U.S. patent application number 14/662139 was filed with the patent office on 2015-07-09 for preventive or therapeutic agent for sensitized t cell-mediated diseases comprising il-6 antagonist as an active ingredient.
The applicant listed for this patent is CHUGAI SEIYAKU KABUSHIKI KAISHA. Invention is credited to Masahiko MIHARA.
Application Number | 20150191540 14/662139 |
Document ID | / |
Family ID | 13374532 |
Filed Date | 2015-07-09 |
United States Patent
Application |
20150191540 |
Kind Code |
A1 |
MIHARA; Masahiko |
July 9, 2015 |
PREVENTIVE OR THERAPEUTIC AGENT FOR SENSITIZED T CELL-MEDIATED
DISEASES COMPRISING IL-6 ANTAGONIST AS AN ACTIVE INGREDIENT
Abstract
A preventive or therapeutic agent for sensitized T cell-mediated
diseases comprising an interleukin-6 (IL-6) antagonist, for example
an antibody directed against IL-6 receptor, an antibody directed
against IL-6, an antibody directed against gp130, and the like.
Inventors: |
MIHARA; Masahiko; (Shizuoka,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CHUGAI SEIYAKU KABUSHIKI KAISHA |
Tokyo |
|
JP |
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|
Family ID: |
13374532 |
Appl. No.: |
14/662139 |
Filed: |
March 18, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11340412 |
Jan 25, 2006 |
9017677 |
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14662139 |
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09381598 |
Sep 20, 1999 |
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PCT/JP98/01217 |
Mar 20, 1998 |
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11340412 |
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Current U.S.
Class: |
530/388.1 |
Current CPC
Class: |
A61P 37/06 20180101;
A61K 2039/505 20130101; A61P 37/00 20180101; C07K 2317/76 20130101;
C07K 16/2866 20130101; C07K 16/248 20130101 |
International
Class: |
C07K 16/24 20060101
C07K016/24 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 21, 1997 |
JP |
9-68467 |
Claims
1. A preventive or therapeutic agent for sensitized T cell-mediated
diseases comprising an interleukin-6 (IL-6) antagonist as an active
ingredient.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of copending U.S. Ser.
No. 11/340,412 filed 25 Jan. 2006, now allowed, which is a
continuation of U.S. Ser. No. 09/381,598 having an international
filing date of 20 Mar. 1998, abandoned, which is the national phase
of PCT application PCT/JP98/01217 having an international filing
date of 20 Mar. 1998, which claims benefit of Japanese patent
application No. 9-68467 filed 21 Mar. 1997. The contents of the
above patent applications are incorporated by reference herein in
their entirety.
TECHNICAL FIELD
[0002] The present invention relates to a preventive or therapeutic
agent for sensitized T cell-mediated diseases comprising an
interleukin-6 (IL-6) antagonist as an active ingredient. The
present invention also relates to an inhibitor of sensitized T
cells comprising an interleukin-6 (IL-6) antagonist as an active
ingredient. Furthermore, the present invention relates to a
suppressive agent of sensitized T cells comprising an antibody
directed against IL-6 receptor as an active ingredient.
BACKGROUND ART
[0003] IL-6 is a cytokine which is also called B cell stimulating
factor 2 (BSF2) or interferon 132. IL-6 was discovered as a
differentiation factor involved in the 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 various
functions of 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 et al., J. Exp. Immunol. 18: 1253-1258,
1988).
[0004] IL-6 transmits its biological activity through two types of
proteins on the cell. One of them is IL-6 receptor, a
ligand-binding protein with a molecular weight of about 80 kD, to
which IL-6 binds. IL-6 receptor occurs not only in a membrane-bound
form that penetrates through and is expressed on the cell membrane
but also as a soluble IL-6 receptor consisting mainly of the
extracellular region.
[0005] The other protein is a membrane-bound protein gp 130 having
a molecular weight of about 130 kD that is involved in signal
transduction. IL-6 and IL-6 receptor form the IL-6/IL-6 receptor
complex which, after binding to gp130, transmits its biological
activity to the cell (Taga, T. et al., J. Exp. Med. (1987) 166,
967).
[0006] IL-6 antagonist is a substance that inhibits the
transduction of biological activity of IL-6. As the IL-6
antagonist, there have been known so far antibody directed against
IL-6 (anti-IL-6 antibody), antibody directed against IL-6 receptor
(anti-IL-6 receptor antibody), and antibody directed against gp130
(anti-gp130 antibody). In addition, there are also known IL-6
antagonists that are disclosed in the International Patent
Application WO 95-00852, the International Patent Application WO
95-11303, the International Patent Application WO 96-34104, the
International Patent Application WO 96-18648, the International
Patent Application WO 96-17869, Japanese Unexamined Patent
Publication (Kokai) No. 7(1995)-324097, and Japanese Unexamined
Patent Publication (Kokai) No. 8(1996)-311098.
[0007] Anti-IL-6 receptor antibody has been described in several
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, U.S.
Pat. No. 5,216,28). A humanized PM-1 antibody was obtained by
grafting the complementarity determining regions (CDRs) of a mouse
antibody PM-1 (Hirata et al., J. Immunology (1989) 143, 2900-2906),
to a human antibody (the International Patent Application WO
92-19759).
[0008] On the other hand, in many autoimmune diseases and allergic
diseases, there are T cells that recognize specific antigens
(sensitized T cells) and these sensitized T cells are known to be
involved in the pathology of such diseases. For example, there are
known the presence of sensitized T cells that are directed to
myelin basic protein in multiple sclerosis (Zhang, J. et al., J.
Exp. Med (1994) 179, 973-984), S antigen in uveitis (Nussenblatt,
R. B. et al., Am. J. Ophthalmol (1980) 89, 173-179), thyroglobulin
in chronic thyroiditis, foods and acarids for atopic dermatitis
(Kubota, Y. et al., J. Dermatol (1993) 20, 85-87, Kondo, N. et al.,
J. Allergy Clin. Immunol (1993) 91, 658-668), bacteria, viruses,
fungi, etc. in delayed hypersensitivity, and metal, Japanese
lacquer, etc. in contact dermatitis, and the like.
[0009] Furthermore, it is also possible to induce pathological
states similar to those in humans by immunizing an animal with
these antigens or by introducing antigen-specific sensitized T
cells into a non-immunized animal. Based on these facts, it is
thought that sensitized T cells play an important role in the above
diseases. Currently, steroids and/or immunosuppressive agents are
used for the treatment of these diseases, but they are symptomatic
treatments and require administration for a long period of time,
which eventually poses the problem of side effects.
[0010] It has not been known so far that IL-6 antagonists as
described above exhibit a suppressive effect on sensitized T cells
and a therapeutic effect on the diseases in which sensitized T
cells are involved.
Disclosure of the Invention
[0011] It is an object of the present invention to provide a
therapeutic agent for sensitized T cell-mediated diseases said
agent being free of the above-mentioned drawbacks.
[0012] Thus, the present invention relates to a preventive or
therapeutic agent for sensitized T cell-mediated diseases
comprising an IL-6 antagonist as an active ingredient.
[0013] The present invention also relates to a preventive or
therapeutic agent for sensitized T cell-mediated diseases
comprising an antibody directed against IL-6 receptor as an active
ingredient.
[0014] The present invention also relates to a preventive or
therapeutic agent for sensitized T cell-mediated diseases
comprising a monoclonal antibody directed against IL-6 receptor as
an active ingredient.
[0015] The present invention also relates to a preventive or
therapeutic agent for sensitized T cell-mediated diseases
comprising a monoclonal antibody directed against human IL-6
receptor as an active ingredient.
[0016] The present invention also relates to a preventive or
therapeutic agent for sensitized T cell-mediated diseases
comprising a monoclonal antibody directed against mouse IL-6
receptor as an active ingredient.
[0017] The present invention also relates to a preventive or
therapeutic agent for sensitized T cell-mediated diseases
comprising PM-1 antibody as an active ingredient.
[0018] The present invention also relates to a preventive or
therapeutic agent for sensitized T cell-mediated diseases
comprising MR16-1 antibody as an active ingredient.
[0019] The present invention also relates to a preventive or
therapeutic agent for sensitized T cell-mediated diseases
comprising an antibody directed against IL-6 receptor having the
constant region (C region) of human antibody as an active
ingredient.
[0020] The present invention also relates to a preventive or
therapeutic agent for sensitized T cell-mediated diseases
comprising a chimeric antibody or a humanized antibody directed
against IL-6 receptor as an active ingredient.
[0021] The present invention also relates to a preventive or
therapeutic agent for sensitized T cell-mediated diseases
comprising humanized PM-1 antibody as an active ingredient.
[0022] The present invention also relates to a preventive or
therapeutic agent for multiple sclerosis, uveitis, chronic
thyroiditis, delayed hypersensitivity, contact dermatitis, or
atopic dermatitis comprising the above IL-6 antagonist as an active
ingredient.
[0023] The present invention also relates to an suppressive agent
of sensitized T cells comprising IL-6 antagonist as an active
ingredient.
[0024] The present invention also relates to an suppressive agent
of sensitized T cells comprising an antibody directed against IL-6
receptor as an active ingredient.
BRIEF EXPLANATION OF DRAWINGS
[0025] FIG. 1 shows a suppressive action on the mouse delayed-type
foot pad edema reaction by MR16-1 after the simultaneous
administration of MR16-1 as the sensitization by tubercle
bacillus.
EMBODIMENT FOR CARRYING OUT THE INVENTION
[0026] 1. IL-6 Antagonist
[0027] IL-6 antagonists for use in the present invention may be of
any origin, any kind, and any form, as long as they have a
suppressive effect on sensitized T cells, a preventive or
therapeutic effect for diseases in which sensitized T cells are
involved.
[0028] IL-6 antagonists block signal transduction by IL-6 and
inhibit the biological activity of IL-6. As the IL-6 antagonist,
there can be mentioned anti-IL-6 antibody, anti-IL-6 receptor
antibody, anti-gp130 antibody, altered IL-6, or partial peptides of
IL-6 or IL-6 receptor.
[0029] 1-1. Anti-IL-6 Antibody
[0030] 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, a mammalian
origin, are preferred. Monoclonal antibodies of a mammalian origin
include those produced by a hybridoma and recombinant antibody
produced by a host which has been transformed with an expression
vector containing genetically engineered antibody genes. These
antibodies, via binding to IL-6, inhibit the binding of IL-6 to
IL-6 receptor, and thereby blocks signal transduction of the
biological activity of IL-6 into the cell.
[0031] Examples of such antibodies include MH166 (Matsuda et al.,
Eur. J. Immunol. (1988) 18, 951-956) and SK2 antibody (Sato, K. et
al., The 21st Nihon Mennekigakkai Soukai (General Meeting of the
Japan Immunology Society), Academic Record (1991) 21, 166) and the
like.
[0032] 1-1-1. Preparation of IL-6
[0033] An anti-IL-6 antibody-producing hybridoma can be basically
constructed using a known procedure as described below. Thus, IL-6
may be used as a sensitizing antigen and is immunized in the
conventional method of immunization. The immune cells thus obtained
are fused with known parent cells in the conventional cell fusion
process, and then monoclonal antibody-producing cells are screened
by the conventional screening method to prepare the desired
hybridoma.
[0034] Specifically, anti-IL-6 antibody may be obtained in the
following manner. For example, a human antigen used as the
sensitizing antigen can be obtained using the IL-6 gene
sequence/amino acid sequence disclosed in Eur. J. Biochem (1987)
168, 543, J. Immunol. (1988) 140, 1534, or Argic. Biol. (1990) 54,
2685.
[0035] After a suitable host cell was transformed by inserting the
IL-6 gene sequence into a known expression vector system, the IL-6
protein of interest is purified from the host cell or the culture
supernatant thereof. The purified IL-6 protein can be used as a
sensitizing antigen. Alternatively, a fusion protein of the IL-6
protein and another protein may be used as a sensitizing
antigen.
[0036] 1-2. Anti-IL-6 Receptor Antibody
[0037] 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 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 with an expression
vector containing genetically engineered antibody genes. The
antibodies, via binding to IL-6 receptor, inhibit the binding of
IL-6 to IL-6 receptor, and thereby block the transduction of the
biological activity of IL-6 into the cell.
[0038] Examples of such antibodies include MR16-1 antibody (Saito,
et al., J. Immunology (1993) 147, 168-173), PM-1 antibody (Hirata,
et al., J. Immunology (1989) 143, 2900-2906), or 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.
[0039] 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 July 10, 1990 with the National
Institute of Bioscience and Human Technology, Agency of Industrial
Science and Technology, of 1-3, Higashi 1-chome, Tsukuba-shi,
Ibaraki, Japan, as FERM BP-2998. And 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 March 13, 1997 with the National Institute of Bioscience and
Human Technology, Agency of Industrial Science and Technology, of
1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan, as FERM
BP-5875.
[0040] 1-2-1. Preparation of IL-6 Receptor
[0041] Hybridomas producing a monoclonal antibody can be basically
prepared using a known procedure as described below. Thus, IL-6
receptor is used as a sensitizing antigen and is immunized
according to the conventional method of immunization. The immune
cells thus obtained are fused with known parent cells in the
conventional cell fusion process, and then monoclonal
antibody-producing cells may be screened by the conventional
screening method to prepare the desired hybridoma.
[0042] Specifically, anti-IL-6 receptor antibody may be prepared in
the following manner. For example, the human IL-6 receptor used as
the sensitizing antigen for obtaining antibody can be obtained
using the IL-6 receptor gene sequence/amino acid sequence disclosed
in European Patent Application EP 325474, and the mouse IL-6
receptor can be obtained using that disclosed in Japanese
Unexamined Patent Publication (Kokai) 3(1991)-155795.
[0043] There are two types of IL-6 receptor proteins: 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
substantially of the extracellular region of the IL-6 receptor
bound to the cell membrane, and thereby is different from the
membrane-bound IL-6 receptor in that the latter lacks the
transmembrane region or both of the transmembrane region and the
intracellular region.
[0044] After the gene sequence of IL-6 receptor was 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.
The IL-6 receptor protein thus purified may be used as the
sensitizing antigen. Alternatively, cells that are expressing IL-6
receptor protein or a fusion protein of the IL-6 receptor protein
and another protein may be used as the sensitizing antigen.
[0045] E. coli that has a plasmid pIBIBSF2R containing 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 National Institute of Bioscience and Human
Technology, Agency of Industrial Science and Technology, of 1-3,
Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan, as FERM BP-2232.
[0046] 1-3. Anti-gp130 Antibody
[0047] 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, 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 with an expression vector containing
genetically engineered antibody genes. The antibodies, via binding
to gp130, inhibit the binding of IL-6/IL-6 receptor complex to
gp130, and thereby block the transduction of the biological
activity of IL-6 into the cell.
[0048] Examples of such antibodies include AM64 antibody (Japanese
Unexamined Patent Publication (Kokai) 3(1991)-219894), 4B11
antibody and 2H4 antibody (US5571513), B-S 12 antibody and B-P8
antibody (Japanese Unexamined Patent Publication (Kokai)
8(1996)-291199).
[0049] 1-3-1. Preparation of gp130
[0050] A monoclonal antibody-producing hybridoma can be basically
constructed using a known procedure as described below. Thus, gp130
may be used as a sensitizing antigen and is immunized in the
conventional method of immunization. The immune cells thus obtained
are fused with known parent cells in the conventional cell fusion
process, and then the monoclonal antibody-producing hybridomas are
screened by the conventional screening method to prepare the
desired hybridoma.
[0051] Specifically, monoclonal antibody may be obtained in the
following manner. For example, gp130 used as the sensitizing
antigen can be obtained using the IL-6 receptor gene sequence/amino
acid sequence disclosed in European Patent Application EP
411946.
[0052] After a suitable host cell was transformed by inserting the
gp130 gene sequence into a known expression vector system, the
gp130 protein of interest is purified from the host cell or from
the culture supernatant thereof. The purified gp130 protein can be
used as the sensitizing antigen. Alternatively, cells that are
expressing IL-6 receptor protein or a fusion protein of the gp 130
protein and another protein may be used as the sensitizing
antigen.
[0053] 1-4. Preparation of Antibody-Producing Hybridoma
[0054] Though mammals to be immunized with the sensitizing antigen
are not specifically limited, they are preferably selected in
consideration of their compatibility with the parent cell for use
in cell fusion. They generally include, but not limited to, rodents
such as mice, rats, hamsters and the like.
[0055] Immunization of animals with a sensitizing antigen is
carried out using a known method. A general method, for example,
involves the intraperitoneal or subcutaneous administration of a
sensitizing antigen to the mammal. Specifically, a sensitizing
antigen which has been diluted and suspended in an appropriate
amount of phosphate buffered saline (PBS) or physiological saline
etc. is mixed, as desired, with an appropriate amount of a common
adjuvant, for example Freund's complete adjuvant. After being
emulsified, it is preferably administered to a mammal for several
times every 4 to 21 days. Alternatively a suitable carrier may be
used at the time of immunization of the sensitizing antigen.
[0056] After immunization and the confirmation of the increase in
the desired antibody levels in the serum, the immune cells are
taken out from the mammal and are subjected to cell fusion, in
which preferred immune cells include, in particular, the spleen
cells.
[0057] The 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)
(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), 5194 (Trowbridge, I. S.,
J. Exp. Med. (1978) 148: 313-323), R210 (Galfre, G. et al., Nature
(1979) 277: 131-133) and the like.
[0058] Cell fusion between the above immune cells and the 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.
[0059] 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, in addition, an adjuvant such as dimethyl
sulfoxide etc. may be added as desired to enhance efficiency of the
fusion.
[0060] The preferred ratio of the immune cells and the myeloma
cells to be used 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 RPMI1640 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.
[0061] 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 about 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 the sequential addition of a
suitable culture liquid and centrifugation to remove the
supernatant, cell fusion agents etc., which are undesirable for the
growth of the hybridoma, can be removed.
[0062] Said hybridoma is selected by culturing in the conventional
selection medium, for example, the HAT culture medium (a culture
liquid containing hypoxanthine, aminopterin, and thymidine).
Culturing in said HAT culture medium is continued generally for a
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 that produce the desired antibody
are screened and monclonally cloned.
[0063] 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 desired antigen or
desired antigen-expressing cells, and the resulting sensitized B
lymphocytes are fused with a human myeloma cell, for example U266,
to obtain the desired human antibody having the activity of binding
to desired antigen or desired antigen-expressing cells (see
Japanese Post-examined Patent Publication (Kokoku) No.
1(1989)-59878). Furthermore, a transgenic animal having a
repertoire of all human antibody genes is immunized with the
antigen or the antigen-expressing cells to obtain the desired human
antibody in the method described above (see International Patent
Application WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO
96/34096 and WO 96/33735).
[0064] 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.
[0065] In order to obtain monoclonal antibodies from said
hybridoma, there can be mentioned 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
administered to 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.
[0066] Specifically the hybridoma producing anti-IL-6 receptor
antibody can be constructed using the method disclosed in Japanese
Unexamined Patent Publication (Kokai) 3(1989)-139293. It can be
conducted by a method in which the PM-1 antibody-producing
hybridoma that was internationally deposited under the provisions
of the Budapest Treaty as FERM BP-2998 on Jul. 10, 1990 with the
National Institute of Bioscience and Human Technology, Agency of
Industrial Science and Technology, of 1-3, Higashi 1-chome,
Tsukuba-shi, Ibaraki, Japan, is intraperitoneally injected to
BALB/c mice (manufactured by CLEA Japan) to obtain the ascites from
which the PM-1 antibody is purified, or: a method in which said
hybridoma is cultured in a suitable culture medium such as the
RPMI1640 medium containing 10% bovine fetal serum and 5%
BM-Condimed H1 (manufactured by Boehringer Mannheim), the hybridoma
SFM medium (manufactured by GIBCO-BRL), the PFHM-II medium
(manufactured by GIBCO-BRL) and the like, and the PM-1 antibody can
be purified from the supernatant.
[0067] 1-5. Recombinant Antibody
[0068] A recombinant antibody which was produced by the recombinant
gene technology in which an antibody gene was cloned from the
hybridoma and integrated into a suitable vector which was then
introduced into a host can be used in the present invention as
monoclonal antibody (see, for example, Carl, A. K., Borrebaeck, and
James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, published in
the United Kingdom by MACMILLAN PUBLISHERS LTD. 1990).
[0069] Specifically, mRNA encoding the variable (V) region of the
desired antibody is isolated from the hybridoma producing the
antibody. The isolation of mRNA is conducted by preparing total RNA
using, for example, a known method such as the guanidine
ultracentrifuge method (Chirgwin, J. M. et al., Biochemistry (1979)
18, 5294-5299), the AGPC method (Chmczynski, P. et al., (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).
[0070] 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; Belyaysky, A. et al.,
Nucleic Acids Res. (1989) 17, 2919-2932) that employs polymerase
chain reaction (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.
[0071] Once the 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, the 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.
[0072] In order to produce the antibody for use in the present
invention, the antibody gene is integrated as described below 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 may be transformed
into a host cell and the antibody can then be expressed
therein.
[0073] 1-6. Altered Antibody
[0074] In accordance with the present invention, artificially
altered recombinant antibody such as chimeric antibody and
humanized antibody can be used for the purpose of lowering
heterologous antigenicity against humans. These altered antibody
can be produced using known methods.
[0075] 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 96/02576). Using this known
method, chimeric antibody useful for the present invention can be
obtained.
[0076] For example, the plasmid that contains DNA encoding the L
chain V region or the H chain V region of chimeric PM-1 antibody
was designated as pPM-k3 or pPM-h1, respectively, and E. coli
having the plasmid has been internationally deposited under the
provisions of the Budapest Treaty as NCIMB 40366 and NCIMB 40362,
respectively, on February 11, 1991 with the National Collections of
Industrial and Marine Bacteria Limited (see International Patent
Application WO 92-19759).
[0077] Humanized antibody which is also called reshaped human
antibody has been made by grafting the complementarity determining
regions (CDRs) of antibody of a mammal other than the human, for
example mouse antibody, into the CDRs of a 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 96/02576).
[0078] 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 by
the PCR technique. The DNA thus obtained is ligated to the DNA
encoding the C region of human antibody and then is integrated 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).
[0079] For the FR of human antibody ligated through CDR, the
complementarity determining region that forms a favorable antigen
binding site is selected. When desired, amino acids in the
framework region of the antibody variable region may be substituted
so that the complementarity determining region of reshaped human
antibody may form an appropriate antigen biding site (Sato, K. et
al., Cancer Res. (1993) 53, 851-856).
[0080] For chimeric antibody or humanized antibody, the C region of
human antibody is used. As the C region of human antibody, there
can be mentioned C.gamma., and C.gamma.1, C.gamma.2, C.gamma.3, and
C.gamma.4, for example, can be used. The C region of human antibody
may be modified to improve the stability of antibody or the
production thereof.
[0081] Chimeric antibody consists of the variable region of
antibody derived from a mammal other than the human and the C
region derived from human antibody, whereas humanized antibody
consists of the complementarity determining region of antibody
derived from a mammal other than the human and the framework region
(FR) and the C region of antibody derived from human antibody.
Accordingly, antigenicity thereof in the human body has been
reduced so that they are useful as the active ingredient of the
therapeutic agents of the present invention.
[0082] A preferred embodiment of the humanized antibody for use in
the present invention includes humanized PM-1 antibody (see
International Patent Application WO 92-19759).
[0083] 1-7. Expression and Production
[0084] Antibody genes constructed as described above may be
expressed and obtained in a known method. In the case of mammalian
cells, expression may be accomplished using a DNA in which a
commonly used useful promoter, the antibody gene to be expressed,
and the poly A signal at 3' downstream thereof have been operably
linked or a vector containing said DNA. Examples of the
promoter/enhancer include human cytomegalovirus immediate early
promoter/enhancer.
[0085] Additionally, as the promoter/enhancer which can be used for
expression of antibody for use in the present invention, there are
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.).
[0086] For example, expression may be readily accomplished by the
method of Mulligan et al. (Nature (1979) 277, 108) when SV40
promoter/enhancer is used, or by the method of Mizushima et al.
(Nucleic Acids Res. (1990) 18, 5322) when HEF1.alpha.
promoter/enhancer is used.
[0087] In the case of E. coli, expression may be conducted by
operably linking a commonly used useful promoter, a signal sequence
for antibody secretion, and the 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. (Nature (1098) 341, 544-546; FASEB J. (1992) 6,
2422-2427) may be used when lacz promoter is used, and the method
of Better et al. (Science (1988) 240, 1041-1043) may be used when
araB promoter is used.
[0088] As the 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) 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).
[0089] 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 the 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.
[0090] For the production of antibody for use in the present
invention, any production system can be used. The production system
of antibody preparation comprises the in vitro or the in vivo
production system. As the in vitro production system, there can be
mentioned a production system which employs eukaryotic cells and
the production system which employs prokaryotic cells.
[0091] When the 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 Nicotiana tabacum, which
is subjected to callus culture. Known fungal cells include yeasts
such as the genus Saccharomyces, more specifically Saccharomyces
cerevisiae, or filamentous fungi such as the genus Aspergillus,
more specifically Aspergillus niger.
[0092] When the prokaryotic cells are used, there are the
production systems which employ bacterial cells. Known bacterial
cells include Escherichia coli (E. coli), and Bacillus
subtilis.
[0093] 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, DMEM, MEM,
RPMI1640, and IMDM 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.
[0094] 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.
[0095] As mammals, goats, pigs, sheep, mice, and cattle can be used
(Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). Also, as
insects, silkworms can be used.
[0096] When plants are used, tabacco, for example, can be used.
[0097] Antibody genes are introduced into these animals or plants,
and the antibodies are produced in such animals or plants, and
recovered. For example, an antibody gene is inserted into the
middle of the gene encoding protein which is inherently produced in
the milk such as goat 13 casein to prepare fusion genes. DNA
fragments containing the fusion gene into which the antibody gene
has been inserted are injected into a goat embryo, and the embryo
is introduced into a female goat. The desired antibody is obtained
from the milk produced by the transgenic goat born to the goat who
received the embryo or offsprings 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).
[0098] When silkworms are used, baculovirus into which the desired
antibody gene has been inserted is infected to the silkworm, and
the desired antibody can be obtained from the body fluid of the
silkworm (Susumu, M. et al., Nature (1985) 315, 592-594). Moreover,
when tabacco 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 infected to tabacco such as
Nicotiana tabacum to obtain the desired antibody from the leaves of
the tabacco (Julian, K.-C. Ma et al., Eur. J. Immunol. (1994) 24,
131-138).
[0099] When antibody is produced in in vitro or in vivo production
systems, as described above, DNA encoding the heavy chain (H chain)
or the light chain (L chain) of antibody may be separately
integrated into an expression vector and the hosts are transformed
simultaneously, or DNA encoding the H chain and the L chain may be
integrated into a single expression vector and the host is
transformed therewith (see International Patent Application WO
94-11523).
[0100] 1-8. Modified Antibody
[0101] Antibodies for use in the present invention may be antibody
fragments or modified versions thereof as long as they are
preferably used. 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.
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 in Enzymology
(1989) 178, 476-496, Academic Press, Inc.; Plueckthun, A. and
Skerra, A., Methods in Enzymology (1989) 178, 476-496, Academic
Press, Inc.; Lamoyi, E., Methods in Enzymology (1986) 121, 652-663;
Rousseaux, J. et al., Methods in Enzymology (1986) 121, 663-669;
Bird, R. E. et al., TIBTECH (1991) 9, 132-137).
[0102] 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.
[0103] 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.
[0104] Once DNAs encoding scFv are constructed, an expression
vector containing them and a host transformed with said expression
vector can be obtained by the conventional methods, and scFv can be
obtained using the resultant host by the conventional methods.
[0105] 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
claim of the present application encompasses these antibody
fragments.
[0106] As modified antibodies, antibodies associated with various
molecules such as polyethylene glycol (PEG) can be used. "Antibody"
as used in the claim of the present application 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.
[0107] 1-9. Separation and Purification of Antibody
[0108] 1-9-1. Separation and Purification of Antibody
[0109] Antibodies produced and expressed as described above can be
separated from the inside or outside of the host cell and then may
be purified to homogeneity. Separation and purification of the
antibody for use in the present invention may be accomplished by
affinity chromatography. As the column used for such affinity
chromatography, there can be mentioned Protein A column and Protein
G column. Examples of the carriers used in the Protein A column are
Hyper D, POROS, Sepharose F. F. and the like. Alternatively,
methods for separation and purification conventionally used for
proteins can be used without any limitation. Separation and
purification of the antibody for use in the present invention may
be accomplished by combining, as appropriate, chromatography other
than the above-mentioned affinity chromatography, filtration,
ultrafiltration, salting-out, dialysis and the like. Chromatography
includes, for example, ion exchange chromatography, hydrophobic
chromatography, gel-filtration and the like. These chromatographies
can be applied into HPLC. Alternatively, reverse-phase
chromatography can be used.
[0110] 1-9-2. Determination of Antibody Concentration
[0111] The concentration of antibody obtained in the above 2-1 can
be determined by the measurement of absorbance or by the
enzyme-linked immunosorbent assay (ELISA) and the like. Thus, when
absorbance measurement is employed, the antibody for use in the
present invention or a sample containing the antibody 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 the ELISA method is used,
measurement is conducted as follows. Thus, 100 .mu.l of goat
anti-human IgG (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.
[0112] After blocking, 100 .mu.l each of appropriately diluted
antibody for use in the present invention or a sample containing
the antibody, or 100 .mu.l of human IgG (manufactured by CAPPEL) as
the standard is added, and incubated at room temperature for 1
hour. 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 the measurement of absorbance at 405 nm using the
MICROPLATE READER Model 3550 (manufactured by Bio-Rad) to calculate
the concentration of the desired antibody.
[0113] 1-10. IL-6 Antagonists Other than Antibodies
[0114] The altered IL-6 for use in the present invention has an
activity of binding to IL-6 receptor and does not transmit the
biological activity of IL-6. Thus, the altered IL-6, though it
competes with IL-6 for binding to IL-6 receptor, does not transmit
the biological activity of IL-6, and thereby it blocks signal
transduction by IL-6.
[0115] Altered IL-6 may be constructed through the introduction of
mutation by replacing amino acid residues of the amino acid
sequence of IL-6. IL-6, the source of the altered IL-6, may be of
any origin, but when the antigenicity is to be considered, it is
preferably human IL-6. Specifically, the secondary structure of
IL-6 is predicted using a known molecular modeling program of the
amino acid sequence, for example WHATIF (Vriend et al., J. Mol.
Graphics (1990), 8, 52-56), and the overall effects of the amino
acid residue to be replaced is evaluated. After an appropriate
amino acid residue was determined, mutation is introduced by the
commonly used polymerase chain reaction (PCR) method using a vector
containing the base sequence encoding human IL-6 gene thereby to
obtain a gene encoding altered IL-6. This is then integrated, as
desired, into an appropriate expression vector, from which altered
IL-6 can be obtained according to the expression, production and
purification of said recombinant antibody.
[0116] Specific examples of the altered IL-6 are disclosed in
Brakenhoff et al., J. Biol. Chem. (1994) 269, 86-93, and Savino et
al., EMBO J. (1994) 13, 1357-1367, WO 96-18648, and WO
96-17869.
[0117] The IL-6 partial peptide or the IL-6 receptor partial
peptide for use in the present invention has an activity of binding
to IL-6 receptor or IL-6, respectively, and does not transmit the
biological activity of IL-6. Thus, the IL-6 partial peptide or the
IL-6 receptor partial peptide binds to IL-6 receptor or IL-6,
respectively, and thereby capture it. As a result, they do not
transmit the biological activity of IL-6, and block signal
transduction of IL-6.
[0118] The IL-6 partial peptide or the IL-6 receptor partial
peptide is a peptide comprising some or all of the amino acid
sequence of the region involved in the binding to IL-6 and IL-6
receptor in the amino acid sequence of IL-6 or IL-6 receptor. Such
a peptide generally comprises 10-80, preferably 20-50, more
preferably 20-40 amino acid residues.
[0119] The IL-6 partial peptide or the IL-6 receptor partial
peptide can be constructed by specifying the region involved in the
binding to IL-6 and IL-6 receptor in the amino acid sequence of
IL-6 or IL-6 receptor, and by producing some or all of the amino
acid sequence by a conventional method such as a genetic
engineering technology or a peptide synthesis method.
[0120] In order to prepare the IL-6 partial peptide or the IL-6
receptor partial peptide by a genetic engineering technology, the
DNA sequence encoding the desired peptide is integrated into an
expression vector, from which the peptide can be obtained according
to the expression, production, and purification of said recombinant
antibody.
[0121] Preparation of the IL-6 partial peptide or the IL-6 receptor
partial peptide by the peptide synthesis method can be effected
using a method commonly used in peptide synthesis such as the solid
phase synthesis or the liquid phase synthesis. Specifically the
method described in Zoku-iyakuhin no Kaihatsu (Sequel to
Development of Pharmaceuticals), Vol. 14, Peputido Gousei (Peptide
Synthesis), edited by Haruaki Yajima, Hirokawa Shoten, 1991, may be
used. The solid phase synthesis method used includes, for example,
a reaction in which an amino acid corresponding to the C-terminal
of the peptide to be synthesized is coupled to a support which is
insoluble in organic solvents, and then an amino acid in which
.alpha.-amino group or a side chain functional group has been
protected with an appropriate protecting group is condensed one
amino acid at a time from the C-terminal to the N-terminal
direction, and a reaction in which said protecting group of the
.alpha.-amino group of the amino acid or the peptide coupled to the
resin is eliminated is alternately repeated to elongate the peptide
chain. The solid phase peptide synthesis methods are divided into
the Boc method and the Fmoc method depending on the type of
protecting group to be used.
[0122] After the synthesis of the desired peptide is complete, the
peptide chain is cleaved from the support via a deprotection
reaction. For cleavage from the peptide chain, hydrogen fluoride or
trifuluoromethane sulfonate in the Boc method and TFA in the Fmoc
method are generally used. In the Boc method, for example, the
above peptide resin is treated in hydrogen fluoride in the presence
of anisole. Subsequently, the protecting group is eliminated and
the peptide is recovered by cleaving from the support. By
lyophilizing this, crude peptide can be obtained. On the other
hand, in the Fmoc method, TFA, for example, is used in a manner
similar to the above to effect the deprotection reaction and the
cleavage reaction of the peptide from the support.
[0123] The crude peptide thus obtained can be applied to HPLC for
its separation and purification. Its elution can be carried out in
a water-acetonitrile solvent system that is commonly used for
protein purification under an optimum condition. The fraction
corresponding to the peak of the profile of the chromatography
obtained is collected and lyophilized. The peptide fraction thus
purified is identified by subjecting it to the analysis of
molecular weight by mass spectroscopic analysis, the analysis of
amino acid composition, or the analysis of amino acid sequence, and
the like.
[0124] Specific examples of the IL-6 partial peptide or the IL-6
receptor partial peptide are disclosed in Japanese Unexamined
Patent Publication (Kokai) 2(1990)-188600, Japanese Unexamined
Patent Publication (Kokai) 7(1995)-324097, Japanese Unexamined
Patent Publication (Kokai) 8(1996)-311098, and U.S. Pat. No.
5,210,075.
[0125] 2. Confirmation of the Activity of IL-6 Antagonist
[0126] The activity of the IL-6 antagonist for use in the present
invention can be evaluated using a conventionally known method. For
example, IL-6 is added to the IL-6-dependent cell MH60.BSF2 and the
activity can be evaluated using the incorporation of
.sup.3H-thymidine into the IL-6-dependent cell in the coexistence
of the IL-6 antagonist as an index. Alternatively, evaluation can
be effected by adding .sup.125I-labeled IL-6 and an excess amount
of non-labeled IL-6 to the U266, an IL-6 receptor-expressing cell,
and adding the IL-6 antagonist at the same time and then by
determining the .sup.125I-labeled IL-6 bound to the IL-6
receptor-expressing cell.
[0127] 3. Confirmation of Therapeutic Effects
[0128] In order to confirm the effects accomplished by the present
invention, the IL-6 antagonist for use in the present invention may
be given to an animal that has been sensitized with T cells via
challenge or to an animal to which sensitized T cells have been
introduced, and the suppressive effects on the sensitized T cells
are evaluated.
[0129] As the sensitizing antigen to be given to the animal, for
example, tubercle bacillus can be used.
[0130] As the animal to be immunized, animals generally used in the
experiments can be used such as mice, rats, rabbits, and the like.
The effect of the present invention to be evaluated can be
confirmed by observing the delayed inflammatory reaction induced by
challenging the same antigen to an animal that was given the
antigen.
[0131] As described the examples below, in the mouse delayed foot
pad edema reaction, the administration of anti-IL-6 receptor
antibody resulted in suppression of the delayed inflammatory
reaction was observed. Since it was known that sensitized T cells
are involved in the delayed foot pad edema reaction, it was
revealed that IL-6 antagonists such as anti-IL-6 receptor antibody
exert an inhibitory effect on the sensitized T cells.
[0132] 4. Route of administration and pharmaceutical
preparation
[0133] The preventive or therapeutic agents for sensitized T
cell-mediated diseases of the present invention may be
administered, either systemically or locally, by a parenteral
route, for example intravenous injection such as drip infusion,
intramuscular injection, intraperitoneal injection, and
subcutaneous injection. The method of administration may be chosen,
as appropriate, depending on the age and the conditions of the
patient. The effective dosage is chosen from the range of 0.01 mg
to 100 mg per kg of body weight per administration. Alternatively,
the dosage in the range of 1 to 1000 mg, preferably 5 to 50 mg per
patient may be chosen.
[0134] The preventive or therapeutic agents for sensitized T
cell-mediated diseases of the present invention may contain
pharmaceutically acceptable carriers or additives depending on the
route of administration. Examples of such carriers or additives
include water, a pharmaceutical 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, a
pharmaceutically acceptable surfactant and the like.
[0135] Additives used are chosen from, but not limited to, the
above or combinations thereof depending on the dosage form.
[0136] The subject diseases to be prevented or treated of the
present invention are diseases in which sensitized T cell are
involved. Specifically, they include delayed hypersensitivity,
chronic thyroiditis, uveitis, atopic dermatitis, contact
dermatitis, or multiple sclerosis. The preventive or therapeutic
agents of the present invention are useful as preventive or
therapeutic agents for diseases in which these sensitized T cells
are involved.
EXAMPLES
[0137] The present invention will now be explained in more details
with reference to the working examples, reference examples, and
experimental examples. It should be noted, however, that the
present invention is not limited to them in any way.
Example 1
Inhibitory Effects on Delayed Foot Pad Reaction
[0138] Dry dead cells of Mycobacterium butyricum were added to 2.5
mg/ml in the Freund's incomplete adjuvant to prepare an emulsion,
0.2 ml of which was then subcutaneously injected to C57BL/6 male
mice for challenge. On day 14, 10 mg of dry dead cells of
Mycobacterium butyricum suspended in physiological saline were
subcutaneously injected to the right foot pad of the animal to
evoke the reaction. Twenty four hours later, the weights of the
left and the right foot pad were measured and the difference in the
weights were used as an index of the strength of the reaction.
[0139] MR16-1 antibody at 0.125 mg, 0.5 mg, or 2 mg was
intraperitoneally given only once simultaneously with the
challenge. The control group received rat IgG (KH-5) having the
same isotype and the unsensitized mouse group received
physiological saline in a similar manner. The result is shown in
FIG. 1.
[0140] The one time administration of MR16-1 antibody on the day of
challenge inhibited the delayed foot pad edema reaction in a
dose-dependent manner.
Reference Example 1
Preparation of Human Soluble IL-6 Receptor
[0141] Soluble IL-6 receptor was prepared by the PCR method using a
plasmid pBSF2R.236 containing cDNA that encodes IL-6 receptor
obtained according to the method of Yamasaki et al., Science (1988)
241, 825-828. Plasmid pBSF2R.236 was digested with a restriction
enzyme Sph I to obtain the cDNA of IL-6 receptor, which was then
inserted into mp 18 (manufactured by Amersham). Using a synthetic
oligoprimer designed to introduce a stop codon into the cDNA of
IL-6 receptor, a mutation was introduced into the cDNA of IL-6
receptor by the PCR method using the in vitro Mutagenesis System
(manufactured by Amersham). The procedure resulted in the
introduction of a stop codon to the amino acid at position 345, and
gave the cDNA encoding soluble IL-6 receptor.
[0142] In order to express the cDNA of soluble IL-6 receptor in CHO
cells, it was ligated to plasmid pSV (manufactured by Pharmacia) to
obtain plasmid pSVL344. The cDNA of soluble IL-6 receptor that was
cleaved with Hind III-Sal I was inserted to plasmid pECEdhfr
containing the cDNA of dhfr to obtain plasmid pECEdhfr344 that can
be expressed in the CHO cells.
[0143] Ten .mu.g of plasmid pECEdhfr344 was transfected to a
dhfr-CHO cell line DXB-11 (Urland et al., Proc. Natl. Acad. Sci.
U.S.A. (1980) 77, 4216-4220) by the calcium phosphate method (Chen
et al., Mol. Cell. Biol. (1987) 7, 2745-2751). The transfected CHO
cells were cultured in a nucleoside-free cc MEM selection medium
containing 1 mM glutamin, 10% dialyzed FCS, 100 U/ml penicillin,
and 100 .mu.g/ml streptomycin.
[0144] The selected CHO cells were screened by the limiting
dilution method to obtain a single CHO cell clone. The CHO cell
clone was amplified in 20 nM to 200 nM methotrexate (MTX) to obtain
a CHO cell line 5E27 that produces human soluble IL-6 receptor. The
CHO cell line 5E27 was cultured in an Iscov-modified Dulbecco's
medium (IMDM, manufactured by Gibco) containing 5% FBS. The culture
supernatant was collected and the concentration of soluble IL-6
receptor in the culture supernatant was determined by ELISA.
Reference Example 2
Preparation of Anti-Human IL-6 Antibody
[0145] Ten .mu.g of the recombinant IL-6 (Hirano et al., Immunol.
Lett., 17:41, 1988) was immunized to BALB/c mice together with
Freund's complete adjuvant, and this was repeated every week until
anti-IL-6 antibody could be detected in the serum. Immune cells
were extracted from local lymph node and were then fused with a
myeloma cell line P3U1 using polyethylene glycol 1500. Hybridomas
were selected according to the method of Oi et al. (Selective
Methods in Cellular Immunolgy, W. H. Freeman and Co., San
Francisco, 351, 1980) that employs the HAT medium, and the
hybridoma that produces anti-human IL-6 antibody was
established.
[0146] The hybridoma that produces anti-human IL-6 antibody was
subjected to IL-6 binding assay as follows. Thus, a 96-well
microtiter plate made of flexible polyvinyl (manufactured by
Dynatech Laboratories, Inc., Alexandria, Va.) was coated with 100
.mu.l of goat anti-mouse Ig (10 manufactured by Cooper Biomedical,
Inc., Malvern, Pa.) in 0.1 M carbonate-hydrogen carbonate buffer,
pH9.6, overnight at 4.degree. C. Subsequently, the plate was
treated with PBS containing 1% bovine serum albumin (BSA) at room
temperature for 2 hours.
[0147] After washing in PBS, 100 .mu.l of the hybridoma culture
supernatant was added to each well, and then was incubated
overnight at 4.degree. C. The plate was washed, .sup.125I-labled
recombinant IL-6 was added to each well to a concentration of 2000
cpm/0.5 ng/well, and then radioactivity of each well after washing
was determined by a gamma counter (Beckman Gamma 9000, Beckman
Instruments, Fullerton, Calif.). Of 216 hybridoma clones, 32 were
positive in the IL-6 binding assay. From these clones, stable
MH166.BSF2 was finally obtained. Anti-IL-6 antibody MH166 produced
by said hybridoma has a subtype of IgG1 .kappa..
[0148] Then, the IL-6-dependent mouse hybridoma clone MH166.BSF2
was used to examine a neutralizing activity with respect to the
growth of the hybridoma by MH166. MH166.BSF2 cells were aliquoted
to 1.times.10.sup.4/200 .mu.l/well, and samples containing MH166
antibody were added thereto, cultured for 48 hours, 15.1 Ci/mM
.sup.3H-thymidine (New England Nuclear, Boston, Mass.) was added,
and the culturing was continued for further 6 hours. The cells were
placed on a glass filter paper and were treated by the automatic
harvester (Labo Mash Science Co., Tokyo, Japan). As the control,
rabbit anti-IL-6 antibody was used.
[0149] As a result, MH166 antibody inhibited the incorporation of
.sup.3H-thymidine of MH166.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. (J. Immunol., 143, 2900-2906, 1989) was bound to
CNBr-activated Sepharose 4B (manufactured by Pharmacia Fine
Chemicals, Piscataway, N.J.) according to the attached regimen and
IL-6 receptor (Science (1988) 241, 825-828) was purified. A human
myeloma cell line U266 was solubilized with 1 mM p-para-aminophenyl
methane sulphonyl fluoride hydrochloride (manufactured by Wako
Chemicals) containing 1% digitonin (manufactured by Wako
Chemicals), 10 mM triethanolamine (pH 7.8) and 0.15 M NaCl
(digitonin buffer), and mixed with MT18 antibody bound to Sepharose
4B beads. Then, the beads were washed six times with the digitonin
buffer, to prepare the partially purified IL-6 receptor.
[0151] BALB/c mice were immunized four times every ten days with
the above partially purified IL-6 receptor obtained from
3.times.10.sup.9 U266 cells, and then a hybridoma was prepared
using a standard method. The hybridoma culture supernatant from the
growth-positive positive well was tested for its activity of
binding to IL-6 receptor according to the method described below.
5.times.10.sup.7 U266 cells were labeled with .sup.35S-methionine
(2.5 mCi) and were solubilized with the above digitonin buffer. The
solubilized U266 cells were mixed with a 0.04 ml volume of MT18
antibody bound to Sepharose 4B beads, and then were washed six
times with the digitonin buffer. .sup.35S-methionine-labeled IL-6
receptor was eluted with 0.25 ml of the digitonin buffer (pH 3.4)
and was neutralized in 0.025 ml of 1M Tris (pH 7.4).
[0152] 0.05 ml of the hybridoma culture supernatant was mixed with
0.01 ml of Protein G Sepharose (manufactured by Pharmacia). After
washing, Sepharose was incubated with 0.005 ml .sup.35S-labeled
IL-6 receptor solution prepared as described above. The
immunoprecipitate was analyzed by SDS-PAGE to investigate the
hybridoma culture supernatant that reacts with IL-6 receptor. As a
result, the reaction positive hybridoma clone PM-1 was established.
The antibody produced from the hybridoma PM-1 has a subtype of
IgG1.kappa..
[0153] The inhibitory activity of IL-6 binding of the antibody
produced by the hybridoma PM-1 to human IL-6 receptor was studied
using the human myeloma cell line U266. A human recombinant IL-6
was prepared from E. coli (Hirano et al., Immunol. Lett., 17:41,
1988), and was labeled with .sup.125I using the Bolton-Hunter
reagent (New England Nuclear, Boston, Mass.) (Taga, T. et al., J.
Exp. Med. (1987) 166, 967). 4.times.10.sup.5 U266 cells were
cultured with the 70% (v/v) culture supernatant of hybridoma PM-1
together with 14,000 cpm of .sup.125I-labeled IL-6 in the presence
of 100-fold excess of unlabeled IL-6 for one hour at room
temperature. Seventy .mu.l of the sample was layered on 300 .mu.l
FCS in a 400 .mu.l microfuge polyethylene tube. After
centrifugation, the radioactivity of the cell was determined.
[0154] 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
[0155] A monoclonal antibody directed against mouse IL-6 receptor
was prepared according to the method described in Saito, et al., J.
Immunol. (1993) 147, 168-173.
[0156] The CHO cells that produce mouse soluble IL-6 receptor were
cultured in the IMDM liquid medium containing 10% FCS. From the
culture supernatant, mouse soluble IL-6 receptor was purified using
mouse soluble IL-6 receptor RS 12 (see Saito, et al., supra) and an
affinity column fixed to Affigel 10 gel (Biorad).
[0157] The mouse soluble IL-6 receptor (50 .mu.g) thus obtained was
mixed with Freund's complete adjuvant, which was then injected to
the abdomen of Wistar rats (Japan Charles River). From 2 weeks the
animals were boosted with Freund's incomplete adjuvant. On day 45,
the rats were sacrificed, and the spleen cells at about
2.times.10.sup.8 were fused with 1.times.10.sup.7 mouse myeloma
cells P3U1 using 50% PEG1500 (Boehringer Mannheim) according to the
conventional method, and then were screened by the HAT culture
medium.
[0158] After the culture supernatant was added to the plate coated
with rabbit anti-rat IgG antibody (Cappel), mouse soluble IL-6
receptor was reacted. Subsequently, using rabbit anti-mouse IL-6
receptor antibody and alkaline phosphatase-labeled sheep
anti-rabbit IgG, hybridomas producing antibody directed against
mouse soluble IL-6 receptor were screened by ELISA. After antibody
production was confirmed, the hybridoma clones were subscreened
twice to obtain a single hybridoma clone. The clone was designated
as MR16-1.
[0159] The neutralizing activity of the antibody produced by the
hybridoma on signal transduction of mouse IL-6 was examined by
3H-thymidine incorporation using MH60.BSF2 cells (Matsuda et al.,
J. Immunol. (1988) 18, 951-956). To a 96-well plate, MH60.BSF2
cells were prepared at 1.times.10.sup.4 cells/200 .mu.l/well. To
the plate were added mouse IL-6 and MR16-1 antibody or RS12
antibody at 12.3-1000 ng/ml, and then were cultured at 37.degree.
C. and 5% CO.sub.2 for 44 hours and then 1 .mu.Ci/well of
.sup.3H-thymidine was added. After 4 hours, the incorporation of
.sup.3H-thymidine was measured. As a result, MR16-1 antibody
suppressed the incorporation of .sup.3H-thymidine of the MH60.BSF2
cells.
[0160] Thus, it was demonstrated that the antibody produced by the
hybridoma MR16-1 inhibits the binding of IL-6 to IL-6 receptor.
INDUSTRIAL APPLICABILITY
[0161] In accordance with the present invention, it was shown that
IL-6 antagonists such as anti-IL-6 receptor antibody has a
suppressive effect on the sensitized T cells. Thus, it was
indicated that IL-6 antagonists are useful as a therapeutic agent
for multiple sclerosis, uveitis, chronic thyroiditis, delayed
hypersensitivity, contact dermatitis, or atopic dermatitis.
[0162] Reference to the microorganisms deposited under the Patent
Cooperation Treaty, Rule 13-2, and the name of the Depository
institute [0163] Depository institute [0164] Name: the National
Institute of Bioscience and Human
[0165] Technology, Agency of Industrial Science and Technology
[0166] Address: 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan
[0167] Microorganism (1)
[0168] Indication: Rat-mouse hybridoma MR16-1
[0169] Deposition number: FERM BP-5875
[0170] Deposition date: Mar. 13, 1997 [0171] Microorganism (2)
[0172] Indication: HB 101-pIBIBSF2R
[0173] Deposition number: FERM BP-2232
[0174] Deposition date: Jan. 9, 1989 [0175] Microorganism (3)
[0176] Indication: PM1
[0177] Deposition number: FERM BP-2998
[0178] Deposition date: Jul. 12, 1989 [0179] Depository organ
[0180] Name: National Collection of Industrial and Marine
[0181] Bacteria Limited [0182] Address: 23 St Machar Drive Aberdeen
AB2 IRY [0183] Microorganism (4)
[0184] Indication: Escherichia coli DH5.alpha.-pPM-k3
[0185] Deposition number: NCIMB 40366
[0186] Deposition date: Feb. 12, 1991 [0187] Microorganism (5)
[0188] Indication: Escherichia coli DH5.alpha.-pPM-h1
[0189] Deposition number: NCIMB 40362
[0190] Deposition date: Feb. 12, 1991
* * * * *