U.S. patent application number 14/986884 was filed with the patent office on 2016-07-07 for therapeutic agent for chronic arthritides diseases of childhood-related diseases.
This patent application is currently assigned to Chugai Seiyaku Kabushiki Kaisha. The applicant listed for this patent is Chugai Seiyaku Kabushiki Kaisha. Invention is credited to Masahiro Iwamoto, Seiji Minota, Takako Miyamae, Norihiro Nishimoto, Shumpei Yokota, Kazuyuki Yoshizaki.
Application Number | 20160194401 14/986884 |
Document ID | / |
Family ID | 26612973 |
Filed Date | 2016-07-07 |
United States Patent
Application |
20160194401 |
Kind Code |
A1 |
Yoshizaki; Kazuyuki ; et
al. |
July 7, 2016 |
THERAPEUTIC AGENT FOR CHRONIC ARTHRITIDES DISEASES OF
CHILDHOOD-RELATED DISEASES
Abstract
A therapeutic agent for chronic arthritides diseases of
childhood-related diseases, for example chronic arthritides
diseases of childhood, Still's disease and the like, comprising an
interleukin-6 (IL-6) antagonist as an active ingredient.
Inventors: |
Yoshizaki; Kazuyuki;
(Ashiya-shi, JP) ; Nishimoto; Norihiro; (Osaka,
JP) ; Iwamoto; Masahiro; (Oyama-shi, JP) ;
Minota; Seiji; (Tokyo, JP) ; Yokota; Shumpei;
(Chigasaki-shi, JP) ; Miyamae; Takako;
(Yokohama-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chugai Seiyaku Kabushiki Kaisha |
Tokyo |
|
JP |
|
|
Assignee: |
Chugai Seiyaku Kabushiki
Kaisha
Tokyo
JP
|
Family ID: |
26612973 |
Appl. No.: |
14/986884 |
Filed: |
January 4, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13064953 |
Apr 28, 2011 |
9255145 |
|
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14986884 |
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11704233 |
Feb 9, 2007 |
7955598 |
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13064953 |
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10473165 |
Sep 29, 2003 |
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PCT/JP02/03312 |
Apr 2, 2002 |
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11704233 |
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Current U.S.
Class: |
424/133.1 ;
424/143.1; 424/172.1 |
Current CPC
Class: |
A61K 9/0019 20130101;
A61P 37/02 20180101; C07K 16/248 20130101; Y10S 514/825 20130101;
A61K 39/3955 20130101; C07K 2317/76 20130101; C07K 2317/24
20130101; C07K 2317/14 20130101; A61P 43/00 20180101; C07K 16/2866
20130101; A61K 45/06 20130101; A61P 29/00 20180101; A61P 19/02
20180101; A61K 2039/505 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 9/00 20060101 A61K009/00; A61K 45/06 20060101
A61K045/06; A61K 39/395 20060101 A61K039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 2, 2001 |
JP |
2001-103627 |
Apr 6, 2001 |
JP |
2001-109131 |
Claims
1-15. (canceled)
16. A method for treatment of adult-onset Sill's disease,
comprising administering a subcutaneous injection of an antibody
against human IL-6 receptor to a human patient who needs said
treatment, wherein the antibody against the IL-6 receptor is an
antibody which inhibits binding of IL-6 to the IL-6 receptor by
binding to the IL-6 receptor.
17. The method of claim 16, wherein the antibody against IL-6
receptor is a monoclonal antibody.
18. The method of claim 16, wherein the antibody against L-6
receptor is recombinant.
19. The method of claim 17, wherein the monoclonal antibody against
IL-6 receptor is the PM-1 antibody (FERM BP-2998) or a humanized or
chimeric version thereof.
20. The method of claim 16, wherein treatment further comprises
administration of methotrexate, non-steroidal anti-inflammatory
drugs (NSAIDs), or corticosteroids.
21. A method for treatment of systemic-onset type juvenile
rheumatoid arthritis, comprising administering an intravenous
injection of an antibody against human IL-6 receptor to a human
patient who needs said treatment, wherein the antibody against the
IL-6 receptor is an antibody which inhibits binding of IL-6 to the
IL-6 receptor by binding to the IL-6 receptor, wherein the antibody
against human IL-6 receptor is administered in a dose of 1-20 mg/kg
body weight.
22. The method of claim 21, wherein the antibody against human IL-6
receptor is administered in a dose of 8-12 mg/kg body weight.
23. The method of claim 21, wherein the antibody against human IL-6
receptor is administered once every two weeks.
24. The method of claim 21, wherein the antibody against IL-6
receptor is a monoclonal antibody.
25. The method of claim 21, wherein the antibody against IL-6
receptor is recombinant.
26. The method of claim 24, wherein the monoclonal antibody against
IL-6 receptor is the PM-1 antibody (FERM BP-2998) or a humanized or
chimeric version thereof.
27. The method of claim 21, wherein treatment further comprises
administration of one or more of methotrexate, a non-steroidal
anti-inflammatory drug (NSAID), or a corticosteroid.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This Application is a Continuation of application Ser. No.
13/064,953, filed Apr. 28, 2011, which is a Divisional of
application Ser. No. 11/704,233, filed Feb. 9, 2007, which is a
Divisional of application Ser. No. 10/473,165, which entered the
U.S. national phase on Sep. 29, 2003, from PCT/JP02/03312 filed
Apr. 2, 2002, which claims priority from Japanese patent
applications 2001-109131 filed Apr. 6, 2001 and 2001-103627 filed
Apr. 2, 2001. The entire contents of the aforementioned
applications are incorporated herein by reference
FIELD OF THE INVENTION
[0002] The present invention relates to a therapeutic agent for
"chronic arthritides diseases of childhood-related diseases"
comprising an interleukin-6 (IL-6) antagonist as an active
ingredient. Chronic arthritides diseases of childhood-related
diseases include chronic arthritides diseases of childhood, Still's
disease and the like.
BACKGROUND ART
[0003] IL-6 is a cytokine called B-cell stimulating factor 2 (BSF2)
or interferon .beta.2. IL-6 was discovered as a differentiation
factor responsible for activation of B-lymphatic cells (Hirano, T.
et al., Nature (1986) 324, 73-76). Thereafter, it was found to be a
multifunctional cytokine that influences the function of various
cells (Akira, S. et al., Adv. in Immunology (1993) 54, 1-78). IL-6
has been reported to induce the maturation of T lymphatic cells
(Lotz, M. et al., J. Exp. Med. (1988) 167, 1253-1258).
[0004] IL-6 propagates its biological activity through two proteins
on the cell. One is a ligand-binding protein, IL-6 receptor, with a
molecular weight of about 80 kD to which IL-6 binds (Taga T. et
al., J. Exp. Med. (1987) 166, 967-981; Yamasaki, K. et al., Science
(1987) 241, 825-828). IL-6 receptor exists not only in a
membrane-bound form that penetrates and is expressed on the cell
membrane but also as a soluble IL-6 receptor consisting mainly of
the extracellular region.
[0005] The other is non-ligand-binding membrane-bound protein gp130
with a molecular weight of about 130 kD that takes part in signal
transduction. IL-6 and IL-6 receptor form an IL-6/IL-6 receptor
complex, to which gp130 is bound, and thereby the biological
activity of IL-6 is propagated into the cell (Taga et al., Cell
(1989) 58, 573-581).
[0006] IL-6 antagonists are substances that inhibit the
transduction of IL-6 biological activities. Up to now, there have
been known antibodies to IL-6 (anti-IL-6 antibodies), antibodies to
IL-6 receptor (anti-IL-6 receptor antibodies), antibodies to gp130
(anti-gp130 antibodies), reshaped IL-6, IL-6 or IL-6 receptor
partial peptides, and the like.
[0007] Antibodies to IL-6 receptor have been described in a number
of reports (Novick D. et al., Hybridoma (1991) 10, 137-146; Huang,
Y. W. et al., Hybridoma (1993) 12, 621-630; International Patent
Application WO 95-09873; French Patent Application FR 2694767;
United States Patent U.S. Pat. No. 5,216,128). A humanized PM-1
antibody was obtained by implanting the complementarity determining
region (CDR) of a mouse antibody PM-1 (Hirata et al., J. Immunology
(1989), one of anti-IL-6 receptor antibodies, 143, 2900-2906) into
a human antibody (International Patent Application WO
92-19759).
[0008] Chronic arthritides diseases of childhood are diseases
comprising mainly chronic arthritis that develops at less than 16
years of age and is the most prevalent disease among the collagen
diseases that develop in children. Unlike rheumatoid arthritis (RA)
in adults, they are not considered to be a homogeneous disease and
have a variety of disease types, and therefore they tend to be
dealt with as a disease entity different from rheumatoid arthritis
in adults.
[0009] As the name of chronic arthritides diseases of childhood,
"juvenile rheumatoid arthritis (JRA)" has been used in Japan
according to the diagnostic criteria in the United States, whereas
in Europe the term "juvenile chronic arthritis (JCA)" is mainly
used. Recently, terms such as idiopathic chronic arthritis (ICA)
and juvenile idiopathic arthritis (JIA) have been used.
[0010] The disease types of chronic arthritides diseases of
childhood have been categorized in various ways. According to the
American College of Rheumatology (ACR), they are divided, as
arthritic diseases that develop in children less than 16 years old
and persist for six weeks or longer, into three disease types: 1)
systemic onset JRA, 2) polyarticular, 3) pauciarticular (ARA
classification) (JRA Criteria Subcommittee of the Diagnostic and
Therapeutic Criteria Committee of the American Rheumatism
Association Arthritis Rheum 20 (Suppl): 195, 1977). In Europe, the
European League Against Rheumatism (EULAR) has made a
classification that states that, though it differs from the above
ARA classification in that the duration of arthritis is three
months or more and arthritis due to psoriasis, ankylosing
spondylitis etc. has been excluded, the three disease types are
similar (Bulletin 4, Nomenclature and classification of Arthritis
in Children. Basel, National Zeitung AG, 1977).
[0011] Recently, a revision of the classification has been
attempted, and the International League of Associations for
Rheumatology (ILAR) proposed in 1995 a classification plan of
Idiopathic Arthritides of Childhood (Fink C W, Proposal for the
development of classification criteria for idiopathic arthritides
of childhood. J. Rheumatol., 22: 1566 (1995)), and in 1997 the
revision was proposed as an ILAR plan (Southwood T R, Classifying
childhood arthritis, Ann. Rheum. Dis. 56: 79 (1997)). This
classification provides division into: 1) systemic arthritis, 2)
polyarthritis RF positive, 3) polyarthritis RF negative, 4)
oligoarthritis, 5) extended oligoarthritis, 6) enthesitis related
arthritis, 7) psoriatic arthritis, and 8) others.
[0012] Furthermore, the present inventors have proposed a method of
classifying chronic arthritides diseases of childhood into:
[0013] 1) primary chronic arthritides of childhood
[0014] (1) SPRASH syndrome (SPRASH: spiking fever, pericarditis,
rash, arthritis, splenomegaly, hepatomegaly)
[0015] Starts with relaxation heat and efflorescence, and serositis
and hepatomegaly are observed with concomitant onset of
simultaneous or delayed arthritis, but at times arthritis may not
be observed.
[0016] (2) idiopathic chronic arthritides of childhood
[0017] No underlying diseases are present, and arthritis is the key
pathology. [0018] a) rheumatoid factor (RF)-positive type [0019] b)
anti-nuclear antibody (ANA)-positive type [0020] c) RF/ANA-negative
type
[0021] 2) secondary chronic arthritides of childhood
[0022] Genetic or nongenetic original diseases are accompanied by
arthritis (Shunpei Yokota, "Advances in recent therapeutic methods
for chronic arthritides diseases of childhood", Rheumatism, 39: 860
(1999)).
[0023] It has been reported that various cytokines are involved in
chronic arthritides diseases of childhood. In particular, it is
thought that imbalance in inflammatory cytokines IL-1, IL-6, IL-12
and TNF-.alpha., and anti-inflammatory cytokines IL-1ra (IL-1
receptor antagonist), IL-10, IL-13, sTNFR (soluble TNF receptor) is
associated with the disease.
[0024] For the treatment of chronic arthritides diseases of
childhood, nonsteroidal anti-inflammatory drugs, corticosteroids,
antirheumatic drugs (gold compounds etc.), immunosuppressants,
methotrexate (MTX etc.) have been used. However, as the therapeutic
effects differ with the patients, the development of more effective
therapeutic regimens is being awaited.
[0025] Still's disease, first described by the British pediatrician
Dr. Still in 1897, was reported to have a clinical picture clearly
different from that of rheumatoid arthritis in adults and is a
disease seen in children to adults (especially in adolescence and
the main symptoms include fever, erythema, arthritis, serositis and
the like. Among them, adult-onset type is designated as adult onset
Still's disease. In Still's disease, rheumatoid factor is usually
negative.
[0026] In children, Still's disease is another name of the systemic
type of juvenile rheumatoid arthritis (juvenile rheumatoid
arthritis (JRA), JCA (juvenile chronic arthritis), juvenile
idiopathic arthritis (JIA)) which is a chronic arthritis developing
in children at less than 16 years old. For the causes of Still's
disease, environmental factors such as a virus, host factors such
as HLA, and immunological abnormalities have been reported, but the
etiology is still obscure.
[0027] Still's disease in adults and that in children are
considered to be almost the same disease, though there are minor
differences in clinical feature in addition to the age when the
disease develops. Still's disease in children refers to JRA of the
systemic type as described above. However, JRA and rheumatoid
arthritis (RA) in adults are clinically different in many ways and
are dealt with as different diseases, and therefore Still's disease
in adults is often dealt with as an independent disease entity
among the rheumatic diseases.
[0028] As diagnostic criteria for Still's disease in adults, there
have been known those by Yamaguchi (Journal of Rheumatology 19(3):
424-30, 1992), Reginato (Seminars in Arthritis & Rheumatism
17(1): 39-57, 1987), Cush (Rheumatology Grand Rounds, University of
Pittsburgh Medical Center; Jan. 30, 1984), Goldman (Southern
Medical Journal 73: 555-563, 1980) and the like.
[0029] On the relationship between Still's disease and cytokines,
association with cytokines such as IL-1, IL-2, IL-4, IL-6, IL-7,
IL-8, IL-10, TNF-.alpha., and IFN-.gamma. has been reported, and
among them, inflammatory cytokines such as IL-1, IL-6, TNF-.alpha.,
and IFN-.gamma. have been implicated in the pathology of Still's
disease.
[0030] With respect to IL-6, de Benedetti et al. reported that
serum levels of IL-6 are elevated in Still's disease in children
(Arthritis Rheum. 34: 1158, 1991), and that a large amount of
IL-6/soluble IL-6 receptor (sIL-6R) complex is present in the serum
of patients with Still's disease in children and a correlation can
be seen between this complex level and CRP values (J. Clin. Invest.
93: 2114, 1994). Furthermore, Rooney et al. have reported that
plasma levels of IL-6 and TNF-.alpha. are elevated in patients with
Still's disease in children (Br. J. Rheumatol. 34: 454, 1995).
[0031] As a method of treating Still's disease, nonsteroidal
anti-inflammatory drugs, corticosteroids, antirheumatic drugs (gold
compounds etc.), immunosuppressants, gamma globulin formulations,
methotrexate (MTX etc.) have been used. However, as the therapeutic
effects differ with the patients, the development of more effective
therapeutic regimens is being sought after.
DISCLOSURE OF THE INVENTION
[0032] Thus, the present invention provides a novel therapeutic
agent for chronic arthritides diseases of childhood-related
diseases, said agent being of a type different from the
conventional therapeutic agents for chronic arthritides diseases of
childhood-related diseases. In accordance with the present
invention, chronic arthritides diseases of childhood-related
diseases included chronic arthritides diseases of childhood and
Still's disease.
[0033] After intensive and extensive study to solve the above
problems, the present inventors have found that an interleukin-6
(IL-6) antagonist has an effect of treating chronic arthritides
diseases of childhood-related diseases, and have completed the
present invention.
[0034] Thus, the present invention provides a therapeutic agent for
chronic arthritides diseases of childhood-related diseases
comprising an interleukin-6 (IL-6) antagonist as an active
ingredient.
[0035] More specifically, the present invention provides a
therapeutic agent for chronic arthritides diseases of childhood
comprising an interleukin-6 (IL-6) antagonist as an active
ingredient.
[0036] The present invention also provides a therapeutic agent for
Still's disease comprising an interleukin-6 (IL-6) antagonist as an
active ingredient.
BEST MODE FOR CARRYING OUT THE INVENTION
[0037] The above IL-6 antagonist is preferably an antibody against
IL-6 receptor, and preferably a monoclonal antibody against human
IL-6 receptor or a monoclonal antibody against mouse IL-6 receptor.
As the above monoclonal antibody against human IL-6 receptor, there
can be illustrated PM-1 antibody, and as the above monoclonal
antibody against mouse IL-6 receptor, there can be illustrated
MR16-1 antibody.
[0038] The above antibody is preferably a chimeric antibody, a
humanized antibody or a human antibody and, for example, is a
humanized PM-1 antibody.
[0039] Chronic arthritides diseases of childhood which are the
subject of treatment with a therapeutic agent of the present
invention include all diseases in the above ARA, EULAR, and ILAR
classifications, and the classification by the present inventors.
With the advance in the serological diagnostic methods and the
advance in therapeutic methods, the disease type classification of
chronic arthritides diseases of childhood is now undergoing a
review on a global scale and it can be said to be in a state of
uncertainty. Preferred treatment subjects, for the therapeutic
agent of the present invention, are: in the ARA classification,
systemic onset, polyarticular, and pauciarticular; in the EULAR
classification, systemic onset, polyarticular, and oligoarticular;
in the ILAR classification, systemic onset, polyarticular (RF
positive), polyarticular (RF negative), oligoarthritis, and
extended oligoarthritis; and, in the classification by the present
inventors, primary chronic arthritides of childhood (SPRASH
syndrome, idiopathic chronic arthritides of childhood (a.
rheumatoid factor (RF)-positive type, b. anti-nuclear antibody
(ANA)-positive type, c. RF/ANA-negative type)), and as most
preferred subjects of treatment are: in the ARA classification,
systemic onset and polyarticular; in the EULAR classification,
systemic onset and polyarticular; in the ILAR classification,
systemic onset, polyarticular (RF positive), polyarticular (RF
negative), and extended oligoarthritis; and, in the classification
by the present inventors, primary chronic arthritides of childhood
(SPRASH syndrome, idiopathic chronic arthritides of childhood (a.
rheumatoid factor (RF)-positive type, b. anti-nuclear antibody
(ANA)-positive type)). More preferred subjects of treatment are: in
the ARA classification, systemic onset and polyarticular; in the
EULAR classification, systemic onset and polyarticular; in the ILAR
classification, systemic onset, polyarticular (RF positive), and
extended oligoarthritis; and, in the classification by the present
inventors, primary chronic arthritides of childhood (SPRASH
syndrome, idiopathic chronic arthritides of childhood (a.
rheumatoid factor (RF)-positive type)).
[0040] IL-6 antagonists for use in the present invention may be of
any origin, any type, and any form, as long as they exhibit
therapeutic effects on chronic arthritides diseases of
childhood-related diseases.
[0041] IL-6 antagonists are substances that block signal
transduction by IL-6 and inhibit the biological activity of IL-6.
IL-6 antagonists are substances that preferably have an inhibitory
action on the binding to any of IL-6, IL-6 receptor or gp130. As
IL-6 antagonists, there can be mentioned, for example, anti-IL-6
antibody, anti-IL-6 receptor antibody, anti-gp130 antibody,
reshaped IL-6, soluble reshaped IL-6 receptor, or partial peptides
of IL-6 or IL-6 receptor, as well as low molecular weight
substances that exhibit activities similar to them.
[0042] Anti-IL-6 antibodies for use in the present invention can be
obtained as polyclonal or monoclonal antibodies using a known
method. As the anti-IL-6 antibodies for use in the present
invention, monoclonal antibodies of, in particular, mammalian
origin are preferred. Monoclonal antibodies of a mammalian origin
include those produced by a hybridoma and those produced by a host
which has been transformed by gene engineering technology with an
expression vector containing the antibody gene. These antibodies,
via binding to IL-6, block the binding of IL-6 to IL-6 receptor,
and thereby block the propagation of biological activity of IL-6
into the cell.
[0043] Examples of such antibodies include MH166 antibody (Matsuda,
et al., Eur. J. Immunology (1988) 18, 951-956), or SK2 antibody
(Sato, et al., The 21st General Meeting of the Japanese Society for
Immunology, Gakujutu Kiroku (1991) 21, 166) etc.
[0044] A hybridoma that produces anti-IL-6 antibody can be
basically constructed using a known procedure as described bellow.
Thus, IL-6 is used as a sensitizing antigen, which is immunized in
the conventional method of immunization, and the immune cells thus
obtained are fused with known parent cells in a conventional cell
fusion process, followed by a conventional screening method to
screen monoclonal antibody-producing cells.
[0045] Specifically, anti-IL-6 antibodies may be obtained in the
following manner. For example, human IL-6 to be used as the
sensitizing antigen for obtaining antibody can be obtained using
the IL-6 gene/amino acid sequence disclosed in Eur. J. Biochem.
(1987) 168, 543-550; J. Immunol. (1988) 140, 1534-1541, or Agr.
Biol. Chem. (1990) 54, 2685-2688.
[0046] After the gene sequence of IL-6 was inserted into a known
expression vector to transform a suitable host cell, the IL-6
protein of interest may be purified from the host cell or a culture
supernatant thereof by a known method, and the purified IL-6
protein may be used as the sensitizing antigen. Alternatively, a
fusion protein of the IL-6 protein and another protein may be used
as the sensitizing antigen.
[0047] Anti-IL-6 receptor antibodies for use in the present
invention can be obtained as polyclonal or monoclonal antibodies
using a known method. As the anti-IL-6 receptor antibodies for use
in the present invention, monoclonal antibodies of, in particular,
a mammalian origin are preferred. Monoclonal antibodies of a
mammalian origin include those produced by a hybridoma and those
produced by a host which has been transformed by gene engineering
technology with an expression vector containing the antibody gene.
These antibodies, via binding to IL-6, block the binding of IL-6 to
IL-6 receptor, and thereby block the propagation of biological
activity of IL-6 into the cell.
[0048] Examples of such antibodies include MR16-1 antibody (Tamura,
T. et al., Proc. Natl. Acad. Sci. USA (1993) 90, 11924-11928), PM-1
antibody (Hirata, Y. et al., J. Immunology (1989) 143, 2900-2906),
AUK12-20 antibody, AUK64-7 antibody or AUK146-15 antibody
(International Patent Application WO 92-19759), and the like. Among
them, PM-1 antibody is most preferred.
[0049] Incidentally, the hybridoma cell line which produces PM-1
antibody has been internationally deposited under the provisions of
the Budapest Treaty as PM-1 on Jul. 12, 1988 with the International
Patent Organism Depository of the National Institute of Industrial
Science and Technology (Central 6, 1-1-1 Higashi, Tsukuba City,
Ibaraki Pref., 305-5466 Japan) as FERM BP-2998. Also, the hybridoma
cell line which produces MR16-1 antibody has been internationally
deposited under the provisions of the Budapest Treaty as Rat-mouse
hybridoma MR16-1 on Mar. 13, 1997 with the International Patent
Organism Depository of the National Institute of Industrial Science
and Technology (Central 6, 1-1-1 Higashi, Tsukuba City, Ibaraki
Pref., 305-5466 Japan) as FERM BP-5875.
[0050] A hybridoma that produces anti-IL-6 receptor monoclonal
antibody can, basically, be constructed using a known procedure as
described bellow. Thus, IL-6 receptor is used as a sensitizing
antigen, which is immunized in the conventional method of
immunization, and the immune cells thus obtained are fused with
known parent cells in a conventional cell fusion process, followed
by a conventional screening method to screen monoclonal
antibody-producing cells.
[0051] Specifically, anti-IL-6 receptor antibodies may be obtained
in the following manner. For example, human IL-6 receptor used as
the sensitizing antigen for obtaining antibody can be obtained
using the IL-6 receptor gene/amino acid sequence disclosed in
European Patent Application No. EP 325474, and mouse IL-6 receptor
can be obtained using the IL-6 receptor gene/amino acid sequence
disclosed in Japanese Unexamined Patent Publication (Kokai) No.
3-155795.
[0052] There are two types of IL-6 receptor: IL-6 receptor
expressed on the cell membrane, and IL-6 receptor detached from the
cell membrane (Soluble IL-6 Receptor; Yasukawa et al., J. Biochem.
(1990) 108, 673-676). Soluble IL-6 receptor antibody is composed of
the substantially extracellular region of IL-6 receptor bound to
the cell membrane, and is different from the membrane-bound IL-6
receptor in that the former lacks the transmembrane region or both
of the transmembrane region and the intracellular region. IL-6
receptor protein may be any IL-6 receptor, as long as it can be
used as a sensitizing antigen for preparing anti-IL-6 receptor
antibody for use in the present invention.
[0053] After a gene encoding IL-6 receptor has been inserted into a
known expression vector system to transform an appropriate host
cell, the desired IL-6 receptor protein may be purified from the
host cell or a culture supernatant thereof using a known method,
and the IL-6 receptor protein thus purified may be used as the
sensitizing antigen. Alternatively, cells that express IL-6
receptor protein or a fusion protein of IL-6 receptor protein and
another protein may be used as the sensitizing antigen.
[0054] Escherichia coli (E. coli) containing a plasmid pIBIBSF2R
that comprises cDNA encoding human IL-6 receptor has been
internationally deposited under the provisions of the Budapest
Treaty as HB101-pIBIBSF2R on Jan. 9, 1989 with the International
Patent Organism Depository of the National Institute of Industrial
Science and Technology (Central 6, 1-1-1 Higashi, Tsukuba City,
Ibaraki Pref., 305-5466 Japan) as FERM BP-2232.
[0055] Anti-gp130 antibodies for use in the present invention can
be obtained as polyclonal or monoclonal antibodies using a known
method. As the anti-gp130 antibodies for use in the present
invention, monoclonal antibodies of, in particular, mammalian
origin are preferred. Monoclonal antibodies of a mammalian origin
include those produced by a hybridoma and those produced by a host
which has been transformed by gene engineering technology with an
expression vector containing the antibody gene. These antibodies,
via binding to gp130, block the binding of gp130 to the IL-6/IL-6
receptor complex, and thereby block the propagation of biological
activity of IL-6 into the cell.
[0056] Examples of such antibodies include AM64 antibody (Japanese
Unexamined Patent Publication (Kokai) No. 3-219894), 4B11 antibody
and 2H4 antibody (U.S. Pat. No. 5,571,513), B-S12 antibody and B-P8
antibody (Japanese Unexamined Patent Publication (Kokai) No.
8-291199) etc.
[0057] A hybridoma that produces anti-gp130 antibody can be
basically constructed using a known procedure as described below.
Thus, gp130 is used as a sensitizing antigen, which is immunized in
the conventional method of immunization, and the immune cells thus
obtained are fused with known parent cells in a conventional cell
fusion process, followed by a conventional screening method to
screen monoclonal antibody-producing cells.
[0058] Specifically, monoclonal antibodies may be obtained in the
following manner. For example, gp130 used as the sensitizing
antigen for obtaining antibody can be obtained using the gp130
gene/amino acid sequence disclosed in European Patent Application
No. EP 411946.
[0059] The gene sequence of gp130 may be inserted into a known
expression vector, and said vector is used to transform a suitable
host cell. From the host cell or a culture supernatant therefrom,
the gp130 protein of interest may be purified by a known method,
and the purified IL-6 protein may be used as the sensitizing
antigen. Alternatively, cells expressing gp130, or a fusion protein
of the gp130 protein and another protein may be used as the
sensitizing antigen.
[0060] Preferably, mammals to be immunized with the sensitizing
antigen are selected in consideration of their compatibility with
the parent cells for use in cell fusion and they generally include,
but are not limited to, rodents such as mice, rats and
hamsters.
[0061] Immunization of animals with a sensitizing antigen is
carried out using a known method. A general method, for example,
involves intraperitoneal or subcutaneous administration of a
sensitizing antigen to the mammal. Specifically, a sensitizing
antigen, which was diluted and suspended in an appropriate amount
of phosphate buffered saline (PBS) or physiological saline etc., is
mixed with an appropriate amount of a common adjuvant such as
Freund's complete adjuvant. After being emulsified, it is
preferably administered to a mammal several times every 4 to 21
days. Additionally, a suitable carrier may be used at the time of
immunization of the sensitizing antigen.
[0062] After the immunization and confirmation of an increase in
the desired antibody levels in the serum by a conventional method,
immune cells are taken out from the mammal and are subjected to
cell fusion. As preferred immune cells that are subjected to cell
fusion, there can be specifically mentioned spleen cells.
[0063] Mammalian myeloma cells as the other parent cells which are
subjected to cell fusion with the above-mentioned immune cells
preferably include various known cell lines such as P3x63Ag8.653
(Kearney, J. F. et al., J. Immunol. (1979) 123, 1548-1550),
P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978)
81, 1-7), NS-1 (Kohler, G. and Milstein, C., Eur. J. Immunol.
(1976) 6, 511-519), MPC-11 (Margulies, D. H. et al., Cell (1976) 8,
405-415), SP2/0 (Shulman, M. et al., Nature (1978) 276, 269-270),
FO (de St. Groth, S. F. et al., J. Immunol. Methods (1980) 35,
1-21), S194 (Trowbridge, I. S., J. Exp. Med. (1978) 148, 313-323),
R210 (Galfre, G. et al., Nature (1979) 217, 131-133) and the like,
which may be used as appropriate.
[0064] Cell fusion between the above immune cells and myeloma cells
may be essentially conducted in accordance with a known method such
as is described in Milstein et al. (Kohler, G. and Milstein, C.,
Methods Enzymol. (1981) 73, 3-46) and the like.
[0065] More specifically, the above cell fusion is carried out in
the conventional nutrient broth in the presence of, for example, a
cell fusion accelerator. As the cell fusion accelerator, for
example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like
may be used, and an adjuvant such as dimethyl sulfoxide may be
added as desired to enhance the efficiency of fusion.
[0066] The preferred ratio of the immune cells and the myeloma
cells for use is, for example, 1 to 10 times more immune cells than
the myeloma cells. Examples of culture media to be used for the
above cell fusion include, for example, RPMI 1640 medium and MEM
culture medium suitable for the growth of the above myeloma cell
lines, and the conventional culture medium used for this type of
cell culture and, besides, a serum supplement such as fetal calf
serum (FCS) may be added.
[0067] In cell fusion, predetermined amounts of the above immune
cells and the myeloma cells are mixed well in the above culture
liquid, to which a PEG solution previously heated to about
37.degree. C., for example a PEG solution with a mean molecular
weight of 1000 to 6000, is added at a concentration of 30 to 60%
(w/v) and mixed to obtain the desired fusion cells (hybridomas).
Then, by repeating a sequential addition of a suitable culture
liquid and centrifugation to remove the supernatant, cell fusion
agents etc., that are undesirable for the growth of the hybridoma,
can be removed.
[0068] Said hybridoma is selected by culturing in the conventional
selection medium, for example, HAT culture medium (a culture liquid
containing hypoxanthine, aminopterin, and thymidine). Culturing in
said HAT culture medium is continued generally for the period of
time sufficient to effect killing of the cells other than the
desired hybridoma (non-fusion cells), generally several days to
several weeks. The conventional limiting dilution method is
conducted in which the hybridomas producing the desired antibody
are screened and cloned.
[0069] In addition to obtaining the above hybridoma by immunizing
an animal other than the human with an antigen, it is also possible
to sensitize human lymphocytes in vitro with the desired antigen
protein or antigen-expressing cells, and the resulting sensitized
B-lymphocytes are fused with a myeloma cell, for example U266,
having the ability of dividing permanently to obtain a hybridoma
that produces the desired human antibody having the activity of
binding to the desired antigen or antigen-expressing cells
(Japanese Post-examined Patent Publication (Kokoku) 1-59878).
Furthermore, a transgenic animal having a repertoire of human
antibody genes is immunized with the antigen or antigen-expressing
cells to obtain the desired human antibody according to the
above-mentioned method (see International Patent Application WO
93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096 and WO
96/33735).
[0070] The monoclonal antibody-producing hybridomas thus
constructed can be subcultured in the conventional culture liquid,
or can be stored for a prolonged period of time in liquid
nitrogen.
[0071] In order to obtain monoclonal antibodies from said
hybridoma, there can be used a method in which said hybridoma is
cultured in the conventional method and the antibodies are obtained
as the supernatant, or a method in which the hybridoma is implanted
into and grown in a mammal compatible with said hybridoma and the
antibodies are obtained as the ascites. The former method is
suitable for obtaining high-purity antibodies, whereas the latter
is suitable for a large scale production of antibodies.
[0072] For example, an anti-IL-6 receptor antibody-producing
hybridoma can be polypeptide by a method disclosed in Japanese
Unexamined Patent Publication (Kokai) No. 3-139293. There may be
used a method in which The PM-1 antibody-producing hybridoma that
has been internationally deposited under the provisions of the
Budapest Treaty on Jul. 12, 1988 with the International Patent
Organism Depository of the National Institute of Industrial Science
and Technology (Central 6, 1-1-1 Higashi, Tsukuba City, Ibaraki
Pref., 305-5466 Japan) as FERM BP-2998 is intraperitoneally
injected to BALB/c mice to obtain ascites, from which ascites PM-1
antibody may be purified, or a method in which the hybridoma is
cultured in a RPMI 1640 medium containing 10% bovine fetal serum,
5% BM-Codimed H1 (manufactured by Boehringer Mannheim), the
hybridoma SFM medium (manufactured by GIBCO BRL), the PFHM-II
medium (manufactured by GIBCO BRL) or the like, from the culture
supernatant of which PM-1 antibody may be purified.
[0073] In accordance with the present invention, as monoclonal
antibody, there can be used a recombinant antibody that was
produced by cloning an antibody gene from a hybridoma and the gene
is then integrated into an appropriate vector, which is introduced
into a host to produce the recombinant antibody using gene
recombinant technology (see, for example, Borrebaeck, C. A. K. and
Larrick, J. W., THERAPEUTIC MONOCLONAL ANTIBODIES, published in the
United Kingdom by MACMILLAN PUBLISHERS LTD. 1990).
[0074] Specifically, mRNA encoding the variable region (V region)
of the antibody is isolated from the cell that produces the
antibody of interest, for example a hybridoma. The isolation of
mRNA is conducted by preparing total RNA by a known method such as
the guanidine ultracentrifuge method (Chirgwin, J. M. et al.,
Biochemistry (1979) 18, 5294-5299), the AGPC method (Chomczynski,
P. et al., Anal. Biochem. (1987) 162, 156-159), and then mRNA is
purified from the total RNA using the mRNA Purification kit
(manufactured by Pharmacia) and the like. Alternatively, mRNA can
be directly prepared using the Quick Prep mRNA Purification Kit
(manufactured by Pharmacia).
[0075] cDNA of the V region of antibody may be synthesized from the
mRNA thus obtained using a reverse transcriptase. cDNA may be
synthesized using the AMV Reverse Transcriptase First-strand cDNA
Synthesis Kit and the like. Alternatively, for the synthesis and
amplification of cDNA, the 5'-Ampli FINDER RACE Kit (manufactured
by Clontech) and the 5'-RACE method (Frohman, M. A. et al., Proc.
Natl. Acad. Sci. U.S.A. (1988) 85, 8998-9002; Belyavsky, A. et al.,
Nucleic Acids Res. (1989) 17, 2919-2932) which employs PCR may be
used. The desired DNA fragment is purified from the PCR product
obtained and may be ligated to vector DNA. Moreover, a recombinant
vector is constructed therefrom and then is introduced into E. coli
etc., from which colonies are selected to prepare the desired
recombinant vector. The base sequence of the desired DNA may be
confirmed by a known method such as the dideoxy method.
[0076] Once DNA encoding the V region of the desired antibody has
been obtained, it may be ligated to DNA encoding the constant
region (C region) of the desired antibody, which is then integrated
into an expression vector. Alternatively, DNA encoding the V region
of the antibody may be integrated into an expression vector which
already contains DNA encoding the C region of the antibody.
[0077] In order to produce antibody for use in the present
invention, the antibody gene is integrated into an expression
vector so as to be expressed under the control of the expression
regulatory region, for example an enhancer and/or a promoter.
Subsequently, the expression vector is transformed into a host cell
and the antibody can then be expressed therein.
[0078] In accordance with the present invention, artificially
altered recombinant antibodies such as chimeric antibody, humanized
antibody and human antibody can be used for the purpose of lowering
heterologous antigenicity against humans. These altered antibody
can be produced using known methods.
[0079] Chimeric antibody can be obtained by ligating the thus
obtained DNA encoding the V region of antibody to DNA encoding the
C region of human antibody, which is then integrated into an
expression vector and introduced into a host for production of the
antibody therein (see European Patent Application EP 125023, and
International Patent Application WO 92-19759). Using this known
method, chimeric antibody useful for the present invention can be
obtained.
[0080] Plasmids containing the L chain V region or the H chain V
region of chimeric PM-1 antibody have each been designated as
pPM-k3 and pPM-h1, respectively, and E. coli having a respective
plasmid has been internationally deposited under the provisions of
the Budapest Treaty as NCIMB40366 and NCIMB40362 on Feb. 11, 1991
with the National Collections of Industrial and Marine Bacteria
Limited.
[0081] Humanized antibody which is also called reshaped human
antibody has been made by implanting the complementarity
determining region (CDR) of antibody of a mammal other than the
human, for example mouse antibody, into the CDR of human antibody.
The general recombinant DNA technology for preparation of such
antibodies is also known (see European Patent Application EP 125023
and International Patent Application WO 92-19759).
[0082] Specifically, a DNA sequence which was designed to ligate
the CDR of mouse antibody with the framework region (FR) of human
antibody is synthesized from several divided oligonucleotides
having sections overlapping with one another at the ends thereof.
The DNA thus obtained is ligated to DNA encoding the C region of
human antibody and then is incorporated into an expression vector,
which is introduced into a host for antibody production (see
European Patent Application EP 239400 and International Patent
Application WO 92-19759).
[0083] For the FR of human antibody ligated through CDR, the CDR
that has a favorable antigen-binding site is selected. When
desired, amino acids in the FR of antibody V region may be
substituted so that the CDR of humanized antibody may form an
appropriate antigen biding site (Sato, K. et al., Cancer Res.
(1993) 53, 851-856).
[0084] As the C region of human antibody, there can be used, for
example, C.gamma.1, C.gamma.2, C.gamma.3, or C.gamma.4. The C
region of human antibody may also be modified in order to improve
the stability of antibody and of the production thereof.
[0085] Chimeric antibody consists of the V region of antibody of a
human origin other than humans and the C region of human antibody,
and humanized antibody consists of the complementarity determining
region of antibody of a human origin other than humans and the
framework region and the C region of human antibody, with their
antigenicity in the human body being decreased, and thus are useful
as antibody for use in the present invention.
[0086] As a preferred embodiment of humanized antibody for use in
the present invention, there can be mentioned humanized PM-1
antibody (see International Patent Application WO 92-19759).
[0087] As a method of obtaining human antibody, in addition to
those described above, there is known a method of obtaining human
antibody by means of panning. For example, the variable region of
human antibody is expressed on the surface of a phage by the phage
display method as a single chain antibody (scFv) to select a phage
that binds to the antigen. By analyzing the gene of the phage
selected, the DNA sequence encoding the variable region of the
human antibody that binds to the antigen can be identified. Once
the DNA sequence of scFv that binds to the antigen has been
clarified, said sequence can be used to prepare a suitable
expression vector and human antibody can be obtained. These methods
are already known and can be found in WO 92/01047, WO 92/20791, WO
93/06213, WO 93/11236, WO 93/19172, WO 95/01438, and WO
95/15388.
[0088] Antibody genes constructed as mentioned above may be
expressed and obtained in a known manner. In the case of mammalian
cells, expression may be accomplished using a DNA in which a
commonly used useful promoter, an antibody gene to be expressed,
and the poly A signal have been operably linked at 3' downstream
thereof, or a vector containing it. As the promoter/enhancer, for
example, there can be mentioned human cytomegalovirus immediate
early promoter/enhancer.
[0089] Additionally, as the promoter/enhancer which can be used for
expression of antibody for use in the present invention, there can
be used viral promoters/enhancers such as retrovirus, polyoma
virus, adenovirus, and simian virus 40 (SV40), and
promoters/enhancers derived from mammalian cells such as human
elongation factor 1.alpha. (HEF1.alpha.).
[0090] For example, expression may be readily accomplished by the
method of Mulligan et al. (Mulligan, R. C. et al., Nature (1979)
277, 108-114) when SV40 promoter/enhancer is used, and by the
method of Mizushima, S. et al. (Mizushima, S. and Nagata, S.,
Nucleic Acids Res. (1990) 18, 5322) when HEF1.alpha.
promoter/enhancer is used.
[0091] In the case of E. coli, expression may be conducted by
operably linking a commonly used promoter, a signal sequence for
antibody secretion, and an antibody gene to be expressed, followed
by expression thereof. As the promoter, for example, there can be
mentioned lacz promoter and araB promoter. The method of Ward et
al. (Ward, E. S. et al., Nature (1989) 341, 544-546; Ward, E. S. et
al., FASEB J. (1992) 6, 2422-2427) may be used when lacz promoter
is used, and the method of Better et al. (Better, M. et al.,
Science (1988) 240, 1041-1043) may be used when araB promoter is
used.
[0092] As a signal sequence for antibody secretion, when produced
in the periplasm of E. coli, the pelB signal sequence (Lei, S. P.
et al., J. Bacteriol. (1987) 169, 4379-4383) can be used. After
separating the antibody produced in the periplasm, the structure of
the antibody is appropriately refolded before use (see, for
example, WO 96-30394).
[0093] As the origin of replication, there can be used those
derived from SV40, polyoma virus, adenovirus, bovine papilloma
virus (BPV), and the like. Furthermore, for amplification of the
gene copy number in the host cell system, expression vectors can
include, as selectable markers the aminoglycoside transferase (APH)
gene, the thymidine kinase (TK) gene, E. coli xanthine
guaninephosphoribosyl transferase (Ecogpt) gene, the dihydrofolate
reductase (dhfr) gene, and the like.
[0094] For the production of antibody for use in the present
invention, any production system can be used, and the production
systems of antibody preparation comprise the in vitro or the in
vivo production system. As the in vitro production systems, there
can be mentioned a production system which employs eukaryotic cells
and the production system which employs prokaryotic cells.
[0095] When eukaryotic cells are used, there are the production
systems which employ animal cells, plant cells, and fungal cells.
Known animal cells include (1) mammalian cells such as CHO cells,
COS cells, myeloma cells, baby hamster kidney (BHK) cells, HeLa
cells, and Vero cells, (2) amphibian cells such as Xenopus oocytes,
or (3) insect cells such as sf9, sf21, and Tn5. Known plant cells
include, for example, those derived from the Nicotiana tabacum
which is subjected to callus culture. Known fungal cells include
yeasts such as genus Saccharomyces, more specifically Saccharomyces
cereviceae, or filamentous fungi such as the Aspergillus family,
more specifically Aspergillus niger.
[0096] When prokaryotic cells are used, there are the production
systems which employ bacterial cells. Known bacterial cells include
Escherichia coli, and Bacillus subtilis.
[0097] By introducing, via transformation, the gene of the desired
antibody into these cells and culturing the transformed cells in
vitro, the antibody can be obtained. Culturing is conducted in the
known methods. For example, as the culture liquid for mammalian
cells, DMEM, MEM, RPMI1640, IMDM and the like can be used, and
serum supplements such as fetal calf serum (FCS) may be used in
combination. In addition, antibodies may be produced in vivo by
implanting cells into which the antibody gene has been introduced
into the abdominal cavity of an animal, and the like.
[0098] As in vivo production systems, there can be mentioned those
which employ animals and those which employ plants. When animals
are used, there are the production systems which employ mammals and
insects.
[0099] As mammals, goats, pigs, sheep, mice, and cattle can be used
(Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). Also, as
insects, silkworms can be used and, in the case of plants, tobacco,
for example, can be used.
[0100] Antibody genes are introduced into these animals and plants,
in which the genes are produced and then collected. For example,
antibody genes are inserted into the middle of the gene encoding
protein which is inherently produced in the milk such as goat
.beta. casein to prepare fusion genes. DNA fragments containing the
fusion gene into which the antibody gene has been inserted are
injected to a goat embryo, and the embryo is introduced into a
female goat. The desired antibody is obtained from the milk
produced by a transgenic goat produced by the goat that received
the embryo or the offspring thereof. In order to increase the
amount of milk containing the desired antibody produced by the
transgenic goat, hormones may be given to the transgenic goat as
appropriate (Ebert, K. M. et al., Bio/Technology (1994) 12,
699-702).
[0101] When silkworms are used, the silkworm is infected with a
baculovirus into which desired antibody gene has been inserted, and
the desired antibody can be obtained from the body fluid of the
silkworm (Maeda, S. et al., Nature (1985) 315, 592-594). Moreover,
when tobacco is used, the desired antibody gene is inserted into an
expression vector for plants, for example pMON 530, and then the
vector is introduced into a bacterium such as Agrobacterium
tumefaciens. The bacterium is then used to infect tobacco such as
Nicotiana tabacum to obtain the desired antibody from the leaves of
the tobacco (Julian, K.-C. Ma et al., Eur. J. Immunol. (1994) 24,
131-138).
[0102] When antibody is produced in an in vitro or in vivo
production systems, as mentioned above, DNA encoding the heavy
chain (H chain) or light chain (L chain) of antibody is separately
incorporated into an expression vector and the hosts are
transformed simultaneously, or DNA encoding the H chain and the L
chain of antibody is integrated into a single expression vector and
the host is transformed therewith (see International Patent
Application WO 94-11523).
[0103] Antibodies for use in the present invention may be fragments
of antibody or modified versions thereof as long as they are
preferably used in the present invention. For example, as fragments
of antibody, there may be mentioned Fab, F(ab')2, Fv or
single-chain Fv (scFv) in which Fv's of H chain and L chain were
ligated via a suitable linker.
[0104] Specifically antibodies are treated with an enzyme, for
example, papain or pepsin, to produce antibody fragments, or genes
encoding these antibody fragments are constructed, and then
introduced into an expression vector, which is expressed in a
suitable host cell (see, for example, Co, M. S. et al., J. Immunol.
(1994) 152, 2968-2976; Better, M. and Horwitz, A. H., Methods
Enzymol. (1989) 178, 476-496; Plucktrun, A. and Skerra, A., Methods
Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986)
121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121,
663-669; Bird, R. E. et al., TI BTECH (1991) 9, 132-137).
[0105] scFv can be obtained by ligating the V region of H chain and
the V region of L chain of antibody. In the scFv, the V region of H
chain and the V region of L chain are preferably ligated via a
linker, preferably a peptide linker (Huston, J. S. et al., Proc.
Natl. Acad. Sci. U.S.A. (1988) 85, 5879-5883). The V region of H
chain and the V region of L chain in the scFv may be derived from
any of the above-mentioned antibodies. As the peptide linker for
ligating the V regions, any single-chain peptide comprising, for
example, 12-19 amino acid residues may be used.
[0106] DNA encoding scFv can be obtained using DNA encoding the H
chain or the H chain V region of the above antibody and DNA
encoding the L chain or the L chain V region of the above antibody
as the template by amplifying the portion of the DNA encoding the
desired amino acid sequence among the above sequences by the PCR
technique with the primer pair specifying the both ends thereof,
and by further amplifying the combination of DNA encoding the
peptide linker portion and the primer pair which defines that both
ends of said DNA be ligated to the H chain and the L chain,
respectively.
[0107] Once DNAs encoding scFv are constructed, an expression
vector containing them and a host transformed with said expression
vector can be obtained by a conventional method, and scFv can be
obtained using the resultant host by a conventional method.
[0108] These antibody fragments can be produced by obtaining the
gene thereof in a similar manner to that mentioned above, and by
allowing it to be expressed in a host. "Antibody" as used in the
present invention encompasses these antibody fragments.
[0109] As modified antibodies, antibodies associated with various
molecules such as polyethylene glycol (PEG) can be used. "Antibody"
as used in the present invention encompasses these modified
antibodies. These modified antibodies can be obtained by chemically
modifying the antibodies thus obtained. These methods have already
been established in the art.
[0110] Antibodies expressed and produced as described above can be
separated from inside or outside of the cell or from the host and
then may be purified to homogeneity. Separation and purification of
antibody for use in the present invention may be accomplished by
affinity chromatography. As the column used for affinity
chromatography, there can be mentioned Protein A column and Protein
G column. Examples of carriers for use in Protein A column include,
for example, Hyper D, POROS, Sepharose F.F. (Pharmacia) and the
like. In addition, commonly used methods of separation and
purification for proteins can be used, without any limitation.
[0111] Chromatography other than the above affinity chromatography,
filters, gel filtration, salting out, dialysis and the like may be
selected and combined as appropriate, in order to separate and
purify the antibodies for use in the present invention.
Chromatography includes, for example, ion exchange chromatography,
hydrophobic chromatography, gel-filtration and the like. These
chromatographies can be applied to high performance liquid
chromatography (HPLC). Also, reverse phase HPLC (rpHPLC) may be
used.
[0112] The concentration of antibody obtained as above can be
determined by measurement of absorbance or by ELISA and the like.
Thus, when absorbance measurement is employed, the antibody
obtained is appropriately diluted with PBS(-) and then the
absorbance is measured at 280 nm, followed by calculation using the
absorption coefficient of 1.35 OD at 1 mg/ml. When ELISA is used,
measurement is conducted as follows. Thus, 100 .mu.l of goat
anti-human IgG antibody (manufactured by TAGO) diluted to 1
.mu.g/ml in 0.1 M bicarbonate buffer, pH 9.6, is added to a 96-well
plate (manufactured by Nunc), and is incubated overnight at
4.degree. C. to immobilize the antibody. After blocking, 100 .mu.l
each of appropriately diluted antibody for use in the present
invention or samples containing the antibody, or human IgG
(manufactured by CAPPEL) as the standard is added, and incubated at
room temperature for 1 hour.
[0113] After washing, 100 .mu.l of 5000-fold diluted alkaline
phosphatase-labeled anti-human IgG antibody (manufactured by BIO
SOURCE) is added, and incubated at room temperature for 1 hour.
After washing, the substrate solution is added and incubated,
followed by measurement of absorbance at 405 nm using the
MICROPLATE READER Model 3550 (manufactured by Bio-Rad) to calculate
the concentration of the desired antibody.
[0114] Reshaped IL-6 for use in the present invention is a
substance that has an activity of binding with IL-6 receptor and
that does not propagate the biological activity of IL-6. Thus,
though reshaped IL-6 competes with IL-6 for binding to IL-6
receptor, it does not propagate the biological activity of IL-6,
and therefore reshaped IL-6 blocks signal transduction by IL-6.
[0115] Reshaped IL-6 may be prepared by introducing mutations by
replacing amino acid residues of the amino acid sequence of IL-6.
IL-6 from which reshaped IL-6 is derived may be of any origin, but
it is preferably human IL-6 considering antigenicity etc.
[0116] Specifically, the secondary structure of the amino acid
sequence of IL-6 may be estimated using a known molecular modeling
program such as WHATIF (Vriend et al., J. Mol. Graphics (11990) 8,
52-56), and its effect on the overall amino acid residues to be
replaced is evaluated. After determining suitable amino acid
residues, mutation may be introduced using a vector containing a
base sequence encoding human IL-6 gene as a template in a commonly
used PCR method so as to replace amino acids, and thereby to obtain
a gene encoding reshaped IL-6. This may be integrated, as
appropriate, into a suitable expression vector to obtain reshaped
IL-6 according to the above-mentioned methods for expression,
production, and purification of recombinant antibody.
[0117] Specific examples of reshaped IL-6 has been disclosed in
Brakenhoff et al., J. Biol. Chem. (1994) 269, 86-93, Saviono et
al., EMBO J. (1994) 13, 1357-1367, WO 96-18648 and WO 96-17869.
[0118] Partial peptides of IL-6 or partial peptides of IL-6
receptor for use in the present invention are substances that have
an activity of binding to IL-6 receptor or IL-6, respectively, and
that do not propagate the biological activity of IL-6. Thus,
partial peptides of IL-6 or partial peptides of IL-6 receptor bind
to and capture IL-6 receptor or IL-6, respectively, so as to
inhibit specifically the binding of IL-6 to IL-6 receptor. As a
result, they do not allow propagating of the biological activity of
IL-6, and thereby block signal transduction by IL-6.
[0119] Partial peptides of IL-6 or partial peptides of IL-6
receptor are peptides are peptides comprising part or all of the
amino acid sequence involved in the binding of IL-6 and IL-6
receptor in the amino acid sequences of IL-6 or IL-6 receptor. Such
peptides comprise usually 10-80 amino acid residues, preferably
20-50 amino acid residues, and more preferably 20-40 amino acid
residues.
[0120] Partial peptides of IL-6 or partial peptides of IL-6
receptor specify the regions involved in the binding of IL-6 and
IL-6 receptor in the amino acid sequence of IL-6 or IL-6 receptor,
and part or all of the amino acid sequence can be prepared by a
commonly known method such as gene engineering technology or
peptide synthesis.
[0121] In order to prepare partial peptides of IL-6 or partial
peptides of IL-6 receptor by gene engineering technology, a DNA
sequence encoding the desired peptide can be integrated into an
expression vector so that they may be obtained according to the
above-mentioned methods for expression, production, and
purification of recombinant antibody.
[0122] In order to prepare partial peptides of IL-6 or partial
peptides of IL-6 receptor by peptide synthesis, a commonly used
method in peptide synthesis such as solid-phase synthesis or
liquid-phase synthesis can be used.
[0123] Specifically, methods described in "Zoku Iyakuhinno
Kaihatsu, Vol. 14: Peptide Synthesis" edited by Haruaki Yajima,
Hirokawa Shoten, 1991, can be used. As the solid-phase synthesis,
there can be used a method in which an amino acid corresponding to
the C-terminal of the peptide to be synthesized is bound to a
support insoluble in organic solvents, and then a reaction in which
amino acids of which .alpha.-amino group and a side chain
functional group has been protected with a suitable protecting
group is condensed one by one in the direction of from the
C-terminal to the N-terminal and a reaction in which said
protecting group of the .alpha.-amino group of the amino acid or
the peptide bound to the resin is eliminated therefrom are
alternately repeated to extend the peptide chain. The solid-phase
peptide synthesis is roughly divided in the Boc method and the Fmoc
method depending on the type of protecting groups used.
[0124] After thus synthesizing the peptide of interest, a
deprotecting reaction or a cleavage reaction of the peptide chain
from the support may be performed. For the cleavage reaction of
peptide chains, the Boc method employs hydrogen fluoride or
trifluoromethanesulfonic acid, or the Fmoc method usually employs
TFA. In the Boc method, the above protected peptide resin is
treated in the presence of anisole in hydrogen fluoride.
Subsequently, the elimination of the protecting group and the
cleavage from the support may be performed to collect the peptide.
Lyophilization of this yields crude peptide. On the other hand, in
the Fmoc method, the deprotection reaction and the cleavage
reaction of the peptide chain from the support may be performed in
a manner similar to the one mentioned above.
[0125] The crude peptide obtained may be subjected to HPLC to
separate and purify it. In its elution, a water-acetonitrile
solvent commonly used in protein purification may be used under an
optimal condition. Fractions corresponding to the peaks of the
chromatographic profile are harvested and then lyophilized. For the
peptide fractions thus purified, molecular weight analysis by mass
spectroscopy, analysis of amino acid composition, or analysis of
amino acid sequence is performed for identification.
[0126] Specific examples of IL-6 partial peptides and IL-6 receptor
partial peptides have been disclosed in Japanese Unexamined Patent
Publication (Kokai) No. 2-188600, Japanese Unexamined Patent
Publication (Kokai) No. 7-324097, Japanese Unexamined Patent
Publication (Kokai) No. 8-311098, and U.S. Pat. Publication U.S.
Pat. No. 5,210,075.
[0127] The inhibitory activity of IL-6 signal transduction by IL-6
antagonist of the present invention can be evaluated using a
commonly known method. Specifically, IL-6-dependent human myeloma
line (S6B45, KPMM2), human Lennert T lymphoma line KT3, or
IL-6-dependent HN60.BSF2 cells are cultured, to which IL-6 is
added, and at the same time, in the presence of IL-6 antagonist,
the incorporation of .sup.3H labeled thymidine by the IL-6
dependent cells is determined. Alternatively, .sup.125I-labeled
IL-6 and IL-6 antagonist, at the same time, are added, and then
.sup.125I-labeled IL-6 that bound to the IL-6-ecpressing cells is
determined for evaluation. In the above assay system, in addition
to the group in which the IL-6 antagonist is present, a negative
control group in which contains no IL-6 antagonist is set up, and
the results obtained in both are compared to evaluate the
IL-6-inhibiting activity by IL-6 antagonist.
[0128] As shown in Examples below, as therapeutic effects was
observed by administration of anti-IL-6 receptor antibody to
children suffering from chronic arthritis, IL-6 antagonists such as
anti-IL-6 receptor antibody were shown to have a therapeutic effect
for chronic arthritides of childhood-related diseases.
[0129] Subjects to be treated in the present invention are mammals.
The subject mammals to be treated are preferably humans.
[0130] Therapeutic agents for chronic arthritides of
childhood-related diseases of the present invention may be
administered orally or parenterally and systemically or locally.
For example, intravenous injection such as drip infusion,
intramuscular injection, intraperitoneal injection, subcutaneous
injection, suppositories, enema, oral enteric coated tablets, and
the like may be selected, and the dosage regimen may be selected as
appropriate depending on the age and conditions of the patient. The
effective dose is chosen from the range of 0.01 mg to 100 mg per kg
of body weight per administration. Alternatively, the dosage of 1
to 1000 mg, preferably 5 to 50 mg per patient may be selected.
[0131] Preferable dose and method for administering is, for example
in the case of anti-IL6 receptor antibody, an effective dose that
provides free antibody in the blood, and specifically, 0.5 mg to 40
mg, and preferably 1 mg to 20 mg per 1 kg body weight per month
(four weeks), which is administered at once, or divided to several
parts and administered, for example, twice/week, once/week,
once/two weeks, once/four weeks, etc, for example intravenously for
example by dripping, or subcutaneously. Administering schedule may
be adjusted by elongating intervals from twice/week or once/week to
once/two weeks, once/three weeks, once/four weeks, etc, dependent
on observation of symptoms, and blood test profile.
[0132] Therapeutic agents for chronic arthritides of
childhood-related diseases of the present invention may contain
pharmaceutically acceptable carriers and additives depending on the
route of administration. Examples of such carriers or additives
include water, a pharmaceutically acceptable organic solvent,
collagen, polyvinyl alcohol, polyvinylpyrrolidone, a carboxyvinyl
polymer, carboxymethylcellulose sodium, polyacrylic sodium, sodium
alginate, water-soluble dextran, carboxymethyl starch sodium,
pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum Arabic,
casein, gelatin, agar, diglycerin, propylene glycol, polyethylene
glycol, Vaseline, paraffin, stearyl alcohol, stearic acid, human
serum albumin (HSA), mannitol, sorbitol, lactose, pharmaceutically
acceptable surfactants and the like. Actual additives used are
chosen from, but not limited to, the above or combinations thereof
depending on the dosage form.
EXAMPLES
[0133] The present invention will be explained more specifically
below with reference to Working Examples and Reference Examples,
but it is to be noted that the present invention is not limited by
these examples in any way.
Working Example 1
[0134] A patient (5 years old, male) with systemic-onset type
juvenile rheumatoid arthritis having the following history was
subjected to a MRA (humanized anti-IL-6 receptor antibody)
treatment.
History Before the Treatment
[0135] Symptoms developed with relaxation heat (one-peak fever at
about 40.degree. C. for consecutive days), arthralgia at both
knees, and anthema. After diagnosing based on leukocytosis,
negative anti-nuclear antibody, negative rheumatoid factor,
increased erythrocyte sedimentation rate, high CRP levels, etc., an
aspirin administration was started, but no improvement in
relaxation heat and arthralgia was observed and the general
condition was aggravated. Thus, it was changed to an oral bolus
administration of steroid (prednisolone 30 mg/day) to see an
improvement in various symptoms. However, with the gradual decrease
in prednisolone, symptoms recurred at 10 mg/day, the patient was
rehospitalized, subjected to a methylprednisolone (mPSL) pulse
therapy and plasmapheresis, and furthermore the combined use of
cyclosporin A (Cs A) was carried out with no improvement. Symptoms
were severe (leukocyte count 25400/.mu.l, CRP 11.2 mg/dL), and a
plasmapheresis+mPSL pulse therapy+Cs A was carried out and the
patient entered into remission. As an aftertreatment,
prednisolone+froben was given to control relaxation heat, and a
decline in fever was clinically noted, but inflammation-related
hematology tests remained at high values (CRP>5 mg/dL), and
after discharge from hospital, relaxation heat was periodically
observed, but treatment and observation were continued mainly on an
ambulatory basis. However, the patient started to complain of
backache that was aggravated by fever, and after close examination
using MBI etc., destructive damages were noted in the fourth and
the fifth thoracic vertebras suggesting that they are compression
fracture. Due to the necessity of relief to the thoracic vertebras,
bed rest was continued for about one year, and accordingly muscles
of the lower limbs have markedly weakened which rendered walking
completely impossible. The triadic therapy of
prednisolone+froben+Cs A was continued but CRP never dropped to 5
mg/dL or lower.
The Result of Treatment
[0136] Administration started at 2 mg/kg. Since no side effects
were seen, the dosage was increased to 4 mg/kg in a once per week
administration. Fever that had been noted until then disappeared
quickly, and about two weeks later CRP became negative. General
malaise was cleared, and the patient somewhat improved. It became
possible to decrease prednisolone gradually and has decreased to 1
mg/day.
[0137] From the above results, MRA was found to be effective for
the treatment of chronic arthritides diseases of childhood of which
symptoms could not be controlled even with nonsteroidal
anti-inflammatory drugs such as aspirin and froben, long term bolus
steroids (for example prednine and medrol), and immunosuppressants
such as cyclosporin A and methotrexate. Therefore, it can be said
that IL-6 antagonist, in particular anti-IL-6 receptor antibody, is
effective as a therapeutic agent for chronic arthritides diseases
of childhood, specifically the systemic onset type of the ARA
classification, the systemic onset type of the EULAR
classification, the systemic onset type of the ILAR classification,
and the SPRASH syndrome of the present inventors'
classification.
Working Example 2
[0138] A 22-year old female. In April 1998, erythema punctatum
appeared at the femur, the recordial region, and fingers, and in
May, arthralgia at the shoulder, the elbow and the knee, and fever
between 38 and 39.degree. C. appeared. Though nonsteroidal
anti-inflammatory drugs (NSAIDs) were started, fever persisted, and
in July, with leukocyte count at 18100/.mu.l, CRP at 18.3 mg/dl,
and serum ferritin at 440 ng/ml, the patient was diagnosed as
having adult Still's disease. From early January 2000, fever
between 39 and 40.degree. C. and arthralgia appeared, which were
believed to be a flare-up of adult Still's disease (CRP 15.8 mg/dl,
ferritin, 205.8 ng/ml).
[0139] Since it was difficult to reduce the dosage of steroids,
methotrexate (MTX) and cyclosporin A (Cs A) were used in
combination, but this could not control the progress of the
disease, which aggravated breathing, and thus the patient was
placed under the control of artificial respiration. Though the
disease was somewhat improved by a steroid therapy, a treatment
with humanized anti-IL-6 receptor antibody (MRA) was started
because of the complication of severe osteoporosis. MRA (200 mg)
was intravenously drip-infused for every two weeks. The
inflammatory reaction became negative on day 6 after the
administration, and decreases in the amount of corticosteroids
progressed smoothly, and no severe side effects were observed.
[0140] From the above results, MRA was found to be effective for
the treatment of adult Still's disease of which symptoms could not
be controlled even with the combined use of MTX and CsA. Therefore,
it can be said that IL-6 antagonist, in particular anti-IL-6
receptor antibody, is effective as a therapeutic agent for Still's
disease, specifically adult Still's disease.
Reference Example 1
Preparation of Human Soluble IL-6 Receptor
[0141] Using a plasmid pBSF2R.236 containing cDNA that encodes IL-6
receptor obtained by the method of Yamasaki et al. (Yamasaki et
al., Science (1988) 241, 825-828), soluble IL-6 receptor was
prepared by the PCR method. The plasmid pBSF2R.236 was digested
with a restriction enzyme Sph I to obtain IL-6 receptor cDNA, which
was inserted into mp18 (manufactured by Amersham). Using a
synthetic primer designed to introduce a stop codon into IL-6
receptor cDNA, mutation was introduced into IL-6 receptor cDNA by
the PCR method in an in vitro mutagenesis system (manufactured by
Amersham). By this procedure, the stop codon was introduced at the
position of amino acid 345, and cDNA encoding soluble IL-6 receptor
was obtained.
[0142] In order to express soluble IL-6 receptor in CHO cells, it
was ligated to a plasmid pSV (manufactured by Pharmacia) to obtain
a plasmid pSVL344. Soluble IL-6 receptor cDNA digested with
HindIII-SalI was inserted into a plasmid pECEdhfr containing the
cDNA of dhfr to obtain a CHO cell-expressing plasmid
pECEdhfr344.
[0143] Ten .mu.g of plasmid pECEdhfr344 was transfected to a
dhfr-CHO cell line DXB-11 (Urlaub, G. et al., Proc. Natl. Acad.
Sci. USA (1980) 77, 4216-4220) by the calcium phosphate
precipitation method (Chen, C. et al., Mol. Cell. Biol. (1987) 7,
2745-2751). The transfected CHO cells were cultured for three weeks
in a nucleoside-free .alpha.MEM selection medium containing 1 mM
glutamine, 10% dialyzed FCS, 100 U/ml penicillin and 100 .mu./ml
streptomycin.
[0144] The selected CHO cells were screened by the limiting
dilution method to obtain a single CHO cells clone. The CHO cell
clone was amplified with 20 nM-200 nM of methotrexate to
investigate a human soluble IL-6 receptor-producing CHO cell line
5E27. The CHO cell line 5E27 was cultured in a Iscov modified
Dulbecco medium (IMDM, manufactured by Gibco) supplemented with 5%
FBS. The culture supernatant was collected and the concentration of
soluble IL-6 receptor in the culture supernatant was determined by
ELISA. The result confirmed the presence of soluble IL-6 receptor
in the culture supernatant.
Reference Example 2
Preparation of Anti-Human IL-6 Antibody
[0145] Ten .mu.g of tissue-type IL-6 (Hirano et al., Immunol. Lett.
(1988) 17, 41) was used with Freund's complete adjuvant to immunize
BALB/c mice, and this was repeated every week until anti-IL-6
antibody can be detected in the serum. Immune cells were removed
from the local lymph nodes, and were fused with a myeloma cell line
P3U1 using polyethylene glycol 1500. Hybridomas were selected by
the method of Oi et al. (Selective Methods in Cellular Immunology,
W.H. Freeman and Co., San Francisco, 351, 19080) using the HAT
culture medium to establish a hybridoma producing anti-human IL-6
antibody.
[0146] The hybridoma producing anti-human IL-6 antibody was
subjected to an IL-6 binding assay in the following manner. Thus, a
96-well microtiter plate (manufactured by Dynatech Laboratories,
Inc., Alexandria, Va.) made of flexible polyvinyl was coated
overnight with 100 .mu.l of goat anti-mouse Ig (10 .mu.l/ml,
manufactured by Cooper Biomedical, Inc., Malvern, Pa.) in 0.1 M
carbonate hydrogen carbonate buffer (pH 9.6) at 4.degree. C. Then,
the plate was treated in 100 .mu.l of PBS containing 1% bovine
serum albumin (BSA) at room temperature for 2 hours.
[0147] After the plate was washed in PBS, 100 .mu.l of the
hybridoma culture supernatant was added to each well, and incubated
overnight at 4.degree. C. After washing the plate,
.sup.125I-labelled recombinant type IL-6 was added to each well to
2000 cpm/0.5 ng/well, and after washing, radioactivity of each well
was measured by a gamma counter (Beckman Gamma 9000, Beckman
Instruments, Fullerton, Calif.). Of 216 hybridoma clones, 32
hybridoma clones were positive in the IL-6 binding assay. From
among these clones, finally MH166.BSF2, a stable clone, was
selected. Anti-IL-6 antibody MH166 has a subtype of IgG1 .kappa.
type.
[0148] Then, using a IL-6-dependent mouse hybridoma clone
MH60.BSF2, a neutralizing activity with regard to the growth of the
hybridoma by MH166 antibody was investigated. MH60.BSF2 cells were
aliquoted to 1.times.10.sup.4/200 .mu.l/well, to which a sample
containing MH166 antibody was added, and cultured for 48 hours.
After adding 0.5 .mu.Ci/well of .sup.3H-thymidine (New England
Nuclear, Boston, Mass.), culturing was continued for further six
hours. The cells were placed on a glass filter paper, and were
treated by an automated harvester (Labo Mash Science Co., Tokyo,
Japan). As the control, rabbit anti-IL-6 antibody was used.
[0149] As a result, MH166 antibody inhibited .sup.3H-thymidine
incorporation by MH60.BSF2 cells induced by IL-6 in a dose
dependent manner. This revealed that MH166 antibody neutralizes the
activity of IL-6.
Reference Example 3
Preparation of Anti-Human IL-6 Receptor Antibody
[0150] Anti-IL-6 receptor antibody MT18 prepared by the method of
Hirata et al. (Hirata, Y. et al., J. Immunol. (1989) 143,
2900-2906) was conjugated to a CNBr-activated Sepharose 4B
(manufactured by Pharmacia Fine Chemicals, Piscataway, N.J.) to
purify IL-6 receptor (Yamasaki et al., Science (1988) 241,
825-828). A human myeloma cell line U266 was solubilized with 1 mM
p-paraaminophenylmethanesulfonyl fluoride hydrochloride
(manufactured by Wako Pure Chemicals) (digitonin buffer) containing
1% digitonin (manufactured by Wako Pure Chemicals), 10 mM
triethanolamine (pH 7.8), and 0.15 M NaCl, and was mixed with MT18
antibody conjugated to Sepharose 4B beads. Subsequently, the beads
were washed six times in the digitonin buffer to prepare a
partially purified IL-6 receptor.
[0151] BALB/c mice were immunized with the above partially purified
IL-6 receptor obtained from 3.times.10.sup.9 U266 cells four times
every ten days, and then a hybridoma was prepared according to a
standard method. The culture supernatant of the hybridoma from
growth-positive wells were examined for the biding activity to IL-6
receptor in the following manner. 5.times.10.sup.7 U266 cells were
labelled with .sup.35S-methionine (2.5 mCi), and were solubilized
with the above digitonin buffer. The solubilized U266 cells were
mixed with 0.04 ml of MT18 antibody conjugated to Sepharose 4B
beads, and then washed for six times in the digitonin buffer. Using
0.25 ml of the digitonin buffer (pH 3.4),
.sup.35S-methionine-labeled IL-6 receptor was eluted, which was
neutralized with 0.025 ml of 1M Tris, pH 7.4.
[0152] 0.05 ml of the hybridoma culture supernatant was mixed with
0.01 ml Protein G Sepharose (manufactured by Pharmacia). After
washing, the Sepharose was incubated with 0.005 ml solution of
.sup.35S-labeled IL-6 receptor solution. The immunoprecipitated
substances were analyzed by SDS-PAGE to study the culture
supernatant of hybridoma that reacts with IL-6 receptor. As a
result, a reaction-positive hybridoma clone PM-1 was established.
Antibody produced from the hybridoma PM-1 had the IgG1 .kappa.
subtype.
[0153] The activity of the antibody produced by the hybridoma PM-1
to inhibit the binding of IL-6 to IL-6 receptor was evaluated using
a human myeloma cell line U266. Human recombinant IL-6 was prepared
from E. coli (Hirano et al., Immunol. Lett. (1988) 17, 41-45), and
was labeled with .sup.125I using the Bolton-Hunter reagent (New
England Nclear, Boston, Mass.) (Taga et al., J. Exp. Med. (1987)
166, 967-981).
[0154] 4.times.10.sup.5 U266 cells were cultured with a culture
supernatant of 70%(v/v) hybridoma PM-1 and 14000 CPM of
.sup.125I-labeled IL-6 for one hour. Seventy microliters of a
sample was layered onto 300 .mu.l of FCS in a 400 .mu.l microfuge
polyethylene tube, centrifuged, and then the radioactivity of the
cells were measured.
[0155] The result revealed that the antibody produced by the
hybridoma PM-1 inhibits the binding of IL-6 to IL-6 receptor.
Reference Example 4
Preparation of Anti-Mouse IL-6 Receptor Antibody
[0156] A monoclonal antibody against mouse IL-6 receptor was
prepared by the method of Saito, T. et al., J. Immunol. (1991) 147,
168-173.
[0157] CHO cells that produce soluble mouse IL-6 receptor were
cultured in an IMDM culture medium supplemented with 10% FCS. From
the culture supernatant, soluble mouse IL-6 receptor was purified
using an affinity column in which anti-mouse IL-6 receptor antibody
RS12 (see the above Saito, T. et al.) was immobilized to the
Affigel 10 gel (manufactured by Biorad).
[0158] Fifty .mu.g of soluble mouse IL-6 receptor thus obtained was
mixed with Freund's complete adjuvant, which was intraperitoneally
injected to the abdomen of Wistar rats. Two weeks later, the rats
received booster immunization with Freund's incomplete adjuvant. On
day 45, spleen cells were removed from the rats, and
2.times.10.sup.8 of the cells were subjected to cell fusion with
1.times.10.sup.7 mouse myeloma cells P3U1 with 50% PEG1500
(manufactured by Boehringer Mannheim) using a standard method, and
the hybridoma were then screened with the HAT medium.
[0159] After adding the culture supernatant to a plate coated with
rabbit anti-rat IgG antibody (manufactured by Cappel), soluble
mouse IL-6 receptor was reacted thereto. Then, using an ELISA
method employing rabbit anti-mouse IL-6 receptor antibody and
alkaline phosphatase-labelled sheep anti-rabbit IgG, hybridomas
that produce antibodies against soluble mouse IL-6 receptor were
screened. The hybridoma clones for which antibody production was
confirmed were subjected to subscreening twice to obtain a single
hybridoma clone. This clone was designated as MR16-1.
[0160] A neutralizing activity in signal transduction of mouse IL-6
by the antibody produced by this hybridoma was examined using
.sup.3H-thymidine incorporation that employs MH60.BSF2 cells
(Matsuda, T. et al., J. Immunol. (1988) 18, 951-956). To a 96-well
plate, MH60.BSF2 cells were prepared to 1.times.10.sup.4 cells/200
.mu.l/well. To this plate were added 10 .mu.g/ml of mouse IL-6 and
MR16-1 antibody or RS12 antibody at 12.3-1000 ng/ml, and cultured
at 37.degree. C. in 5% CO.sub.2 for 44 hours, followed by the
addition of 1 .mu.Ci/well of .sup.3H-thymidine. Four hours later,
the incorporation of .sup.3H-thymidine was measured. As a result,
MR16-1 antibody inhibited the .sup.3H-thymidine incorporation by
MH60.BSF2 cells.
[0161] Thus, it was revealed that antibody produced by the
hybridoma MR16-1 (FERM BP-5874) inhibits the binding of IL-6 to
IL-6 receptor.
* * * * *