U.S. patent application number 10/349023 was filed with the patent office on 2003-07-17 for polypeptides.
This patent application is currently assigned to KABUSHIKI KAISHA HAYASHIBARA SEIBUTSU KAGAKU KENKYUJO. Invention is credited to Kurimoto, Masashi, Okura, Takanori, Torigoe, Kakuji.
Application Number | 20030133919 10/349023 |
Document ID | / |
Family ID | 27301588 |
Filed Date | 2003-07-17 |
United States Patent
Application |
20030133919 |
Kind Code |
A1 |
Torigoe, Kakuji ; et
al. |
July 17, 2003 |
Polypeptides
Abstract
Disclosed are a polypeptide (including that in soluble form) as
receptor for a novel cytokine, i.e., interleukin-18, a DNA encoding
the polypeptide, and the uses of the polypeptide including
pharmaceutical and neutralizer to interleukin-18. Pharmaceuticals
with the polypeptide is useful to treat and prevent autoimmune and
allergic disease because it suppresses and regulates excessive
immunoreaction.
Inventors: |
Torigoe, Kakuji; (Okayama,
JP) ; Okura, Takanori; (Okayama, JP) ;
Kurimoto, Masashi; (Okayama, JP) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.
624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
US
|
Assignee: |
KABUSHIKI KAISHA HAYASHIBARA
SEIBUTSU KAGAKU KENKYUJO
2-3, 1-chome, Shimoishii
Okayama-shi
JP
|
Family ID: |
27301588 |
Appl. No.: |
10/349023 |
Filed: |
January 23, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10349023 |
Jan 23, 2003 |
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09556972 |
Apr 24, 2000 |
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6559298 |
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09556972 |
Apr 24, 2000 |
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08996338 |
Dec 22, 1997 |
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6087116 |
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Current U.S.
Class: |
424/93.21 ;
435/366; 435/455 |
Current CPC
Class: |
C07K 14/7155 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
424/93.21 ;
435/455; 435/366 |
International
Class: |
A61K 048/00; C12N
005/08; C12N 015/85 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 12, 1997 |
JP |
74697/1997 |
Jul 28, 1997 |
JP |
215488/1997 |
Oct 9, 1997 |
JP |
291837/1997 |
Claims
What is claimed is:
1. A method for treating diseases associated with excessive IL-18
induced immunoreaction, comprising: transforming effector cells
with an isolated DNA comprising a nucleotide sequence encoding an
IL-18 binding polypeptide capable of binding IL-18; and introducing
the transformed effector cells into a subject to provide adoptive
immunogene therapy to treat diseases associated with excessive
IL-18 induced immunoreaction.
2. The method according to claim 1, further comprising
proliferating the transformed effector cells in vitro prior to
introducing the transformed effector cells into a subject.
3. The method according to claim 2, wherein the effector cells are
tumor cells collected from the subject to be treated.
4. The method according to claim 2, wherein the effector cells are
lymphocytes collected from the subject to be treated.
5. A method for treating diseases associated with excessive IL-18
induced immunoreaction, comprising administering to a subject in
need thereof an isolated DNA comprising a nucleotide sequence
encoding an IL-18 binding polypeptide capable of binding IL-18.
6. An isolated DNA comprising a nucleotide sequence encoding an
IL-18 binding polypeptide derived from an IL-18 receptor, wherein
said IL-18 receptor comprises the amino acid sequence of SEQ ID NO:
21.
7. The isolated DNA according to claim 6, wherein said nucleotide
sequence encoding said IL-18 binding polypeptide comprises a part
or a whole of the nucleotide sequence of SEQ ID NO: 4 or a
complementary sequence thereto.
8. The isolated DNA according to claim 7, wherein said nucleotide
sequence encoding said IL-18 binding polypeptide is selected from
the group consisting of SEQ ID NO: 2, SEQ ID NO: 11, and
complementary sequences thereto.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to a novel receptor protein which
recognizes a cytokine, more particularly, to a novel polypeptide
which recognizes interleukin-18 (hereinafter abbreviated as
"IL-18").
[0003] 2. Description of the Prior Art
[0004] IL-18 is a type of cytokine or substance which mediates
signal transduction in immune system. As seen in Japanese Patent
Kokai Nos. 27,189/96 and 193,098/96 and Haruki Okamura et al.,
Nature, Vol. 378, No. 6,552, pp. 88-91 (1995), IL-18 was
provisionally designated as "interferon-gamma inducing factor"
immediately after its discovery: This designation was changed later
into "IL-18" in accordance with the proposal in Shimpei Ushio et
al., The Journal of Immunology, Vol. 156, pp. 4,274-4,279 (1996).
IL-18 in mature form consists of 157 amino acids and possesses
properties of inducing in immunocompetent cells the production of
interferon-gamma (hereinafter abbreviated as "IFN-.gamma.") which
is known as useful biologically-active protein, as well as of
inducing and enhancing the generation and cytotoxicity of killer
cells. Energetic studies are now in progress to develop and realize
various uses of IL-18 in pharmaceuticals such as antiviral,
antimicrobial, antitumor and anti-immunopathic agents which have
been in great expectation because of these properties of IL-18.
[0005] As described above, in nature, cytokines including IL-18 are
produced and secreted as substances responsible for signal
transduction in immune system. Therefore, excessive amounts of
cytokines may disturb the equilibria in immune system when they are
produced or administered in the body of mammals. The surface of
usual mammalian-cells may bear certain sites or "receptors" which
are responsible for recognition of cytokines: Secreted cytokines
transduce no signal in cells till they are bound to the receptors.
In normal immune system, there would be definite equilibria between
respective cytokines and their receptors. Thus, in this field, with
the purpose of developing and realizing IL-18 as pharmaceuticals,
in addition to the clarification of physiological activities of
IL-18, an expedited establishment of mass production and
characterization of IL-18 receptor (hereinafter abbreviated as
"IL-18R") have been in great expectation.
SUMMARY OF THE INVENTION
[0006] In view of the foregoing, the first object of this invention
is to provide a polypeptide as IL-18R which can be easily prepared
on a large scale.
[0007] The second object of this invention is to provide uses of
such polypeptide as pharmaceuticals.
[0008] The third object of this invention is to provide a DNA which
encodes the polypeptide.
[0009] The fourth object of this invention is to provide a process
to prepare the polypeptide.
[0010] The fifth object of this invention is to provide an agent to
neutralize IL-18 using the polypeptide.
[0011] The sixth object of this invention is to provide a method to
neutralize IL-18 using the polypeptide.
[0012] We energetically and extensively screened various means
which might attain these objects, eventually resulting in the
finding that a substance which recognized IL-18 was present in L428
cell, a type of lymphoblastoid cell derived from a patient with
Hodgkin's disease. We isolated and characterized this substance,
revealing that its nature was proteinaceous, as well as that it
well recognized and bound IL-18 even when in isolated form. It was
also found that the IL-18R thus identified was efficacious in
treatment and prevention of various diseases resulting from
excessive immunoreaction, such as autoimmune diseases, because in
mammals including human, IL-18R recognized and neutralized IL-18
which activated immune system. Further, we have energetically
studied L428 cell using as probe some partial amino acid sequences
of the IL-18R, resulting in obtainment of a DNA which did encode
IL-18R. We confirmed that a polypeptide obtained by bringing such
DNAs into expression in artificial manner well recognized IL-18 and
shared some essential physiological activities with the IL-18R
separated from L428 cell, as well as that it was preparable in
desired amounts by recombinant DNA techniques using such DNA. Thus
we accomplished this invention.
[0013] More particularly, this invention attains the first object
with a polypeptide as IL-18R, which is obtainable through gene
expression.
[0014] This invention attains the second object with an agent for
IL-18R susceptive diseases, which contains as effective ingredient
such polypeptide.
[0015] This invention attains the third object with a DNA which
encodes the polypeptide.
[0016] This invention attains the forth object with a process to
prepare polypeptide, comprising bringing into expression a DNA
which encodes the polypeptide, and collecting the resultant
polypeptide.
[0017] This invention attains the fifth object with an agent to
neutralize IL-18, which contains as effective ingredient the
polypeptide.
[0018] This invention attains the sixth object with a method to
neutralize IL-18, characterized by allowing the polypeptide to act
on IL-18.
[0019] L428 cell, which is feasible in this invention, have been
deposited in the Patent Microorganism Depository, National
Institute of Bioscience and Human-Technology, Agency of Industrial
Science and Technology, 1-3, Higashi 1 chome, Tsukuba-shi,
Ibaraki-ken, 305, Japan, under the accession number of "FERM
BP-5777" on and after Dec. 24, 1996.
BRIEF EXPLANATION OF THE ACCOMPANYING DRAWINGS
[0020] FIG. 1 shows that the monoclonal antibody MAb #117-10C binds
to L428 cells and IL-18R while competing with IL-18.
[0021] FIG. 2 is an image of intermediate tone given on display,
which shows IL-18R on gel electrophoresis visualized by the Western
blotting method using the monoclonal antibody MAb #117-10C.
[0022] FIG. 3 shows the inhibitory action of the monoclonal
antibody MAb #117-10C on the activity of IL-18.
[0023] FIG. 4 is the chromatogram obtained by applying to IL-18R an
immunoaffinity chromatography using the monoclonal antibody MAb
#117-10C.
[0024] FIG. 5 is the peptide map of IL-18R.
[0025] FIG. 6 shows, the structure of the recombinant DNA
"pcDNA/HuIL-18R" of this invention.
[0026] FIG. 7 shows the structure of the recombinant DNA
"pEFHIL18R-14" of this invention.
[0027] FIG. 8 shows the structure of the recombinant DNA
"pEFHIL18RD1-2-H" of this invention.
[0028] FIG. 9 shows the structure of the recombinant DNA
"pEFHIL18RD1-H" of this invention.
[0029] FIG. 10 shows the structure of the recombinant DNA
"pEFMIL18RSHT" of this invention.
[0030] Throughout the Figures, the symbol "Pcmv" indicates the
cytomegalo virus promotor; "EF1.alpha.P", the elongation factor
promotor; "IL-18R cDNA", the cDNA encoding the polypeptide of this
invention; "EFHIL18R-14 cDNA", the cDNA encoding the soluble
polypeptide of human origin according to this invention;
"HIL18RD1-2-H cDNA", the cDNA encoding the soluble polypeptide of
human origin according to this invention; "HIL18RD1-H cDNA", the
cDNA encoding the soluble polypeptide of human origin according to
this invention; and "EFMIL18RSHT cDNA", the cDNA encoding the
soluble polypeptide of mouse origin according to this
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0031] This invention relates to a polypeptide as IL-18R, which is
obtainable through gene expression. The polypeptide of human origin
according to this invention usually contains as partial amino acid
sequence(s) one or more amino acid sequences of SEQ ID NOs: 12 to
19: As a whole; it contains a part or whole of the amino acid
sequence of SEQ ID NO: 20. While the polypeptide of mouse origin
according to this invention usually contains a part or whole of the
amino acid sequence of SEQ ID NO: 21. Thus, the wording
"polypeptide" as referred to in this invention shall include, in
addition to those which wholly contain the amino-acid sequence of
either SEQ ID NO: 20 or 21, for example, those which contain the
same amino acid sequence but with addition of one or more amino
acids, in particular, those which contain one or more amino acids
linked to the C- and/or N-termini in SEQ ID NO: 20 or 21; those
which contain the same amino acid sequence as in SEQ ID NOs: 20 and
21 but with deletion of one or more amino acids, in particular,
soluble polypeptides which contain the amino acid sequences of SEQ
ID NOs: 22 to 25; and those which contain either of the amino acid
sequences as described above but with a saccharide chain, as far as
they are obtainable through gene expression and possess the
essential functions of IL-18R. As to IL-18, those of human and
mouse origins commonly consisting of 157 amino acids have been
documented: Human IL-18 bears the amino acid sequence of SEQ ID NO:
26 (where the amino acid with symbol "Xaa" represents either
isoleucine or threonine), while mouse counterpart, the amino acid
sequence of SEQ ID NO: 27 (where the amino acid with symbol "Xaa"
represents either methionine or threonine).
[0032] The polypeptide of this invention is usually prepared by
applying recombinant DNA techniques, more particularly, by bringing
into expression in artificial manner a DNA which encodes the
polypeptide, and collecting the resultant polypeptide. This
invention provides, in addition to a DNA which encodes the
polypeptide, a process to prepare the polypeptide using recombinant
DNA techniques: By practicing such a process according to this
invention, desired amounts the polypeptide can be easily
obtained.
[0033] The DNA which is used in this invention are those which
originating natural sources, those which can be obtained by
artificially modifying them and those which can be obtained through
chemical synthesis, provided that they do encode the polypeptide.
Generally, in this field, in case of artificially expressing DNAs
which encode polypeptides, one may replace one or more nucleotides
in the DNAs with different nucleotides and/or link an appropriate
nucleotide sequence to the DNAs, with purpose of improving their
expression efficiency and/or the physiological and physicochemical
properties of the polypeptides. Such modifications are feasible in
the DNA of this invention of course: For example, one can link to
the 5'- and 3'-termini of the DNA as described above recognition
sites for appropriate restriction enzymes, initiation and
termination codons, promoters and/or enhancers, as far as the final
polypeptide products do retain desired physiological activities.
Thus, the wording "DNA" as referred to in this invention shall
mean, in addition to those which encode any polypeptides as
described above, those which are complementary thereto, and further
those where one or more nucleotides have been replaced with
different nucleotides while conserving the amino acid sequence.
[0034] To obtain such a DNA from natural sources, for example,
mammalian cells including epithelial cells, endothelial cells,
interstitial cells, chondrocytes, monocytes, granulocytes,
lymphocytes, neurocytes and their established cell lines of human
and mouse origins are screened with oligonucleotides as probe which
can be prepared with reference to the amino acid sequences of SEQ
ID NOs: 12 to 25. Examples of preferred cells are cell lines which
are obtained by establishing hemopoietic cells including
lymphocytes, in particular, JM cells, HDLM-2 cells, MOLT-16 cells
and PEER cells described in Jun Minowada, Cancer Review, Vol. 10,
pp. 1-18 (1988), and lymphoblastoid cells such as L428 cell (FERM
BP-5777), KG-1 cell (ATCC CCL-246) and U-937 cells (ATCC CRL-1593).
The human and mouse DNAs obtained in this way usually contain a
part or whole of respective nucleotide sequences of SEQ ID NOs: 1
and 2. For example, as shown in SEQ ID NO: 7, the DNA obtained from
L428 cell, a type of lymphoblastoid cell derived from a patient
with Hodgkin's disease, consists of the nucleotide sequence of SEQ
ID NO: 1 encoding the amino acid sequence of SEQ ID NO: 20, and
another nucleotide sequence encoding signal peptide which is linked
to the 5'-terminal in the nucleotide sequence of the SEQ ID NO: 1.
Soluble polypeptides with the amino acid sequences of SEQ ID NOs:
22 to 25 are usually encoded by respective nucleotide sequences of
SEQ ID NOs: 3 to 6, which are usually used in a form where, as
shown in the nucleotide sequences of SEQ ID NOs: 8 to 11, a
nucleotide sequence encoding signal peptide is linked to the
5'-terminal in the nucleotide sequences of SEQ ID NOs: 3 to 6. Such
a DNA can be also obtained through usual chemical synthesis, and in
any case, DNAs can be amplified to desired levels by PCR method
once they become available. By the way, the amino acid sequences of
SEQ ID NOs: 20 and 21 are described along with the amino acid
sequences for signal peptides in P. Parnet et al., The Journal of
Biological Chemistry, Vol. 271, pp. 3,967-3,970 (1996): This paper
however makes neither suggestion nor teaching that the polypeptides
with the amino acid sequences of SEQ ID NOs: 20 and 21 do function
as IL-18R.
[0035] Such DNA expresses the polypeptide when introduced into an
appropriate host of microbe, animal or plant origin. The DNA of
this invention is usually prepared into a recombinant DNA prior to
introduction into host. Such recombinant DNA, which consists of the
DNA of this invention and an autonomously replicable vector, can be
easily prepared with usual recombinant DNA techniques, provided
that the DNA is available. Examples of vectors which can receive
the DNA of this invention are plasmid vectors including pKK223-3,
pCDNAI/Amp, BCMGSNeo, pcDL-SR.alpha., pKY4, pCDM8, pCEV4, pME18S
and pEF-BOS. Autonomously replicable vectors usually comprises
other nucleotide sequences, for example, promotor, enhancer,
replication origin, terminator of transcription, splicing sequence
and/or selection marker which facilitate the expression of the DNA
of this invention in particular hosts. Expression of the DNA
becomes artificially regulatable upon external stimuli when it is
used in combination with either heat shock protein promotor or
interferon a promotor as disclosed in Japanese Patent Kokai No.
163,368/95 by the same applicant.
[0036] Conventional methods are feasible in the insertion of the
DNA of this invention into such vector. More particularly, a gene
with the DNA of this invention and an autonomously replicable
vector are first digested with restriction enzyme and/or
ultrasonication, then the resultant DNA and vector fragments are
ligated. Ligation of DNA and vector fragments become much easier
when genes and vectors are digested with restriction enzymes
specific to particular nucleotides, for example, AccI, BamHI,
BstXI, EcoRI, HindIII, NotI, PstI, SacI, SalI, SmaI, SpeI, XbaI and
XhoI. To ligate DNA and vector fragments, they are first annealed,
if necessary, then exposed to DNA ligase in vivo or in vitro. The
recombinant DNA thus obtained is unlimitedly replicable in hosts of
microbe and animal origins. Such recombinant DNA is introduced into
an appropriate host, prior to use in preparation of the
polypeptide. Although conventional hosts of microbe, animal and
plant origins are feasible in this invention, it is preferable to
choose a host of yeast or mammalian origin in case that the final
use of the polypeptide is pharmaceuticals. Examples of host cells
of mammalian origin are epithelial cell, interstitial cell and
hemopoietic cell of human, monkey, mouse and hamster origins, in
particular, 3T3 cell (ATCC CCL-92), C127I cell (ATCC CRL-1616),
CHO-K1 cell (ATCC CCL-61), CV-1 cell (ATCC CCL-70), COS-1 cell
(ATCC CRL-1650), HeLa cell (ATCC CCL-2), MOP-8 cell (ATCC CRL-1709)
and their mutant strains. To introduce the DNA of this invention
into such a host, one can employ conventional methods, for example,
DEAE-dextran method, calcium phosphate transfection method,
electroporation method, lipofection method, microinjection method
and viral infection method using retrovirus, adenovirus,
herpesvirus and vaccinia virus. To select among the resultant
transformants a clone which is capable of producing the
polypeptide, the transformants are cultivated on culture medium,
followed by selecting one or more clones where production of the
polypeptide is observed. Recombinant DNA techniques using host
cells of mammalian origin are detailed, for example,
Jikken-Igaku-Bessatsu, Saibo-Kogaku Handbook (The handbook for the
cell engineering), edited by Toshio KUROKI, Masaru TANIGUCHI and
Mitsuo OSHIMURA, published by Yodosha. Co., Ltd., Tokyo, Japan
(1992), and Jikken-Igaku-Bessatsu, Biomanual Series 3,
Idenshi-Cloning-Jikken-Ho (The experimental methods for the gene
cloning), edited by Takashi YOKOTA and Kenichi ARAI; published by
Yodosha Co., Ltd., Tokyo, Japan (1993).
[0037] The transformant thus obtained produces and secretes the
polypeptide inside and/or outside the host cell when cultivated on
culture medium. Such cultivation is feasible with conventional
culture media directed to cultivation of transformants, which are
usually composed by adding to a bufferized water as base inorganic
ions such as sodium ion, potassium ion, calcium ion, phosphoric ion
and chloric ion; minor elements, carbon sources, nitrogen sources,
amino acids and vitamins which meet to the metabolism of particular
hosts; and, if necessary, sera, hormones, cell growth factors and
cell adhesion factors. Particular media are, for example, 199
medium, DMEM medium, Ham's F12 medium, IMDM medium, MCDB 104
medium, MCDB 153 medium, MEM medium, RD medium, RITC 80-7 medium,
RPMI-1630 medium, RPMI-1640 medium and WAJC 404 medium. One can
obtain a culture product containing the polypeptide by inoculating
on such a culture medium a transformant in an amount of
1.times.10.sup.4-1.times.10.sup.7 cells/ml, preferably,
1.times.10.sup.5-1.times.10.sup.6 cells/ml, and subjecting the
transformant to suspension or monolayer culture at around
37.degree. C. for 1 day to 1 week, preferably, 2 to 4 days while
replacing the culture medium with a fresh preparation, if
necessary. The culture product thus obtained usually contains about
1 .mu.g/l to 1 mg/l polypeptide, dependently of the type of
transformant and cultivation conditions.
[0038] The culture product obtained in this. way is first subjected
to ultrasonication, cell-lytic enzyme and/or detergent to disrupt
cells, if necessary, then polypeptides are separated from the cells
or cell debris by filtration and centrifugation, followed by
purification. In the purification, a culture product which has been
separated from cell or cell debris is subjected to conventional
methods common in purification of biologically-active proteins, for
example, salting-out, dialysis, filtration, concentration,
fractional precipitation, ion-exchange chromatography, gel
filtration chromatography, adsorption chromatography, isoelectric
focusing chromatography, hydrophobic chromatography, reversed phase
chromatography, affinity chromatography, gel electrophoresis and
isoelectric focusing gel electrophoresis which are used in
combination, if necessary. The purified polypeptide is then
concentrated and lyophilized into liquid or solid to meet to its
final use. The IL-18 and monoclonal antibody, disclosed in Japanese
Patent Kokai No. 193,098/96 and Japanese Patent Application No.
356,426/96 by the same applicant, are very useful in purification
of the polypeptide: Immunoaffinity chromatographies using these do
yield a high-purity preparation of the polypeptide with minimized
costs and labors.
[0039] The polypeptide of this invention exhibits a remarkable
efficacy in treatment and prevention of various diseases resulting
from excessive immunoreaction because in mammals including human,
the polypeptide recognizes and binds IL-18 which may activate
immune system. Immune system, which is in nature to defend living
bodies from harmful foreign substances, may cause unfavorable
results in living bodies because of its nature. When mammals
receive a graft of organ, for example, skin, kidney, liver, heart
and bone marrow, the rejection reaction and immunoreaction against
alloantigen may activate T-cells, resulting in the occurrence of
inflammation and proliferation of lymphocytes. Similar phenomena
are observed in case that host receives the invasion by
heteroantigens, for example, allergens, which are not recognized as
self. In autoimmune diseases, allergic reactions are induced by
substances which must be recognized as self. The polypeptide of
this invention exhibits a remarkable efficacy in treatment and
prevention of various diseases resulting from such an
immunoreaction because the polypeptide suppresses or regulates the
immunoreaction when administered in mammals including human. Thus,
the wording "susceptive diseases" as referred to in this invention
shall mean all the diseases resulting from augmented immunoreaction
which can be treated and/or prevented by the direct or indirect
action of IL-18R: Particular susceptive diseases are, for example,
rejection reactions associated with a graft of organ as described
above, autoimmune and allergic diseases including pernicious
anemia, atrophic gastritis, insulin-resistant diabetes, Wegener
granulomatosis, discoid lupus erythematosus, ulcerative colitis,
cold agglutinin-relating diseases, Goodpasture's syndrome, primary
biliary cirrhosis, sympathetic ophtalmitis, hyperthyroidism,
juvenile onset type diabetes, Sjogren syndrome, autoimmune
hepatitis, autoimmune hemolytic anemia, myasthenia gravis, systemic
scleroderma, systemic lupus erythematosus, polyleptic cold
hemoglobinuria, polymyositis, periarteritis nodosa, multiple
sclerosis, Addison's disease, purpura hemorrhagica, Basedow's
disease, leukopenia, Behcet's disease, climacterium praecox,
rheumatoid arthritis, rheumatopyra, chronic thyroiditis, Hodgkin's
disease, HIV-infections, asthma, atopic dermatitis, allergic
nasitis, pollinosis and apitoxin-allergy. In addition, the
polypeptide of this invention is efficacious in treatment and
prevention of septic shock which results from production or
administration of excessive IFN-.gamma..
[0040] Thus, the agent for susceptive disease, which contains as
effective ingredient the polypeptides of this invention, would find
a variety of uses as anti-autoimmune-diseases, anti-allergies,
anti-inflammatories, immunosuppressants, hematopoietics,
leukopoietics, thrombopoietics, analgesics and antipyretics
directed to treatment and/or prevention of susceptive diseases as
illustrated in the above. The agent according to this invention is
usually prepared into liquid, suspension, paste and solid forms
which contain the polypeptide in an amount of 0.00001-100 w/w %,
preferably, 0.0001-20 w/w %, dependently on the forms of agents as
well as on the types and symptoms of susceptive disease.
[0041] The agent for susceptive diseases according to this
invention includes those which are solely composed of the
polypeptide, as well as including those in composition with, for
example, one or more physiologically-acceptable carriers,
excipients, diluents, adjuvants, stabilizers and, if necessary,
other biologically-active substances: Examples of such stabilizer
are proteins such as serum albumins and gelatin; saccharides such
as glucose, sucrose, lactose, maltose, trehalose, sorbitol,
maltitol, mannitol and lactitol; and buffers which are mainly
composed of phosphate or succinate. Examples of the
biologically-active substances usable in combination are FK506,
glucocorticoid, cyclophosphamide, nitrogen mustard,
triethylenethiophosphoramide, busulfan, pheniramine mustard,
chlorambucil, azathioprine, 6-mercaptopurine, 6-thioguanine,
6-azaguanine, 8-azaguanine, 5-fluorouracil, cytarabine,
methotrexate, aminopterin, mitomycin C, daunorubicin hydrochloride,
actinomycin D, chromomycin A.sub.3, bleomycin hydrochloride,
doxorubicin hydrochloride, cyclosporin A, L-asparaginase,
vincristine, vinblastine, hydroxyurea, procarbazine hydrochloride,
adrenocortical hormone and auri colloid; receptor antagonists to
cytokines other than IL-18, for example, antibodies respectively
against interleukin-1 receptor protein, interleukin-2 receptor
protein, interleukin-5 receptor protein, interleukin-6 receptor
protein, interleukin-8 receptor protein and interleukin-12 receptor
protein; and antagonists respectively against TNF-.alpha. receptor,
TNF-.beta. receptor, interleukin-1 receptor, interleukin-5 receptor
and interleukin-8 receptor.
[0042] The agent for susceptive diseases according to this
invention includes pharmaceuticals in minimal dose unit: The
wording "pharmaceutical in minimal dose unit" represents those
which are prepared into a physically united form suitable for
prescription and also allowed to contain the polypeptide in an
amount corresponding to its single dose or multiple (up to 4-fold)
or divisor (up to {fraction (1/40)}) thereof: Examples of such form
are injection, liquid, powder, granule, tablet, capsule,
sublingual, ophthalmic solution, nasal drop and suppository. The
agent for susceptive diseases according to this invention can be
administrated through both oral and parenteral routes to exhibit in
each case a remarkable efficacy in treatment and prevention of
susceptive diseases. More particularly, the polypeptide is
administered through oral route or parenteral route such as
intradermal, subcutaneous, intramuscular or intravenous route at a
dose of about 1 .mu.g/time/adult to about 1 g/time/adult,
preferably, about 10 .mu.g/time/adult to about 100 mg/time/adult 1
to 4 times/day or 1 to 5 times/week over 1 day to 1 year.
[0043] The DNA which encodes the polypeptide of this invention is
useful in "gene therapies". Particularly, in usual gene therapies,
the DNA of this invention is first inserted in a vector derived
from virus such as retrovirus, adenovirus or adeno-associated virus
and, alternatively, embedded in either cationic- or membrane
fusible-liposomes, then the inserted or embedded DNA is directly
injected in a patient with an IL-18 susceptive disease and,
alternatively, introduced into lymphocytes, which have been
collected from the patient, and implanted in the patient. In
adoptive immuno gene therapies, by introducing the DNA of this
invention into effector cells similarly as in the usual gene
therapies, the cytotoxicity of effector cells against tumors and
virus-infected cells is enhanced and this would strengthen adoptive
immunotherapy. In tumor vaccine gene therapy, tumor cells, which
have been extracted from a patient, are introduced with the DNA of
this invention similarly as in the usual gene therapies, allowed to
proliferate in vitro to a prescribed level and then
self-transplanted to the patient: The transplanted tumor cells act
as vaccine in the body of the patient, exhibiting a strong and
antigen-specific antitumor immunity. Thus, the DNA of this
invention exhibits a remarkable efficacy in gene therapies for
various diseases including, for example, malignant tumors, vial
diseases, infections and autoimmune diseases, as well as in
suppression of rejection reaction and excessive immunoreaction
associated with grafts of organs and allergic diseases. General
procedures for gene therapies are detailed in
Jikken-Igaku-Bessatsu, Biomanual UP Series,
Idenshichiryo-no-Kisogijutsu (Basic techniques for the gene
therapy), edited by Takashi SHIMADA, Izumi SAITO, and Keiya OZAWA,
published by Yodosha Co., Ltd., Tokyo, Japan (1996).
[0044] Further, the polypeptide of this invention is useful in
affinity chromatography and labelled assay directed to purification
and detection of IL-18 because the polypeptide bears properties of
recognizing and binding IL-18. In addition, the polypeptide of this
invention, in particular, that in soluble form is useful in
screening in vivo or in vitro agonists and antagonists to IL-18.
Furthermore, the agent to neutralize IL-18 containing as effective
ingredient the polypeptide and the method to neutralize IL-18 where
IL-18 is exposed to the polypeptide are useful in treatment of
various diseases which result from production and administration of
excessive IL-18.
[0045] The following Examples are to illustrate the way of
practicing this invention. The techniques employed in Examples 1 to
3 are common in this field as detailed, for example,
Jikken-Igaku-Bessatsu, Saibo-Kogaku Handbook (The handbook for the
cell engineering), edited by Toshio KUROKI, Masaru TANIGUCHI and
Mitsuo OSHIMURA, published by Yodosha. Co., Ltd., Tokyo, Japan
(1992), and Jikken-Igaku-Bessatsu, Biomanual Series 3,
Idenshi-Cloning-Jikken-Ho (The experimental methods for the gene
cloning), edited by Takashi YOKOTA and Kenichi ARAI, published by
Yodosha Co., Ltd., Tokyo, Japan (1993).
EXAMPLE 1
Preparation and Characterization of IL-18R
Example 1-1
Preparation of IL-18R
[0046] Newborn hamsters were intraperitoneally injected with an
anti-lymphocyte antibody of rabbit origin to suppress their
possible immunoreaction, subcutaneously injected at their dorsal
areas with about 5.times.10.sup.5 cell/animal of L428 cells (FERM
BP-5777), a type of lymphoblastoid cell derived from a patient with
Hodgkin's disease, and fed in usual manner for 3 weeks. The tumor
masses, subcutaneously occurred, about 10 g each, were extracted,
disaggregated and washed in usual manner in serum-free RPMI-1640
medium (pH 7.4), thus obtaining proliferated cells.
[0047] The proliferated cells were added with a mixture solution
(volume ratio of 9:1) of 0.83 w/v % NH.sub.4Cl and 170 mM Tris-HCl
buffer (pH 7.7) in an amount 10-fold larger than the wet weight of
the cells, stirred and collected by centrifugation at 2,000 rpm for
10 minutes. The cells were then suspended in an appropriate amount
of phosphate buffered saline (hereinafter abbreviated as "PBS"),
stirred, collected by centrifugation at 2,000 rpm, resuspended to
give a cell density of about 1.times.10.sup.8 cells/ml in 10 mM
Tris-HCl buffer (pH 7.2) with 1 mM MgCl.sub.2 and disrupted with
"POLYTRON", a cell disrupter commercialized by Kinematica AG,
Littau/Lucerne, Switzerland. The resultant was added with 10 mM
Tris-HCl buffer (pH 7.2) containing both 1 mM MgCl.sub.2 and 1M
sucrose to give a final sucrose concentration of 0.2M, and
centrifuged at 1,000 rpm to collect the supernatant which was then
centrifuged at 25,000 rpm for 60 minutes, followed by collecting
the precipitate. The precipitate was added with adequate amounts of
12 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
(hereinafter abbreviated as "CHAPS"), 10 mM
ethylenediaminetetraacetatic acid (hereinafter abbreviated as
"EDTA") and 1 mM phenylmethylsulfonylflu- oride, stirred at
4.degree. C. for 16 hours, and centrifuged at 25,000 rpm for 60
min, followed by collecting the supernatant.
[0048] The supernatant was charged to a column of "WHEAT GERM
LECTIN SEPHAROSE 6B", a gel product for affinity chromatography
commercialized by Pharmacia LKB Biotechnology AB, Uppsala, Sweden,
pre-equilibrated in PBS with 12 mM CHAPS, and the column was washed
with PBS containing 12 mM CHAPS, and then charged with PBS
containing both 0.5 M N-acetyl-D-glucosamine and 12 mM CHAPS while
monitoring the protein content in the eluate with the absorbance of
ultraviolet at a wave length of 280 nm. The fractions with an
absorbance of 0.16-0.20 were collected and pooled, thus obtaining
about 25 liters of aqueous solution with a protein content of about
1 mg/ml per 10.sup.12 starting cells.
[0049] A small portion of the solution was sampled, added with 4 ng
human IL-18which had been .sup.125I-labelled in usual manner,
incubated at 4.degree. C. for 1 hour, added with appropriate
amounts of "POLYETHYLENE GLYCOL 6000", polyethylene glycol
preparation with an averaged molecular weight of 6,000 daltons,
commercialized by E. Merck, Postfach, Germany, and allowed to stand
under ice-chilling conditions for 30 minutes to effect binding
reaction. The reaction product was centrifuged at 6,000 rpm for 5
minutes and the resultant precipitate was collected to determine
the level of radioactivity. In parallel, there was provided another
sections as control in which 3 .mu.g non-labelled human IL-18 was
used along with .sup.125I-labelled human IL-18 and treated
similarly as above. Comparison with control revealed that the
radioactivity of the precipitate from the sample solution was
significantly higher. This indicated that the aqueous solution
obtained in the above did contain IL-18R and the I-18R recognized
and bound IL-18 when exposed to IL-18.
Example 1-2
Binding Ability to Monoclonal Antibody
[0050] L428 cells (FERM BP-5777) were suspended in RPMI-1640 medium
(pH 7.4), supplemented with 0.1 v/v % bovine serum albumin and also
containing 0.1 v/v % NaN.sub.3, to give a cell density of
4.times.10.sup.7 cells/ml, while monoclonal antibody MAb#117-10C
specific to human IL-18R, obtained by the method described in
Japanese Patent Application No. 356,426/96 by the same applicant,
was dissolved in another preparation of RPMI-1640 medium
supplemented with 0.1 w/v % bovine serum albumin to give different
concentrations of 0.019 .mu.g/ml, 0.209 .mu.g/ml, 2.3 .mu.g/ml,
25.3 .mu.g/ml and 139.5 .mu.g/ml.
[0051] Fifty microliter aliquots of the cell suspension prepared in
the above were mixed with 50 .mu.l of either solution with
different monoclonal antibody concentrations, agitated at 4.degree.
C. for 2 hours, added with 50 .mu.l of RPMI-1640 medium
supplemented with 0.1 v/v % bovine serum albumin and also
containing 4 ng .sup.125I-labelled human IL-18 prepared in usual
manner, and agitated at the same temperature for an additional 30
minutes. Subsequently, each cell suspension was added with 200
.mu.l mixture solution (volume ratio 1:1) of dibutylphthalate and
diocthylphtalate and centrifuged at 10,000 rpm and 20.degree. C.
for 5 minutes, followed by collecting the resultant precipitates
containing the cells which were then determined for radioactivity
using "MODEL ARC-300", a gamma-ray counter commercialized by Aloka
Co., Ltd, Tokyo, Japan.
[0052] In parallel, there were provided additional two sections
where the monoclonal antibody was neglected, while 4 ng
.sup.125I-labelled human IL-18 was treated similarly as in the
sample testing section with or without 4 micrograms of non-labelled
human IL-18 (hereinafter referred to as "non-specific binding
section" and "whole binding section" respectively). The levels of
radioactivity found in "non-specific binding section" and "whole
binding section" were put in Formula 1 together with that found in
the sample testing section to calculate percent inhibition. The
results were as shown in FIG. 1.
Formula 1
[0053] 1 Percent Inhibition = ( Whole binding ) - ( Testing ) (
Whole binding ) - ( Non - specific binding ) .times. 100
[0054] Fifty microliter aliquots of an IL-18R in aqueous solution
obtained by the method in Example 1-1 were added with 50 .mu.l
solution with different concentrations for monoclonal antibody MAb
#117-10C prepared similarly as above, agitated at 4.degree. C. for
2 hours, added with 4 ng .sup.125I-labelled human IL-18, and
agitated at 4.degree. C. for an. additional 30 minutes.
Subsequently, each mixture was added with 50 .mu.l of 4 mg/ml
.gamma.-globulin, allowed to stand under ice-chilling conditions
for 30 minutes, added with 250 .mu.l of PBS with 20 w/v %
polyethylene glycol, allowed to stand under ice-chilling conditions
for an additional 30 minutes, and centrifuged at 6,000 rpm at
4.degree. C. for 5 minutes, followed by collecting the resultant
precipitates which were then determined for radioactivity similarly
as above.
[0055] At the same time, there were provided additional two
sections where the monoclonal antibody was neglected, while 4 ng of
.sup.125I-labelled human IL-18 were treated similarly as in the
sample testing section with or without 4 .mu.g of non-labelled
human IL-18 (hereinafter referred to as "whole binding section" and
"non-specific binding section" respectively). The levels of
radioactivity found in these two section were put in Formula 1
together in that found in the sample testing section to calculate
percent inhibition. The results were as shown in FIG. 1.
[0056] As seen in FIG. 1, in both cases of using L428 cell and
IL-18R in solution, the binding of IL-18 to L428 cell and IL-18R
were inhibited much more as the concentration of monoclonal
antibody MAb #117-10C elevated. This indicated that the monoclonal
antibody MAb #117-10C was bound to the possible IL-18R on the
surface of L428 cell in a fashion competing with IL-18, as well as
that the aqueous solution obtained by the method in Example 1 did
contain a protein capable of recognizing IL-18 or IL-18R and the
monoclonal antibody MAb #117-10C specifically reacted with the
IL-18R.
Example 1-3
Western Blotting
[0057] A portion of the IL-18R in aqueous solution obtained by the
method in Example 1 was sampled, added with 2/3 volume of a mixture
solution of 2.5 w/v % sodium dodecyl sulfate and 50 v/v % glycerol,
incubated at 37.degree. C. for 1 hour, and separated into
respective proteinaceous components on conventional SDS-PAGE using
10-20% gradient gel but using no reducing agent. The proteinaceous
components on the gel were transferred in usual manner to a
nitrocellulose membrane which was then soaked for 1 hour in an
appropriate amount of 50 mM Tris-HCl buffer (pH 7.5) with 10
.mu.g/ml of monoclonal antibody MAb #117-10C obtained by the
methods described in Japanese Patent Application No. 356,426/96 by
the same applicant, 10 v/v % "BLOCK ACE", an immobilizing agent
commercialized by Dainippon Seiyaku Co., Ltd., Osaka, Japan, and
0.05 v/v % "TWEEN 20", a detergent commercialized by City Chemical
Corp., New York, U.S.A., and washed in 50 mM Tris-HCl buffer (pH
7.5) with 0.05 v/v % Tween 20 to remove the remaining antibody. The
membrane was then soaked in Tris-HCl buffer (pH 7.5) with an
appropriate amount of an anti-mouse immunoglobulin antibody of
rabbit origin prelabelled with horse radish peroxidase, 10 v/v %
"BLOCK ACE" and 0.05 v/v % "TWEEN 20" for 1 hour to effect
reaction, washed in 50 mM Tris-HCl buffer (pH 7.5) with 0.05 v/v %
"TWEEN 20" and developed using "ECL kit", a kit for development
commercialized by Amersham Corp., Arlington Heights, U.S.A.
[0058] At the same time, there was provided another section without
the monoclonal antibody MAb #117-10C as control and it was treated
similarly as above. The molecular weight markers were bovine serum
albumin (67,000 daltons), ovalbumin (45,000 daltons), carbonic
anhydrase (30,000 daltons), trypsin inhibitor (20,100 daltons) and
.alpha.-lactoalbumin (14,000 daltons). The results were as shown in
FIG. 2.
[0059] In the gel electrophoresis in FIG. 2, Lane 2 (with
monoclonal antibody) bore a distinct band of IL-18R which was never
found in Lane 3 (without monoclonal antibody).
Example 1-4
Inhibition of IL-18 Activity
[0060] KG-1 cells (ATCC CCL246), an established cell line derived
from a patient with acute myelogenous leukemia, were suspended in
RPMI-1640 medium (pH 7.2), supplemented with 10 v/v % fetal bovine
serum and also containing 100 .mu.g/ml kanamycin and 18.8 mM
Na.sub.2HPO.sub.4, to give a cell density of 1.times.10.sup.7
cells/ml, added with monoclonal antibody MAb #117-10C, obtained by
the method described in Japanese Patent Application No. 356,426/96
by the same applicant, to give a concentration of 10 .mu.g/ml and
incubated at 37.degree. C. for 30 minutes.
[0061] The KG-1 cells in suspension were distributed on 96-well
microplate to give respective amounts of 50 .mu.l/well, added with
50 .mu.l of human IL-18 which had been dissolved in a fresh
preparation of the same medium to give respective concentrations of
0 ng/ml, 1.56 ng/ml, 3.12 ng/ml, 6.25 ng/ml, 12.5 ng/ml and 25
ng/ml, further added with 50 .mu.l/well of 5 .mu.g/ml
lipopolysaccharide in a fresh preparation of the above medium, and
incubated at 37.degree. C. for 24 hours, after which each
supernatant was collected and determined for IFN-.gamma. content by
conventional enzyme immunoassay. In parallel, there were provided
additional sections without the monoclonal antibody MAb #117-10C
for respective IL-18 concentrations as control and they were
treated similarly as above. The results were as shown in FIG. 3.
The IFN-.gamma. contents in FIG. 3 were calibrated with reference
to the standardized IFN-.gamma. preparation Gg23-901-530 available
from the International Institute of Health, USA, and expressed in
the International Unit (IU).
[0062] The results in FIG. 3 indicated that the presence of
monoclonal antibody MAb #117-10C inhibited the induction of
IFN-.gamma. by IL-18 in KG-1 cell as immunocompetent cell. This
also indicated that monoclonal antibody MAb #117-10C blocked the
IL-18R on the surface of KG-1 cell in a fashion competing with
IL-18, thus preventing the signal transduction of IL-18 to KG-1
cell.
Example 1-5
Purification of IL-18R
[0063] Seventy-eight milligrams of a monoclonal antibody MAb
#117-10C, obtained by the method described in Japanese Patent
Application No. 356,426/96 by the same applicant, was dissolved in
an appropriate amount of distilled water and the solution was
dialyzed against borate buffer (pH 8.5) with 0.5M NaCl at 4.degree.
C. for 16 hours. Thereafter, in usual manner, an appropriate amount
of "CNBr-ACTIVATED SEPHAROSE 4B", a CNBr-activated gel,
commercialized by Pharmacia LKB Biotechnology AB, Uppsala, Sweden,
was added to the dialyzed solution and allowed to react at
4.degree. C. for 18 hours under gentle stirring conditions to
immobilize the monoclonal antibody MAb #117-10C on the gel.
[0064] The gel was packed into column in a plastic cylinder,
equilibrated with 2 mM CHAPS, charged with an IL-18R in aqueous
solution obtained by the method in Example 1-1, and applied with
PBS with 12 mM CHAPS to remove non-adsorbed components. The column
was then applied with 35 mM ethylamine containing 2 mM CHAPS (pH
10.8) while collecting the eluate in every 8 ml fractions which
were then checked for presence of IL-18R by the method in Example
1-1 using .sup.125I-labelled human IL-18. The chromatogram obtained
in this operation was as shown in FIG. 4.
[0065] As seen in FIG. 4, IL-18R was eluted in a single sharp peak
when immunoaffinity chromatography using monoclonal antibody MAb
#117-10C was applied to a mixture of IL-18R and contaminants such
as the aqueous solution of IL-18R in Example 1-1. The fractions
corresponding to this single peak were collected, pooled and
lyophilized, thus obtaining a purified IL-18R in solid form.
[0066] Thereafter, a portion of the purified IL-18R was sampled,
incubated in PBS at 100.degree. C. for 5 minutes, and determined
for residual activity by the method in Example 1-2, resulting in no
binding to IL-18 which proved that IL-18R was inactivated by
heating. This would support that the nature of this receptor is
proteinaceous.
[0067] Further, a portion of the purified IL-18R obtained in the
above was dissolved in an appropriate amount of PBS, dialyzed
against PBS at ambient temperature overnight, added with an
appropriate amount of .sup.125I-labelled human IL-18 prepared by
the method in Example 1-1 and 1 mM "BS.sup.3", a polymerizing agent
commercialized by Pierce, Rockford, U.S.A., and allowed to stand at
0.degree. C. for 2 hours to form a conjugate of IL-18R and
.sup.125I-labelled human IL-18. The reaction mixture was added with
Tris-HCl buffer (pH 7.5), allowed to stand at 0.degree. C. for an
additional 1 hour to suspend the conjugation reaction, separated
into respective proteinaceous components on SDS-PAGE using a set of
molecular weight markers and dithiothreitol as reducing agent, and
subjected to autoradiogram analysis.
[0068] The apparent molecular weight for this conjugate of IL-18R
and .sup.125I-labelled human IL-18 was about 50,000 to 200,000
daltons when estimated with reference to the mobility of molecular
weight markers on the autoradiogram. Since the molecular weight of
IL-18 is about 20,000 daltons, the molecular weight of IL-18R can
be estimated about 30,000-180,000 daltons on the assumption that
IL-18R binds one human IL-18 molecule.
Example 1-6
Peptide Mapping of IL-18R
[0069] A purified IL-18R obtained by the method in Example 1-5 was
electrophoresed on SDS-PAGE using 7.5 w/v % gel with 2 w/v %
dithiothreitol as reducing agent, and the gel was then soaked for 5
minutes in a mixture solution of 40 v/v % aqueous methanol and 1
v/v % acetic acid with 0.1 w/v % Coomassie Brilliant Blue for
development, and soaked for an additional 2 hours for destaining in
the same solution but without Coomassie Brilliant Blue, after which
the stained part in the gel, molecular weight of 80,000-110,000
daltons, was cut off, added with 50 v/v % aqueous acetonitrile
containing 0.2 M (NH.sub.4).sub.2CO.sub.3 and repeatedly agitated
at ambient temperature. Thereafter, the gel slices were
lyophilized, added with 0.2M (NH.sub.4).sub.2CO.sub.3 (pH 8.0),
allowed to stand for 5 minutes to effect swelling, added with
appropriate amounts of 1 mM hydrochloric acid with 0.1 .mu.g/.mu.l
"SEQUENCING GRADE MODIFIED TRYPSIN", a reagent of trypsin
commercialized by Promega Corp., Madison, U.S.A., and 0.2 M
(NH.sub.4).sub.2CO.sub.3 (pH 8.9), and allowed to react at
37.degree. C. overnight. After suspending with 10 v/v % aqueous
acetic acid solution, the reaction mixture was added with a mixture
solution of 0.1 v/v % trifluoroacetic acid and 60 v/v % aqueous
acetonitrile and agitated at ambient temperature, after which the
resultant supernatant was collected, concentrated in vacuo and
centrifugally filtered, thus obtaining a concentrate with peptide
fragments.
[0070] The concentrate was charged to ".mu.RPC C2/C18-SC2.1/10", a
column for high-performance liquid chromatography commercialized by
Pharmacia LKB Biotechnology AB, Uppsala, Sweden, pre-equilibrated
with 0.065 v/v % trifluoroacetic acid, and then applied at a flow
rate of 100 .mu.l/min with 0.055 v/v % trifluoroacetic acid
containing 80 v/v % aqueous acetonitrile under liner gradient of
acetonitrile increasing from 0 to 80 v/v over 160 minutes
immediately after application of the eluent. While monitoring the
absorbance at a wavelength of 240 nm, the eluate was fractioned to
separately collect respective peptide fragments which eluted about
45, 50, 55, 58, 62, 72, 75 and 77 minutes after application of the
eluent. The peptide fragments (hereinafter referred to as "peptide
fragment 1", "peptide fragment 2", "peptide fragment 3", "peptide
fragment 4", "peptide fragment 5", "peptide fragment 6", "peptide
fragment 7" and "peptide fragment 8" in the order of elution) were
analyzed in usual manner for amino acid sequence using "MODEL
473A", a protein sequencer commercialized by Perkin-Elmer Corp.,
Norwalk, U.S.A, revealing that the peptide fragments 1 to 8 bore
the amino acid sequences of SEQ ID NOs: 12 to 19 respectively. The
peptide map obtained-by this operation was as shown in FIG. 5.
EXAMPLE 2
Preparation of DNA
Example 2-1
Preparation of Total RNA
[0071] In usual manner, L428 cells (FERM BP-5777) were suspended in
RPMI-1640 medium (pH 7.2) supplemented with 10 v/v % fetal bovine
serum, and proliferated at 37.degree. C. while scaling up the
cultivation. When the cell density reached a prescribed level, the
proliferated cells were collected, suspended in 10 mM sodium
citrate (pH 7.0) containing both 6M guanidine isothiocyanate and
0.5 w/v % sodium N-laurylsarcosinate, and then disrupted with a
homogenizer.
[0072] Aliquots of 0.1M EDTA (pH 7.5) containing 5.7M CsCl.sub.2
were placed in 35 ml-reaction tubes, poured with the cell
disruptant obtained in the above in layer over the EDTA in each
tube, and subjected to ultracentrifugation at 20.degree. C. at
25,000 rpm for 20 hours to collect the RNA fraction. The RNA
fraction was distributed in 15 ml-centrifugation tubes, added with
an equivolume each of a mixture solution of chloroform/1-butanol
(volume ratio 4:1), agitated for 5 minutes and centrifuged at
4.degree. C. at 10,000 rpm for 10 minutes, after which the aqueous
layer was collected, added with 2.5-fold volume of ethanol and
allowed to stand at -20.degree. C. for 2 hours to precipitate the
total RNA. The precipitate was collected, washed with 75 v/v %
aqueous ethanol, and then dissolved in 0.5 ml of sterilized
distilled water to obtain a solution of the total RNA originating
from L428 cell.
Example 2-2
Preparation of mRNA
[0073] An aqueous solution containing total RNA solution obtained
by the method in Example 2-1 was added with 0.5 ml of 10 mM
Tris-HCl buffer (pH 7.5), containing both 1 mM EDTA and 0.1 w/v %
sodium N-laurylsarcosinate, to bring the total volume to 1 ml. The
mixture solution was added with 1 ml of "OLIGOTEX.TM.-dT30
<SUPER>", a latex with an oligonucleotide of (dT).sub.30
commercialized by Japan Roche K. K., Tokyo, Japan, reacted at
65.degree. C. for 5 minutes and rapidly cooled in an ice-chlling
bath. Thereafter, the reaction mixture was added with 0.2 ml of 5
mM NaCl, incubated at 37.degree. C. for 10 minutes, centrifuged at
10,000 rpm for 10 minutes to collect the resultant precipitate in
pellet form which was then suspended in 0.5 ml of sterilized
distilled water and incubated at 65.degree. C. for 5 minutes to
desorb the mRNA from the latex. The obtained solution was added
with an appropriate amount of ethanol, and the resultant
precipitate was collected and lyophilized to obtain a solid of
mRNA.
Example 2-3
Preparation of DNA Fragment Encoding Polypeptide
[0074] Four microliters of 25 mM MgCl.sub.2, 2 .mu.l of 100 mM
Tris-HCl buffer (pH 8.3) containing 500 mM KCl, 1 .mu.l of 25 mM
dNTP mix, 0.5 .mu.l of 40 units/.mu.l ribonuclease inhibitor and 1
.mu.l of 200 units/.mu.l reverse transcriptase were placed in a 0.5
ml-reaction tube, added with 10 ng of an mRNA, obtained by the
method in Example 2-2, along with an appropriate amount of random
hexanucleotides, and added with sterilized distilled water to bring
the total volume of 20 .mu.l. The obtained mixture was incubated
first at 42.degree. C. for 20 minutes, then at 99.degree. C. for 5
minutes to suspend the reaction, thus obtaining a reaction mixture
containing a first strand cDNA.
[0075] Twenty microliters of the reaction mixture was added with 1
.mu.l of 2.5 units/.mu.l "CLONED Pfu POLYMERASE", a DNA polymerase
commercialized by Stratagene Cloning Systems, California, U.S.A.,
10 .mu.l of the reaction buffer and 1 .mu.l of 25 mM dNTP mix, both
commercialized by Stratagene Cloning Systems, added with 0.1 .mu.g
each of oligonucleotides as sense and antisense primers having
respective nucleotide sequences as shown with
5'-TCAGTCGACGCCACCATGAATTGTAGAGAA-3' and
5'-GAAGCGGCCGCATCATTAAGACTCGGAAAGAAC-3' which had been prepared on
the basis of the amino acid sequence described in P. Parnet et al.,
The Journal of Biological Chemistry, Vol. 271, pp. 3967-3970
(1996), added with sterile distilled water to bring the total
volume to 100 .mu.l. The resultant mixture was subjected first to
3-time cycles of incubating at 95.degree. C. for 1 minute,
42.degree. C. for 2 minutes and 72.degree. C. for 3 minutes in the
given order, then to 35-time cycles of incubating at 95.degree. C.
for 1 minute, 60.degree. C. for 2 minutes and 72.degree. C. for 3
minutes in the given order to effect PCR reaction.
[0076] Fifty nanograms of the obtained PCR product was added with 1
ng of "pCR-Script Cam SK(+)", a plasmid vector commercialized by
Stratagene Cloning Systems, California, U.S.A., and then subjected
to ligation reaction at 16.degree. C. for 2 hours using "DNA
LIGATION KIT VERSION 2", a DNA ligation kit commercialized by
Takara Syuzo, Co., Ltd., Otsu, Shiga, Japan, to insert the DNA
fragment of the PCR product in the plasmid vector. A portion of the
reaction product was sampled and used in usual manner to transform
"XL1-BLUE MRF' KAN", an Escherichia coli strain commercialized by
Stratagene Cloning Systems, California, U.S.A.
EXAMPLE 3
Preparation of Recombinant DNA
[0077] A transformant obtained by the method in Example 2-3 was
inoculated in LB medium containing 30 .mu.g/ml chloramphenicol and
cultivated at 37.degree. C. for 18 hours, after which the cells
were collected from the culture and treated in usual manner to
obtain the plasmid DNA. After confirming by the dideoxy method that
the plasmid DNA contained the nucleotide sequence of SEQ ID NO: 7,
the plasmid DNA was exposed to both restriction enzymes NotI and
SalI, and 100 ng of the obtained DNA fragment was added with 10 ng
of "pcDNAI/Amp", a plasmid vector with a modified multiple cloning
site, commercialized by Invitrogen Corporation, San Diego, U.S.A.,
which had been predigested with both restriction enzymes NotI and
XhoI, and subjected to ligation reaction at 16.degree. C. for 2
hours using "LIGATION KIT VERSION 2", a ligation kit commercialized
by Takara Syuzo Co., Ltd., Otsu, Shiga, Japan. A portion of the
reaction product was sampled and introduced in usual manner into
"XL1-BLUE MRF' KAN", a strain of Escherichia coli commercialized by
Stratagene Cloning Systems, California, U.S.A., to obtain a
transformant "cDNA/HuIL-18R" which contained a recombinant DNA
"pcDNA/HuIL-18R" of this invention. The recombinant DNA
"pcDNA/HuIL-18R" was analyzed in usual manner, revealing that in
the recombinant DNA, a DNA "IL-18R cDNA", which contained the
nucleotide sequence of SEQ ID NO: 1 encoding the polypeptide of
this invention, was linked downstream the cytomegalo virus promotor
Pcmv, as shown in FIG. 6.
EXAMPLE 4
Preparation of Transformant
[0078] A transformant "cDNA/HuIL-18R" obtained by the method in
Example 3 was inoculated in LB medium (pH 7.5) containing 100
.mu.g/ml ampicillin and cultured at 37.degree. C. for 18 hours,
after which the cells were collected from the culture and treated
in usual manner to obtain the plasmid DNA. Separately, COS-1 cell
(ATCC CRL-1650), a fibroblastic cell line derived from a kidney of
African green monkey was proliferated in usual manner, and 20
micrograms of the plasmid DNA obtained in the above was introduced
by conventional electroporation method into 1.times.10.sup.7 COS-1
cells to obtain transformant cells which contained the DNA of this
invention.
EXAMPLE 5
Preparation of Polypeptide
[0079] DMEM medium (pH 7.2) supplemented with 10 v/v % fetal bovine
serum was distributed in flat-bottomed culture bottles, inoculated
with transformant cells, obtained by the method in Example 4, to
give a cell density of 1.times.10.sup.5 cells/ml, and cultured at
37.degree. C. in 5 v/v % CO.sub.2 incubator for 3 days. After
removing the supernatant from the culture, PBS containing both 5 mM
EDTA and 0.02 w/v % NaN.sub.3 was placed in the culture bottles to
desorb the proliferated cells.
[0080] After washing in PBS, the proliferated cells were rinsed in
a buffer containing 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl.sub.2 and
0.1 mM EDTA (hereinafter referred to as "hypotonic buffer"), and
suspended in a fresh preparation of the hypotonic buffer to give a
cell density of 2.times.10.sup.7 cells/ml. The cell suspension was
homogenized with a Dounce-type homogenizer under ice-chilling
conditions, and the resultant homogenate was centrifuged at 15,000
rpm at 5 minutes to remove both cell nuclei and intact cells, and
dialyzed overnight against PBS containing 2 mM CHAPS.
[0081] The dialyzed product was charged to a column of immobilized
monoclonal antibody MAb #117-10C, prepared by the method in Example
1-5, which was then applied with PBS containing 12 mM CHAPS to
remove non-adsorbed components. Thereafter, the column was applied
with 35 mM ethylamine (pH 10.8) containing 2 mM CHAPS while
collecting and fractionating the eluate, was applied to the column,
and the eluate was fractionally collected. Each fraction was then
checked for presence of the polypeptide of human origin by the
method in Example 1-1 using .sup.125I-labelled human IL-18,
selected and pooled to obtain per 10.sup.8 starting cells about 2
ml of an aqueous solution which contained a polypeptide with the
amino acid sequence of SEQ ID NO: 20. The protein content in the
solution was about 10 .mu.g/ml.
[0082] The polypeptide thus obtained was studied for
physicochemical properties by the methods in Example 1. As the
result, the polypeptide obtained in this Example contained each
amino acid sequence in SEQ ID NOs: 12 to 19 as partial amino acid
sequences, as well as exhibiting physiological activities which
were similar to those of the IL-18R from L428 cell.
EXAMPLE 6
Soluble Polypeptide from Human Origin
Example 6-1
Preparation of Recombinant DNA
[0083] One nanogram of a recombinant DNA "pcDNA/HuIL-18R" obtained
by the method in Example 3, 10 .mu.l of 10.times.PCR buffer and 1
.mu.l of 25 mM dNTP mix were placed in 0.5 ml-reaction tube, added
with 1 microliter of 2. units/microliter Pfu DNA polymerase, added
with appropriate amounts of oligonucleotides as sense and antisense
primers having respective nucleotide sequences as shown with
5'-TCAGTCGACGCCACCATGAATTGTAGAGAATTA-3- ' and
5'-GAAGCGGCCGCATCATTATCTTGTGAAGACGTG-3', and with sterile distilled
water to bring the total volume to 100 .mu.l. The resultant mixture
was subjected first to 3-time cycles of incubating at 94.degree. C.
for 1 minute, 42.degree. C. for 2 minutes in and 72.degree. C. for
3 minutes in the given order, then to 35-time cycles of incubating
at 94.degree. C. for 1 minute, 60.degree. C. for 2 minutes and
72.degree. C. for 3 minutes in the given order to effect PCR
reaction.
[0084] Fifty nanograms of the obtained PCR product was added with 1
ng of "pCR-SCRIPT SK(+)", a plasmid vector commercialized by Takara
Syuzo Co. Ltd., Otsu, Shiga, Japan, and reacted using "DNA LIGATION
KIT VERSION 2", a DNA ligation kit commercialized by Takara Shuzo
Co. Ltd., Otsu, Shiga, Japan, at 16.degree. C. for 2 hours to
insert the DNA fragment as the PCR product into the plasmid vector.
A portion of the reaction product was sampled and "XL1-BLUE MRF'
KAN", a strain of Escherichia coli commercialized by Stratagene
Cloning Systems, California, U.S.A., was transformed therewith in
usual manner.
[0085] The transformant obtained in the above was inoculated in LB
medium (pH 7.5) containing 100 .mu.g/ml ampicillin and cultivated
at 37.degree. C. for 18 hours, after which the cells were collected
from the culture and treated in usual manner to obtain the plasmid
DNA. After confirming by the dideoxy method that the plasmid DNA
contained the nucleotide sequence of SEQ ID NO: 10, the plasmid DNA
was exposed to both restriction enzymes NotI and SalI, and 100 ng
of the resultant DNA fragment was added with long of "pEF-BOS", a
plasmid vector prepared in accordance with the method described in
S. Mizushima, Nucleic Acid Research, Vol. 18, No. 17, pp. 5,332
(1990) with slight modification and also predigested with both
restriction enzymes NotI and XhoI, and subjected to ligation
reaction using "LIGATION KIT VERSION 2", a DNA ligation kit
commercialized by Takara Shuzo Co., Ltd., Otsu, Shiga, Japan, at
16.degree. C. for 2 hours. A portion of the reaction product was
sampled and introduced in usual manner into "XL1-BLUE MRF' KAN", a
strain of Escherichia coli commercialized by Stratagene Cloning
Systems, California, U.S.A., thus obtaining a transformant
"EFHIL18R-14" which contained a recombinant DNA "pEFHIL18R-14" of
this invention. The recombinant DNA "pEFHIL18R-14" was analyzed in
usual manner, revealing that in the recombinant DNA, a cDNA
"EFHIL18R-14 cDNA", which contained the nucleotide sequence of SEQ
ID NO: 6 encoding the polypeptide of this invention, was located
downstream the elongation factor 1 promotor EF1.alpha.P as shown in
FIG. 7.
Example 6-2
Preparation of Transformant
[0086] A transformant "EFHIL18R-14" obtained by the method. in
Example 6-1 was inoculated in LB medium (pH 7.5) containing 100
.mu.g/ml ampicillin and cultivated at 37.degree. C. for 18 hours,
after which the cells were collected from the culture and treated
in usual manner to obtain the plasmid DNA. Separately, COS-1 cell
(ATCC CRL-1650), a fibroblastoid cell line derived from a kidney of
African green monkey, was proliferated in usual manner, and 20
micrograms of the plasmid DNA obtained in the above was introduced
by conventional electroporation method into 1.times.10.sup.7 COS-1
cells to obtain transformant cells which contained the DNA of this
invention.
Example 6-3
Preparation of Soluble Polypeptide
[0087] "ASF104", a serum-free nutrient culture medium
commercialized by Ajinomoto Co., Inc., Tokyo, Japan, was
distributed in flat-bottomed culture bottles, inoculated with
ransformant cells, obtained by the method in Example 6-2, to givee
a cell density of 1.times.10.sup.5 cells/ml, and cultured in usual
manner at 37.degree. C. in 5 v/v % CO.sub.2 incubator for 3 days.
The supernatant was collected from the culture and charged to a
column of an immobilized monoclonal antibody #117-10C prepared by
the method in Example 1-5, after which the column was applied first
with PBS containing 12 mM CHAPS to remove non-adsorbed components,
then with 35 mM ethylamine (pH 10.8) containing 2 mM CHAPS while
collecting and fractionating the eluate. Each fraction was checked
for presence of human soluble polypeptide by the method in Example
1-1 using .sup.125I-labelled human IL-18, selected and pooled to
obtain per 10.sup.8 starting cells about 2 ml of an aqueous
solution which contained a polypeptide with the amino acid sequence
of SEQ ID NO: 22. The protein content in the solution was about 10
.mu.g/ml.
[0088] The soluble polypeptide thus obtained was studied for
physicochemical properties by the method in Example 1. As the
result, the soluble polypeptide obtained in this Example contained
each amino acid sequences in SEQ ID NOs: 12 to 17 and 19 as partial
sequences, as well as exhibiting physiological activities which
were similar to the IL-18R from L428 cell.
EXAMPLE 7
Soluble Polypeptide of Human Origin
[0089] One nanogram of an recombinant DNA "pEFHIL18R-14" obtained
by the method in Example 6-1, 10 .mu.l of 10.times.PCR buffer and 1
.mu.l of 25 mM dNTP mix were placed in 0.5 ml-reaction tube, added
with 1 .mu.l of 2.5units/.mu.l Pfu DNA polymerase, further added
with appropriate amounts of oligonucleotides as sense and antisense
primers having respective nucleotide sequences as shown with
5'-TCAGTCGACGCCACCATGAATTGTAGAG-3' and
5'-GAAGCGGCCGCTCATTAGTGATGGTGATGGTGATGTGCAACATGGTTAAGCTT-3', and
filled up to 100 .mu.l with sterile distilled water. The resultant
mixture was subjected first to 3-time cycles of incubating at
94.degree. C. for 1 minute, 42.degree. C. for 2 minutes and
72.degree. C. for 1 minute in the given order, then to 35-time
cycles of incubating at 94.degree. C. for 1 minute, 64.degree. C.
for 1 minute and 72.degree. C. for 1 minute in the given order to
effect PCR reaction, thus obtaining a DNA fragment which consisted
of the nucleotide sequence of SEQ ID NO: 5, a digestion site for
restriction enzyme SalI and a Kozak's sequence both linked to the
5'-terminal of the nucleotide sequence of SEQ ID NO: 5, and a
digestion site for restriction enzyme NotI and a nucleotide
sequence encoding (His).sub.6 tag both linked to the 3'-terminal of
the nucleotide sequence of SEQ ID NO: 5. This DNA fragment was
introduced similarly as in Example 6-1 in "XL1-Blue MRF' Kan", a
strain of Escherichia coli commercialized by Stratagene Cloning
Systems, California, U.S.A., to obtain a transformant which
contained a recombinant DNA "pEFHIL18RD1-2-H" according to this
invention. Analysis of the recombinant DNA in usual manner
confirmed that in this recombinant DNA a cDNA "HIL18RD1-2-H", which
contained the nucleotide sequence of SEQ ID NO: 5 encoding the
polypeptide of this invention, was located downstream the
elongation factor promotor EF1.alpha.P as shown in FIG. 8.
[0090] The recombinant DNA "pEFHIL18RD1-2-H" was introduced in
COS-1 cells similarly as in Example 6-2 using the transformant thus
obtained, and the COS-1 cells were then cultivated similarly as in
Example 6-3. The supernatant of the resultant culture was
concentrated with membrane filtration, and charged on a column of
"Ni-NTA Spin Kit", a gel product for affinity chromatography
commercialized by QIAGEN GmbH, Hilden, Germany, which was then
applied with PBS containing 20 mM imidazole to remove the
non-adsorbed fractions. Thereafter, the column was applied with PBS
containing 250 mM imidazole, and the eluate was collected in
fractions while checking the presence of human soluble polypeptide
in each fraction by the method in Example 1-1 using
.sup.125I-labelled human IL-18, after which the fractions with the
polypeptide were collected and pooled, thus obtaining about 2 ml of
an aqueous solution containing the polypeptide with the amino acid
sequence of SEQ ID NO: 23 per starting 10.sup.8 cells. The protein
content in the solution was about 10 .mu.g/ml.
[0091] The soluble polypeptide thus obtained was studied for
physicochemical properties by the method in Example 1. As the
result, the soluble polypeptide obtained in this Example contained
a part or whole of each amino acid sequences in SEQ ID NOs: 14 to
16 and 19 as partial amino acid sequences, as well as exhibiting
physiological activities which were similar to those of IL-18R from
L428 cell.
EXAMPLE 8
Soluble Polypeptide of Human Origin
[0092] A transformant containing a recombinant DNA "pEFHIL18RD1-H"
according to this invention was prepared similarly as in Example 7,
except that sense and antisense primers were replaced with
oligonucleotides having respective nucleotide sequences as shown
with 5'-TCAGTCGACGCCACCATGAATTGTAGAG-3' and 5'-
GAAGCGGCCGCTCATTAGTGATGGTGATGG- TGATGTCTTTCAGTGAAACAGCT-3'.
Analysis of the recombinant DNA in usual manner confirmed that in
the recombinant DNA a cDNA "HIL18RD1-H", which contained the
nucleotide sequence of SEQ ID NO: 3 encoding the polypeptide of
this invention, was located downstream the elongation factor
promotor EF1.alpha.P as shown in FIG. 9. Thereafter, similarly as
in Example 7, the recombinant DNA was introduced in COS-1 cells and
brought into expression, thus obtaining about 2 ml of an aqueous
solution containing a polypeptide with the amino acid sequence of
SEQ ID NO: 24 per 10.sup.8 starting cells. The protein content in
the solution was about 10 .mu.g/ml.
[0093] The polypeptide of this invention thus obtained were studied
for physicochemical properties by the method in Example 1. As the
result, the soluble polypeptide obtained in this Example contained
each amino acid sequences of SEQ ID NOs: 14 and 15 as partial amino
acid sequences, as well as exhibiting physiological activities
which were similar to those of the IL-18R from L428 cell.
EXAMPLE 9
Soluble Polypeptide of Mouse Origin
Example 9-1
Preparation of Recombinant DNA
[0094] A reaction product containing a first strand cDNA was
obtained by subjecting an mRNA, prepared in usual manner from mouse
liver, in place with that from L428 cell to the same reaction to
synthesize first strand cDNA as in Example 2-3. The reaction
product was treated by the same PCR method. as in Example 2-3,
except that the sense and antisense primers were replaced with
oligonucleotides having respective nucleotide sequence as shown
with 5'-TCAGTCGACGCCACCATGCATCATGAAGAA-3' and
5'-GAAGCGGCCGCATCATTAGTGATGGTGATGGTGATGTGTAAAGACATGGCC-3', which
had been prepared on the basis of the amino acid sequence described
in P. Parnet et al., The Journal of Biological Chemistry, Vol. 271,
pp. 3,967-3,970 (1996) and also the nucleotide sequence of SEQ ID
NO: 1: This operation gave a DNA fragment which comprised the
nucleotide sequence of SEQ ID NO: 11, a digestion site for
restriction enzyme SalI linked to the 5'-terminal in the nucleotide
sequence of the SEQ ID NO: 11, and a cleavage site for restriction
enzyme NotI and a nucleotide sequence encoding (His).sub.6 tag both
linked to the 3'-terminal in the nucleotide sequence of the SEQ ID
NO: 11.
[0095] According to the method in Example 6-1, this DNA fragment
was introduced into "XL1-BLUE MRF' KAN", a strain of Escherichia
coli commercialized by Stratagene Cloning Systems, California,
U.S.A., to transform. After a plasmid DNA was collected from the
transformant and confirmed to contain the nucleotide sequence of
SEQ ID NO: 11, the plasmid DNA was introduced into "XL1-BLUE MRF'
KAN", a strain of Escherichia coli strain commercialized by
Stratagene Cloning Systems, California, U.S.A., to obtain a
transformant "EFMIL18RSHT" which contains a recombinant DNA
"pEFMIL18RSHT" according to this invention. Analysis in usual
manner confirmed that in the recombinant DNA "pEFMIL18RSHT" a cDNA
"EFMIL18RSHT cDNA", which contained the nucleotide sequence of SEQ
ID NO: 4 encoding the polypeptide of this invention, was linked to
downstream of the elongation factor 1 promotor EF1.alpha.P, as
shown in FIG. 8.
Example 9-2
Preparation of Transformant and Soluble Polypeptide
[0096] According to the method in Example 6-2, a plasmid DNA was
collected from a transformant "EFMIL18RSHT" obtained by the method
in Example 9-1, and introduced into COS-1 cells to obtain
transformant cells which contained a DNA encoding a soluble
polypeptide of mouse origin.
[0097] "ASF104", a serum-free nutrient culture medium
commercialized by Ajinomoto Co., Inc., Tokyo, Japan, was
distributed in flat-bottomed culture bottles, inoculated with the
transformed COS-1 cells to give a cell density of
1.times.10.sup.5cells/ml, and cultivated in usual manner at
37.degree. C. in 5 v/v % CO.sub.2 incubator for 3 days. The
supernatant was collected from the resultant culture and charged to
a column of "Ni-NTA", a gel product for affinity chromatography,
commercialized by QIAGEN GmbH, Hilden, Germany, after which the
column was applied first with PBS containing 20 mM imidazole to
remove non-adsorbed components, then with PBS containing 250 mM
imidazole while collecting and fractionating the eluate. The
fractions were checked for presence of mouse soluble polypeptide by
the method in Example 1-1 using .sup.125I-labelled mouse IL-18,
selected and pooled, thus obtaining per 10.sup.8 starting cells
about 2 ml of an aqueous solution which contained a polypeptide
with the amino acid sequence of SEQ ID NO: 25. The protein content
in the solution was about 100 .mu.g/ml. The soluble polypeptide
thus obtained was studied in accordance with the method in Example
1, revealing that it efficiently neutralized mouse IL-18.
EXAMPLE 10
Liquid Agent
[0098] Either polypeptide obtained by the method in Examples 5 to 8
was separately dissolved in aliquots of physiological saline
containing as stabilizer 1 w/v % "TREHAOSE", a powdered crystalline
trehalose commercialized by Hayashibara Co., Ltd., Okayama, Japan,
to give respective concentration of 1 mg/ml, and the resultant
mixtures were separately and sterilely filtered with membrane in
usual manner to obtain four distinct liquid agents.
[0099] The products, which are excellent in stability, are useful
as injection, ophthalmic solution and collunarium in treatment and
prevention of susceptive diseases including autoimmune
diseases.
EXAMPLE 11
Dried Injection
[0100] One hundred milligrams of either polypeptide obtained by the
methods in Example 5 to 8 was separately dissolved in aliquots of
physiological saline containing 1 w/v % sucrose as stabilizer, the
resultant solutions were separately and sterilely filtered with
membrane, distributed in vials in every 1 ml aliquot, lyophilized
and sealed in usual manner to obtain four distinct pulverized
agents.
[0101] The products, which are excellent in stability, are useful
as dried injection in treatment and prevention of susceptive
diseases including autoimmune diseases.
EXAMPLE 12
Ointment
[0102] "HI-BIS-WAKO 104", a carboxyvinylpolymer commercialized by
Wako Pure Chemicals, Tokyo, Japan, and "TREHAOSE", a powdered
crystalline trehalose commercialized by Hayashibara Co., Ltd.,
Okayama, Japan, were dissolved in sterilized distilled water to
give respective concentrations of 1.4 w/w % and 2.0 w/w %, and
either polypeptide obtained by the methods in Examples 5 to 8 was
separately mixed with aliquots of the resultant solution to
homogeneity, and adjusted to pH 7.2 to obtain four distinct paste
agents containing about 1 mg/g of the polypeptide of this invention
each.
[0103] The products, which are excellent in both spreadablity and
stability, are useful as ointment in treatment and prevention of
susceptive diseases including autoimmune diseases.
EXAMPLE 13
Tablet
[0104] Aliquots of "FINETOSE", a pulverized anhydrous crystalline
alpha-maltose commercialized by Hayashibara Co., Ltd., Okayama,
Japan, were separately admixed with either polypeptide, obtained by
the methods in Examples 5 to 8, and aliquots of "LUMIN" as cell
activator,
[bis-4-(1-ethylquinoline)][.gamma.-4'-(1-ethylquinoline)]
pentamethionine cyanine, to homogeneity, and the resultant mixtures
were separately tableted in usual manner to obtain four distinct
types of tablets, about 200 mg each, containing about 1 mg/tablet
of the polypeptide of this invention and also 1 mg/tablet of LUMIN
each.
[0105] The products, which are excellent in swallowability and
stability and also bears an cell activating property, are useful as
tablet in treatment and prevention of susceptive diseases including
autoimmune diseases.
EXPERIMENT
Acute Toxicity Test
[0106] In usual manner, a variety of agents, obtained by the
methods in Examples 8 to 11, were percutaneously or orally
administrated or intraperitoneally injected to 8 week-old mice. As
the result, the LD.sub.50 of each sample was proved about 1 mg or
higher per body weight of mouse in terms of the amount of the
polypeptide, regardless of administration route. This does support
that the polypeptide of this invention is safe when incorporated in
pharmaceuticals directed to use in mammals including human.
[0107] As explained above, this invention is based on the discovery
of a novel receptor protein which recognizes IL-18. The polypeptide
of this invention exhibits a remarkable efficacy in relief of
rejection reaction associated with grafts of organs and also in
treatment and prevention of various disease resulting from
excessive immunoreaction because the polypeptide bears properties
of suppressing and regulating immunoreaction in mammals including
human. Further, the polypeptide of this invention is useful in
clarification of physiological activities of IL-18, establishment
of hybridoma cells which are capable of producing monoclonal
antibodies specific to IL-18R, and also affinity chromatography and
labelled assay to purify and detect IL-18. In addition, the
polypeptide of this invention, in particular, that in soluble form
is useful in screening in vivo and in vitro agonists and
antagonists to IL-18. The polypeptide of this invention, which
bears these outstanding usefulness, can be easily prepared in
desired amounts by the process according to this invention using
recombinant DNA techniques.
[0108] This invention, which exhibits these remarkable effects,
would be very significant and contributive to the art.
Sequence CWU 0
0
* * * * *