Title: Treatment of cerebral disorders by inhibition of
IL-8 binding to receptor
United States Patent: 6,497,878
Issued: December 24, 2002
Inventors: Yamashita; Junkoh (Kanazawa, JP); Ikeda; Kiyonobu
(Kanazawa, JP); Matsumoto; Tetsuya (Kanazawa, JP); Matsushima; Kouji (Matsudo,
Assignee: Chugai Seiyaku Kabushiki Kaisha (Tokyo, JP)
Appl. No.: 171629
Filed: October 19, 1998
PCT Filed: October 30, 1997
PCT NO: PCT/JP97/01406
371 Date: April 23, 1997
Methods and compositions for treating or preventing cerebral stroke,
cerebral infarction, cerebral edema, reperfusion injury and increased
cerebral vascular permeability which employ an agent that prevents the
binding of IL-8 to its receptor are disclosed.
MODE FOR CARRYING OUT THE INVENTION
1. IL-8 Binding-inhibition Agents
An IL-8 binding-inhibition agent for use in the present invention may be
of any origin, any kind, and any form, as long as it has a therapeutic or
preventive effect on cerebral stroke, cerebral edema, reperfusion injury
of cerebral ischemia, or increased cerebral vascular permeability.
An IL-8 binding-inhibition agent for use in the present invention is a
substance that inhibits the binding of IL-8 to IL-8 receptor. Specifically
it is a substance that blocks signal transmission by IL-8 by binding to
IL-8, which in turn inhibits the biological activity of IL-8.
Human IL-8 undergoes different processing at the N-terminal end. However,
as a target of an IL-8 binding-inhibition agent for use in the present
invention, the number of amino acid residues of IL-8 is not limited as
long as it retains the biological activity of IL-8.
On the other hand, human IL-8 receptors occur as those referred to as IL-8
receptor A (.alpha. or 2) and those referred to as IL-8 receptor B (.beta.
or 1). However, as the receptor the binding of IL-8 to which is inhibited
by an IL-8 binding-inhibition agent for use in the present invention, its
type does is not limited as long as it induces the biological activity of
As an IL-8 binding-inhibition agent for use in the present invention,
anti-IL-8 antibody is most preferred and it only requires the confirmation
of its preventive or therapeutic effects according to the following
2. Anti-IL-8 Antibody
Anti-IL-8 antibodies for use in the present invention may be of any
origin, any kind (monoclonal or polyclonal), and any form, as long as it
has a preventive or therapeutic effect against cerebral stroke, cerebral
edema, reperfusion injury of cerebral ischemia, or increased cerebral
Anti-IL-8 antibodies for use in the present invention can be obtained as
polyclonal or monoclonal antibodies using known methods. As the anti-IL-8
antibodies for use in the present invention, monoclonal antibodies of, in
particular, mammalian origin, are preferred. Monoclonal antibodies of
mammalian origin include those produced by hybridomas or hosts which have
been transformed with expression vectors containing genetically engineered
antibody genes. The antibody, via binding to IL-8, blocks the binding to
IL-8 receptor expressed on neutrophils etc. and thereby inhibits signal
transmission of IL-8, and is therefore an antibody which inhibits the
biological activity of IL-8.
Examples of such antibodies include WS-4 antibody (Ko, Y. et al., J.
Immunol. Methods (1992) 149, 227-235) and DM/C7 antibody (Mulligan, M. S.
et al., J. Immunol. (1993) 150, 5585-5595), Pep-1 antibody and Pep-3
antibody (International Patent Application WO 92/04372), or 6G4.2.5
antibody and A5.12.14 antibody (International Patent Application WO
95/23865; Boylan, A. M. et al., J. Clin. Invest. (1992) 89, 1257-1267).
Among them, WS-4 antibody is most preferred.
Incidentally, the hybridoma cell line which produces WS-4 antibody has
been internationally deposited under the provisions of the Budapest Treaty
as mouse hybridoma WS-4 on Apr. 17, 1996 with the National Institute of
Bioscience and Human-Technology, Agency of Industrial Science and
Technology, of 1-3, Higashi 1-chome, Tsukuba city, Ibaraki pref., Japan,
as FERM BP-5507.
IL-8 used as the sensitizing antigen for obtaining antibody can be
obtained using the respective IL-8 gene/amino acid sequence as disclosed
in Matsushima, K. et al., J. Exp. Med. (1988) 167, 1883-1893 for human
IL-8, in Yoshimura, T. and Johnson, D. G., J. Immunol. (1993) 151,
6225-6236 for guinea pig IL-8, in Goodman, R. B. et al., Biochemistry
(1992) 31, 10483-10490 for porcine IL-8, in Harada, A. et al., Int.
Immunol. (1993) 5, 681-690 for rabbit IL-8, in Ishikawa, J. et al., Gene
(1993) 131, 305-306 for canine IL-8, Seow, H. F. et al., Immunol. Cell
Biol. (1994) 72, 398-405 for sheep IL-8, and Villinger, F. et al, J.
Immunol. (1995) 155, 3946-3954 for monkey IL-8.
It is known that human IL-8 is produced in a variety of cells and
undergoes different processing at the N-terminal end (Leonard, E. J. et
al., Am. J. Respir. Cell. Mol. Biol. (1990) 2, 479-486). Though an IL-8
that has 79, 77, 72, 71, 70 or 69 amino acid residues has been known so
far, the number of amino acid residues is not limited in any way so long
as the IL-8 can be used as the antigen for obtaining anti-IL-8 antibody
which is used in the present invention.
The gene sequence of IL-8 is inserted into a known expression vector to
transform an appropriate host cell. From the host cell or the culture
supernatant thereof, the desired IL-8 protein is purified using a known
method, and the purified IL-8 protein may be used as the sensitization
3. Antibody-producing Hybridoma
Hybridomas producing monoclonal antibodies can be basically constructed
using a known procedure as described bellow. Thus, IL-8 is 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 screened by the
conventional screening method to screen monoclonal antibody-producing
Preferably mammals to be immunized with the sensitization antigen are
selected in consideration of their compatibility with the parent cells for
use in cell fusion generally and they generally include, but are not
limited to, rodents such as mice, rats, hamsters, and the like.
Immunization of animals with a sensitization antigen is carried out using
known methods. A general method, for example, includes intraperitoneal or
subcutaneous administration of a sensitization antigen to the mammal.
Specifically, a sensitization 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 for several times every 4 to 21 days.
Additionally a suitable carrier may be used at the time of immunization of
the sensitization antigen.
After the immunization and confirmation of the increase in the desired
antibody levels in the serum by a conventional method, 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.
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 P3 (P3x63Ag8.653) (Kearney, J. F. et al.,
J. Immunol. (1979) 123, 1548-1550), P3x63Ag8U.1 (Yelton, D. E. et al.,
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), F0 (de St. Groth, S. F. and Scheidegger,
D., 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) 277,
131-133) and the like.
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. (Galfre, G. and Milstein, C., Methods Enzymol.
(1981) 73, 3-46) and the like.
More specifically, the above cell fusion is carried out in the
conventional nutrient medium 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 assistant agent such as dimethyl sulfoxide etc. may be added as
desired to enhance the efficiency of the fusion.
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
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.
In cell fusion, predetermined amounts of the above immune cells and the
myeloma cells are mixed wellin the above culture medium, to which a PEG
solution previously heated to about 37oC., for example the 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 the sequential addition of a
suitable culture medium and centrifugation to remove the supernatant, cell
fusion agents etc. which are undesirable for the growth of the hybridoma
can be removed.
The hybridoma is selected by culturing in a conventional selection medium,
for example, HAT culture medium (a culture medium containing hypoxanthine,
aminopterin, and thymidine). Culturing in the 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 monoclonally
In addition to obtaining the above hybridoma by immunizing non-human
animals with an antigen, it is also possible to sensitize human
lymphocytes in vitro with IL-8, and the resulting sensitized lymphocytes
are fused with a myeloma cell, for example U266, having the ability to
divide permanently to obtain the desired human antibody having the
activity of binding to IL-8 (Japanese Examined Patent Publication (Kokoku)
No. 1-59878). Furthermore, a transgenic animal having a repertoire of
human antibody genes is immunized with the antigen IL-8 to obtain
anti-IL-8 antibody-producing cells, which are immortalized and then used
to obtain human antibody to IL-8 (see International Patent Application WO
92/03918, WO 93/12227, WO 94/02602, WO 94/25585, WO 96/33735 and WO
The monoclonal antibody-producing hybridomas thus constructed can be
subcultured in the conventional culture medium, or can be stored for a
prolonged period of time in liquid nitrogen.
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 transplanted 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.
4. Recombinant Antibody
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, Borrebaeck, C. A. K. and Larrick, J. W., THERAPEUTIC MONOCLONAL
ANTIBODIES, published in the United Kingdom by MACMILLAN PUBLISHERS LTD.
Specifically, mRNA encoding the variable region (V-region) of anti-IL-8
antibody is isolated from the hybridoma producing anti-IL-8 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 (Chomczynski,
P. and Sacchi, N., Anal. Biochem. (1987) 162, 156-159), and then mRNA is
purified from the total RNA using a mRNA Purification kit (Pharmacia) and
the like. Alternatively, mRNA can be directly prepared using a QuickPrep
mRNA Purification Kit (Pharmacia).
cDNA of the V region of antibody may be synthesized from the obtained mRNA
using a reverse transcriptase. cDNA may be synthesized using the AMV
Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Kogyo),
and the like. Alternatively, for the synthesis and amplification of cDNA,
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) using the 5'-Ampli FINDER RACE Kit (Clontech) and 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., which is selected to
prepare the desired recombinant vector. The base sequence of the desired
recombinant DNA may be confirmed by a known method such as the dideoxy
nucleotide chain termination method.
Once the DNA encoding the V regions of the desired anti-IL-8 antibody have
been obtained, it may be ligated to DNA encoding the constant regions (C
regions) 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.
In order to produce anti-IL-8 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 is then expressed
The antibody gene may be expressed by integrating separately DNA encoding
a heavy chain (H chain) or a light chain (L chain) of the antibody into an
expression vector and co-transforming host cell, or by integrating DNA
encoding an H chain and an L chain into a single expression vector and
transforming host cell (International Patent Application WO 94/11523).
5. Altered Antibody
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.
Chimeric antibody can be obtained by ligating the thus obtained DNA
encoding the V region of antibody other than human 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.
E. coli having the plasmid containing the L chain or the H chain of
chimeric WS-4 antibody has been internationally deposited under the
provisions of the Budapest Treaty as Escherichia coli DHS
(HEF-chWS4L-g.kappa.) and Escherichia coli JM109 (HEF-chWS4H-g.gamma.1) on
Jul. 12, 1944 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-4739 and FERM BP-4740,
Humanized antibody which is also called reshaped human antibody has been
made by transplanting 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 96/02576).
Specifically, a DNA sequence which was designed to ligate the CDRs of
mouse antibody with the framework region (FRs) of human antibody is
synthesized from several divided oligonucleotides having sections
overlapping with one another at the ends thereof, and the oligonucleotides
are then synthesized into one integrated DNA by PCR method. The DNA thus
obtained is ligated to a DNA encoding a 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 96/02576).
For the FRs of a human antibody being ligated with CDRs, the CDRs that
have a favorable antigen-binding site are selected. When desired, amino
acids in FRs of antibody V region may be substituted so that CDRs of
humanized antibody may form an appropriate antigen biding site (Sato, K.
et al., Cancer Res. (1993) 53, 851-856).
For chimeric antibody and humanized antibody, a C region of human antibody
may be used depending on the purpose. For example, C.gamma.1, C.gamma.2,
C.gamma.3, and C.gamma.4 can be used. The C region of human antibody may
also be modified in order to improve the stability of antibody and of the
production thereof. For example, when the subclass IgG4 of antibody is
chosen, the amino acid sequence CPSCP of part of the hinge region of IgG4
can be converted to the amino acid sequence CPPCP of the hinge region of
IgG1 to resolve the structural instability of IgG4 (Angal, S. et al., Mol.
Immunol. (1993) 30, 105-108).
Chimeric antibody consists of V regions of antibody derived from a mammal
other than the human and C regions derived from human antibody, whereas
humanized antibody consists of the CDRs of antibody derived from a mammal
other than the human and the FRs and the C region of antibody derived from
human antibody. Accordingly, since the amino acid sequences derived from a
mammal other than the human are reduced to a minimum in the above
antibodies, antigenicity thereof in the human body is reduced so that they
are useful as an active ingredient of preventive or therapeutic agents of
the present invention.
A preferred embodiment of humanized antibody for use in the present
invention includes humanized WS-4 antibody (see International Patent
Application WO 96/02576). In the humanized WS-4 antibody, CDRs of the WS-4
antibody derived from a mouse have been ligated to FRs of the human
antibody REI for the L chain, and to the FRl-3 of the human antibody VDH26
and the FR4 of the human antibody 4B4 for:the H chain, and part of the
amino acid residues of the FR has been substituted to obtain
E. coli having plasmid coding for an L chain or an H chain of humanized
WS-4 antibody has been deposited under the provisions of the Budapest
Treaty as Escherichia coli DH5 (HEF-RVLa-qK) and Escherichia coli JM109
(HEF-RVHg-g.gamma.1) on Jul. 12, 1994 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-4738 and FERM BP-4741, respectively.
6. Modified Antibody
Antibodies for use in the present invention may be fragments of antibody
or modified versions thereof as long as they bind to IL-8 and thereby
inhibit the activity of IL-8. 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 Howitzer, A. H., Methods
Enzymol. (1989) 178, 476-496; Pluckthun, 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. and Walker, B. W., Trends Biotechnol. (1991) 9, 132-137).
scFv can be obtained by ligating a V region of an H chain and a V region
of an L chain of an antibody. In the scFv, a V region of an H chain and a
V region of an 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). A V region of an H chain and a V region of an 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.
DNA encoding scFv can be obtained using a DNA encoding an H chain or an H
chain V region of the above antibody and a DNA encoding an L chain or an 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,
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.
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.
As modified antibodies, anti-IL-8 antibody conjugated 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 obtained antibodies. These methods have already been
established in the art.
7. Expression and Production of Anti-IL-8 Antibody
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 an expression vector containing a conventionally
used useful promoter, an antibody gene to be expressed, and DNA in which
the poly A signal has been operably linked at 3' downstream thereof.
Examples of the promoter/enhancer include human cytomegalovirus immediate
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 so forth and promoters/enhancers derived from
mammalian cells such as human elongation factor 1 (HEF1.alpha.) and so
For example, expression may be readily accomplished by the method of
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.
(Nucleic Acids Res. (1990) 18, 5322) when HEF1.alpha. promoter/enhancer is
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. As the promoter, for example, there can be
mentioned lacz promoter and araB promoter. The method of Ward, E. S. et
al. (Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427) may be used
when lacz promoter is used, and the method of Better, M. et al. (Science
(1988) 240, 1041-1043) may be used when araB promoter is used.
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, International Patent
Application WO 96/30394).
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.
For the production of antibody for use in the present invention, any
production system can be used, and 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.
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 the
Nicotiana genus, more specifically cells derived from Nicotiana tabacum
which is subjected to callus culture. Known fungal cells include (1)
yeasts such as the Saccharomyces genus, more specifically Saccharomyces
cerevisiae, or (2) filamentous fungi such as the Aspergillus genus, more
specifically Aspergillus niger.
When the prokaryotic cells are used, there are the production systems
which employ bacterial cells. Known bacterial cells include Escherichia
coli, and Bacillus subtilis.
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 medium 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.
As further 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.
As mammals, goats, pigs, sheep, mice, and cattle can be used (Glaser, V.,
SPECTRUM Biotechnology Applications, 1993). Also as insects, silkworms can
When plants are used, tobacco, for example, can be used.
Antibody genes are introduced into these animals or plants, and antibodies
are produced in such animals or plants, and 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 the transgenic goat born to the goat who
received the embryo or offsprings thereof. In order to increase the amount
of milk produced 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).
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 (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 infected to tobacco
such as Nicotiana tabacum to obtain the desired antibody from the leaves
of the tobacco (Ma, J. K. et al., Eur. J. Immunol. (1994) 24, 131-138).
When antibody is produced in in vitro or in vivo production systems, as
mentioned above, DNA encoding the H chain or 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 (International Patent Application WO 94/11523).
8. Separation and Purification of Antibody
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 such affinity chromatography, there can be mentioned
Protein A column and Protein G column. Examples of the column employing
Protein A column are Hyper D, POROS, Sepharose F. F. (Pharmacia) and the
Alternatively, methods for separation and purification conventionally used
for proteins can be used without any limitation. Separation and
purification of antibody may be accomplished by combining, as appropriate,
chromatography columns other than the above-mentioned affinity
chromatography, filters, ultraconcentration, salting-out, dialysis and the
like (Antibodies: A Laboratory Manual, Ed Harlow and David Lane, Cold
Spring Harbor Laboratory, 1988). Chromatography other than affinity
chromatography includes, for example, ion exchange chromatography,
hydrophobic chromatography, gel-filtration and the like (Strategies for
Protein Purification and Characterization: A Laboratory Course Manual, Ed
Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996).
9. Measurement of Antibody Concentration
The concentration of antibody obtained in the above 8 can be determined by
measurement of absorbance or by the enzyme-linked immunosorbent assay
(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, though different with different species and
subclasses, 1.4 OD at 1 mg/ml in the case of human antibody. When the
ELISA method is used, measurement is conducted as follows. Thus, 100 .mu.l
of goat anti-human IgG antibody diluted to 1 .mu.g/ml in 0.1 M bicarbonate
buffer, pH 9.6, is added to a 96-well plate (Nunc), and is incubated
overnight at 4oC. to immobilize the antibody. After blocking, 100
.mu.l each of appropriately diluted antibody of the present invention or
samples containing the antibody, or 100 l of human IgG of a known
concentration as the concentration 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 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 (Bio-Rad) to calculate the
concentration of the desired antibody based on the absorbance of the
concentration standard IgG.
10. Confirmation of Activity
Activity of an IL-8 binding-inhibition agent for use in the present
invention can be confirmed by the method described below or a commonly
For example, a known method can be used for the measurement of the
antigen-binding activity (Antibodies: A Laboratory Manual. Ed Harlow and
David Lane, Cold Spring Harbor Laboratory, 1988) and the ligand receptor
binding-inhibition activity (Harada, A. et al., Int. Immunol. (1993) 5,
681-690) of anti-IL-8 antibody for use in the present invention.
As methods for determining the antigen-binding activity of anti-IL-8
antibody for use in the present invention, there can be used ELISA, EIA
(enzyme immunoassay), RIA (radioimmunoassay), or the fluorescent antibody
method. When ELISA is employed, for example, IL-8 is added to a 96-well
plate onto which polyclonal antibody against IL-8 has been immobilized,
and then samples containing the desired anti-IL-8 antibody, for example a
culture supernatant of anti-IL-8 antibody-producing cells or purified
antibody is added thereto. Secondary antibody that recognizes the desired
anti-IL-8 antibody labeled with an enzyme such as alkaline phosphatase is
added, and the plate is incubated, washed, and then the enzyme substrate
such as p-nitrophenyl phosphate is added, and the absorbance is measured
to evaluate the antigen-binding activity.
As methods for measuring the inhibition activity of ligand receptor
binding of the anti-IL-8 antibody for use in the present invention, the
conventional Cell ELISA or the ligand receptor binding assay can be used.
In the case of Cell ELISA, for example, blood cells or cancer cells
expressing IL-8 receptors such as neutrophils are cultured in a 96-well
plate to allow the cells to adhere there onto, which is then immobilized
with paraformaldehyde etc. Alternatively, the membrane fractions of cells
expressing IL-8 receptors are prepared and 96-well plates on which the
fractions have been immobilized are prepared. To this are added a sample
containing the desired anti-IL-8 antibody, for example a culture
supernatant of anti-IL-8 antibody-producing cells or purified antibody,
and IL-8 which is labeled with a radioisotope such as 125 I, and then
the plate is incubated, washed, and radioactivity is measured to determine
the amount of IL-8 bound to the IL-8 receptor and thereby to evaluate the
inhibition activity of ligand receptor binding of anti-IL-8 antibody.
In the inhibition assay of IL-8 binding to IL-8 receptors on the cells,
blood cells or cancer cells expressing IL-8 receptors such as neutrophils
are separated by means of centrifugation etc. to prepare a cell
suspension. A solution of IL-8 labeled with a radioisotope such as
125 I, or a mixture of unlabeled IL-8 and labeled IL-8, and a
solution comprising anti-IL-8 antibody whose concentration has been
adjusted are added to the cell suspension. After incubating for a given
period of time, the cells are separated, and the radioactivity of the
labeled IL-8 bound onto the cell is measured.
As methods for measuring the neutrophil chemotaxis inhibiting ability of
anti-IL-8 antibody for use in the present invention, a known method using
chemotaxis chambers such as the one described by Grob, P. M. et al. (J.
Biol. Chem. (1990) 265, 8311-8316) can be, used.
Specifically, anti-IL-8 antibody is diluted with a culture medium such as
RPMI 1640, DMEM, MEM, or IMDM, and then a concentration-adjusted IL-8 is
added thereto, which is dispensed into the bottom layer of the chamber
partitioned up,and down by filters. Subsequently, a prepared cell
suspension, for example a neutrophil suspension, is added to the upper
layer of the chamber and then allowed to stand for a given period of time.
Since migrating cells will adhere to the bottom surface of the filter
attached to the chamber, the number of cells adhered thereto can be
measured by a method using a stain or fluorescent antibody etc. Also,
visual examination under the microscope or automatic measurement using a
counting device can also be employed.
11. Method of Administration and Pharmaceutical Preparation
Therapeutic agents that contain as an active ingredient an IL-8
binding-inhibition agent such as anti-IL-8 antibody of the present
invention may be administered, either orally or parenterally, or either
systemically or locally.
For example a proteinaceous IL-8 binding-inhibition agent such as
anti-IL-8 antibody of the present invention may be administered by
intravenous injection such as drip infusion, intramuscular injection,
intraperitoneal injection, subcutaneous injection, intraspinal injection,
and the like either systemically or locally. The method of administration
may be chosen, as appropriate, depending on the age and the conditions of
An IL-8 binding-inhibition agent such as anti-IL-8 antibody may be
administered to a patient suffering from a disease in an amount sufficient
to treat the disease and the symptoms of complications thereof or to
prevent them at least partially. For example, the effective dosage is
chosen from the range of 0.01 mg to 1000 mg per kg of body weight per
administration. Alternatively, the dosage in the range of 5 to 2000
mg/body per patient may be chosen. However, the dosage of a preventive or
therapeutic agent containing an IL-8 binding-inhibition agent such as
anti-IL-8 antibody of the present invention is not limited to these
The timing of administration may be, after the onset of cerebral stroke,
cerebral edema, or after reperfusion injury of cerebral ischemia.
Alternatively, the timing may be at the time of reperfusion after temporal
blocking of cerebral blood flow or when reperfusion is expected in a
reperfusion therapy such as the thrombolytic therapy, or it may be
administered when increased vascular permeability is estimated.
Preventive or therapeutic agents that contain as the active ingredient an
IL-8 binding-inhibition agent such as anti-IL-8 antibody of the present
invention may be formulated into a pharmaceutical preparation (Remington's
Pharmaceutical Science, latest edition, Mark Publishing Company, Easton,
U.S.A.), and may further contain pharmaceutically acceptable carriers or
Examples of such carriers or pharmaceutical 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, agar, polyethylene glycol, diglycerin,
glycerin, prdpylene glycol, Vaseline, paraffin, stearyl alcohol, stearic
acid, human serum albumin (HSA), mannitol, sorbitol, lactose,
pharmaceutically acceptable surfactants and the like.
Actual additives are chosen from, but not limited to, the above or
combinations thereof depending on the dosage form of a preventive or
therapeutic agent of the present invention.
When anti-IL-8 antibody is used as a parenteral injection, purified
anti-IL-8 antibody may be dissolved in a solvent, for example,
physiological saline, a buffer, a glucose solution, etc., to which are
added an anti-adsorption agent such as Tween 80, Tween 20, gelatin, human
serum albumin etc. Alternatively, a lyophilized agent which is
reconstituted prior to use may be used, and as an excipient for
lyophilization, sugar alcohols and sugars such as mannitol, glucose etc.
can be used.
12. Confirmation of preventive or therapeutic effects
For confirmation of preventive or therapeutic effects on cerebral
infarction, cerebral edema, or reperfusion injury of cerebral ischemia,
the focal cerebral ischemia model or the global cerebral ischemia model is
used (Keiji Sano, ed., "Nosocchu Jikkenn Handbook (Handbook of Cerebral
Apoplexy Experiments)," IPC, 43-51, 1990). Effects on reperfusion injury
may be investigated by a transient occlusion in which blood flow is
resumed after blocking cerebral blood flow for a given period of time in
respective models. When no perfusion is required on the order hand,
permanent occlusion is performed in which blockage of cerebral blood flow
Though animals used generally include monkeys, dogs, cats, rabbits, rats,
mice, Mongolian gerbils, and the like, any species of animal can be used
as long as the expression of IL-8 has been confirmed in the animal for the
purpose of confirming the preventive or therapeutic effects of an IL-8
binding-inhibition agent such as anti-IL-8 antibody of the invention. In
this regard, there can be mentioned rabbits, guinea pigs, pigs, dogs,
sheep, monkeys, and the like.
Methods for producing focal cerebral ischemia models are broadly divided
into those in which incoming cerebral vessels are compression-occluded
from the outside and those in which emboli are injected. Specifically,
there. can be mentioned: a method in which, for one or several strings of
intracranial arteries or carotid arteries that send the blood stream to
the brain such as the middle cerebral artery, the anterior cerebral
artery, the posterior communicating cerebral artery, the internal carotid
artery, the external carotid artery, and the tibial artery, the target
artery is surgically cauterized within the framework that it does not
cause global ischemia; a method in which the target artery is occluded
with an artery clip; a method in which the target artery is ligated; or a
method in which a photosensitive dye is intravenously given and a laser
light is irradiated to the target artery to produce thrombi, and the like.
Alternatively, blood coagulation factors, clots, or air is given to
The method of creating the global ischemia model may comprise blocking
simultaneously arteries such as the internal carotid artery, the external
carotid artery, and the tibial artery at sites relatively proximal to the
heart. As the method of blocking the artery, there can be mentioned a
method in which the target artery is cauterized, a method in which the
target artery is clipped with an artery clip, a plug method in which
emboli are placed in the target artery, and the like.
In any method selected from the above, an IL-8 binding-inhibition agent
such as anti-IL-8 antibody is administered at any timing such as before or
immediately after ischemia, after ischemia for a given period of time,
immediately before or immediately after reperfusion, or after reperfusion
for a given period of time. After ischemial for a given period of time, or
reperfusion for a given period of time, cerebral circulation, cerebral
metabolism, and neurological functions are measured, and after the animal
was sacrificed neurological pathology, infarct size, edema, increased
vascular permeability and the like are evaluated.
As the method for determining cerebral circulation, there are mentioned,
for example, the hydrogen clearance method, the thermocouple method, the
laser Doppler method, and the like (Keiji Sano, ed., "Nosocchu Jikkenn
Handbook (Handbook of Cerebral Apoplexy Experiments)," IPC, 193-240,
As the method for quantifying the infarct size, for example, the following
method can be mentioned. After removing the brain, it is cut into slices
with a fixed thickness. The sliced brain tissue is stained with
2,3,5-triphenyltetrazolium chloride (TTC) or the Nissl's stain to
distinctively quantify the injured areas, or thin sections are prepared
which are then histopathologically distinguished and quantified using the
hematoxylin-eosin stain (Keiji Sano, ed., "Nosocchu Jikkenn Handbook
(Handbook of Cerebral Apoplexy Experiments)," IPC, 587-623, 1990).
As the method for quantifying edema, for example, the following method can
be mentioned: the method in which after the brain is removed, the specific
gravity of a fixed amount of the tissue is determined using the density
gradient; the method in which water content of the brain tissue is
determined using the weight ratio of the wet weight and the dry weight; or
the method in which it is evaluated by the nuclear magnetic resonance
method (Keiji Sano, ed., "Nosocchu Jikkenn Handbook (Handbook of Cerebral
Apoplexy Experiments)," IPC, 630-635, 1990).
As the method for quantifying increased vascular permeability, for
example, the following method can be mentioned. Thirty minutes before
sacrificing the animal used for the experiment, a given concentration of
Evans blue solution is intravenously administered. After removing the
brain, it is cut into slices with a fixed thickness. The area stained blue
by Evans blue in the sliced brain tissue is quantified. (Keiji Sano, ed.,
"Nosocchu Jikkenn Handbook (Handbook of Cerebral Apoplexy Experiments),"
IPC, 693-705, 1990). Additionally, it is also possible to determine
cerebral edema with increased vascular permeability as an index, since
increased vascular permeability also induces cerebral edema.
As the method for creating the model of subarachnoid hemorrhage, there are
a method of injecting blood or a substance that is capable of inducing
angiospasm into the subarachnoid cavity of an animal for which the
expression of IL-8 has been confirmed or placing them after opening the
head, or a method of allowing the animal to bleed by mechanically sticking
a needle into or cutting cerebral vessels, etc. (Keiji Sano, ed., "Nosocchu
Jikkenn Handbook (Handbook of Cerebral Apoplexy Experiments)," IPC,
As the model of cerebral hemorrhage, there are the intracranial blood
injection model, the intracranial microbaloon swelling model, and the like
(Keiji Sano, ed., "Nosocchu Jikkenn Handbook (Handbook of Cerebral
Apoplexy Experiments)," IPC, 134-138, 1990).
Claim 1 of 8 Claims
What is claimed is:
1. A method to treat cerebral infarction, which method comprises
systemically administering to a subject in need of such treatment an
amount of an anti-IL-8 antibody effective to treat the cerebral
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