100 pmol/.mu.l,
Sequence Table, Sequence ID No. 9), 1 .mu.l of primer, hOBM SalR2
(100 pmol/.mu.l, Sequence Table, Sequence ID No. 14), and 0.5 .mu.l
of ExTaq (5 u/.mu.l) (Takara Shuzo Co.) were mixed and reacted
(PCR) in a micro centrifugel tube. After the reaction at 95.degree.
C. for 5 minutes, at 50.degree. C. for one second, at 55.degree. C.
for one minute, at 74.degree. C. for one second, and at 72.degree.
C. for 5 minutes, a cycle reaction consisting of a reaction at
96.degree. C. for one minute, at 50.degree. C. for one second, at
55.degree. C. for one minute, at 74.degree. C. for one second, and
at 72.degree. C. for 3 minutes, was repeated 25 times. From the
total reaction mixture DNA fragment (750 bp) was purified. The
whole amount of purified DNA fragment was digested with restriction
enzymes SaII (Takara Shuzo Co.) and BspHI (New England Bilabs Co.),
and subjected to electrophoresis on a 1% agarose gel to obtain
purified DNA fragment (Fragment 1, about 320 bp). The fragment was
dissolved in 20 .mu.l of sterilized distilled water. In the same
manner, DNA fragment (Fragment 2, about 450 bp) obtained by
digesting 4 .mu.g of pUC19hOBM with restriction enzymes BamHI, and
BspHI (Takara Shuzo Co.) and DNA fragment (Fragment 3, about 3.6
kb), obtained by digesting 2 .mu.g of pTrXFus (InVitrogen Co.) with
restriction enzymes BamHI, and SaII (Takara Shuzo Co.) were
respectively purified and dissolved in 20 .mu.l of sterilized
distilled water. The QIAEXII gel extraction kit was used for
purification of the DNA fragments. Fragments 1-3 were ligated by
incubating at 16.degree. C. for 2.5 hours using DNA ligation kit
ver. 2 (Takara Shuzo Co.). Using the ligation reaction, Escherichia
coli GI724 (Invirogen Co.) was transformed according to the method
described in the Instruction Manual of ThioFusion Expression System
(Invirogen Co.). A microorganism strain with plasmid in which the
hOBM cDNA fragment is fused in frame to thioredoxin gene was
selected from the resulting ampicillin resistant transformants by
analysis of DNA restriction map obtained by digestion with
restriction enzyme and by determination of DNA sequence. The
microorganism strain thus obtained was named GI724/pTrxhOBM.
[0262] (2) Expression of Trx-hOBM in Escherichia coli
[0263] GI724/pTrxhOBM and GI724 containing pTrxFus (GI724/pTrxFus)
were respectively cultured six hours with shaking at 30.degree. C.
in 2 ml of RMG-Amp medium (0.6% Na.sub.2HPO.sub.4, 0.3%
KH.sub.2PO.sub.4, 0.05% NaCl, 0.1% NH.sub.4Cl, 2% casamino acid, 1%
glycerol, 1 mM MgCl.sub.2, 100: g/ml ampicillin, pH 7.4). The broth
(0.5 ml) was added to 50 ml of Induction medium (0.6%
Na.sub.2HPO.sub.4, 0.3% KH.sub.2PO.sub.4, 0.05% NaCl, 0.1%
NH.sub.4Cl, 0.2% casamino acid, 0.5% glucose, 1 mM MgCl.sub.2, 100
.mu.g/ml ampicillin, pH 7.4) and cultured with shaking at
30.degree. C. When OD.sub.600nm reached about 0.5, L-tryptophan was
added to a final concentration of 0.1 mg/ml, followed by culturing
with shaking at 30.degree. C. for an additional 6 hours. The
culture broth was centrifuged at 3000.times.g to collect the cells,
which were then suspended in 12.5 ml of PBS. The suspension was
subjected to an ultrasonic generator (Ultrasonics Co.) to disrupt
the cells. The disrupt cells were centrifuged at 7000.times.g for
30 minutes to obtain a supernatant liquid as a soluble protein
fraction. Ten .mu.l of this soluble protein fraction was subjected
to SDS polyacrylamide (10%) electrophoresis under reducing
conditions. As a result, as shown in FIG. 23, a band with a
molecular weight of 40,000 which was not detected in the soluble
protein fraction of GI724/pTrxFus was found in the soluble protein
fraction of GI724/pTrxhOBM. Accordingly, it was confirmed that a
fusion protein (Trx-hOBM) of thioredoxin and human OBM was
expressed in Escherichia coli.
[0264] (3) Binding capability of Trx-hOBM to OCIF
[0265] Binding of the expressed Trx-hOBM to OCIF was confirmed
according to the following experiment. Anti-thioredoxin antibody
(Invirogen Co.) which was diluted 5000-fold with 10 mM sodium
hydrogen carbonate solution was added to a 96-well immunoplate
(Nunc Co.) in the amount of 100 .mu.l per well. After being allowed
to stand overnight at 4.degree. C., the liquid in the wells was
discarded. Two hundred Il of a solution prepared by diluting Block
Ace.TM. (Snow Brand Milk Products Co., Ltd.) two-fold with PBS
(BA-PBS) was added to each well. After being allowed to stand for
one hour at room temperature, the wells were washed three times
with P-PBS. The soluble protein fractions originating from the
above-described GI724/pTrxhOBM or GI724/pTrxFus, each diluted with
BA-PBS in various concentrations were added to each well in the
amount of 100 .mu.l. After being allowed to stand for two hours at
room temperature, each well was washed three times with P-PBS and
charged with 100 .mu.l of OCIF (100 ng/ml) which was diluted with
BA-PBS. After being allowed to stand for two hours at room
temperature, each well was washed three times with P-PBS and
charged with 100 .mu.l of peroxidase-labeled anti-OCIF antibody
(described in WO 96/26217) which was diluted 2,000-fold with
BA-PBS. After being allowed to stand for two hours at room
temperature, each well was washed six times with P-PBS and charged
with 100 .mu.l of TMB solution. After being allowed to stand for
about 10 minutes at room temperature, each well was charged with
100 .mu.l of termination solution (Stopping Reagent). Absorbance of
each well at 450 nm was measured by a microplate reader. The
results are shown in FIG. 24. There was no difference in the
absorbance between the sample with the soluble protein fraction
originating from GI724/pTrxFus added thereto and the sample without
the addition of this soluble protein fraction. On the other hand,
the absorbance increased in the samples to which the soluble
protein fraction originating from GI724/pTrxhOBM was added in
proportion to the concentration of the soluble protein fraction.
The results of the other experiment wherein the dilution rate of
the soluble protein fraction was maintained constant (1%) while
adding OCIF diluted with BA-PBS in different concentrations (0-100
ng/ml) are shown in FIG. 25. It can be seen that the absorbance
remained low at any concentrations of OCIF in samples using a
soluble protein fraction originating from GI724/pTrxFus, whereas
the absorbance increased in proportion to the OCIF concentration in
the samples to which the soluble protein fraction originating from
GI724/pTrxhOBM was added. Based on these results, it was confirmed
that Trx-hOBM which is produced from GI724/pTrxhOBM has a
capability of binding to OCIF.
[0266] (4) Large-scale cultivation of Escherichia coli which
produces Trx-hOBM GI724/pTrxhOBM cells were spread on RMG-Amp agar
(0.6% Na.sub.2HPO.sub.4, 0.3% KH.sub.2PO.sub.4, 0.05% NaCl, 0. 1%
NH.sub.4Cl, 2% casamino acid, 1.5% agar, pH 7.4) using a platinum
transfer 100 p. The cells were cultured overnight at 30.degree. C.
The cultured cells were suspended in 10 ml of Induction medium. The
suspension was added (5 ml for each) to two 2 1 Erlenmeyer flasks
containing 500 ml of Induction medium and cultured at 30.degree. C.
with shaking. When the OD.sub.600nm reached about 0.5, L-tryptophan
was added to a final concentration of 0.1 mg/ml. Culturing with
shaking was continued for six hours at 30.degree. C. The culture
broth was centrifuged for 20 minutes at 3000.times.g to collect the
cells, which were suspended in 160 ml of PBS. The suspension was
subjected to an ultrasonic generator (Ultrasonics Co.) to disrupt
the cells. The supernatant liquid was centrifuged for 30 minutes at
7000.times.g to obtain a soluble protein fraction.
[0267] (5) Preparation of OCIF-immobilized affinity column
[0268] Two g of TSKgel AF-Tolesyl Toyopal 650 (Tosoh Corp.) and 40
ml of 1.0 M potassium phosphate buffer (pH 7.5) containing 35.0 mg
of recombinant OCIF, which was prepared according to the method
described in WO 96/26217, were mixed. The mixture was gently shaken
overnight at 4.degree. C. to effect a coupling reaction. The
reaction mixture was centrifuged to remove the supernatant. To
inactivate excess active residues, 40 ml of 0.1 M Tris-HCl buffer
(pH 7.5) was added to the precipitated carrier and the mixture was
gently shaken at room temperature for one hour. The carrier in a
column was washed with 0.1 M glycine-HCl buffer (pH 3.3) containing
0.01% Polysorbate 80 and 0.2 M NaCl and 0.1 M sodium citrate buffer
(pH 2.0) containing 0.01% Polysorbate 80 and 0.2 M NaCl. The
carrier in the column was equilibrated by charging twice with 10 mM
sodium phosphate buffer (pH 7.4) containing 0.01% Polysorbate
80.
[0269] (6) Purification of Trx-hOBM using OCIF-immobilized affinity
column
[0270] Unless otherwise indicated, purification of Trx-hOBM was
carried out at 4.degree. C. The above-mentioned OCIF-immobilized
affinity carrier (10 ml) and the soluble protein fraction (120 ml)
prepared in Example 25(4) were mixed. The mixture was gently shaken
overnight at 4.degree. C. in four 50 ml centrifugel tubes using a
rotor. An Econo-columnTm (internal diameter: 1.5 cm, length: 15 cm,
manufactured by BioRad Co.) was filled with the carrier in the
mixture. The column was charged with 300 ml of PBS containing 0.01%
Polysorbate 80, 100 ml of 10 mM sodium phosphate buffer (pH 7.0)
containing 0.01% Polysorbate 80 and 2.0 M NaCl, and 100 ml of 0.1 M
glycine-HCl buffer (pH 3.3) containing 0.01 % Polysorbate 80 and
0.2 M NaCl, in that order. Next, proteins adsorbed in the column
were eluted with 0.1 M sodium citrate buffer (pH 2.0) containing
0.01% Polysorbate 80 and 0.2 M NaCl. The eluate was collected in 5
ml portions. Each fraction thus collected was immediately
neutralized with addition of 10% volume of 2 M Tris buffer solution
(pH 8.0). Presence or absence of Trx-hOBM in the eluted fractions
was determined according to the method previously described in
Example 25(3) (the binding capability to OCIF). The fractions
containing Trx-hOBM were collected and purified further.
[0271] (7) Purification of Trx-hOBM by gel filtration
[0272] About 25 ml of Trx-hOBM fractions obtained in Example 25(6)
was concentrated to about 0.5 ml by centrifuging using Centriplus
10 and Centricon 10 (Amicon Co.). This sample was applied to a
Superose 12 HR 10/30 column (1.0.times.30 cm, Pharmacia Co.)
previously equilibrated with PBS containing 0.01% Polysorbate 80.
For the separation, PBS containing 0.01% Polysorbate 80 was used as
a mobile phase at a flow rate of 0.25 ml/min. The eluate from the
column was collected in 0.25 ml portions. The Trx-hOBM in the
thus-collected fractions was detected by the same method as
previously described in the Example 25(3) and SDS-PAGE. Fractions
containing purified Trx-hOBM were collected and the protein
concentration of Trx-hOBM was determined. The measurement of the
protein concentration was carried out using bovine serum albumin as
a standard substance using DC-Protein assay kit (BioRad Co.).
Example 26
Osteoclast formation-inducing activity of hOBM
[0273] phOBM and pcDL-SR .alpha. 296 were respectively transfected
into COS-7 cells using Lipofectamine (Gibco Co.). The cells were
cultured for one day in DMEM containing 10% FCS, trypsinized,
plated on cover slips (15 mm round shape, manufactured by Matsunami
Co.) in 24-well plates at 5.times.10.sup.4 cells per well, and
cultured for 2 days. The culture plate was washed once with PBS.
The cells were fixed with PBS containing 1% paraformaldehyde at
room temperature for 8 minutes. The plate on which the fixed cells
were attached was washed 6 times with PBS, then 700 .mu.l of mouse
spleen cells suspended at 1.times.10.sup.6/ml in .alpha.-MEM
containing 10.sup.-8 M active-form vitamin D.sub.3, 10.sup.-7 M
dexamethasone, and 10% fetal bovine serum were added to each well.
Millicell PCF (Millipore Co.) was set in each well and a suspension
of ST2 cells in the above-mentioned culture medium
(4.times.10.sup.4/ml) were added, 700 .mu.l per well, into the
Millicell PCF followed by incubation at 37.degree. C. for 6 days.
After the culture, the Millicell PCF was removed, the plate was
washed once with PBS, and the cells were fixed with acetone-ethanol
solution (50:50) for one minute. Then, the cells exhibiting
tartaric acid-resistant acid phophatase activity (TRAP), which is a
specific marker for osteoclast, were selectively stained using
LEUKOCYTE ACID PHOSPHATASE kit (Sigma Co.). As a result of
microscopic observation, TRAP-positive cells were not detected in
the wells in which COS-7 cells transfected with pcDL-SR .alpha.296
were fixed. In contrast, 65.+-.18 (average.+-.standard deviation,
n=3) TRAP positive cells were observed in the wells in which COS-7
cells transfected with phOBM were fixed. Moreover, expression of
calcitonin receptor was confirmed by the fact that
.sup.125I-labeled salmon calcitonin (Amersham Co.) specifically
bound to these TRAP positive cells. Based on these findings, it has
been proven that human OBM, which is the protein encoded by cDNA of
the present invention, has osteoclast formation-inducing
activity.
Example 27
Osteoclast formation-inducing activity of Trx-hOBM and
secreted-form human OBM
[0274] Mouse spleen cells were suspended in .alpha.-MEM containing
10.sup.-8 M active-form vitamin D.sub.3, 10.sup.-7 M dexamethasone,
and 10% fetal bovine serum at a concentration of
2.times.10.sup.6/ml. The suspension was added to a 24 well plate in
the amount of 350 .mu.l per well. Each well was then charged with
350 .mu.l of a solution prepared by diluting purified Trx-hOBM with
the above-mentioned culture medium (40 ng/ml), 350 .mu.l of
solution prepared by 10-fold diluting a conditioned medium which
was produced by culturing 293-EBNA cells, onto which pCEPshOBM or
pCEP4 were transfected, in a culture medium IMDM-10% FCS, with
above-mentioned culture medium, or 350 .mu.l only of the
above-mentioned culture medium. The Millicell PCF (Mollipore Co.)
was placed on each well, to which 600 .mu.l of ST2 cells which were
suspended in the above-mentioned culture medium
(4.times.10.sup.4/ml) were added. After cultured for six days, the
Millicell PCF was removed. The plate was washed once with PBS and
the cells were fixed by acetone-ethanol solution (50:50) for one
minute. Then, the cells exhibiting the activity of tartaric acid
resistant acidic phophatase (TRAP activity) were selectively
stained using LEUKOCYTE ACID PHOSPHATASE kit (Sigma Co.). The
results of microscopic observation revealed that no cells
exhibiting the TRAP activity were detected in the wells to which no
Trx-hOBM was added, whereas 115.+-.19 (average.+-.standard
deviation, n=3) TRAP-positive cells were observed in the wells to
which Trx-hOBM was added. Similarly, while no cells exhibiting TRAP
activity were detected in the wells to which conditioned medium of
293-EBNA cells transfected with pCEP4 had been added, 125.+-.23
(average.+-.standard deviation, n=3) TRAP positive cells were
observed in the wells to which conditioned medium of 293-EBNA cells
transfected with pCEPshOBM had been added. Moreover, expression of
calcitonin receptor was confirmed by the fact that
.sup.125I-labeled salmon calcitonin (Amersham Co.) specifically
binds to these TRAP positive cells. These results have proven that
Trx-hOBM and secreted-form hOBM exhibit osteoclast
formation-inducing activity.
Example 28
Preparation of polyclonal antibody
[0275] Mouse sOBM or human sOBM used as an immunogen was prepared
according to the method described above. Especially, mouse sOBM
cDNA (cDNA (Sequence ID No. 18) encoding mouse sOBM (Sequence ID
No. 16) which does not have the membrane binding region of the
mouse OBM due to absence of the amino acids from the N-terminal
down to the 72nd amino acid) or human sOBM cDNA (cDNA (Sequence ID
No. 19) encoding human sOBM (Sequence ID No. 17) which does not
have the membrane binding region of human OBM due to absence of the
amino acids from the N-terminal down to the 71st amino acid) was
ligated with a Hind III/EcoRV fragment (5.2 kb) of the expression
vector pSec TagA (InVitrogen Co.) including the nucleotide sequence
encoding a signal peptide of .kappa.-chain of immunoglobulin,
together with an EcoRI/PmaCl fragment (0.32 kb) of OBM cDNA, using
a ligation kit ver. 2 (Takara Shuzo Co.). Escherichia coli DH5
.alpha. was transformed with the reaction product. The plasmids
obtained from the resulting ampicillin resistant strains were
purified by the alkali SDS and digested with an restriction enzyme
to select a plasmid with 0.6 Kb and 0.32 kb fragments inserted into
pSec TagA. The sequence of this plasmid was identified using the
Dyedeoxyterminator Cycle Sequencing FS kit (product of Perkin Elmer
Co.). As a result, it was confirmed that this plasmid has a
sequence encoding mouse or human sOBM. After plasmid was digested
with restriction enzymes NheI/XhoI, a fragment (1.0 kb)
corresponding to secretion form OBM cDNA was recovered by agarose
gel electrophoresis. This fragment was inserted into an NheI/XhoI
fragment (10.4 kb) of the expression vector pCEP4 (InVitrogen Co.)
using a ligation kit. Escherichia coli DH5 .alpha. was transformed
using the reaction product. Plasmids were purified by the alkali
SDS from the resulting ampicillin resistant strains. Analyzing
these plasmids by digesting with a restriction enzyme, Escherichia
coli possessing a secretion type OBM expression plasmid (pCEP sOBM)
having the objective structure was selected. The Escherichia coli
strain having the pCEP sOBM was cultured and pCEP sOBM was purified
using a Qiafilter plasmid midy kit (Qiagen Co.). Next, 293-EBNA
cells were suspended in IMDM (IMDM-10% FCS) containing 10% FCS and
plated onto a 24-well p late coated with collagen (product of
Sumitomo Bakelite Co., Ltd.) at a cell density of 2.times.10.sup.5
cells/2 ml/well. After culturing overnight, the cells were
tranformed with 1 .mu.l g of pCEP sOBM or pCEP4 using 4 .mu.l of
Lipofectamine (Gibco Co.) and further cultured for two days in 0.5
ml of serum-free IMDM or IMDM-10% FCS. The culture supernatant was
recovered. A cell line with high productivity of recombinant mouse
soluble OBM (msOBM) or human soluble OBM (hsOBM) was screened as
follows. Sodium bicarbonate was added to the culture supernatant
which is assumed to contain msOBM or hsOBM to a final concentration
of 0.1 M. One hundred .mu.l of the culture supernatant was added to
each well in 96-well immunoplates (Nunc Co.) and allowed to stand
overnight at 4.degree. C., thereby msOBM or hsOBM in the culture
supernatant was immobilized on each well. To each well, 200 .mu.l
of Block AceTM (Snow Brand Milk Products Co., Ltd.) solution
diluted four-fold with PBS (B-PBS) was added and the plates were
allowed to stand for two hours at room temperature. After washing
each well in the plates three times with PBS (P-PBS) containing
0.1% Polysorbate 20, 100 .mu.l of each recombinant OCIF (rOCIF)
solution (3-100 ng/ml) diluted serially with P-PBS was added to
each well in the plates. The plates were allowed to stand for two
hours at 37.degree. C. After washing the plates three times with
PBS, 100 .mu.l of a peroxidase-labeled anti-OCIF polyclonal
antibody (WO 96/26217) diluted with B-PBS was added to each well.
After allowing to stand for two hours at 37.degree. C., the wells
were washed six times with P-PBS. Then, 100 .mu.l of TMB solution
(TMB Soluble Reagent, High Sensitivity, ScyTek Co.) was added to
each well in the plates and the plates were allowed to stand at
room temperature for about 10 minutes, subsequently the reaction
was terminated by adding 100 .mu.l of a stopping solution (Stopping
Reagent, ScyTek Co.) to each well. Absorbance at 450 nm of each
well was measured using a microplate reader. It was confirmed that
the absorbance increased remarkably in proportion to concentration
of the added OCIF in the plates in which msOBM or hsOBM in the
culture supernatant of the cell line producing msOBM or hsOBM was
immobilized therein.
[0276] The cell line that exhibited a high rate of increase in
absorbance was selected as a a strain with high productivity.
Thus-related 293-EBNA cells with high productivity of msOBM or
hsOBM were cultured on a large scale in an IMDM medium containing
5% FCS, using 25 T-flasks (T-225). After the cell reached to
confluent, a fresh culture medium was added to each T-225 flask in
the amount of 100 ml per flask and cells were cultured for 3-4
days, to collect the culture supernatant. These procedures were
repeated four times to obtain 10 L of the culture supernatant
containing msOBM or hsOBM. Purified msOBM (10 mg) or hsOBM (12 mg),
which shows homogeneous band (molecular weight: 32 kDa) on
SDS-polyacrylamide gel electrophoresis, were obtained from the
culture supernatant by means of affinity chromatography on an
OCIF-immobilized column and gel filtration chromatography according
to the method described in examples 25(6) and 25(7). Each
thus-obtained purified preparation was used as an antigen for
immunization. Each protein antigen obtained was dissolved in
phosphate buffered saline (PBS) to a concentration of 200 .mu.g/ml
and emulsified with an equivalent volume of Freund's complete
adjuvant. One ml of the emulsion was subcutaneously immunized to
each of three Japanese white rabbits about once every week. A
booster injection was given when the antibody titer reached a peak.
Whole blood was collected 10 days thereafter. The serum was diluted
two-fold with a binding buffer for protein A sepharose
chromatography (BioRad Co.) and applied to a protein A column
equilibrated with the same buffer. After washing the column
extensively with the same buffer, the anti-sOBM antibody adsorbed
to the column was eluted with an elution buffer (BioRad Co.) or 0.1
M glycine-HCl buffer, pH 3.0. To neutralize the eluate immediately,
the eluate was fractionated using test tubes containing a small
amount of 1.0 M Tris-HCl (pH 8.0). The eluate was dialyzed against
PBS overnight at 4.degree. C. The antibody content in the antibody
solution was measured by the Lowry method using bovine IgG as a
standard protein. Thus, about 10 mg of purified immunoglobulin
(IgG) containing the polyclonal antibody of the present invention
per 1 ml of rabbit antiserum was obtained.
Example 29
Measurement of OBM and sOBM by ELISA using polyclonal antibody
[0277] A sandwich ELISA was constructed using the rabbit anti-human
sOBM polyclonal antibody obtained in Example 28 as the solid phase
antibody and enzyme-labeled antibody. Peroxidase (POD)-labeled
antibody was prepared according to the method of Ishikawa (Ishikawa
et al., J. Imunoassay, Vol. 4, 209-327, 1983).
[0278] The anti-human sOBM polyclonal antibody obtained in the
Example 28 was dissolved in a 0.1 M NaHCO.sub.3 to a concentration
of 2 .mu.g/ml. One hundred .mu.l of the resulting solution was
added to each well in 96-well immunoplates (Nunc Co.), which was
then allowed to stand at room temperature overnight. Next, 200
.mu.l of 50% Block Ace.TM. (Snow Brand Milk Co., Ltd.) was added to
each well and the plates were allowed to stand for one hour at room
temperature. The wells were washed three times with PBS containing
0.1% Polysorbate 20 (washing buffer).
[0279] Human OBM was expressed according to the method of Example
26 and purified according to the method of Example 2. The purified
human OBM and the purified human sOBM prepared in example 28 were
serially diluted with the first reaction buffer (0.2 M Tris-HCl
buffer, pH 7.2, containing 40% Block Ace and 0.1% Polysorbate 20),
respectively, and 100 .mu.l of the diluted solution was added to
each well in the plates. The plates were allowed to stand at room
temperature for two hours, and washed three times with the
above-mentioned washing buffer. Subsequently, 100 .mu.l of
POD-labeled anti-human sOBM polyclonal antibody diluted 1000-fold
with the second reaction buffer (0.1 M Tris-HCl buffer, pH 7.2,
containing 25% Block Ace and 0.1% Polysorbate 20) was added to each
well in the plates. After the plates were allowed to stand at room
temperature for two hours, each well was washed three times with
the washing buffer. Next, 100:1 of enzyme substrate solution (TMB,
ScyTek Co.) was added to each well in the plates, and the plates
were allowed to stand for 10 minutes, followed by the addition of
100:1 of a reaction termination solution (Stopping reagent, ScyTek
Co.) to stop the enzyme reaction. The absorbance at 450 m of each
well was measured using a microplate reader. The results are shown
in FIG. 26. The sandwich ELISA using a rabbit anti-human sOBM
polyclonal antibody recognized almost equally human sOBM (molecular
weight, about 32 kDa) and human OBM (molecular weight, about 40
kDa), with a measurement sensitivity of about 12.5.times.10.sup.-3
pmol/ml (human OBM: about 500 pg/ml, human sOBM: about 400 pg/ml).
The measurement of mouse sOBM and mouse OBM by ELISA using the
rabbit anti-mouse sOBM polyclonal antibody obtained in the Example
28 was able to be carried out in the same manner. It was confirmed
that an extremely small amount of mouse sOBM or mouse OBM can be
measured with almost the same sensitivity as described above.
[0280] As mentioned above, the anti-human sOBM polyclonal antibody
of the present invention prepared in the Example 28 can equally
recognize both the human sOBM and human OBM antigens. Therefore,
the antibody was named anti-human OBM/sOBM polyclonal antibody.
Similarly, the anti-mouse sOBM polyclonal antibody prepared in the
Example 28 can equally recognize both the mouse sOBM and mouse OBM
antigens. This antibody was therefore named anti-mouse OBM/sOBM
polyclonal antibody.
Example 30
Preparation of monoclonal antibody
[0281] The purified human sOBM prepared in the Example 28 was used
as the antigen for immunization. The purified human sOBM was
dissolved in physiological saline solution to a concentration of 10
.mu.g/ml and emulsified by mixing with an equivalent volume of
Freund's complete adjuvant. The emulsion was intraperitoneally
administered to BALB/c mice at a dose of 200 .mu.l three times,
once a week, to immunize mice. Next, the equivalent volume of the
Freund's complete adjuvant was added to a physiological saline
solution containing 5 .mu.g/ml of human sOBM and the mixture was
sufficiently emulsified. This emulsion was injected
intraperitoneally to BALB/c mice at a dose of 200 .mu.l, once a
week for four weeks for immunization. One week after the fourth
immunization, 100 .mu.l of a physiological saline solution
containing 10: g/ml of human sOBM was intravenously administered to
the BALB/c mice as a booster. After three days, the spleen was
extracted and spleen cells were separated. The spleen cells were
fused with mouse myeloma cells, P3x63-Ag8.653 according to a
conventional method (Koehler, G. and Milstein, C., Nature, 256, 495
(1975)). The suspended fused cells were cultured for 10 days in an
HAT medium containing hypoxanthine, aminopterin, and thymidine.
After the myeloma cells were dead and hybridomas appeared, the HAT
medium was replaced with an aminopterin-free HAT medium, and the
cell culture was continued.
Example 31
Selection of hybridoma and cloning
[0282] Appearance of hybridomas was recognized 10 days after cell
fusion in Example 30. Monoclonal antibodies recognizing the human
sOBM with high affinity and hybridomas producing these antibodies
were selected according to the following procedure using the
improved solid phase ELISA which is described below. In addition,
to select the anti-OBM monoclonal antibody which recognizes both
human sOBM and mouse sOBM, mouse sOBM prepared in the Example 27
was used in addition to human sOBM as the antigen for the solid
phase ELISA. The human sOBM and mouse sOBM were respectively
dissolved in a 0.1 M sodium bicarbonate solution at a concentration
of 5 .mu.g/ml. Fifty ml of each antigen solution was added to each
well in 96-well immunoplates (Nunc Co.). The plates were allowed to
stand at 4.degree. C. overnight to immobilize the antigens. The
antigen solution in each well was discarded. Each well was then
filled with 200 .mu.l of 50% Block Ace.TM. (Snow Brand Milk
Products Co., Ltd.) and allowed to stand at room temperature for
one hour. After each well was washed with phosphate buffered saline
solution (PBS-P) containing 0.1% Polysorbate 20, 40 .mu.l of calf
serum (Hiclone Inc.) was added to each well. Subsequently, 10 .mu.l
of each hybridoma culture supernatant was added to each well and
each well was incubated at room temperature for two hours in the
presence of 80% calf serum. The object of the solid phase ELISA in
the presence of 80% calf serum is to select a hybridoma which
produce an antibody which can detect a very small amount of human
sOBM or mouse sOBM even in a solution containing high concentration
of protein and in the presence of an immunoreaction interfering
substance derived from serum, i.e. a hybridoma which can produce an
antibody with a high affinity for human sOBM or mouse sOBM. After
the reaction at room temperature for two hours, the plates were
washed with PBS-P and subsequently, 50 .mu.l of peroxidase-labeled
anti-mouse IgG (KPL Co.) diluted 5000-fold with physiological
saline solution containing 25% Block Ace was added to each well.
After the reaction at room temperature for two hours, the plate was
washed three times with PBS-P. After the addition of 50 .mu.l of an
enzyme substrate solution (TMB, ScyTek Co.) to each well, the
reaction was continued at room temperature for five minutes. The
enzymatic reaction was stopped by the addition of 50 .mu.l of a
termination solution (stopping reagent, ScyTek Co.). Hybridomas
which produce antibodies recognizing human sOBM or mouse sOBM were
selected by measuring absorbance at 450 nm of each well using a
microplate reader (Immune Reader NJ200.TM., Nippon InterMed Co.).
Hybridomas producing antibodies exhibiting particularly high
absorbance (OD.sub.450nm) were selected. Cloning of these
hybridomas by a limiting dilution method was repeated 3 to 5 times
to establish stable hybridomas. Hybridomas exhibiting particularly
high antibody productivity were selected among the established
antibody-producing hybridoma clones.
Example 32
Production and purification of monoclonal antibody
[0283] The antibody-producing hybridomas obtained in the Example
31, i.e. high affinity antibody-producing hybridoma which
recognizes human sOBM and hybridoma which produces an antibody
showing cross-reactivity to the mouse sOBM were cultured,
respectively. Each hybridoma was implanted intraperitoneally to
BALB/c mice (1.times.10.sup.6 cells per mouse) to which pristan
(Aldorich Co.) was administered one week previously. After about
2-3 weeks, accumulated ascites were collected. The monoclonal
antibody, which recognizes human sOBM of the present invention or
both the human sOBM and mouse sOBM in the ascites, was purified
according to the purification method of anti-OBM/sOBM polyclonal
antibodies using a Protein A column described in the Example 28.
The purified monoclonal antibody was thus obtained from the ascites
by Protein A column chromatography (Pharmacia Co.).
Example 33
Antigen specificity of monoclonal antibody
[0284] The antigen specificity of a monoclonal antibody which
specifically recognizes human sOBM and the monoclonal antibody
exhibiting cross-reactivity to both the human sOBM and mouse sOBM
was investigated using human sOBM, human intact OBM having a
membrane binding region, mouse sOBM, and mouse intact OBM having a
membrane binding region. More than thirty kinds of monoclonal
antibody were obtained. The results on several representative
antibodies are shown in Table 1. As a result, it was found that
most anti-human sOBM monoclonal antibodies which specifically
recognize human sOBM also recognize the human intact OBM having a
membrane binding region, but not the mouse OBM and the mouse intact
OBM which has a membrane binding region. On the other hand, it was
found that only a few monoclonal antibodies recognizing both the
human sOBM and mouse sOBM were obtained and that these antibodies
exhibit cross-reactivity to both the human OBM and mouse OBM. These
results show that there are common antigen-recognizing sites,
namely common epitopes, in both the human OBM and mouse OBM. Based
on the fact that the anti-human sOBM monoclonal antibody prepared
using the human sOBM as an immune antigen also equally recognizes
human OBM having a membrane binding region, anti-human sOBM
monoclonal antibody was named the anti-human OBM/sOBM monoclonal
antibody. TABLE-US-00002 TABLE 1 Antigen Antibody hsOBM hOBM MsOBM
mOBM H-OBM 1 + + - - H-OBM 2 + + - - H-OBM 3 + + - - H-OBM 4 + + -
- H-OBM 5 + + - - H-OBM 6 + + - - H-OBM 7 + + - - H-OBM 8 + + - -
H-OBM 9 + + + + H-OBM 10 + + - - H-OBM 11 + + - - H-OBM 12 + + - -
H-OBM 13 + + + + H-OBM 14 + + - - hsOBM: human soluble OBM, hOBM:
human membrane bonding type OBM, msOBM: mouse soluble OBM, mOBM:
mouse membrane bonding type OBM
Example 34
Determination of class and subclass of monoclonal antibody
[0285] The class and subclass of the monoclonal antibody of the
present invention were determined by the immunoglobulin class and
subclass analysis kit (Amersham Co.) according to the protocol
indicated. The results on representative monoclonal antibodies are
shown in Table 2. As shown in Table 2, the majority of anti-human
OBM/sOBM monoclonal antibodies were IgG.sub.1, the others were
IgG.sub.2a and IgG.sub.2b. Light chains for all antibodies were
.kappa. chains. TABLE-US-00003 TABLE 2 Antibody IgG.sub.1
IgG.sub.2a IgG.sub.2b IgG.sub.3 IgA .kappa. H-OBM 8 - + - - - +
H-OBM 9 + - - - - + H-OBM 10 + - - - - + H-OBM 11 + - - - - + H-OBM
12 - - + - - + H-OBM 13 + - - - - + H-OBM 14 + - - - - +
Example 35
Measurement of the dissociation constant (K.sub.d value) of
monoclonal antibody
[0286] The dissociation constant of the monoclonal antibody was
measured according to a known method (Betrand Friguet et al.:
Journal of Immununological Methods, 77, 305-319, 1986). That is,
the purified antibody obtained in the Example 32 was diluted with
0.4 M Tris-HCl buffer (a primary buffer, pH 7.4) containing 40%
Block Ace and 0.1% Polysorbate 20 to give a concentration of 5
ng/ml. The solution was mixed with an equivalent volume of a
diluted solution of purified human soluble OBM (hsOBM) obtained in
Example 28 in the primary buffer with a concentration range of 6.25
to 10: g/ml. The mixture was allowed to stand for 15 hours at
4.degree. C. to bind the hsOBM to the monoclonal antibody. After 15
hours, the antibody not bound to the hsOBM (10 .mu.g/ml, 100
.mu.l/well) was measured using an immobilized solid phase ELISA to
calculate the dissociation constant of the monoclonal antibody to
the hsOBM. In addition, affinity to msOBM of an antibody, which is
a monoclonal antibody for the hsOBM and also exhibits the
cross-reactivity to mouse soluble OBM (msOBM), was measured
according to the same method except for using msOBM instead of the
hsOBM. Dissociation constant of antibodies, which exhibit high
affinity to each antigen and are useful for enzymatic immunoassay
and binding assay, are shown in Table 3. TABLE-US-00004 TABLE 3
Antibody Subclass Antigen Dissociation constant Kd (M) H-OBM 1
IgG.sub.1 (.kappa.) hsOBM 1 .times. 10.sup.-11 < kd < 1
.times. 10.sup.-10 H-OBM 4 IgG.sub.1 (.kappa.) hsOBM 1 .times.
10.sup.-11 < kd < 1 .times. 10.sup.-10 H-OBM 9 IgG.sub.1
(.kappa.) hsOBM 1 .times. 10.sup.-9 < kd < 1 .times.
10.sup.-8 H-OBM 9 IgG.sub.1 (.kappa.) msOBM 1 .times. 10.sup.-8
< kd < 1 .times. 10.sup.-7
[0287] As a result, the dissociation constants (Kd) of H-OBM 1 and
H-OBM 4 which are the antibodies specific to human soluble OBM
(hsOBM) were in the order of 10.sup.-11 M, indicating the high
affinity to hsOBM. On the other hand, the Kd value of the antibody
H-OBM 9 which recognizes both the hsOBM and mouse soluble OBM
(msOBM) was in the order of 10.sup.-8 M to msOBM and in the order
of 10.sup.-9 M to hsOBM. In addition, the dissociation constant of
the other antibody which recognizes both antigens in the Table 1,
i. e. the dissociation constant of H-OBM 13 for each antigen, was
the same as that of H-OBM 9, and these two antibodies belong to the
same subclass. These findings suggest the possibility that they are
the identical antibodies which recognize the same epitope of each
antigen.
Example 36
Measuring method of human OBM and sOBM by sandwich ELISA using
anti-human OBM/sOBM monoclonal antibodies
[0288] A sandwich ELISA was constructed using the two high affinity
monoclonal antibodies obtained in Example 35, H-OBM 1 and H-OBM 4,
respectively as a solid phase antibody and an enzyme-labeled
antibody. Labeling of the antibody was carried out using a
maleimide activated-peroxidase kit (Piers Co.). The antibody, H-OBM
1, was dissolved in a 0.1 M sodium bicarbonate solution to a
concentration of 10 .mu.g/ml, and 100 .mu.l of the solution was
added to each well in 96-well immunoplates (Nunc company). After
being allowed to stand overnight at 4.degree. C. to immobilize the
antibody, the solution was discarded and 300 .mu.l of 50% Block
Ace.TM. solution was added to each well in the plates. Each well in
the plates was blocked by allowing to stand at room temperature for
two hours. After blocking, the plates were washed with phosphate
buffered saline containing 0.1% Polysorbate 20 (PBS-P). Human OBM
(hOBM) and human soluble OBM (hsOBM) were respectively diluted with
0.4 M Tris-HCl buffer, pH 7.4, containing 40% Block Ace.TM. (Snow
Brand Milk Products Co., Ltd.) and 0.1% Polysorbate 20 (Wako Pure
Chemicals Co., Ltd.) (the first reaction buffer) to prepare test
samples with various concentrations. These test samples with
different concentrations were added to each well in the amount of
100 .mu.l per well and reacted to the antibody, H-OBM 1 immobilized
on each well by incubating at room temperature for two hours. After
two hours, the plates were washed with PBS-P. Next, 100 .mu.l of a
solution of POD-labeled H-OBM 4 antibody in 0.2 M Tris-HCl buffer,
pH 7.4, containing 25% Block Ace.TM. and 0.1% Polysorbate 20 (the
second reaction buffer) was added to each well, followed by further
incubating at room temperature for two hours. The plates were then
washed with PBS-P and 100 .mu.l of an enzyme substrate solution
(TMB, ScyTek Co.) was added to each well to start enzyme reaction.
The enzyme reaction was terminated by the addition of 100 .mu.l of
a reaction termination solution (stopping reagent, ScyTek Co.) to
each well. The absorbance of each well at 450 nm was measured using
a microplate reader. The results are shown in FIG. 27.
[0289] As a result, it was confirmed that the sandwich ELISA
constructed using the two anti-human OBM/sOBM monoclonal
antibodies, H-OBM 1 and H-OBM 4 with high affinity for human
OBM/sOBM prepared in the Example 35, equally recognizes human OBM
and human sOBM, and is able to measure a very small amount of human
OBM or human sOBM at a quantitative limit of about
1.25.times.10.sup.-3 to 2.5.times.10.sup.-3 pmol/ml (about 50-100
pg/ml for human OBM with a molecular weight of 40 kDa, about 40-80
pg/ml for human sOBM with a molecular weight of 32 kDa). The
hybridomas which produce these two anti-human OBM/sOBM monoclonal
antibodies, H-OBM 1 and H-OBM 4 were named H-OBM1 and H-OBM4,
respectively. The hybridoma producing anti-human OBMIsOBM
monoclonal antibody (H-OBM 9) which recognizes mouse OBM and mouse
sOBM and also has an osteoclast formation-inhibitory activity was
named H-OBM9. These hybridomas were deposited with the National
Institute of Bioscience and Human Technology, the Agency of
Industrial Science and Technology, on Nov. 5, 1993 with Deposition
Nos. FERM BP-6264 (H-OBM 1), FERM BP-6265 (H-OBM 4), and FERM
BP-6266 (H-OBM 9).
Example 37
Measurement of mouse OBM and mouse sOBM using anti-human OBM/sOBM
monoclonal antibody which recognizes mouse OBM and mouse sOBM
[0290] A sandwich ELISA was constructed using the anti-human
OBM/sOBM monoclonal antibody, H-OBM 9, which recognizes mouse OBM
and mouse sOBM obtained as an solid phase antibody in the Examples
33 and 35, and the anti-mouse OBM/sOBM polyclonal antibody as an
enzyme-labeled antibody obtained in the example 28. The mouse OBM
and mouse sOBM were respectively diluted with the first reaction
buffer to give various concentrations in the same manner as in the
Example 35 and then measured sOBM according to the method described
in the Example 36. The results are shown in FIG. 28. As a result,
it was found that mouse OBM and mouse sOBM can be similarly
measured using H-OBM 9 which is the anti-human OBM/sOBM monoclonal
antibody recognizing the mouse OBM and mouse sOBM of the present
invention. As shown by the result of Example 35, this anti-human
OBM/sOBM monoclonal antibody H-OBM 9 has a high dissociation
constant relative to the mouse sOBM, namely it has a comparatively
low affinity to mouse sOBM. The sensitivity in the measurement of
mouse OBM (molecular weight, about 40 kDa) and mouse sOBM
(molecular weight, about 32 kDa) by this ELISA assay was about
25.times.10.sup.-3 pmol/ml (about 1 ng/ml for mouse OBM and about
0.8 ng/ml for mouse sOBM).
Example 38
Osteoclastogenesis-inhibitory activity of anti-OBM/sOBM
antibody
[0291] It is known that osteoclast-like cells (OCL) are induced by
co-culture of mouse spleen cells and ST2 cells (mouse bone
marrow-derived stromal cells; Endocrinology, 125, 1805-1813
(1989)). Capability of the anti-OBM/sOBM antibody to inhibit the
OCL formation when added to the co-culture system was studied.
Because the mouse OBM is expressed in this co-culture system, a
rabbit anti-mouse OBM/sOBM polyclonal antibody which recognizes
mouse OBM and an anti-human OBM/sOBM monoclonal antibody (H-OBM 9)
which recognizes both human OBM and mouse OBM antigens were used as
the antibodies in this example. Seven hundred microliters per well
of each anti-OBM antibody diluted serially with .alpha.-MEM
containing 10% FCS and 350:1/well of male mouse splenocytes
(2.times.10.sup.6/ml) suspended in the same medium described above
were added to each well in a 24-well plate (Nunc). Next, ST2 cells
trypsinized and suspended in the above-mentioned culture medium
containing 4.times.10.sup.-8 M Vitamin D.sub.3 and
4.times.10.sup.-7 M Dexamethazone (8.times.10.sup.4 cells/ml) were
added to each well in the amount of 350 .mu.l/well, followed by
culturing for six days at 37.degree. C. After the plates were
washed once with PBS, cells in each well were fixed with a mixture
of ethanol and acetone (50:50) for one hour at room temperature.
The plates were dried in air, and 500:1 of substrate solution was
added to each well according to the protocol of the LEUKOCYTE ACID
PHOSPHATASE kit (Sigma Co.), followed by incubating for 55 minutes
at 37.degree. C. Only the cells exhibiting the tartaric
acid-resistant acid phophatase activity (TRAP activity), which is a
specific marker for osteoclasts, were stained by this reaction. The
plates were washed once with distilled water and dried in air, and
the number of TRAP-positive cells was counted. The results are
shown in Table 4. As shown in Table 4, both the rabbit anti-mouse
OBM/sOBM polyclonal antibody and the anti-human OBM/sOBM monoclonal
antibody, H-OBM 9, which recognizes mouse OBM inhibited OCL
formation in a dose-dependent manner. These antibodies were found
to possess osteoclastogenesis-inhibitory activity like
osteoclastogenesis-inhibitory factor, OCIF/OPG, and thus are
promising as a therapeutic agent for treating bone metabolism
abnormality symptoms. TABLE-US-00005 TABLE 4 Number of
TRAP-positive multinucleates Amount of Rabbit anti-mouse antibody
OBM/sOBM Mouse anti-human OBM/sOBM (:g/ml) polyclonal antibody
monoclonal antibody (H-OBM 9) 0 1155 .+-. 53 1050 .+-. 45 10 510
.+-. 24 650 .+-. 25 100 10 .+-. 3 15 .+-. 4 (Average .+-. standard
deviation, n = 3)
Example 39
Human osteoclast formation-inducing activity of Trx-OBM
[0292] Monuclear cells were prepared from whole blood collected
from the vein of a healthy adult by density gradient using
Histopaque (Sigma Co.) according to the protocol attached thereto.
The mononuclear cells were suspended at a cell density of
1.3.times.10.sup.6/ml in .alpha.-MEM containing 10.sup.-7 M
Dexamethasone, 200 ng/ml macrophage colony stimulating factor (The
Green Cross Corp.), 10% fetal bovine serum, and purified Trx-OBM
(0-100 ng/ml) obtained in Example 15. The cell suspension was added
to each well in 48-well plates in the amount of 300:1 per well, and
the cells were cultured at 37.degree. C. for three days. After the
culture broth was replaced with the above-mentioned culture medium,
the cells were cultured at 37.degree. C. for four days. The
cultured cells having tartaric acid resistant acid phosphatase
activity (TRAP activity) were selectively stained according to the
method described in Example 5. The number of stained multinucleates
was measured by microscope observation. The results are shown in
FIG. 29. It was confirmed that TRAP-positive multinucleates were
induced in a dose dependent manner by addition of Trx-OBM, while no
TRAP-positive cells were detected in the wells to which Trx-OBM was
not added. Moreover, these TRAP-positive multinucleates were found
positive to vitronectin receptor which is a marker for osteoclasts.
Furthermore, when similar cell culture was carried out on ivory
slices placed on each well in a 48-well plate, pit formation was
observed on the ivory slices only in the presence of Trx-OBM. Based
on these findings, Trx-OBM was formed to have the activity of
inducing human osteoclast formation.
Example 40
Inhibition of bone resorbing activity by anti-OBM/sOBM antibody
[0293] [.sup.45Ca]-CaCl.sub.2 solution (Amersham Co.) was
subcutaneously injected into a ddY mouse (Japan SLC Co.) in the
15th day of pregnancy at a dose of 25 .mu.Ci per mouse to label the
bone of the fetus with .sup.45Ca. Next day, the mouse was
sacrificed to obtain the fetus. The forefoot of the fetus was drawn
and the skin and muscle were removed to obtain the long bones. The
cartilage was removed to obtain the shafts of long bones. The
shafts of long bones were floated one by one in 0.5 ml of culture
medium (BGJb medium (GIBCO BRL company) containing a 0.2% bovine
serum albumin (Sigma Co.) in each well in 24-well plates, and
cultured for 24 hours at 37.degree. C. in 5% CO.sub.2. After the
pre-cultivation, the bones were transferred into various fresh
culture media (0.5 ml), each containing one of four different bone
resorbing factors (vitamin D.sub.3, prostaglandins E.sub.2,
parathyroid hormone, interleukin 1 .alpha.), and normal rabbit IgG
(100 .mu.g/ml; as a control), or the rabbit anti-OBMNsOBM
polyclonal antibody prepared in Example 28, followed by further
cultivation for 72 hours. After the cultivation, the long bones
were placed in 0.5 ml of an aqueous solution of 5% trichloroacetic
acid (Wako Pure Chemicals Co., Ltd.), and allowed to stand at room
temperature for more than 3 hours to decalcify. Five ml of a
scintillator (AQUASOL-2, PACKARD Co.) was added to the culture
broth and the extract of the trichloroacetic acid solution (each
0.5 ml) to measure the radioactivity of .sup.45Ca, whereby the
ratio of the .sup.45Ca which was liberated into the culture broth
by bone resorption was calculated. The results are shown in FIGS.
30 to 33. As a result, vitamin D.sub.3 (10.sup.-8 M) was found to
increase the bone resorbing activity, but the rabbit anti-OBM/sOBM
polyclonal antibody suppressed the bone resorption stimulated by
vitamin D.sub.3 in a concentration-dependent manner, completely
inhibiting the increased bone resorption at a concentration of 100
.mu.g/ml (FIG. 30). Prostaglandins E.sub.2 (10.sup.-6 M) and
parathyroid hormone (100 ng/ml) also increased the bone resorbing
activity. However, addition of 100 .mu.g/ml of the rabbit
anti-OBM/sOBM polyclonal antibody almost completely inhibited the
bone resorption stimulated by prostaglandins E.sub.2 and
parathyroid hormone (FIGS. 31 and 32). On the other hand, normal
rabbit IgG (100 .mu.g/ml), which was used as a positive control,
did not affect the bone resorbing activity induced by
prostaglandins E.sub.2 and parathyroid hormone. Bone resorption was
also increased by interleukin 1 .alpha. (10 ng/ml), but
significantly inhibited by the addition of rabbit anti-OBM/sOBM
polyclonal antibody (100 .mu.g/ml) (FIG. 33). Based on these
results, it is clear that the antibody of the present invention is
a superior substance as a bone resorption inhibitor. The results
obtained by similar experiment using H-OBM 9 which is a mouse
anti-human OBM/sOBM antibody, confirmed that this antibody exhibits
an almost equivalent bone resorption-inhibitory effect as the
rabbit anti-OBM/sOBM polyclonal antibody.
Industrial Applicability
[0294] The present invention provides a novel protein that
specifically binds to osteoclastogenesis-inhibitory factor (OCIF),
a process for preparing the protein, a screening method for a
substance which controls expression of this protein using this
protein, a screening method for a substance which inhibits or
modulates the activity of this protein, a screening method for the
receptor which transmits the activity of this protein by binding
thereto, a pharmaceutical composition which contains the substance
obtained by these screening methods, an antibody for the said
protein, and an agent for treating bone metabolism abnormality
using the antibody.
[0295] Moreover, the present invention provides a DNA encoding a
novel protein (OCIF-binding molecule) which binds to
osteoclastogenesis-inhibitory factor (OCIF), a protein which
possesses an amino acid sequence encoded by the DNA, a method for
preparing the protein specifically binding to OCIF using said DNA
by a genetic engineering technique, and an agent comprising said
protein for treating bone metabolism acatastasia. Furthermore, the
present invention provides a screening method for a substance which
controls expression of the OCIF-binding molecule, a screening
method for a substance which inhibits or modulates the activity of
the OCIF-binding molecule by binding thereto, a screening method
for the receptor which transmits the activity of the OCIF-binding
molecule by binding thereto, and a pharmaceutical composition which
contains the substance obtained by these screening methods.
[0296] Still further, the present invention provides a DNA encoding
a novel human protein capable of binding to
osteoclastogenesis-inhibitory factor, OCIF (human OCIF-binding
molecule, human OBM), a protein containing an amino acid sequence
encoded by the DNA, a process for preparing a protein having
characteristics of specifically binding to OCIF and exhibiting a
biological activity to support and promote the osteoclast
differentiation and maturation by means of genetic engineering
technique, and an agent for treating bone metabolism abnormality
using the protein. Furthermore, the present invention provides a
screening method for a substance which controls expression of the
OCIF-binding molecule, a screening method for a substance which
inhibits or modulates the activity of the OCIF-binding molecule by
binding thereto, a screening method for the receptor which
transmits the biological activity of the OCIF-binding molecule by
binding thereto, a pharmaceutical composition which contains the
substance obtained by these screening methods, an antibody to human
OCIF-binding protein, and an agent for preventing and/or treating
bone metabolism abnormality symptoms using the antibody.
[0297] In addition, the present invention provides antibodies which
recognize both antigens (anti-OBM/sOBM antibodies), one is a
membrane-bound protein which specifically binds to OCIF (OCIF
binding molecule; OBM) and the other a soluble OBM (sOBM) which
does not have a membrane binding region, a process for preparing
the antigen, a method for measuring the OBM and sOBM using these
antibodies, and an agent for preventing and/or treating bone
metabolism abnormality symptoms using the antibody as an effective
component.
[0298] The protein and antibody prepared by the process of the
present invention are useful as medicines and/or reagents for
research and test purposes.
Description of deposited microorganisms
[0299] (1) Name and address of the depository organization to which
microorganism was deposited [0300] Agency of Industrial Science and
Technology [0301] 1-3, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken,
Japan [0302] (postal code 305) [0303] Date of deposition to the
depository organization
[0304] May 23, 1997 [0305] The deposition number
[0306] FERM BP-5953
[0307] (2) Name and address of the depository organization to which
microorganism was deposited [0308] Agency of Industrial Science and
Technology [0309] 1-3, Higashi I-Chome, Tsukuba-shi, Ibaraki-ken,
Japan [0310] (postal code 305)
[0311] Date of deposition to the depository organization [0312]
Aug. 13, 1997
[0313] The deposition number [0314] FERM BP-6058
[0315] (3) Name and address of the depository organization to which
microorganism was deposited [0316] Agency of Industrial Science and
Technology [0317] 1-3, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken,
Japan [0318] (postal code 305)
[0319] Date of deposition to the depository organization [0320]
Nov. 5, 1997 (Original deposition date)
[0321] The deposition number [0322] FERM BP-6264
[0323] (4) Name and address of the depository organization to which
microorganism was deposited [0324] Agency of Industrial Science and
Technology [0325] 1-3, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken,
Japan [0326] (postal code 305)
[0327] Date of deposition to the depository organization [0328]
Nov. 5, 1997 (Original deposition date)
[0329] The deposition number [0330] FEPM BP-6265
[0331] (5) Name and address of the depository organization to which
microorganism was deposited [0332] Agency of Industrial Science and
Technology [0333] 1-3, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken,
Japan [0334] (postal code 305)
[0335] Date of deposition to the depository organization [0336]
Nov. 5, 1997 (Original deposition date)
[0337] The deposition number [0338] FERM BP-6266
Sequence CWU 1
1
19 1 316 PRT Mus musculus 1 Met Arg Arg Ala Ser Arg Asp Tyr Gly Lys
Tyr Leu Arg Ser Ser Glu 1 5 10 15 Glu Met Gly Ser Gly Pro Gly Val
Pro His Glu Gly Pro Leu His Pro 20 25 30 Ala Pro Ser Ala Pro Ala
Pro Ala Pro Pro Pro Ala Ala Ser Arg Ser 35 40 45 Met Phe Leu Ala
Leu Leu Gly Leu Gly Leu Gly Gln Val Val Cys Ser 50 55 60 Ile Ala
Leu Phe Leu Tyr Phe Arg Ala Gln Met Asp Pro Asn Arg Ile 65 70 75 80
Ser Glu Asp Ser Thr His Cys Phe Tyr Arg Ile Leu Arg Leu His Glu 85
90 95 Asn Ala Gly Leu Gln Asp Ser Thr Leu Glu Ser Glu Asp Thr Leu
Pro 100 105 110 Asp Ser Cys Arg Arg Met Lys Gln Ala Phe Gln Gly Ala
Val Gln Lys 115 120 125 Glu Leu Gln His Ile Val Gly Pro Gln Arg Phe
Ser Gly Ala Pro Ala 130 135 140 Met Met Glu Gly Ser Trp Leu Asp Val
Ala Gln Arg Gly Lys Pro Glu 145 150 155 160 Ala Gln Pro Phe Ala His
Leu Thr Ile Asn Ala Ala Ser Ile Pro Ser 165 170 175 Gly Ser His Lys
Val Thr Leu Ser Ser Trp Tyr His Asp Arg Gly Trp 180 185 190 Ala Lys
Ile Ser Asn Met Thr Leu Ser Asn Gly Lys Leu Arg Val Asn 195 200 205
Gln Asp Gly Phe Tyr Tyr Leu Tyr Ala Asn Ile Cys Phe Arg His His 210
215 220 Glu Thr Ser Gly Ser Val Pro Thr Asp Tyr Leu Gln Leu Met Val
Tyr 225 230 235 240 Val Val Lys Thr Ser Ile Lys Ile Pro Ser Ser His
Asn Leu Met Lys 245 250 255 Gly Gly Ser Thr Lys Asn Trp Ser Gly Asn
Ser Glu Phe His Phe Tyr 260 265 270 Ser Ile Asn Val Gly Gly Phe Phe
Lys Leu Arg Ala Gly Glu Glu Ile 275 280 285 Ser Ile Gln Val Ser Asn
Pro Ser Leu Leu Asp Pro Asp Gln Asp Ala 290 295 300 Thr Tyr Phe Gly
Ala Phe Lys Val Gln Asp Ile Asp 305 310 315 2 1538 DNA Mus musculus
2 gccaggacct ctgtgaaccg gtcggggcgg gggccgcctg gccgggagtc tgctcggcgg
60 tgggtggccg aggaagggag agaacgatcg cggagcaggg cgcccgaact
ccgggcgccg 120 cgccatgcgc cgggccagcc gagactacgg caagtacctg
cgcagctcgg aggagatggg 180 cagcggcccc ggcgtcccac acgagggtcc
gctgcacccc gcgccttctg caccggctcc 240 ggcgccgcca cccgccgcct
cccgctccat gttcctggcc ctcctggggc tgggactggg 300 ccaggtggtc
tgcagcatcg ctctgttcct gtactttcga gcgcagatgg atcctaacag 360
aatatcagaa gacagcactc actgctttta tagaatcctg agactccatg aaaacgcagg
420 tttgcaggac tcgactctgg agagtgaaga cacactacct gactcctgca
ggaggatgaa 480 acaagccttt cagggggccg tgcagaagga actgcaacac
attgtggggc cacagcgctt 540 ctcaggagct ccagctatga tggaaggctc
atggttggat gtggcccagc gaggcaagcc 600 tgaggcccag ccatttgcac
acctcaccat caatgctgcc agcatcccat cgggttccca 660 taaagtcact
ctgtcctctt ggtaccacga tcgaggctgg gccaagatct ctaacatgac 720
gttaagcaac ggaaaactaa gggttaacca agatggcttc tattacctgt acgccaacat
780 ttgctttcgg catcatgaaa catcgggaag cgtacctaca gactatcttc
agctgatggt 840 gtatgtcgtt aaaaccagca tcaaaatccc aagttctcat
aacctgatga aaggagggag 900 cacgaaaaac tggtcgggca attctgaatt
ccacttttat tccataaatg ttgggggatt 960 tttcaagctc cgagctggtg
aagaaattag cattcaggtg tccaaccctt ccctgctgga 1020 tccggatcaa
gatgcgacgt actttggggc tttcaaagtt caggacatag actgagactc 1080
atttcgtgga acattagcat ggatgtccta gatgtttgga aacttcttaa aaaatggatg
1140 atgtctatac atgtgtaaga ctactaagag acatggccca cggtgtatga
aactcacagc 1200 cctctctctt gagcctgtac aggttgtgta tatgtaaagt
ccataggtga tgttagattc 1260 atggtgatta cacaacggtt ttacaatttt
gtaatgattt cctagaattg aaccagattg 1320 ggagaggtat tccgatgctt
atgaaaaact tacacgtgag ctatggaagg gggtcacagt 1380 ctctgggtct
aacccctgga catgtgccac tgagaacctt gaaattaaga ggatgccatg 1440
tcattgcaaa gaaatgatag tgtgaagggt taagttcttt tgaattgtta cattgcgctg
1500 ggacctgcaa ataagttctt tttttctaat gaggagag 1538 3 21 DNA
Artificial Sequence Description of Artificial Sequenceprimer 3
aaacgcaaaa aaccagaaag g 21 4 17 DNA Artificial Sequence Description
of Artificial Sequenceprimer 4 gtaaaacgac ggccagt 17 5 17 DNA
Artificial Sequence Description of Artificial Sequenceprimer 5
caggaaacag ctatgac 17 6 22 DNA Artificial Sequence Description of
Artificial Sequenceprimer OBM #8 6 aagccccaaa gtacgtcgca tc 22 7 26
DNA Artificial Sequence Description of Artificial SequenceOBM HF 7
cgaagctttc gagcgcagat ggatcc 26 8 27 DNA Artificial Sequence
Description of Artificial SequenceOBM XR 8 cctctagagt ctatgtcctg
aagtttg 27 9 20 DNA Artificial Sequence Description of Artificial
SequenceOBM3 9 atcagaagac agcactcact 20 10 33 DNA Artificial
Sequence Description of Artificial SequenceOBMSalR2 10 ggggtcgacc
taggacatcc atgctaatgt tcc 33 11 317 PRT Homo sapiens 11 Met Arg Arg
Ala Ser Arg Asp Tyr Thr Lys Tyr Leu Arg Gly Ser Glu 1 5 10 15 Glu
Met Gly Gly Gly Pro Gly Ala Pro His Glu Gly Pro Leu His Ala 20 25
30 Pro Pro Pro Pro Ala Pro His Gln Pro Pro Ala Ala Ser Arg Ser Met
35 40 45 Phe Val Ala Leu Leu Gly Leu Gly Leu Gly Gln Val Val Cys
Ser Val 50 55 60 Ala Leu Phe Phe Tyr Phe Arg Ala Gln Met Asp Pro
Asn Arg Ile Ser 65 70 75 80 Glu Asp Gly Thr His Cys Ile Tyr Arg Ile
Leu Arg Leu His Glu Asn 85 90 95 Ala Asp Phe Gln Asp Thr Thr Leu
Glu Ser Gln Asp Thr Lys Leu Ile 100 105 110 Pro Asp Ser Cys Arg Arg
Ile Lys Gln Ala Phe Gln Gly Ala Val Gln 115 120 125 Lys Glu Leu Gln
His Ile Val Gly Ser Gln His Ile Arg Ala Glu Lys 130 135 140 Ala Met
Val Asp Gly Ser Trp Leu Asp Leu Ala Lys Arg Ser Lys Leu 145 150 155
160 Glu Ala Gln Pro Phe Ala His Leu Thr Ile Asn Ala Thr Asp Ile Pro
165 170 175 Ser Gly Ser His Lys Val Ser Leu Ser Ser Trp Tyr His Asp
Arg Gly 180 185 190 Trp Ala Lys Ile Ser Asn Met Thr Phe Ser Asn Gly
Lys Leu Ile Val 195 200 205 Asn Gln Asp Gly Phe Tyr Tyr Leu Tyr Ala
Asn Ile Cys Phe Arg His 210 215 220 His Glu Thr Ser Gly Asp Leu Ala
Thr Glu Tyr Leu Gln Leu Met Val 225 230 235 240 Tyr Val Thr Lys Thr
Ser Ile Lys Ile Pro Ser Ser His Thr Leu Met 245 250 255 Lys Gly Gly
Ser Thr Lys Tyr Trp Ser Gly Asn Ser Glu Phe His Phe 260 265 270 Tyr
Ser Ile Asn Val Gly Gly Phe Phe Lys Leu Arg Ser Gly Glu Glu 275 280
285 Ile Ser Ile Glu Val Ser Asn Pro Ser Leu Leu Asp Pro Asp Gln Asp
290 295 300 Ala Thr Tyr Phe Gly Ala Phe Lys Val Arg Asp Ile Asp 305
310 315 12 954 DNA Homo sapiens 12 atgcgccgcg ccagcagaga ctacaccaag
tacctgcgtg gctcggagga gatgggcggc 60 ggccccggag ccccgcacga
gggccccctg cacgccccgc cgccgcctgc gccgcaccag 120 ccccctgccg
cctcccgctc catgttcgtg gccctcctgg ggctggggct gggccaggtt 180
gtctgcagcg tcgccctgtt cttctatttc agagcgcaga tggatcctaa tagaatatca
240 gaagatggca ctcactgcat ttatagaatt ttgagactcc atgaaaatgc
agattttcaa 300 gacacaactc tggagagtca agatacaaaa ttaatacctg
attcatgtag gagaattaaa 360 caggcctttc aaggagctgt gcaaaaggaa
ttacaacata tcgttggatc acagcacatc 420 agagcagaga aagcgatggt
ggatggctca tggttagatc tggccaagag gagcaagctt 480 gaagctcagc
cttttgctca tctcactatt aatgccaccg acatcccatc tggttcccat 540
aaagtgagtc tgtcctcttg gtaccatgat cggggttggg ccaagatctc caacatgact
600 tttagcaatg gaaaactaat agttaatcag gatggctttt attacctgta
tgccaacatt 660 tgctttcgac atcatgaaac ttcaggagac ctagctacag
agtatcttca actaatggtg 720 tacgtcacta aaaccagcat caaaatccca
agttctcata ccctgatgaa aggaggaagc 780 accaagtatt ggtcagggaa
ttctgaattc catttttatt ccataaacgt tggtggattt 840 tttaagttac
ggtctggaga ggaaatcagc atcgaggtct ccaacccctc cttactggat 900
ccggatcagg atgcaacata ctttggggct tttaaagttc gagatataga ttga 954 13
27 DNA Artificial Sequence Description of Artificial Sequencehuman
OBM SF 13 ggcgtacgca gagcgcagat ggatcct 27 14 34 DNA Artificial
Sequence Description of Artificial Sequencesynthetic DNA 14
ggggtcgacc atccaggaaa tatcataaca ctcc 34 15 951 DNA Mus musculus 15
atgcgccggg ccagccgaga ctacggcaag tacctgcgca gctcggagga gatgggcagc
60 ggccccggcg tcccacacga gggtccgctg caccccgcgc cttctgcacc
ggctccggcg 120 ccgccacccg ccgcctcccg ctccatgttc ctggccctcc
tggggctggg actgggccag 180 gtggtctgca gcatcgctct gttcctgtac
tttcgagcgc agatggatcc taacagaata 240 tcagaagaca gcactcactg
cttttataga atcctgagac tccatgaaaa cgcaggtttg 300 caggactcga
ctctggagag tgaagacaca ctacctgact cctgcaggag gatgaaacaa 360
gcctttcagg gggccgtgca gaaggaactg caacacattg tggggccaca gcgcttctca
420 ggagctccag ctatgatgga aggctcatgg ttggatgtgg cccagcgagg
caagcctgag 480 gcccagccat ttgcacacct caccatcaat gctgccagca
tcccatcggg ttcccataaa 540 gtcactctgt cctcttggta ccacgatcga
ggctgggcca agatctctaa catgacgtta 600 agcaacggaa aactaagggt
taaccaagat ggcttctatt acctgtacgc caacatttgc 660 tttcggcatc
atgaaacatc gggaagcgta cctacagact atcttcagct gatggtgtat 720
gtcgttaaaa ccagcatcaa aatcccaagt tctcataacc tgatgaaagg agggagcacg
780 aaaaactggt cgggcaattc tgaattccac ttttattcca taaatgttgg
gggatttttc 840 aagctccgag ctggtgaaga aattagcatt caggtgtcca
acccttccct gctggatccg 900 gatcaagatg cgacgtactt tggggctttc
aaagttcagg acatagactg a 951 16 244 PRT Mus musculus 16 Ala Gln Met
Asp Pro Asn Arg Ile Ser Glu Asp Ser Thr His Cys Phe 1 5 10 15 Tyr
Arg Ile Leu Arg Leu His Glu Asn Ala Gly Leu Gln Asp Ser Thr 20 25
30 Leu Glu Ser Glu Asp Thr Leu Pro Asp Ser Cys Arg Arg Met Lys Gln
35 40 45 Ala Phe Gln Gly Ala Val Gln Lys Glu Leu Gln His Ile Val
Gly Pro 50 55 60 Gln Arg Phe Ser Gly Ala Pro Ala Met Met Glu Gly
Ser Trp Leu Asp 65 70 75 80 Val Ala Gln Arg Gly Lys Pro Glu Ala Gln
Pro Phe Ala His Leu Thr 85 90 95 Ile Asn Ala Ala Ser Ile Pro Ser
Gly Ser His Lys Val Thr Leu Ser 100 105 110 Ser Trp Tyr His Asp Arg
Gly Trp Ala Lys Ile Ser Asn Met Thr Leu 115 120 125 Ser Asn Gly Lys
Leu Arg Val Asn Gln Asp Gly Phe Tyr Tyr Leu Tyr 130 135 140 Ala Asn
Ile Cys Phe Arg His His Glu Thr Ser Gly Ser Val Pro Thr 145 150 155
160 Asp Tyr Leu Gln Leu Met Val Tyr Val Val Lys Thr Ser Ile Lys Ile
165 170 175 Pro Ser Ser His Asn Leu Met Lys Gly Gly Ser Thr Lys Asn
Trp Ser 180 185 190 Gly Asn Ser Glu Phe His Phe Tyr Ser Ile Asn Val
Gly Gly Phe Phe 195 200 205 Lys Leu Arg Ala Gly Glu Glu Ile Ser Ile
Gln Val Ser Asn Pro Ser 210 215 220 Leu Leu Asp Pro Asp Gln Asp Ala
Thr Tyr Phe Gly Ala Phe Lys Val 225 230 235 240 Gln Asp Ile Asp 17
246 PRT Homo sapiens 17 Ala Gln Met Asp Pro Asn Arg Ile Ser Glu Asp
Gly Thr His Cys Ile 1 5 10 15 Tyr Arg Ile Leu Arg Leu His Glu Asn
Ala Asp Phe Gln Asp Thr Thr 20 25 30 Leu Glu Ser Gln Asp Thr Lys
Leu Ile Pro Asp Ser Cys Arg Arg Ile 35 40 45 Lys Gln Ala Phe Gln
Gly Ala Val Gln Lys Glu Leu Gln His Ile Val 50 55 60 Gly Ser Gln
His Ile Arg Ala Glu Lys Ala Met Val Asp Gly Ser Trp 65 70 75 80 Leu
Asp Leu Ala Lys Arg Ser Lys Leu Glu Ala Gln Pro Phe Ala His 85 90
95 Leu Thr Ile Asn Ala Thr Asp Ile Pro Ser Gly Ser His Lys Val Ser
100 105 110 Leu Ser Ser Trp Tyr His Asp Arg Gly Trp Ala Lys Ile Ser
Asn Met 115 120 125 Thr Phe Ser Asn Gly Lys Leu Ile Val Asn Gln Asp
Gly Phe Tyr Tyr 130 135 140 Leu Tyr Ala Asn Ile Cys Phe Arg His His
Glu Thr Ser Gly Asp Leu 145 150 155 160 Ala Thr Glu Tyr Leu Gln Leu
Met Val Tyr Val Thr Lys Thr Ser Ile 165 170 175 Lys Ile Pro Ser Ser
His Thr Leu Met Lys Gly Gly Ser Thr Lys Tyr 180 185 190 Trp Ser Gly
Asn Ser Glu Phe His Phe Tyr Ser Ile Asn Val Gly Gly 195 200 205 Phe
Phe Lys Leu Arg Ser Gly Glu Glu Ile Ser Ile Glu Val Ser Asn 210 215
220 Pro Ser Leu Leu Asp Pro Asp Gln Asp Ala Thr Tyr Phe Gly Ala Phe
225 230 235 240 Lys Val Arg Asp Ile Asp 245 18 735 DNA Mus musculus
18 gcgcagatgg atcctaacag aatatcagaa gacagcactc actgctttta
tagaatcctg 60 agactccatg aaaacgcagg tttgcaggac tcgactctgg
agagtgaaga cacactacct 120 gactcctgca ggaggatgaa acaagccttt
cagggggccg tgcagaagga actgcaacac 180 attgtggggc cacagcgctt
ctcaggagct ccagctatga tggaaggctc atggttggat 240 gtggcccagc
gaggcaagcc tgaggcccag ccatttgcac acctcaccat caatgctgcc 300
agcatcccat cgggttccca taaagtcact ctgtcctctt ggtaccacga tcgaggctgg
360 gccaagatct ctaacatgac gttaagcaac ggaaaactaa gggttaacca
agatggcttc 420 tattacctgt acgccaacat ttgctttcgg catcatgaaa
catcgggaag cgtacctaca 480 gactatcttc agctgatggt gtatgtcgtt
aaaaccagca tcaaaatccc aagttctcat 540 aacctgatga aaggagggag
cacgaaaaac tggtcgggca attctgaatt ccacttttat 600 tccataaatg
ttgggggatt tttcaagctc cgagctggtg aagaaattag cattcaggtg 660
tccaaccctt ccctgctgga tccggatcaa gatgcgacgt actttggggc tttcaaagtt
720 caggacatag actga 735 19 741 DNA Homo sapiens 19 gcgcagatgg
atcctaatag aatatcagaa gatggcactc actgcattta tagaattttg 60
agactccatg aaaatgcaga ttttcaagac acaactctgg agagtcaaga tacaaaatta
120 atacctgatt catgtaggag aattaaacag gcctttcaag gagctgtgca
aaaggaatta 180 caacatatcg ttggatcaca gcacatcaga gcagagaaag
cgatggtgga tggctcatgg 240 ttagatctgg ccaagaggag caagcttgaa
gctcagcctt ttgctcatct cactattaat 300 gccaccgaca tcccatctgg
ttcccataaa gtgagtctgt cctcttggta ccatgatcgg 360 ggttgggcca
agatctccaa catgactttt agcaatggaa aactaatagt taatcaggat 420
ggcttttatt acctgtatgc caacatttgc tttcgacatc atgaaacttc aggagaccta
480 gctacagagt atcttcaact aatggtgtac gtcactaaaa ccagcatcaa
aatcccaagt 540 tctcataccc tgatgaaagg aggaagcacc aagtattggt
cagggaattc tgaattccat 600 ttttattcca taaacgttgg tggatttttt
aagttacggt ctggagagga aatcagcatc 660 gaggtctcca acccctcctt
actggatccg gatcaggatg caacatactt tggggctttt 720 aaagttcgag
atatagattg a 741
* * * * *