Title of Invention

A THERAPEUTIC AGENT FOR TREATING RHEUMATOID ARTHRITIS

Abstract There is disclosed a therapeutic agent for treating rheumatoid arthritis, comprising, as an active ingredient, an anti-osteopontin antibody which can inhibit the binding between an integrin recognizing the site of the amino acid sequence RGD and a thrombin-cleaved N-terminal fragment of osteopontin, and which can also inhibit the binding between an integrin recognizing the site of the amino acid sequence SVVYGLR or a corresponding equivalent sequence and a thrombin-cleaved N-terminal fragment of osteopontin, wherein said anti-osteopontin antibody is an antibody raised against the peptide CVDTYDGRGDSVVYGLRS or CVDVPNGRGDSLAYGLR as the antigen or a humanized antibody made by humanization of an antibody which is raised against the peptide CVDTYDGRGDSVVYGLRS or CVDVPNGRGDSLAYGLR as the antigen.
Full Text DESCRIPTION
A THERAPEUTIC AGENT FOR TREATING RHEUMATOID ARTHRITIS
Technical Field of the Invention
The present invention relates to an anti-human
osteopontin antibody and a method for therapeutically treating
autoimmune diseases, rheumatism, and rheumatoid arthritis,
using the antibody.
Background Art
Osteopontin (referred to as"OPN"hereinbelow) is an acidic,
calcium-binding glycoprotein abundant in bone. It has been
known that three types of human OPNisoforms namely osteopontin-a
(referred to as "OPN-a" hereinbelow), osteopontin-b (referred
to as "OPN-b" hereinbelow) and osteopontin-c (referred to as
"OPN-c" hereinbelow), are naturally generated by alternative
splicing (Y. Saitoh et al., (1995) : Laboratory Investigation,
72,55-63). It hasbeenbelieved that among them, the precursor
of OPN-a has an amino acid sequence shown as SQ ID N0.1 below
in the Sequence Listing, where the signal peptide is cleaved
on secretion, so that the mature form OPN-a of I17-N314 isprepared.
Additionally, the mature OPN is cleaved at the C-terminal side
of the 168-th residue arginine with thrombin from a biological
organism into two fragments M-terminal and C-terminal.
OPN described above has various physiologically,
pathologically significant functions, for example cell
adhesion, cell migration, tumorigenesis, immune response and
inhibition of complement-mediated cytolysis. Various types of
receptors on cellular surface mediate the various functions.
OPN has thef RGD sequence therein (for example, OPN-a has the
sequence from the residue at position 159 to the residue at
position 161) . Integrin species recognizing the RGD sequence
such as avb3, avb1 and avb5 are major OPN receptors; specifically,
the integrin species avb3, avbl and avb5 mediate cell adhesion
in vascular smooth muscle cells. Further, avb3 is involved in
the migrations of macrophages, lymphocytes, endothelial cells,
and smooth muscle cells and the like.
Further, research works so far have elucidated that OPN
also binds through the sequence(sWYGLpj to 0.9(31, a4|3l and a4P7
integrin species and that a difference in the mode is also found
such that a4pl binds to both OPN not yet cleaved with thrombin
(non-cleavage-type OPN) and the N-terminal fragment of
thrombin-cleaved OPN (cleavage-type OPN) , while a9pl binds only
to the thrombin-cleavage-type OPN. (Y. Yokosaki et al.,
(1999): The Journal of Biological Chemistry 274, 36328-36334/P.
M. Green et al., (2001): FEBS Letters 503, 75-79/S. T. Barry
et al., (2000): Experimental Cell Research 258, 342-351).
These integrin subunits a9 and a4 or the integrin subunits b1
and P7 are highly similar in terms of amino acid sequence to
each other. Additionally, the integrin species avb1 and avb7
are mainly found in lymphocytes and monocytes, while in
neutrophils, the integrin species are expressed very slightly.
Alternatively, avbl is highly expressed selectively in
neutrophils and has functions essential for neutrophil
migration through VCAM-1 and Tenascin-C. Additionally, the
integrin is also expressed diversely in muscular cells,
epithelial cells and liver cells. As described above, the
cytoplasm domains of the integrin subunits a4 and a9
cooperatively promote leukocyte migration toward inflammatory
sites and aggregation therein, via individual cellular signal
transmission pathways subtly differing from each other, to
enhance their infiltration activities. In such manner, the
integrin subunits are involved in various inflammatory
reactions.
As described above, various types of integrin species
promote leukocyte migration and are thus involved in
inflammatory reactions . Therefore, pharmaceutical substances
inhibiting these integrin activities may potentially have a
possibility as an anti-inflammatory agent. For example, the
integrin avb3 is expressed in osteoclast cells, vascular
endothelial cells and smooth muscle cells and the like. An
anti-avb3 antibody is now under way of development, which will
work to inhibit the binding between the integrin avb3 and various
binding ligands thereof to potentially exert for example an
action to suppress articular damages.
Because receptors of the integrin family commonly emerge
in diverse tissues to provide essential functions for the
control of vital activities, however, the use of antibodies
against integrin for the therapeutic treatment of rheumatoid
arthritis and osteoarthritis may possibly elicit the same
inhibition at other sites and may also cause the occurrence of
side effects.
Additionally, WO 01/71358 discloses a method for
screening for a substance inhibiting the binding between the
a4 integrin and osteopontin and a method for therapeutically
treating inflammatory diseases, using the substance recovered
by such screening.
Various factors have been found to implicate in the
pathogenesis of rheumatoid arthritis. Thus, many reports have
been issued therefor. However, not any of them is reliable.
Further, currently known therapeutic methods thereof are
nosotropic and have not been essentially satisfactory.
Hence, it has been strongly desired to definitely
elucidate the pathogenesis of rheumatoid arthritis and provide
more excellent therapeutics thereof. It is a purpose of the
invention to solve such problems.
Further, rheumatoid arthritis is hardly discriminated
from osteoarthritis. Therefore, it is another purpose of the
invention to provide a diagnostic method thereof.
Disclosure of the Invention
The inventors found that the OPN concentration in each
of the articular cavity fluids of rheumatism patients and
osteoarthritis patients was at a higher value. Additionally,
the inventors found the increase of the ratio of the N-terminal
fragment of the thrombin-cleavage type in the total OPN in
rheumatism patients for the first time. Thus, the inventors
speculated that OPN might be deeply involved in the onset of
these diseases. As described in the following Reference
Example, then, the inventors verified the findings at
experiments using OPN knockout mice.
Further, the inventors prepared antibodies individually
discriminatively recognizing the N-terminal fragment and the
C-terminal fragment from the thrombin-cleaved OPN. Then, the
inventors found at experiments with the antibodies that the
N-terminal fragment of the thrombin-cleaved OPN was at a high
concentration in the articular cavity fluids of patients with
rheumatoid arthritis, in particular.
The inventors focused their attention to the co-presence
of the RGD sequence and the sequence SWYGLR both recognized
by human-type integrin, in the N-terminal fragment at such high
concentration as observed inpatients with rheumatoid arthritis .
Then, the inventors anticipated that an antibody simultaneously
blocking both the sequences would inhibit the binding between
OPN and integrin widely so the antibody might be effective for
the therapeutic treatment of rheumatoid arthritis and
osteoarthritis.
Further, OPN is distributed in kidney, placenta, ovary,
brain, skin and the like, but is mainly expressed in bone tissue.
The inventors considered that for the therapeutic treatment of
rheumatoid arthritis, the binding between OPN and integrin
would preferably be blocked by a method more specific to the
OPN side. Because the diverse integrin species are involved
in inflammation in a cooperative manner, then, the inventors
considered that it would be effective to more widely block the
binding to these diverse integrin species.
Therefore, the inventors prepared an antibody inhibiting
the binding between the RGD sequence of human OPN and integrin
and the binding between the SWYGLR sequence of human OPN and
integrin and then verified the effects thereof at experiments
for cell adhesion and cell migration and the like. Further,
the inventors recovered an antibody against synthetic peptides
corresponding to such inner sequences of murine OPN, to examine
the efficacy of such antibody as a therapeutic agent, using an
arthritis-diseased model in mouse.
More specifically, because murine OPN has the sequences
RGD and SLAYGLR recognizable by murine integrin, which are
located at positions homologous to human OPN in terms of amino
acid sequence, an antibody M5 was recovered as an antibody
simultaneously blocking these sequences. It was verified that
the binding of the antibody M5 with murine OPN and the thrombin
digestion products thereof was inhibited by the peptide GRGDSP
including the sequence RGD site and that the antibody M5 inhibited
the migration of TNF-a-activated monocyte derived from murine
spleen. It was also observed that the antibody M5 had an action
to suppress bone damage when examined in a murine calvaria organ
culture system. Further, it was confirmed that the antibody
exhibited an apparent therapeutic effect, when administered to
a murine collagen arthritis model.
The aforementioned results strongly suggest that the
antibody simultaneously blocking the binding on the sequences
RGD and SWYGLR sites 'with human-type integrin inhibits the
binding between OPN and integrin and is therefore effective for
the therapeutic treatment of rheumatoid arthritis and that the
antibody will possibly be effective not only for rheumatism such
as juvenile articular rheumatism and chronic rheumatism, but
also for psoriatic arthritis and psoriasis. Additionally,
chronic rejections after organ transplantation
characteristically involve vascular and bronchial occlusive
disorders . The histological examination thereof suggests that,
because activation of T cell and macrophage triggers generation
of cytokine and growth factors and disorders of vascular
endothelial cell and because proliferation of vascular smooth
muscle cell elicits fibrogenesis and the like, vascular
occlusion will then possibly occur (P.. Freese et al., (2001) :
Nephrol Dial Transplant, 16, 2401-2406/J. R. Waller et al.,
(2001) : British Journal of Surgery, 88, 1429-1441/S. R.
Lehtonen et al., (2001): Transplantation, 72, 1138-1144). A
report tells that OPN has a function as a protein essential for
macrophage activation and fibrogenesis of vascular smooth
muscle cell (A. O'Regan et al., (2000) : Int J Exp Pathol, 81,
373-390) . Thus, the inventive OPN inhibitory antibody of the
'invention suppresses migration of monocyte and neutrophil,
thereby possibly suppressing a course toward such fibrogenesis .
Thus, the antibody suppresses chronic rejections after organ
transplantation, with the resultant contributions to organ
adhesion. Additionally, the antibody will be effective for the
therapeutic treatment of autoimmune diseases including
systemic autoimmune diseases, erythematodes, uveitis, Behcet
disease, multiple myositis , proliferative glomerulonephritis ,
sarcoidosis and the like.
In other words, the invention provides an anti-osteopontin antibody which can inhibit the
binding between an integrin recognizing the site of the amino acid sequence RGD and N-terminal
fragment of osteopontin, andwhich can also inhibit the binding between an integrin recognizing
the site of the amino acid sequence SVVYGLR or a corresponding equivalent sequence and N-
terminal fragment of osteopontin.
• Further, the invention provides a therapeutic agent of
autoimmune diseases, a therapeutic agent of rheumatism and a
therapeutic agent of rheumatoid arthritis, and these
therapeutic agents contain the anti-osteopontin antibody as the
effective ingredients.
Accordingly, the present invention provides a therapeutic
agent for treating rheumatoid arthritis, comprising, as an active
ingredient, an anti-osteopontin antibody which can inhibit the
binding between an integrin recognizing the site of the amino acid
sequence RGD and a thrombin-cleaved N-terminal fragment of
osteopontin, and which can also inhibit the binding between an
integrin recognizing the site of the amino acid sequence SVVYGLR
or a corresponding equivalent sequence and a thrombin-cleaved N-
terminal fragment of osteopontin, wherein said anti-osteopontin
antibody is an antibody raised against the peptide
CVDTYDGRGDSVVYGLRS or CVDVPNGRGDSLAYGLR as the antigen or a
humanized antibody made by humanization of an antibody which is
raised against the peptide CVDTYDGRGDSVVYGLRS or CVDVPNGRGDSLAYGLR
as the antigen.
Still further, the invention provides a method for
therapeutically treating autoimmune diseases, rheumatism and
rheumatoid arthritis, including administering the anti-osteopontin
antibody to patients with rheumatism and rheumatoid arthritis, to
inhibit the binding between the sequence RGD of osteopontin and
integrin and/or inhibit the binding between the sequence SVVYGLR
and integrin.
Still yet further, the invention provides a diagnostic agent
of rheumatism and a diagnostic method thereof, utilizing the
osteopontin antibody.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:
Fig. 1 shows graphs depicting the inhibition of RGD-
dependent cell adhesion to OPN.
Fig. 2 shows graphs depicting the inhibition of RGD-
dependent and RGD-independent cell adhesion between nOPN and
a9-transformed SW480 cell via the antibody 2K1.
Fig. 3a shows graphs depicting OPN-induced cell
migration.
Fig. 3b shows graphs depicting the suppression of
OPN-induced cell migration via antibodies.
Fig. 4 shows graphs depicting the time course of the change
of arthritis score when the arthritogenic antibody cocktail/LPS
was dosed individually to an OPN gene-defective mouse and normal
mouse.
Fig. 5 shows graphs depicting the comparison of wrist
swelling when the arthritogenic antibody cocktail/LPS was dosed
to the OPN gene-defective mouse and normal mouse, individually.
Fig. 6 shows graphs depicting the adhesion between murine
OPN and NIH3T3 in a concentration-dependent manner.
Fig. 7 shows graphs depicting the inhibition of the
adhesion between murine OPN and NIH3T3 via the peptide GRGDSP.
Fig. 8 shows graphs depicting the inhibition of the
adhesion between murine OPN and NIH3T3 via the antibody M5.
Mode for Carrying out the Invention
The anti-osteopontin antibody (referred to as "OPN
inhibitory antibody" hereinbelow) inhibiting the binding
between an integrin recognizing the sequence RGD and OPN or a
fragment thereof and also inhibiting the binding between an
integrin recognizing the sequence SWYGLR or a corresponding
sequence and OPN or a fragment thereof may be any of antibodies
inhibiting the binding of an integrin recognizing the sequence
RGD, for example avb1, avb3, and avb5 with OPN-a, OPN-b, OPN-c
or an N-terminal fragment thereof and also inhibiting the
binding of an integrin recognizing the sequence SWYGLR, for
example avb1, avb1 and avb7 with OPN-a, OPN-b, OPN-c or an
N-terminal fragment thereof. The sequence SWYGLR or a
corresponding sequence thereof means those described below: the
sequence SWYGLR means the sequence from serine at position 162
to arginine at position 168 in human OPN, while the corresponding
sequence thereof means a SWYGLR-corresponding sequence in the
OPNs of other mammals, which is, for example, swine SWYGLR
identical to the sequence in humans, SVAYGLR in monkey, SLAYGLR
in mouse and rat, SVAYGLK in bovine, and SVAYRLK in rabbit. The
OPN inhibitory antibody of the invention may be any of antibodies
retaining such properties, and the method for preparing the
antibody is not specifically limited. The OPN inhibitory
antibody canbepreparedby using for example OPN-a, OPN-b, OPN-c,
or an N-terminal fragment thereof, or a peptide containing the
amino acid sequence RGDBWYGLR or a corresponding sequence
thereof (referred to as "OPN-related peptide" hereinbelow) as
the antigen. The OPN fragment herein referred to includes OPN
fragments generated by digesting OPN with proteinases and the
like, and is for example a fragment recovered by thrombin
cleavage.
The OPN inhibitory antibody is preferably prepared by
using a peptide containing the sequence RGDSWYGLR as an antigen.
More preferably, the OPN inhibitory antibody is prepared, for
example, by using as an antigen the peptide (VDTYDGRGDSWYGLRS)
containing both the two sequences in a successive sequence,
which starts from valine at position 153 and ends at serine at
position 169 in the case of OPN-a, and subsequently treating
the peptide according to a general method. In order to elevate
the antigenicity, preferably, a product of the OPN-related
peptide bound to a biopolymer compound is used.
For research works of OPN-related diseases, using mouse
as an experimental animal, preferably, an OPN inhibitory
antibody against murine OPN is used. Such antibody is
preferably prepared by using a peptide containing the sequence
RGDSLAYGLR as the antigen.
Examples of the biopolymer compound to be bound to the
OPN-related peptide include for example Macroschisma
hemocyanin (referred to as "KLH" hereinafter), ovalbumin
(referred to as "OVA" hereinafter), bovine serum albumin
(referred to as "BSA" hereinafter), rabbit serum albumin
(referred to as "RSA" hereinafter), and thyroglobulin. Among
them, KLH and thyroglobulin are more preferable.
The OPN-related peptide and the biopolymer compound are
bound together by known methods, for example the mix acid
anhydride process (B. F. Erlanger et al., (1954): J. Biol. Chem.
234, 1090-1094) or the activated ester process (A. E. Karu et
al., (1994): J. Agric. Food Chem. 42, 301-309).
The mix acid anhydride for use in the mix acid anhydride
process can be recovered by subjecting the OPN-related peptide
to general Schotten-Baumann reaction, which is then allowed to
react with a biopolymer compound to prepare the object bound
product of the peptide-polymer compound. The haloformate ester
for use in the mix acid anhydride process includes for example
methyl chloroformate, methyl bromoformate, ethyl chloroformate,
ethyl bromoformate, isobutyl chloroformate and the like. The
ratio of the peptide, the haloformate ester and the polymer
compound to be used according to the method is appropriately
selected in a wide range.
Herein, the Schotten-Baumann reaction is carried out in
the presence of a basic compound. The basic compound for use
in the reaction includes compounds for routine use for
Schotten-Baumann reaction, for example organic bases such as
triethylamine, trimethylamine, pyridine, dimethylaniline,
N-methylmorpholine, diazabicyclononene (DBN),
diazabicycloundecene (DBU), diazabicyclooctane (DABCO) and the
like, and inorganic bases such as potassium carbonate, sodium
carbonate, potassium hydrogen carbonate, sodium hydrogen
carbonate and the like.
Additionally, the reaction is generally progressed at -
20 °C to 100 °C, preferably 0 °C to 50 °C. The reaction time
is about 5 minutes to 10 hours, preferably 5 minutes to 2 hours.
The reaction between the resulting mix acid anhydride and
the biopolymer compound is generally practiced at - 20 °C to
150 °C, preferably 0 °C to 100 °C, for a reaction time of about
5 minutes to 10 hours, preferably 5 minutes to 5 hours. The
mix acid anhydride method is generally carried out in a solvent.
The solvent includes for example any of solvents commonly used
for the mix acid anhydride method, specifically including
halogenated hydrocarbons such as dichloromethane, chloroform,
and dichloroethane; aromatic hydrocarbons such as benzene,
toluene and xylene; ethers such as diethyl ether, dioxane,
tetrahydrofuran, and dimethoxyethane; esters such as methyl
acetate and ethyl acetate; non-protonic polar solvents such as
N, N-dimethylformamide,dimethylsulfoxide, and
hexamethylphosphotriamide; and the like.
Alternatively, the activation ester process is generally
done as follows. Dissolving first the OPN-related peptide in
an organic solvent, for reaction with N-hydroxysuccinimide in
the presence of a coupling agent, an N-hydroxysuccinimide-
activated ester is produced.
As the coupling agent, general coupling agents for
routine use in condensation reaction can be used, including for
example dicyclohexylcarbodiimide, carbonyldiimidazole and
water-soluble carbodiimide. As the organic solvent,
alternatively, for example, N,N-dimethylformamide (DMF),
dimethylsulfoxide and dioxane can be used. The molar ratio of
the peptide and a coupling agent such as N-hydroxysuccinimide
for use in the reaction is preferably 1:10 to 10:1, most
preferably 1:1. The reaction temperature is 0 °C to 50 °C,
preferably 22 °C to 27 °C, while the reaction time is 5 minutes
to 24 hours, preferably one hour to 2 hours. Satisfactorily,
the reaction temperature is a temperature above the individual
melting points and below the individual boiling points.
After the coupling reaction, the reaction solution is
added to a solution dissolving a biopolymer compound therein,
for reaction. In the case that the biopolymer compound has a
free amino group, for example, an acid-amide bond is formed
between the amino group and the carboxyl group of the peptide.
The reaction temperature is 0 °C to 60 °C, preferably 5 °C to
40 °C, and more preferably 22 °C to 27 °C, while the reaction
time is 5 minutes to 24 hours, preferably one hour to 16 hours,
and more preferably one hour to 2 hours.
The reaction product between the OPN-related peptide and
the biopolymer compound as generated by the method is purified
by dialysis or with a desalting column and the like, to recover
the product of the OPN-related peptide bound to the biopolymer
compound (simply referred to as "bound product" hereinafter).
Description now follows hereinbelow about the method for
preparing an antibody, using the bound product thus recovered
as an antigen, and an immunoassay method using the antibody.
For the preparation of the antibody, herein, known methods can
be utilized, appropriately, which are described in for example
Zoku Seikagaku Jikken Koza (Biochemical Experimental Lecture
Series), and Men-eki Seikagaku Kenkyu Ho (Immuno-Biochemistry
Research Method) (Nihon Seikagaku Gakkai hen (Japan Biochemical
Association, ed.)).
In order to prepare a polyclonal antibody using the bound
product in accordance with the invention, an animal is immunized
with the bound product to collect the antibody from the animal.
More specifically, for example, a bound product such as
the OPN-related peptide-thyroglobulin bound product is first
dissolved in sodium phosphate buffer (referred to as "PBS"
hereinafter), which is then mixed with the Freund complete
adjuvant or the Freund incomplete adjuvant, or an auxiliary
agent such as alum. The resulting mixture is used as the
immunogen for immunization of a mammalian animal.
Any animal for routine use in the field can be used as
the animal for immunization, including for example mouse, rat,
rabbit, goat and horse. Additionally, the method for dosing
the immunogen for immunization may be via any of subcutaneous
injection, intraperitoneal injection, intravenous injection,
and intramuscular injection. Subcutaneous injection or
intraperitoneal injection is preferable. Immunization can be
done once or plural times at an appropriate interval, preferably
at an interval of one week to 5 weeks.
According to a general method, then, blood is collected
from the immunized animal, from which serum is separated. By
purifying the polyclonal antibody fraction, the OPN inhibitory
antibody can be recovered.
According to a general method, additionally, an immune
cell recovered by immunizing an animal with the bound product
is fused with myeloma cell to prepare a hybridoma. By
collecting an antibody from a culture of the hybridoma, the OPN
inhibitory antibody can be recovered as a monoclonal antibody.
When the antibody of the invention is intended for use
in the therapeutic treatment of animals including humans,
preference is given to the use of a chimera antibody (see
European Patent Publication EP 0125023) prepared through such
modification by genetic engineering that the resulting OPN
inhibitory antibody might have the same constant region as that
of such antibody for a subject human or animal to be treated
or the use of the antibody having been animalized (see European
Patent Publication EP 0239400 or EP 045126). Otherwise,
preferably, a monoclonal antibody (animal-type antibody for the
animal species) (see European Patent Publication EP 0546073 or
WO 97/07671) is used, as prepared by using a transgenic animal
with an artificially introduced gene involved in the generation
of the antibody in a subject human or animal to be treated.
In the case that the subject to be treated is human and
the OPN inhibitory antibody-generating animal is mouse,
preferably, human mouse chimera antibodies or humanized
antibodies are used. More preferably, a transgenic animal such
as mouse introduced with a human gene involved in the antibody
generation is used to prepare a human-type monoclonal antibody,
for subsequent use. Further, the phage display method may
satisfactorily be used for antibody generation.
The OPN inhibitory antibody thus recovered can be used
in the form of Fv, Fab or F(ab')2 with the antigen recognition
site scissored out of the OPN inhibitory antibody with protease
and the like.
The OPN inhibitory antibody thus recovered is further-
purified, if necessary, which is subsequently formulated into
dosage forms according to a general method, for use in the;
therapeutic treatment of rheumatoid arthritis, rheumatism such
as juvenile articular rheumatism and chronic • rheumatism,
psoriasis arthritis, and psoriasis; the suppression of chronic
rejections after organ transplantation; and the therapeutic
treatment of autoimmune diseases such as systemic autoimmune
diseases, erythematodes, uveitis, Behcet disease, multiple
myositis, skein proliferative nephritis, and sarcoidosis.
The OPN inhibitory antibody of the invention can
preferably be used as a therapeutic agent of rheumatism or a
therapeutic agent of rheumatoid arthritis. Examples of the
dosage forms of these therapeutic agents of rheumatism and the
like include parenteral forms such as injections and infusions,
which are preferably dosed via intravenous injection and
subcutaneous injection (for use as a therapeutic agent of
autoimmune diseases, the examples described above should be
followed). For the formulation, additionally,
pharmaceutically acceptable carriers and additives may be used
within a pharmaceutically acceptable range, depending on the
dosage form.
The amount of the OPN inhibitory antibody to be added for
the formulation varies, depending on the symptomatic severity
and age of a patient, the dosage form, of the formulation to be
used or the binding titer of the OPN inhibitory antibody or the
like. For example, an amount of about 0.1 mg/kg to 100 mg/kg
is satisfactorily used.
Because the OPN inhibitory antibody as the effective ingredient
in the thus recovered therapeutic agent of the invention
strongly binds to the seguences RGD and SWYGLR in OPN, the OPN
inhibitory antibody possibly inhibits the binding between these
OPN regions and integrin, to consequently suppress the
exacerbation of the symptoms of rheumatism, and rheumatoid
arthritis and other autoimmune diseases.
Because the OPN inhibitory antibody of the invention
specifically binds to the OPN side, not to the integrin side,
the antibody potentially never inhibits other significant
functions of integrin, so it is expected that disadvantageous
side effects can be avoided.
Further, the OPN inhibitory antibody of the invention can
also be used for the purpose of screening for a therapeutic agent
of autoimmune diseases. As described above, a compound
inhibiting the binding between the RGD sequence of OPN and
integrin and inhibiting the binding between the SWYGLR
sequence and integrin possibly serves as a therapeutic agent
of autoimmune diseases . Thus, the applicability of a substance
to be screened (test substance) as a therapeutic agent of
autoimmune diseases can be evaluated in a reaction system
prepared by adding the test substance and the OPN inhibitory
antibody in a competitive manner to an assay system in the
presence of given amounts of OPN and integrin, to assay the
extent of the inhibition of the binding between the OPN and
integrin relative to the amount of the OPN inhibitory antibody
used.
Similarly, a compound inhibiting the binding between the
RGD sequence of OPN and integrin and inhibiting the binding
between the SWYGLR sequence and integrin possibly serves as
a therapeutic agent of rheumatism and rheumatoid arthritis.
When the OPN inhibitory antibody is used to compose the same
reaction system as described above, therefore, the reaction
system can be used for screening for rheumatism and rheumatoid
arthritis.
Furthermore, the OPN inhibitory antibody of the invention
can be utilized as a diagnostic agent of rheumatism. As
described above, it is demonstrated that a high concentration
of the N-terminal fragment of thrombin-cleaved OPN is
particularly found in the arthrosis of a patient with rheumatoid
arthritis. Thus, the assay of OPN or the N-terminal fragment
thereof in a sample using the OPN inhibitory antibody can serve
for the diagnosis of rheumatism. As the method therefor, the
following various methods for use as general immunochemical
assay methods ["Hybridoma Method and Monoclonal Antibody",
issued by R&D Planning KK., pp. 30-53, March 5, 1982] are
applicable: radioimmunoassay method (RIA) , ELISA (E. Engvall
etal., (1980) : Methods in Enzymol., 70, 419-439), fluorescent
antibodymethod, plaque method, spot method, aggregationmethod,
Ouchterlony test and the like.
The method can be selected appropriately from various
standpoints. In view of sensitivity, simplicity and the like,
ELISA is preferable. The method more preferably includes
immobilizing the OPN inhibitory antibody of the invention on
a carrier and labeling an antibody recognizing an OPN site
different from that of the OPN inhibitory antibody of the
invention, to detect OPN or the N-terminal fragment thereof.
Thus, such detection method can be used for a diagnostic agent
of rheumatoid arthritis.
The labeling substance for use in the antibody labeling
includes enzymes such as horseradish peroxidase (referred to
as "HRP" hereinafter), alkali phosphatase (referred to as "AP"
hereinafter) and the like, fluorescent substances such as
fluorescein isocyanate, and rhodamine and the like, radioactive
substances such as 32P, 125I and the like, chemiluminescent
substances, and the like. The procedure of sandwich method as
one of more specific detection methods of the OPN isoforms is
described below. Specifically, the procedure includes a first
step (a) of immobilizing the antibody against an OPN isoform
of the invention on a carrier, a second step (b) of blocking
the carrier surface with no immobilized antibody thereon with
amaterialwithno relation with the antigen, for example protein.
The procedure further includes a step (c) of adding a sample
containing various concentrations of the OPN isoform to the
resulting mixture, to generate an OPN isoform-antibody complex,
a step (d) of subsequently adding a labeled anti-OPN isoform
antibody to allow the antibody to bind to the immobilized
antigen-antibody complex, and a final step (e) of assaying the
amount of the label bound to the carrier to determine the amount
of the OPN isoform free in the sample, based on a preliminarily
prepared standard curve.
The carrier used at the step (a) for antibody
immobilization includes, but is not specifically limited to any
carriers for routine use in immunochemical assay methods. The
carrier can include for example polystyrene 96-well microtiter
plate or microtiter plate of amino group-bound type. For
further antibody immobilization, for example, a buffer
containing the antibody is satisfactorily added to and
incubated with the carrier. Known buffers can be used as the
buffer, which is for example 10 mM PBS. The concentration of
the antibody in a buffer may be selected within a wide range,
but generally, the concentration is appropriately about 0.01
to 100 mg/ml and preferably 0.1 to 20 mg/ml • Additionally, the
amount of the buffer is 300 ml/well or less and preferably about
20 to 150 ml/well, when a 96-well microtiter plate is used as
a carrier. Further, the incubation conditions include but are
not specifically limited to overnight incubation at about 4 °C,
which is generally appropriate.
At the step (b) of blocking, further, blocking is done
for the purpose of preventing non-specific adsorption on a
carrier because a part possibly adsorbable on a carrier despite
no relation with the antigen-antibody reaction may potentially
exist in OPN in a sample to be added at the following step. As
the blocking agent, for example, bovine serum albumin (BSA) and
skim milk solution can be used. Otherwise, commercially
available blocking agents such as Block-Ace (manufactured by
Dainippon Pharmaceutical Co ., Ltd. ; Code No . UK-25B) maybe used.
Specifically but not for limitation, blocking is done by adding
for example an appropriate volume of Block-Ace to a part with
the antigen immobilized thereon, for overnight incubation at
about 4 °C and rinsing the resulting part with a buffer. The
buffer includes for example but is not specifically limited to
a buffer of the composition of 10 mM PBS, pH 7.2, C.8 % (w/v)
NaCl, 0.02 % (w/v) KCl, and 0.02 % (v/v) Tween 20.
At the step (c) , then, a sample containing an OPN isoform
is put in contact to the immobilized antibody, to allow the OPN
isoform to be captured on the immobilized antibody and prepare
an immobilized antibody-OPN isoform complex. With no
limitation, the reaction is done at about 37 °C for about one
hour. After the completion of the reaction, the carrier is
rinsed with a buffer, to discard unreactive protein and the like .
A buffer of the composition of 10 mM PBS, pH 7.2 and 0.05 % (v/v)
Tween 20 is preferable as the buffer to be used for the reaction.
At the step (d), further, an immobilized antibody-OPN
isoform-labeled antibody complex is formed by adding a labeled
antibody recognizing another epitope of the OPN isoform
captured on the immobilized antibody. After completion of the
reaction, preferably, the carrier is rinsed with a buffer, to
discard unreactive protein and the like. The buffer described
for the step (c) is used as the buffer for the reaction.
The labeled antibody to be used at the step (d) is required
to recognize an epitope differing from the epitope recognized
by the immobilized antibody at the step (a) . When a polyclonal
antibody recognizing the former-half domain of the OPN isoform
is used as the immobilized antibody, for example, a polyclonal
antibody recognizing the latter-half domain of the OPN isoform
is used as an antibody labeled with a bound enzyme (for example,
HRP or AP or the like) . The use of such antibodies recognizing
different sites as described above enables highly sensitive,
specific assay of an OPN isoform generated by alternative
splicing.
The quantity of the labeled antibody to be used at the
step (d) is preferably about 5,000 to 10,000-fold the quantity
of the immobilized antibody bound to the carrier. Desirably,
the labeled antibody diluted preferably to a final peak
absorbance value of 1.5 to 2.0 at the final assay is used for
reaction. For such dilution, buffers can be used, while the
reaction is done preferably at about 37 °C for about 30 minutes,
followed by rinsing with buffers after completion of the
reaction. But the reaction is not limited to such conditions.
The reactions described above enable the binding of the
antibody-OPN isoform-labeled antibody complex to the carrier.
At the step (e) , finally, a chromogenic substrate
solution reacting with the labeling substance in the
immobilized antibody-OPN isoform-labeled antibody complex is
added for absorbance measurement to calculate the OPN quantity
based on a standard curve.
When an enzyme peroxidase is used as the labeling
substance to label an antibody, for example, a chromogenic
substrate solution containing hydrogen peroxide and 3, 3', 5,
5'-tetramethylbenzine (TMB) or o-phenylenediamine (OPD) can be
used. With no specific limitation, chromogenic reaction is
done by adding a chromogenic substrate solution for reaction
at about 25 °C for about 20 minutes, and subsequently adding
1N sulfuric acid to terminate the enzyme reaction. In the case
that TMB is used, the progress of chromogenic reaction is assayed
on the basis of the absorbance at 450 ran. In the case that an
enzyme AP is used as a labeling substance, alternatively, an
appropriate method includes chromogenic reaction using p-
nitrophenylphosphoric acid (pNPP) as a substrate, the addition
of 2N NaOH to terminate the enzyme reaction and the measurement
of the absorbance at 415 nm.
Using a standard curve preliminarily prepared on the
basis of the absorbance of a reaction solution with addition
of known concentrations of an OPN isoform, the concentration
of the OPN isoform in a sample can be calculated.
The method for detecting the OPN isoform in accordance
with the invention is used for the elucidation of OPN functions,
and the diagnosis and therapeutic treatment of diseases for
which OPN is responsible. One example of the use of the method
includes a detection kit of inflammatory abnormalities by which
rheumatism and rheumatoid arthritis for example can be
discriminated, the kit working for separately detecting the
N-terminal fragment of thrombin-cleaved OPN and non-
cleavage-type OPN, thereby detecting the presence or absence
of any inflammatory abnormalities.
As described above, the N-terminal fragment of
thrombin-cleaved OPN is likely observed at a high concentration
in the articular cavities of patients with rheumatoid arthritis,
in particular. In patients with osteoarthritis, however, the
tendency is significantly low. As described above, the ratio
of the N-terminal fragment occupying OPN in articular cavity
varies in the individual patients. In order to
discriminatively diagnose rheumatism and osteoarthritis,
therefore, the ratio of the N-terminal fragment occupying the
total OPN can be measured, satisfactorily.
As a more specific example, antibodies against individual
peptides of the following three sequences common to all three
OPN isoforms namely OPN-a, OPN-b and OPN-c should be raised.
CVDTYDGRGDSWYGLRS
(C+V153 to S169) (1)
KSKKFRRPDIQYPDATDEC
(K170 to E187+C) (2)
IPVKQADSGSSEEKQC
(117 to Q31+C) (3)
Among them, the sequence (1) is present on the N-terminal
side of the thrombin-cleaved site, and is present common to the
full-length OPN as the thrombin-non-cleavage type and the
N-terminal fragment. Alternatively, the sequence (2) is
present on the C-terminal side of the thrombin-cleaved site and
is present in the full-length OPN of the thrombin-non-cleavage
type but is never contained in the N-terminal fragment. Further,
the sequence (3) corresponds to the amino acid residues at
positions 17 to 31 on the N-terminal side of OPN and is present
common to the full-length OPN as the thrombin-non-cleavage type
and the N-terminal fragment. The diagnosis kit for
discrimination between rheumatism patients and osteoarthritis
patients can be composed of two types of immunodetection
reagents utilizing antibodies individually corresponding to
the three types of sequences. In other words, a first
immunodetection reagent using two types of antibodies against
the peptides represented by the sequences (3) and (2) works for
the assay of thrombin-non-cleavage type OPN commonly recognized
by both the antibodies in a sample. Then, detection can be done
by the same method as the sandwich method, which includes
immobilizing for example an antibody against the peptide of the
sequence (3) on a carrier, allowing the antibody to react with
a sample from a patient, rinsing the carrier, and subsequently
adding an antibody against the peptide of the sequence (2) as
a labeling antibody. In the case of a second immunodetection
reagent, additionally, two antibodies against the peptides
represented by the sequences (1) and (3) are used to assay a
total of the thrombin-non-cleavage-type OPN and the N-terminal
fragment generated by thrombin cleavage in a sample, which are
commonly recognized by both the antibodies. In that case,
detection can be done by the same method as the sandwich method,
which includes immobilizing for example an antibody against the
peptide of the sequence (1) on a carrier, allowing the antibody
to react with a sample from a patient, rinsing the ceirrier, and
subsequently adding an antibody against the peptide of the
sequence (3) as a labeling antibody. Subsequently,, the assay
results of the sample from the same patient with the two types
of the immunodetection reagents are compared together, to
elucidate the ratio of the thrombin-cleavage-generated N-
terminal fragment in the total OPN in the patient, which enables
discrimination between rheumatism and osteoarthritis.
Examples
The invention will now be described in more detail in the
following Examples and Reference Example. But the invention
is not limited to these Examples-.
Example 1
Cloning, construction, purification and reagents for
GST-OPN fusion protein:
Cloning and protein purification were done essentially
according to the method described in the reference (S. Kon et
al., (2000): J. Cell. Biochem. 77: 487-498).
The cDNAs of the human OPN isoforms i.e. OPN-a and OPN-b
were recovered as follows . Using RNAprepared fromNRC-12 cells
of a human kidney cancer cell line as template, cDNA was
synthetically prepared; using the cDNA as template, PCR was done
using the following primers OPN-5 and OPN-3 to recover cDNAs
encoding the full-length human OPN-a and OPN-b individually
including the respective signal peptide regions.
In the manner as described in the reference, then, the
thus cloned cDNAs of OPN-a and OPN-b were inserted in pGEX4T
vector (Amersham Pharmacia Biotech, Tokyo, Japan) so that the
cDNAs might be in the same reading frame as that of the GST gene
(glutathiones-transferase; EC2.5.1.18), for expression in the
form of GST fusion protein, using Escherichia coli JM109 (the
GST-OPN fusion proteins thus recovered are referred to as
"GST-OPN-a" and "GST-OPN-b" hereinbelow).
The cDNA encoding human OPN-c isoform was prepared by
two-step PCR using the OPN-a cDNA as template. At a first step,
PCR was individually done using OPN-5 and the following OPNct-3
primer or the following OPNct-5 and OPN-3 primer; the resulting
two PCR products were mixed together, thermally treated, and
gradually cooled for annealing, followed by addition of an
enzyme for extension. At a second step, subsequently, PCR was
done using the OPN-5 and OPN-3 primers, to recover cDNA encoding
the full-length human OPN-c including the signal peptide region.
The cDNA of the isoform c was integrated in pGEX4T vector by
the same method as for the isoforms a and b, for preparing a
GST fusion protein (referred to as "GST-OPN-c" hereinafter).
OPNct-3:
The cDNA encoding the half moiety at the amino terminal
side (M1-R168) from the thrombin-cleaved site of OPN-a was
recovered by PCR using the OPN-a cDNA as template and OPN-5
together with the following OPNnh-3 primer described below. By
the same method as for the isoforms a and b, the resulting cDNA
was integrated in the pGEX4T vector to prepare a GST protein
(referred to as "GST-Nhalf" hereinbelow) .
The osteopontin protein (hOPN C half) on the carboxyl side
from the thrombin-cleaved site of OPN-a was prepared by two-step
PCR using the OPN-a cDNA as template. At a first step, PCR was
done, individually, using OPN-5, the following OPNch-3 primer,
the following OPNch-5 and the OPN-3 primer. At a second step,
PCR was done using the OPN-5 and OPN-3 primers, to prepare the
OPN protein on the carboxyl side. By the same method as for
the culture supernatant of the CHO/OPN-a cell was subjected to
ion exchange column chromatography using a DEAE-Sepharose CL-6B
column (Amersham Pharmacia Biotech, Tokyo, Japan) and gel
filtration chromatography on an Ultrogel AcA44 column
(manufactured by BioSepra SA), and continuously to reverse-
phase column chromatography on a RESOURCE RPC column (Amersham
Pharmacia Biotech, Tokyo, Japan) . In such manner, purification
was completed.
Various peptides purchased from Sigma Genosis Japan were
used for research works on immune sensitization and bindings;
otherwise, such peptides were recovered by chemical synthesis
by the Fmoc (N-(9-fluorenyl)methoxycarbonyl) process with a
peptide synthesizer (Model 432 A; manufactured by PerkinElmer
Life Science, Inc.) and purification by C18 reverse-phase
column chromatography.
Example 2
Production of monoclonal antibody:
Synthetic peptides corresponding to the inner sequences
of human OPN were prepared, as shown below, which were then used
for immunization.
Peptide 1:
CVDTYDGRGDSWYGLRS (C+V153 to S169)
Peptide 2:
CIDSQELSKVSREFHSH (C+I261 to H276)
33
Specifically, the Peptide 1 has the sequences RGD and
SWYGLR recognizing the avp3 and a9pl integrin receptors,
respectively.
These peptides were bound to thyroglobulin, which were
then used for murine immunization according to a general method.
Continuously, splenocytes were isolated from the immunizedmice,
which were then subjected to cell fusion with a murine myeloma
cell P3-X63-Ag8-653, using polyethylene glycol. According to
the method described in the reference (M. Kinebuchi et al.,
(1991): J. Immunol., 146, 3721-3728), a hybridoma reacting with
each of the peptides used for the immunization was selected.
From mice immunized with the peptides 1 and 2 were
recovered monoclonal antibodies designated 2K1 and 4C1,
respectively. The hybridoma generating the monoclonal
antibody 2K1 was deposited as FERMBP-7883 at the Patent Organism
Depository Center, the National Institute of Advanced
Industrial Science andTechnology (AIST Tsukuba Central 6, 1-1-1,
Higashi, Tsukuba-shi, Ibaraki 305-8566, Japan) on the date of
June 20, 2001. Additionally, the monoclonal antibody 53 (mAb53)
was recovered by immunization with the full-length recombinant
human OPN (D. S. Bautista et al., (1994): J. Biol. Chem., 269,
23280-23285).
Example 3
Reactivity of OPN and thrombin digestion products thereof
with the monoclonal antibodies:
The binding potencies of the monoclonal antibodies 2K1
and 4C1 recovered in the Example 2 to OPN and the thrombin
digestion products thereof were tested by Western blotting
method. It was found that the antibody 2K1 reacted with
GST-OPN-a, GST-OPN-b, GST-OPN-c and GST-Nhalf. The antibody
4C1 reacted with GST-OPN-a, GST-OPN-b, GST-OPN-c and GST-Chalf .
Further, these monoclonal antibodies were not only bound to the
recombinant OPNs of sugar-chain-unbound type as generated in
Escherichia coli but also reacted with the CHO/OPN-a protein
of sugar-chain-bound type and the thrombin digestion products
thereof (referred to as "thrombin-cleaved OPN" hereinafter).
Example 4
Inhibition of cell adhesion to OPN via the monoclonal
antibodies:
It was examined by the following method as to whether or
not the monoclonal antibodies inhibited cell adhesion to OPN.
First, a 96-well plate was precoated with various
concentrations of the CHO/OPN-a at 4 °C overnight, which was
then treated with 0.5 % BSA in PBS under conditions of 37 °C
for 10 minutes, so as to block non-specific adhesion. A human
fibroblast TIG-7 or SW480 cell transformed with the cDNA of an
integrin subunit hereinafter) was suspended in D-MEM containing 0.25 % BSA; 200
ml of the resulting cell suspension (at a cell concentration
of 5 x 104 cells/well) was injected in a 96-well plate precoated
with the CHO/OPN-a or nOPN, in the presence or absence of various
concentrations of the monoclonal antibodies or synthetic
peptides, for incubation at 37 °C for one hour.
The culture medium was discarded from the plate, and all
the wells were rinsed twice with D-MEM containing 0.25 % BSA.
The adherent cells were fixed and stained with 0.5 % crystal
violet in 20 % methanol for 30 minutes.
All the wells were rinsed three times with water, and the
adherent cells were then solubilized into 20 % acetic acid. The
resulting supernatant recovered from each well was analyzed
with an immunoreader, to measure the absorbance at 590 nm to
determine the relative count of the cells adhering to the well.
All the assays were done in a triplicate fashion, and at least
three independent experiments were performed. The values shown
represent mean from three independent experiments.
It has been known that TIG-7 highly adheres to OPN, but
as shown in Fig. 1A, the adhesion is apparently inhibited by
the peptide GRGDSP (100 mg/ml) but not inhibited by a control
peptide (the C-terminal region of K296-N314 in OPN) (100 mg/ml) .
Thus, the adhesion is dependent on RGD. As shown in Fig. 1B,
further, the antibody 2K1 at 200 (mg/ml apparently inhibited the
cell adhesion to OPN. As shown in Fig. 1C, still further, the
effect of 2K1 on the inhibition of cell adhesion is comparative
to the effect exerted by mAb53 and is concentration-dependent.
Still further, 2K1 and mAb53 never inhibit the adhesion of TIG-7
cell to vitronectin (VN) or fibronectin (FN).
Fig. 2 depicts the inhibition of the monoclonal
antibodies on the adhesion of nOPN and vitronectin to the
a9-transformed SW480 cell. As shown in Fig. 2A, the adhesion
between 1 [xg/ml vitronectin and the a9-trans formed SW480 cell
was inhibited by 200 [M GRGDSP peptide(RGD), so the adhesion
is dependent on the RGD. The adhesion of the a9-transformed
SW480 cell to 3 u.g/ml nOPN was inhibited by a combination of
200 |oM GRGDSP and an anti-a9pl monoclonal antibody Y9A2 (A. Wang
etal., (1996): Am. J. Respir. CellMol. Biol., 15, 664-672),
so the adhesion is RGD-dependent and RGD-independent. Fig.2B
additionally shows the effect of 2K1 on the adhesion of the
a9-transformed SW480 cell to nOPN and vitronectin. The
adhesion between the a9-transformed SW480 cell and vitronectin
was never inhibited by 2K1, but the adhesion between the SW480
cell and nOPN was inhibited by 2K1. Consequently, it is
indicated that 2K1 retains the potency of inhibiting RGD-
dependent adhesion.
Example 5
Inhibition of OPN-induced monocyte migration via the
monoclonal antibodies:
Cell migration test using the U937 cell was performed by
using a system ChemoTx101-8 (Neuro Probe Inc. ) . The cell was
adjusted to 2 xlO6 cells/ml with D-MEM containing 0.1 % BSA,
which was then applied to the upper layer on a filter (with a
pore size of 8 mm) , while the OPN protein was added to the lower
layer.
The ChemoTx plate was left to stand in the presence of
5 % C02 at 37 °C for 4 hours. After the plate was left to stand,
the filter was fixed with methanol-, and was then stained with
hematoxylin and eosin (H-E) . The number of the cells migrating
into the back face of the filter was counted with a microscope
(magnification x 400) . The test was done in a triplicate manner,
and the mean was used as data. The results are shown in Fig.
3.
Fig. 3a shows cell migration of the U937 cell toward the
CHO/OPN-a, the thrombin-cleaved OPN and the GST-Nhalf at the
concentrations shown. Additionally, Fig. 3b shows inhibition
assays using the individual OPNs at 10 (j,g/ml, in the presence
or absence of 50 pig/ml 2K1, mAb53 or control murine IgG after
antigen-specific purification.
As shown in Figs. 3a and 3b, the CHO/OPN-a, the
thrombin-cleaved OPN and the GST-Nhalf induce the migration of
the human monocyte U937 in a concentration-dependent manner (A) .
The 2K1 antibody apparently inhibits the monocyte migration
induced by the CHO/OPN-a, the thrombin-cleaved OPN and the
GST-Nhalf. On contrast, mAb53 only inhibits the monocyte
migration induced by the full-length OPN (B).
Reference Example 1
OPN and arthritis induction:
In order to elucidate the OPN function in arthritis, an
OPN gene-defective mouse (S. R. Rittling et al., (1998): J. Bone
and Mminer. Res., 13 (7), 1101-1111) was artificially prepared
according to a general method, for comparative experiments with
normal mouse.
An arthritogenic monoclonal antibody cocktail
commercially available as a substance eliciting arthritis
(under the trade name of a cocktail for arthritis,
Arthrogen-CIA® mAb, Arthritogenic mAb cocktail; manufactured
by Iwai Chemical Pharmaceutical Co., Ltd.) was administered to
the OPN gene-defective mouse (OPN-/-) and a normal mouse (OPN+/+) ,
individually, according to an instruction manual attached to
the product, for arthritis induction. Then, the severity
thereof was observed. For controls, physiological saline was
dosed to the two types of the mice.
Comparison of the severity of arthritis was made on the
basis of arthritis score according to the following standard
and wrist swelling on day 10 post-dosing. The results are shown
in Figs. 4 and 5.
As apparently shown in Fig. 4, the normal mouse dosed with
the arthritogenic antibody cocktail/lipopolysaccharide
(referred to as "LPS" hereinafter) had an increase of the
arthritis score on day 4 and thereafter, until on day 10, the
score reached maximum (12 or more). Alternatively, the
arthritis score of the OPN gene-defective mouse increased on
day 5 and thereafter, but the score was only 4 or less at maximum.
Additionally, any of the groups dosed with physiological saline
had no increase of the arthritis score.
As shown in Fig. 5, further, wrist swelling is apparently
weak in the OPN gene-defective mouse, compared with the normal
mouse, which clearly indicates OPN involvement in arthritis.
Example 6
Inhibitory activity of 2K1 antibody on human peripheral
leukocyte migration:
By the following method, the inhibitory activity of the
2K1 antibody on cytokine-activated human peripheral leukocyte
migration was examined. Table 1 shows the results of the
inhibitory activity on neutrophil migration, while Table 2
shows the results of the inhibitory activity on monocyte
migration.
Experimental method>
At the Ficoll process, a monocyte fraction and a
neutrophil fraction were separated from normal human peripheral
blood (P. M. Daftarian et al., (1996): Journal of Immunology,
157, 12-20) . The intermediate layer between Ficoll and serum
was collected and cultured in a flask at 37 °C for one hour.
The resulting attached cell was used as monocyte. To the
erythrocyte layer remaining after collection of the monocyte
fraction was added a 5-fold volume of 3% dextran-PBS to aggregate
erythrocyte, followed by centrifugation at 150 x g and 4 °C for
5 minutes.
The aggregated erythrocyte was precipitated, while in the
resulting supernatant, neutrophil existed in suspended state.
Then, the fraction was centrifuged at 500 x g and ambient
temperature for 20 minutes, to recover neutrophil. The
monocyte and neutrophil as recovered in such manner were
cultured overnight with human TNF-a (20 ng/mL) for activation.
Then, the resulting activated monocyte and neutrophil were used
for migration experiments.
The migration experiments were done, using a 48-well
micro chemotaxis chamber (manufactured by Neuro Probe Inc.).
After various concentrations of the 2K1 antibody were added to
the thrombin-cleaved OPN and were then preliminarily left to
stand at 37 °C for 15 minutes, the mixtures were added to the
lower chamber (to a final human OPN concentration of 10 mg/mL) .
Placing thereon a polycarbonate filter (pore size of 5 mm),
further, a cell suspension of 50 mL was added to the upper chamber
(2 x 106 cells/mL) .
After culturing in the presence of 5 % C02 at 37 °C for
the isoforms a and b, recombination into pGEX4T vector enabled
the preparation of a GST protein (referred to as "GST-Chalf"
hereinafter).

Various recombinant GST-OPN fusion proteins were
prepared in Escherichia coli by a general method, and were then
purified, using a glutathione-Sepharose column according to the
method described in the reference. Among them, the GST-Nhalf
protein was cleaved at the binding site with a prescission
protease (PreScission; Amersham Pharmacia Biotech, Tokyo,
Japan) , to eliminate the GST protein moiety and thereby recover
a protein (referred to as "nOPN" hereinafter) composed of the
amino-terminal half moiety (I17-R168) of OPN alone.
Alternatively, the cDNA encoding the full-length OPN-
a (M1-N314) was further inserted in pcDNA3.1 (+) vector
(Invitrogen Corporation), for transfection into CHO-K1 cell
(manufactured by Dainippon Pharmaceutical Co ., Ltd.) (referred
to as "CHO/OPN-a cell" hereinafter). The OPN-a of the sugar
chain-bound type (referred to as "CHO/OPN-a" hereinafter) as
recovered from the cell was purified as follows . Specifically,
2 hours, the polycarbonate filter was removed to discard the
cells on the upper surface of the filter; subsequently, the cells
infiltrating into the back face of the filter were stained with
Diff-Quick (manufactured by Baxter, International Inc.). The
stained cells were counted at a magnification x 40. The results
are shown as mean cell counts (cells/mm3) + SD in 6 wells.
Experimental Results>
The 2K1 antibody inhibited the migration of the TNF-
a-activated human peripheral neutrophil and monocyte toward the
thrombin-cleaved OPN.
42
Example 7
Preparation of M5 antibody:
The following synthetic peptide corresponding to the
inner sequence (C+V138 to R153) of murine OPN was prepared for
use in immunization.
M5 peptide:
CVDVPNGRGDSLAYGLR
The peptide was bound to thyroglobulin, for subsequent
use for rabbit immunization according to a general method.
Anti-serum was collected from the immunized rabbit, to prepare
the M5 antibody, using a column packed with the M5 peptide of
the N terminal cysteine bound through disulfide bond to thiol
Sepharose beads (Amersham Pharmacia Biotech, Tokyo, Japan).
Example 8
Reactivity of the M5 antibody with OPN and the thrombin
digestion products thereof:
The binding potency of the M5 antibody recovered in
Example 7 with OPN and the thrombin digestion products thereof
was tested by the Western blotting method. Recombinant murine
OPN of the glycosylated form as generated in CHO cells was used
as the OPN. The M5 antibody reacted with OPN and the thrombin
digestion products thereof.
Example 9
Inhibition of cell adhesion to OPN via the M5 antibody:
It was examined by the method described in the reference
(S. Konetal., (2002): J. Cell. Biochem., 84(2), 420-432) as
to whether or not the M5 antibody might inhibit cell adhesion
to OPN. As the OPN, the full-length murine OPN was used, from
which the GST moiety had preliminarily been removed with
PreScission protease (Amersham Pharmacia Biotech, Tokyo,
Japan) (referred to as "mOPN/de-GST" hereinafter) was used. As
the cell, murine NIH3T3 cell was used.
As shown in Fig. 6, the NIH3T3 cell adheres to mOPN/de-GST
in a concentration-dependent manner. As shown in Fig. 7,
further, the adhesion is apparently inhibited by the peptide
GRGDSP (100 (xg/mL) , so the adhesion depends on RGD. As shown
in Fig. 8, the M5 antibody at 200 u.g/mL apparently inhibited
cell adhesion to OPN.
Example 10
Inhibitory activity of the M5 antibody against murine
spleen-derived monocyte migration:
The inhibitory activity of the M5 antibody against
cytokine-adtivated murine spleen-derived monocyte migration-
was examined by the following method. The results are shown
in Table 3.
Experimental method>
The splenocyte from C57BL/6 mouse was ground with a slide
glass into a single cell, which was then cultured in a flask
at 37 °C for one hour. The resulting adherent cell was used
as monocyte . The monocyte was overnight cultured and activated
with human TNF-oc (20 ng/mL) . The resulting activated monocyte
was used at a migration experiment. The migration experiment
was done by the same method as for the human sample in Example
6 above.
Experimental results>
The M5 antibody inhibited the migration of TNF-a-
activated monocyte derived from murine spleen toward the
thrombin-cleavage-type murine OPN recovered from cleavage of
the full-length murine OPN (manufactured by Genzyme
Corporation) with bovine thrombin (manufactured by Sigma).
Example 11
Action of the M5 antibody on bone damage suppression:
By the following method, the action of the M5 antibody
on bone damage suppression in a murine calvaria organ culture
system was examined. The results are shown in Table 4.
Experimental method>
From a newborn mouse on day 1 after birth was resected
the cranial bone; after size adjustment, a half thereof was
placed in each well of a 24-well plate. To each well was then
added human parathyroid hormone (PTH) (1-34) adjusted with
addition of a D-MEM culture broth (with 10 % bovine serum added)
to a final PTH concentration of 10 nM, to induce bone damage.
The M5 antibody was added to a final concentration of 200 |ig/mL.
After culturing at 37 °C for one week, the quantity of calcium
released from bone into the culture broth was assayed by Calcium
E Test WAKO (Wako Pure Chemical Industries, Ltd.).
Experimental results>
With no PTH addition, calcium was at a value of 7.02 mg/mL.
With PTH addition, however, calcium was at a value of 9 .11 mg/mL .
Hence, it was indicated that calcium release from bone was
promoted. When the M5 antibody was added at a concentration
of 200 |j,g/mL, it was verified that bone absorption was inhibited
by about 7 0 %.
Table 4
** P Example 12
Effect of M5 antibody in mouse collagen arthritis model:
By the following method, the effect of the M5 antibody
in a mouse collagen arthritis model was examined. Table 5 shows
the results about arthritis score; Table 6 shows the results
about leg edema; Table 7 shows the results about body weight
change; and Table 8 shows the results about the change of feed
Experimental method>
For arthritis induction, an arthritogenic antibody
cocktail (under the trade name of a cocktail for arthritis,
Arthrogen-CIA® mAb, Arthritogenic mAb cocktail; manufactured
by Iwai Chemical Pharmaceutical Co., Ltd.) recognizing four
epitopes specific to collagen was used.- To a mouse was
intravenously administered the arthritogenic cocktail; 3 days
later, LPS (100 |ag) was intraperitoneally administered to
thereby elicit arthritis . Arthritis was observed on day 3 after
LPS dosing, which reached maximum on day 6.
Immediately before LPS dosing and 3 days later, the M5
antibdy was intravenously administered at a dose of 40 u.g, 150
(j,g or 400 \xq. As the control group, a rabbit IgG-dosed group
(at a dose of 400 \xq) was arranged. Additionally, the
anti-mouse TNF-cx antibody was intravenously administered at a
dose of 200 |ag/mouse immediately before LPS administration and
3 days later. As the control group, a rat IgG-dosed group (at
a dose of 200 ug) was arranged. Further, MTX was orally given
(at a dose of 3.2 mg/kg) once daily on the very day of LPS dosing
and thereafter. Then, MTX dissolved in 5 ml of 0.5 % methyl
cellulose solution was used. For the control group, 5 ml of
0.5 % methyl cellulose solution was arranged.
Assessment was done per one group of five animals,
concerning four items, namely arthritis score, leg edema, body
weight change and feed intake.
Experimental results>
As shown in Tables 5 to 8, the M5 antibody exerted distinct
suppressive actions on the improvement of arthritis score, the
delay of the onset of arthritis, and the improvement of leg edema
in the mouse arthritis model (therapeutic effect) .• The onset
of arthritis was suppressed in a concentration-dependent manner
at a level such that the effect exceeded the effect of the
anti-mouse TNF-a antibody dosed (at a dose of 200 u.g/mouse) .
In contrast, MTX exerted almost no pharmaceutical efficacy.
In the normal group of the model, furthermore, distinct
body weight decrease by about 3 g was observed in 3 days after
LPS dosing; and the tendency was continued on day 3 to day 6,
although the decrease ratio was more or less reduced. In the
groups dosed with the M5 antibody (150 |j.g, 400 ug) and the group
dosed with the anti-mouse TNF-a antibody, alternatively,
apparent improvement of body weight decrease was observed.
Concerning the feed intake, furthermore, rapid body weight
decrease was observed for any of the pharmaceutical substances
up to day 3 after LPS dosing; on day 3 to day 6, however, the
decrease was improved in the groups dosed with the M5 antibody
and the group dosed with the anti-mouse TNF-a antibody. Table
5 shows the effect on arthritis score; Table 6, the suppressive
effect on leg edema; and Tables 7 and 8 show the effects on body
weight change and feed intake change, respectively.
51
Example 13
Availability of OPN-related fragment peptides:
OPN-related fragment peptides at a state purified by HPLC
chromatography were purchased from Auspep Inc., Parkiville,
Australia. The amino acid sequences thereof are shown in (1)
to (3) .
hOPN5:
Example 14
Preparation of antigens for immunization: •
Products of the OPN-related fragment peptides bound to
thyroglobulin were prepared by the EMCS (N-(6-
maleimidocaproyloxy)-succinimide) process, as follows. For
preparation of such products, the molar ratio of thyroglobulin,
an OPN-related fragment peptide and EMCS was 1:300:400.
4 mg of each of the OPN-related fragment peptides in
Example 13 was dissolved in distilled water of about 1 ml.
Alternatively, 5 mg thyroglobulin dissolved in 1 ml of 0.01 M
phosphate buffer, pH 7.0 and EMCS dissolved at 30 |ng/ul in
dimethylformamide were mixed together, individually at
quantities corresponding to the moles, to prepare a
thyroglobulin-EMCS complex solution. The complex solution was
divided in three portions. To each of the portions was added
the OPN-related fragment peptide solution at a quantity
corresponding to the mole, to thereby prepare a solution of an
EMCS-crosslinked product of the OPN-related fragment peptide
bound to thyroglobulin.
The solution of such bound product was dialyzed, using
PBS, to adjust the concentration of the product to 10 (xg/fxl.
The bound product of the OPN-related fragment peptide and
thyroglobulin was used as an antigen for immunization.
Example 15
Preparation of antigens for screening:
As OPN proteins for screening, fusion proteins between
GST and human OPN isoforms, namely GST-OPN-a, GST-OPN-b and
GST-OPN-c, and fusion proteins between GST and the OPN fragment
on the side of amino group (GST-Nhalf) from the thrombin cleavage
site and the OPN fragment on the side of carboxyl group
(GST-Chalf) from the same thrombin cleavage site were prepared
by the method described in Example 1, for use in the anti-serum
reactivity with OPN.
Example 16
Immune sensitization:
Rabbit was immunized, using as antigens for immunization,
the bound products of the OPN-related fragment peptides and
thyroglobulin prepared in Example 14. Immunization was done,
by boosting 100 |il (100 u.g) of a bound product solution every
one week or every two weeks. The antigens were mixed with the
Freund complete adjuvant for a first immunization and were then
mixed with the Freund incomplete adjuvant for a second
immunization and the following immunizations. After eight
times of immunization, serum was separated from collectedblood,
which was then used as anti-serum.
Example 17
Reactivity of anti-serum with OPN:
The OPN-related fragment peptides prepared in Example 13
were diluted with 0.1 M carbonate buffer, pH 9.5 to 10 (xg/ml,
which were then immobilized at 50 |il/well on a 96-well plate.
After rinsing with PBS and blocking with 0.1 % BSA/PBS/0.05 %
NaN3 solution, a 2-fold serial dilution of the 100-fold dilution
of the anti-serum recovered in Example 16 was placed at 50 ml
in a well, for reaction at 37 °C for 30 minutes.
After termination of the reaction, the well was rinsed
four times with 0.05 % Tween 20-PBS. Then, 50 ml each of
HRP-labeled anti-rabbit IgG (manufactured by IBL Co ., Ltd.) was
added to each well, for reaction at 37 °C for 30 minutes. After
termination of the reaction, 100 |ul each of 0. 05M citrate buffer,
pH 4.5 containing 0.4 mg/ml orthophenylenediamine (OPD) and
aqueous 0.03 % hydrogen peroxide was added to each well. Then,
the plate was left to stand in darkness at ambient temperature
for 15 minutes, for chromogenic reaction. After the
chromogenic reaction, 100 ml of 1N sulfuric acid was added to
each well, to terminate the reaction, for absorbance
measurement at 492 nm.
Using the OPN proteins prepared in Example 15,
alternatively, the reactivity of anti-serum was examined by
Western blotting method. Consequently, anti-sera against the
OPN-related fragment peptides hOPNl and hOPN5 reacted with
GST-OPN-a, GST-OPN-b, GST-OPN-c and GST-Nhalf, but never
reacted with GST-Chalf. Alternatively, anti-serum against the
OPN-related fragment peptide hOPN3 reacted with GST-OPN-a,
GST-OPN-b, GST-OPN-c and GST-Chalf, but never reacted with
GST-Nhalf.
Example 18
Preparation of HRP-bound products of anti-OPN-related fragment
peptides antibodies:
HRP-bound products of the antibodies against the OPN-
related fragment peptides hOPN3 and hOPNl were prepared as
follows. 20 mg of each anti-OPN-related fragment peptide
antibody was digested with pepsin, followed by gel filtration
to purify the F(ab')2 fragment of the anti-OPN-related fragment
peptide antibody. Then, the F (ab') 2 fragment was reduced to Fab'
fragment, by using 2-mercaptoethanol. HRP reacted with EMCS
at 37 °C for 60 minutes, followed by gel filtration to prepare
a HRP-EMCS bound product, which further reacted with the
anti-OPN-related fragment peptide antibody Fab' fragment at 4
°C overnight, followed by gel filtration tc prepare an
EMCS-crosslinked HRP-bound product of the anti-OPN-related
fragment peptide antibody.
Example 19
Construction of sandwich ELISA systems:
From combinations of a sandwich ELISA plate and labeled
antibodies, two types of systems namely 1-3 and 5-1 were prepared.
Specifically, the 1-3 system was prepared as follows. The 10
mg/ml antibody against the OPN-related fragment peptide hOPNl
was added in 100 ml portions to a 96-well ELISA plate. After
overnight reaction at 4 °C, blocking with 10 % BSA/PBS/NaN3
solution was done. The resulting plate at that state was used
as the sandwich ELISA plate. The HRP-bound product of the
antibody against the OPN-related fragment peptide hOPN3
prepared in Example 18 was defined as labeled antibody. As
described above, a combination between the immobilizing plate
using the antibody against hOPNl and the labeled antibody using
the antibody against hOPN3 was defined as system 1-3.
In the same manner, a combination of an immobilizing plate
using the antibody against hOPN5 and a labeled antibody using
the antibody against hOPNl was constructed as system 5-1.
Example 2 0
Osteopontin assay in a test subject by sandwich ELISA
systems:
The OPN protein was assayed as follows. 100 [4,1 of a
solution containing a plasma sample or an articular cavity fluid
sample from a test subject was added to the sandwich ELISAplates
of the systems 1-3 and 5-1, for reaction at 37 °C for one hour.
After reaction, the plates were rinsed four times with 0.05 %
Tween20-PBS, followed by addition of 100 ml each of the labeled
antibodies specific to the individual systems for reaction at
4 °C for 30 minutes. After reaction, the plates were rinsed
six times-with 0.05 % Tween20-PBS, followed by addition of 100
ml of a TMB (tetramethylbenzidine) solution. Then, the
resulting plates were left to stand in darkness at ambient
temperature for 30 minutes. IN sulfuric acid was used to
terminate the reaction, for the assay of the absorbance at 450
nm.
Table 9 shows the OPN values in the articular cavity fluids
of patients (13 cases) with rheumatism as measured by the method;
and Table 10 shows the OPN values in the articular cavity fluids
of patients (12 cases) with osteoarthritis. Additionally,
Table 11 shows the OPN values in the plasmas from rheumatism
patients (16 cases) ; Table 12 shows the OPN values in the plasmas
from osteoarthritis patients (7 cases); and Table 13 shows the
OPN values in the plasmas from normal subjects (6 cases).
As apparently shown in these results, the comparison with
the system 1-3 in terms of plasma OPN value among the patients
with rheumatoid arthritis, the patients with osteoarthritis,
and the normal subjects did not show any significant difference.
However, the comparison with the system 5-1 in terms of plasma
OPN value among the patients with rheumatoid arthritis, the
patients with osteoarthritis, and the normal subjects showed
significantly higher plasma OPN values in the patients with
rheumatoid arthritis and the patients with osteoarthritis than
the OPN value in the normal subjects. The level of significance
was higher in the patients with rheumatoid arthritis. This
indicates that total OPN quantity reflected with the system 5-1
is effective for the diagnosis of the general category of
arthritis.
Alternatively, OPN values in the articular cavity fluids
of the patients with rheumatoid arthritis and the patients with
osteoarthritis are larger than the OPN values in the plasmas
thereof, which strongly indicates local OPN generation.
Additionally, the comparison with the systems 1-3 and 5-1
in terms of OPN value of articular cavity fluid between the
patients with rheumatoid arthritis and the patients with
osteoarthritis showed that the OPN value in the patients with
rheumatoid arthritis was significantly larger than the OPN
value in the patients with osteoarthritis, when any of the
systems 1-3 and 5-1 was used.
As a new indicator, the ratio of OPN values with the
systems 1-3 and 5-1 was examined. The indicator can be used
for the comparison of the ratio of the thrombin-cleaved OPN.
The OPN values in the plasmas and articular cavity fluids from
rheumatoid arthritis patients were 1 or less, and the OPN values
from osteoarthritis patients were 2 or more, so significant
difference was observed. Thus, the OPN values with the systems
1-3/5-1 can be used for a diagnostic method for discriminating
rheumatoid patients from osteoarthritis patients at an early
stage.
Table 9
WE, CLAIM :
1. A therapeutic agent for treating rheumatoid arthritis,
comprising, as an active ingredient, an anti-osteopontin antibody
which can inhibit the binding between an integrin recognizing the
site of the amino acid sequence RGD and a thrombin-cleaved N-
terminal fragment of osteopontin, and which can also inhibit the
binding between an integrin recognizing the site of the amino acid
sequence SVVYGLR or a corresponding equivalent sequence and a
thrombin-cleaved N-terminal fragment of osteopontin, wherein said
anti-osteopontin antibody is an antibody raised against the
peptide CVDTYDGRGDSVVYGLRS or CVDVPNGRGDSLAYGLR as the antigen or
a humanized antibody made by humanization of an antibody which is
raised against the peptide CVDTYDGRGDSVVYGLRS or CVDVPNGRGDSLAYGLR
as the antigen.
2. The therapeutic agent for treating rheumatoid arthritis as
claimed in claim 1, wherein said anti-osteopontin antibody can
inhibit the binding between an a9b1 integrin and a thrombin-
cleaved N-terminal fragment of osteopontin.
3. The therapeutic agent for treating rheumatoid arthritis as
claimed in claim 1, wherein said anti-osteopontin antibody can
inhibit the binding between an a4 integrin and a thrombin-cleaved
N-terminal fragment of osteopontin.
4. The therapeutic agent for treating rheumatoid arthritis as
claimed in claim 1, wherein said anti-osteopontin antibody can
inhibit the binding between an a9b1 integrin and a thrombin-
cleaved N-terminal fragment of osteopontin and the binding between
an a4 integrin and a thrombin-cleaved N-terminal fragment of
osteopontin.
5. The therapeutic agent for treating rheumatoid arthritis as
claimed in anyone of claims 1 to 4, wherein said anti-osteopontin
antibody is a monoclonal antibody.
6. The therapeutic agent for treating rheumatoid arthritis as
claimed in anyone of claims 1 to 4, wherein said anti-osteopontin
antibody is a human type antibody.
There is disclosed a therapeutic agent for treating rheumatoid arthritis, comprising, as an active ingredient, an anti-osteopontin antibody which can inhibit the binding between an integrin recognizing the site of the amino acid sequence RGD and a thrombin-cleaved N-terminal fragment of osteopontin, and which can also inhibit the binding between an integrin recognizing the site of the amino acid sequence SVVYGLR or a corresponding equivalent sequence and a thrombin-cleaved N-terminal fragment of osteopontin, wherein said anti-osteopontin antibody is an antibody raised against the peptide CVDTYDGRGDSVVYGLRS or CVDVPNGRGDSLAYGLR as the antigen or a humanized antibody made by humanization of an antibody which is raised against the peptide CVDTYDGRGDSVVYGLRS or CVDVPNGRGDSLAYGLR as the antigen.

Documents:

1216-KOLNP-2003-CORRESPONDENCE.pdf

1216-KOLNP-2003-FORM 27 1.1.pdf

1216-KOLNP-2003-FORM 27.pdf

1216-KOLNP-2003-FORM-27.pdf

1216-kolnp-2003-granted-abstract.pdf

1216-kolnp-2003-granted-assignment.pdf

1216-kolnp-2003-granted-claims.pdf

1216-kolnp-2003-granted-correspondence.pdf

1216-kolnp-2003-granted-description (complete).pdf

1216-kolnp-2003-granted-drawings.pdf

1216-kolnp-2003-granted-examination report.pdf

1216-kolnp-2003-granted-form 1.pdf

1216-kolnp-2003-granted-form 18.pdf

1216-kolnp-2003-granted-form 3.pdf

1216-kolnp-2003-granted-form 5.pdf

1216-kolnp-2003-granted-form 6.pdf

1216-kolnp-2003-granted-gpa.pdf

1216-kolnp-2003-granted-reply to examination report.pdf

1216-kolnp-2003-granted-specification.pdf


Patent Number 233675
Indian Patent Application Number 1216/KOLNP/2003
PG Journal Number 14/2009
Publication Date 03-Apr-2009
Grant Date 01-Apr-2009
Date of Filing 23-Sep-2003
Name of Patentee IMMUNO-BIOLOGICAL LABORATORIES CO. LTD.
Applicant Address 1091-1, NAKA AZA HIGASHIDA, FUJIOKA-SHI, GUNMA
Inventors:
# Inventor's Name Inventor's Address
1 UEDE TOSHIMITSU 8-2, SHINEIGOJYOU 3-CHOME, KIYOTA-KU, SAPPORO-SHI, HOKKAIDO 044-0835
2 KON SHIGEYUKI C/O IMMUNO-BIOLOGICAL LABORATORIES CO. LTD., 1091-1, NAKA AZA HIGASHIDA, FUJIOKA-SHI, GUNNA 375-0005
3 SAEKI YUKIHIKO 1-21-8, MIZUKIDAI, IZUMI-SHI, OSAKA 594-1118
4 YOKOSAKI YASUYUKI 5-44-801, KAIROUEN 1-CHOME, SAEKI-KU, HIROSHIMA-SHI, HIROSHIMA 731-5135
5 NODA MASAKI 3-11-17-607 EBISUMINAMI, SHIBUYA-KU, TOKYO 150-0022
6 YAMAMOTO NOBUCHIKA C/O FUJISAWA PHARMA-CEUTICAL CO. LTD., 4-7, DOSHOUMACHI 3-CHOME, CHUO-KU, OSAKA-SHI, OSAKA 541-8514
PCT International Classification Number C07K 16/18
PCT International Application Number PCT/JP2002/03382
PCT International Filing date 2002-04-04
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 2001-290700 2001-09-25 Japan
2 2001-107578 2001-04-05 Japan