JPH1045622A - Agent for treatment of chronic rheumatoid arthritis containing anti-il-8 antibody as active component - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject treating agent causing little undesirable side actions such as lesion of digestive tracts caused by the administration over a long period by using an anti-interleukin-8 antibody as an active component. SOLUTION: This treating agent contains anti-interleukin-8 (abbreviated as IL-8) antibody as an active component. The antibody is preferably a monoclonal antibody, especially human-type WS-4, e.g. an antibody produced by a WS-4 antibody-producing hybridoma cell strain (FERM BP-5507), an antibody produced by a host cell transformed by an expression vector containing an antibody gene by a genetic engineering technique, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an anti-interleukin
The present invention relates to a therapeutic agent for rheumatoid arthritis, comprising an antibody-8 (IL-8) antibody as an active ingredient.

[0002]

2. Description of the Related Art IL-8 is a protein belonging to the CXC chemokine subfamily, and was formerly a monocyte-derived neutrophil chemotactic facto.
r), Neutrophil activating protein-1 (neutrophil attractant / a
ctivation protein-1), neutrophil activator (neutrophi
l activating factor). IL-8 is
It is a factor that activates neutrophils and causes neutrophils to acquire the migratory ability. Inflammatory cytokines such as IL-1β and TNFα (Koch,
AE et al., J. Investig. Med. (1995) 43, 28-38;
Larsen, CG et al., Immunology (1989) 68, 31-36)
And mitogens such as PMA and LPS (Yoshimura, T. et al.,
Proc. Natl. Acad. Sci. USA (1987) 84,9233-923
7) and heavy metals such as cadmium (Horiguchi, H. e.
t al., Lymphokine Cytokine Res. (1993) 12, 421-428
) Produced by various cells.

Mononuclear cells isolated from synovial fluid of rheumatoid arthritis patients are compared with IL-8, GRO, MCAF, MIP-1α in peripheral blood mononuclear cells of healthy persons and rheumatoid arthritis patients. ,
It has been reported that chemokines such as MIP-1β are up-regulated (Hosaka, S. et al., Clin. Exp. Immunol.
(1994) 97, 451-457). It is postulated that overexpression of these various chemokines promotes the migration of inflammatory cells into joints, but it is not clear which chemokines play a central role in pathogenesis.

In an in-vitro (in a test tube) experiment,
Since IL-8 activates neutrophils and induces cartilage destruction, some reports suggest that IL-8 may be involved in the pathology of rheumatoid arthritis (Elford, PR and Coope
r, PH, Arthritis Rheum. (1991) 34, 325-332),
It has been reported that there is no correlation between the level of chemotactic activity in synovial fluid from patients with rheumatoid arthritis and IL-8 concentration and that it is due to other factors with neutrophil chemotactic activity (Brennan, F .
M. et al., Eur. J. Immunol. (1990) 20, 2141-214
Four).

In vivo (in vivo), anti-IL-8 antibodies are known to be effective against experimental acute arthritis in animals, and IL-8 antagonists and IL-8 production inhibitors Has been suggested to be useful in the treatment of inflammatory diseases associated with leukocyte infiltration, including acute and chronic arthritis (Akahoshi, T. et al., Lymphokine Cytokine Res.
94) 13, 113-116). However, it was not known at all that anti-IL-8 antibody had a therapeutic effect on rheumatoid arthritis.

[0006]

Until now, anti-rheumatic drugs, non-steroidal anti-inflammatory drugs and steroids have been used for the treatment of rheumatoid arthritis. It has been shown to cause a number of undesirable side effects, such as rash, kidney damage and osteoporosis. Therefore, development of a therapeutic agent for rheumatoid arthritis having few side effects has been awaited. An object of the present invention is to provide a therapeutic agent for rheumatoid arthritis that does not have the above-mentioned disadvantages.

[0007]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that an anti-IL-8 antibody is useful as a therapeutic agent for rheumatoid arthritis, and completed the present invention. That is, the present invention provides a therapeutic agent for rheumatoid arthritis containing an anti-IL-8 antibody as an active ingredient.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION

1. Anti-IL-8 Antibody As long as it has a therapeutic effect on rheumatoid arthritis, the origin, type (monoclonal, polyclonal) and shape of the anti-IL-8 antibody used in the present invention are not limited.

[0009] The anti-IL-8 antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using known means. As the anti-IL-8 antibody used in the present invention, a monoclonal antibody derived from a mammal is particularly preferable. As a mammalian-derived monoclonal antibody,
There are those produced by hybridomas and those produced by hosts transformed with expression vectors containing antibody genes by genetic engineering techniques. By binding to IL-8, this antibody inhibits the binding to the IL-8 receptor expressed on neutrophils and blocks IL-8 signal transduction,
An antibody that inhibits the biological activity of IL-8.

[0010] Such antibodies include the WS-4 antibody (Ko,
Y. et al., J. Immunol.Methods (1992) 149, 227-23
5) and DM / C7 antibodies (Mulligan, MS et al., J. Immuno
l. (1993) 150, 5585-5595), Pep-1 antibody and Pep-3
Antibody (International Patent Application Publication No. WO92 / 04372) or 6G4.
2.5 Antibody and A5.12.14 Antibody (International Patent Application Publication No. WO
95/23865; Boylan, AM et al., J. Clin. Invest.
(1992) 89, 1257-1267). Among these, a particularly preferred antibody is the WS-4 antibody. The WS-4 antibody-producing hybridoma cell line was a Mouse hybr
As idoma WS-4, the Institute of Biotechnology and Industrial Technology (I 1-3, Higashi 1-3-1 Tsukuba, Ibaraki Prefecture)
On 17th, it was deposited internationally under the Budapest Treaty as FERM BP-5507.

2. Hybridomas Producing Antibody A hybridoma producing a monoclonal antibody can be basically produced using a known technique as follows. That is, using IL-8 as a sensitizing antigen, immunizing it according to a normal immunization method, and fusing the obtained immune cells with a known parent cell by a normal cell fusion method,
It can be prepared by screening monoclonal antibody-producing cells by a usual screening method.

Specifically, a monoclonal antibody may be prepared as follows. For example, IL-8 used as a sensitizing antigen for obtaining antibodies is
tsushima, K. et al., J. Exp. Med. (1988) 167, 1883
-1893, Yoshimura, T. a.
nd Johnson, DG, J. Immunol. (1993) 151, 6225-
6236, for porcine IL-8, Goodman, RB et al.,
Biochemistry (1992) 31, 10483-10490, rabbit IL-8
Harada, A. et al, Int. Immunol. (1993) 5,
681-690, for dog IL-8, Ishikawa, J. et al.,
Gene (1993) 131, 305-306 and sheep IL-8
Seow, HF et al., Immunol. Cell Biol. (1994) 72,
398-405, for monkey IL-8, Villinger, F. et a
l., J. Immunol. (1995) 155, 3946-3954.

[0013] Human IL-8 has been reported to be produced in various cells and undergo different processing at the N-terminus (Leonard, EJ et al., Am. J. Respir. Cel).
l. Mol. Biol. (1990) 2, 479-486). So far, 7
I having 9, 77, 72, 71, 70 and 69 amino acid residues
Although L-8 is known, its amino acid residue number is not limited as long as it can be used as an antigen for obtaining the anti-IL-8 antibody used in the present invention. After inserting the gene sequence of IL-8 into a known expression vector system and transforming an appropriate host cell,
The target IL-8 protein may be purified from the host cells or the culture supernatant by a known method, and the purified IL-8 protein may be used as a sensitizing antigen. The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion, and generally is selected from rodents. Animals, for example, mice, rats, hamsters and the like are used.

Immunization of an animal with a sensitizing antigen is carried out according to a known method. For example, as a general method,
This is performed by injecting the sensitizing antigen intraperitoneally or subcutaneously into a mammal. Specifically, the sensitizing antigen was changed to PBS (Phos
phate-Buffered Saline) or an appropriate amount of a suspension diluted with a physiological saline or the like, mixed with an appropriate adjuvant if desired, for example, Freund's complete adjuvant, and emulsified. It is preferred to administer.
In addition, a suitable carrier can be used at the time of immunization of the sensitizing antigen. After immunization as described above, it is confirmed by a conventional method that the level of a desired antibody in serum is increased, and then immunocytes are removed from the mammal and subjected to cell fusion. Cells.

As mammalian myeloma cells as the other parent cells to be fused with the above-mentioned immune cells, various known cell lines, for example, P3 (P3x63Ag8.653) (Kearney,
JF et al., J. Immnol. (1979) 123, 1548-1550
), P3x63Ag8U.1 (Yelton, DE et al., Current To
pics in Microbiology and Immunology (1978) 81, 1-
7), NS-1 (Kohler, G. and Milstein, C., Eur. J. Im
munol. (1976) 6, 511-519), MPC-11 (Margulies, D.
H. et al., Cell (1976) 8, 405-415), SP2 / 0 (Shul
man, M. et al., Nature (1978) 276, 269-270), FO
(De St. Groth, SF and Scheidegger, D., J. Immun
ol. Methods (1980) 35, 1-21), S194 (Trowbridge,
IS, J. Exp. Med. (1978) 148, 313-323), R210
(Galfre, G. et al., Nature (1979) 277, 131-133)
Etc. are preferably used.

The cell fusion of the immune cells and myeloma cells is basically performed by a known method, for example, the method of Milstein et al. (Galfre, G. and Milstein, C., Methods Enzymol.
(1981) 73, 3-46). More specifically, the cell fusion is carried out, for example, in a normal nutrient culture in the presence of a cell fusion promoter. As the fusion promoter, for example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like are used, and if necessary, an auxiliary agent such as dimethyl sulfoxide can be added to enhance the fusion efficiency.

The ratio of the use of the immune cells to the myeloma cells is preferably, for example, 1 to 10 times the number of the immune cells to the myeloma cells. As a culture solution used for the cell fusion, for example, RP suitable for growth of the myeloma cell line
MI1640 culture solution, MEM culture solution, and other normal culture solutions used for this type of cell culture can be used.
Serum replacement such as fetal calf serum (FCS) can also be used in combination.

In the cell fusion, a predetermined amount of the immune cells and myeloma cells are mixed well in the culture solution,
PEG solution heated to a moderate degree, for example, an average molecular weight of 1000-600
Usually, a fused cell (hybridoma) of interest is formed by adding a PEG solution of about 0 at a concentration of 30 to 60% (w / v) and mixing. Subsequently, by repeatedly adding an appropriate culture solution and centrifuging to remove the supernatant, a cell fusion agent or the like unfavorable for hybridoma growth can be removed.

The hybridoma is selected by culturing it in an ordinary selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). The culturing in the HAT culture solution is continued for a time sufficient for cells other than the target hybridoma (non-fused cells) to die, usually several days to several weeks. Then, a usual limiting dilution method is performed, and screening and single cloning of a hybridoma producing the desired antibody are performed.

In addition to obtaining the above-mentioned hybridoma by immunizing a non-human animal with an antigen, human lymphocytes can be in vitro
Sensitized to IL-8, and fused the sensitized lymphocytes with a myeloma cell having permanent dividing ability derived from a human, for example, U266.
It is also possible to obtain a desired human antibody having an activity of binding to E. coli (see Japanese Patent Publication No. 1-59878). Furthermore, a transgenic animal having a human antibody gene repertoire is immunized with IL-8 as an antigen to obtain anti-IL-8 antibody-producing cells, and a human antibody against IL-8 is obtained using the immortalized cells. (International Patent Application Publication No. WO 92/03918, WO
93/12227, WO94 / 02602, WO94 / 25585, WO96 / 33735
And WO 96/34096).

The hybridoma producing the monoclonal antibody thus produced can be subcultured in a normal culture solution, and can be stored for a long time in liquid nitrogen. In order to obtain a monoclonal antibody from the hybridoma, the hybridoma is cultured according to a conventional method, and a method of obtaining the culture supernatant is used, or the hybridoma is transplanted and grown in a mammal compatible with the hybridoma, and the ascites is collected. And the like. The former method is suitable for obtaining high-purity antibodies, while the latter method is suitable for mass production of antibodies.

3. Recombinant Antibody As a monoclonal antibody, an antibody gene is cloned from a hybridoma and inserted into an appropriate vector.
This can be introduced into a host, and a recombinant antibody produced using a gene recombination technique can be used in the present invention (for example, Borrebaeck, CAK and Larrick, JW, THERAPEU
TIC MONOCLONAL ANTIBODIES, Published in the United
Kingdom by MACMILLAN PUBLISHERS LTD, 1990).

Specifically, mRNA encoding the variable region (V region) of the anti-IL-8 antibody is isolated from the hybridoma producing the anti-IL-8 antibody. mRNA can be isolated by a known method, for example, guanidine ultracentrifugation (Chirgwin, JM et al.,
Biochemistry (1979) 18, 5294-5299), AGPC method (Chom
czynski, P. and Sacchi, N., Anal.Biochem. (1987)
162, 156-159), etc., and prepare mRNA.
from total RNA to mRNA using ation Kit (Pharmacia) etc.
Is purified. Also, QuickPrep mRNA Purification Kit
(Pharmacia) to directly prepare mRNA.

An antibody is obtained from the obtained mRNA using reverse transcriptase.
Synthesize V region cDNA. cDNA synthesis is performed using AMV Reverse
Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation) can be used. To synthesize and amplify cDNA, 5'-Ampli FINDER RACE Kit
(Clontech) and polymerase chain reaction (polymerase)
5'-RACE method (Frohma
n, MA et al., Proc. Natl. Acad. Sci. USA (19
88) 85, 8998-9002; Belyavsky, A. et al., Nucleic A
cids Res. (1989) 17, 2919-2932) can be used.

The desired DNA fragment is purified from the obtained PCR product and ligated to a vector DNA. Further, a recombinant vector is prepared from this, introduced into E. coli, etc., and colonies are selected to prepare a desired recombinant vector. The nucleotide sequence of the target DNA is confirmed by a known method, for example, a dideoxynucleotide chain termination method. Once the DNA encoding the V region of the desired anti-IL-8 antibody is obtained, it is ligated to the DNA encoding the desired antibody constant region (C region) and inserted into an expression vector. Alternatively, the DNA encoding the V region of the antibody is
It may be incorporated into an expression vector that already contains the DNA of the region.

To produce the anti-IL-8 antibody used in the present invention, the antibody gene is incorporated into an expression vector so that it is expressed under the control of an expression control region, for example, an enhancer or a promoter. Next, host cells are transformed with this expression vector to express the antibody. Expression of the antibody gene may be performed by separately transforming the DNA encoding the heavy chain (H chain) or light chain (L chain) of the antibody into an expression vector and co-transforming the host cell, or by co-transforming the H chain and L chain. The DNA encoding the strand may be incorporated into a single expression vector to transform host cells (see International Patent Application Publication No.
WO 94/11523).

4. Modified Antibodies In the present invention, a recombinant antibody artificially modified for the purpose of, for example, reducing the xenoantigenicity to humans,
For example, chimeric antibodies, humanized (Humanize)
d) Antibodies can be used. These modified antibodies can be produced using known methods. The chimeric antibody is obtained by ligating a DNA encoding an antibody V region other than a human antibody obtained as described above with a DNA encoding a human antibody C region,
It can be obtained by incorporating this into an expression vector, introducing it into a host and producing it (European Patent Application Publication No. EP 12502).
3, International Patent Application Publication No. WO 96/02576). Using this known method, chimeric antibodies useful in the present invention can be obtained.

Escherichia coli having a plasmid containing the L chain or the H chain of the chimeric WS-4 antibody was isolated from Escherichia coli
li DH5α (HEF-chWS4L-gκ) and Escherichia coli J
As M109 (HEF-chWS4H-gγ1), Research Institute of Biotechnology, Industrial Science and Technology (1-1-3 Higashi, Tsukuba, Ibaraki, Japan)
On July 12, 1994, FERM BP-4739 and FERM respectively
Deposited internationally under the Budapest Treaty as BP-4740. A humanized antibody is also called a reshaped human antibody, and is a complementarity determining region of a non-human mammal, for example, a mouse antibody.
CDR) is transplanted into the complementarity-determining region of a human antibody, and its general gene recombination technique is also known (European Patent Application Publication No. EP 125023, International Patent Application Publication No.
WO 96/02576).

Specifically, a DNA sequence designed to link the CDR of a mouse antibody and the framework region (FR) of a human antibody is replaced with several oligonucleotides having overlapping portions at the ends. Then, the DNA is synthesized and synthesized into a single DNA by PCR. The obtained DNA was converted to DN encoding human antibody C region.
A and then integrated into an expression vector, which is then introduced into a host and produced (European Patent Application Publication No. EP 239400, International Patent Application Publication No. WO 96 /
02576). FR of human antibody linked via CDR
Is selected so that the CDR forms a favorable antigen-binding site. If necessary, the amino acids of FR in the V region of the antibody may be substituted so that the CDRs of the humanized antibody form an appropriate antigen-binding site (Sato, K. et al., Cancer Res.
3) 53, 851-856).

For chimeric antibodies and humanized antibodies, a human antibody C region is used depending on the purpose.
Cγ2, Cγ3, Cγ4 can be used. Also, in order to improve the stability of the antibody or its production, a human antibody
The C region may be modified. For example, subclass antibody
When selecting IgG4, the amino acid sequence CPSCP of the hinge region of IgG4 is replaced with the amino acid sequence CPPCP of the hinge region of IgG1.
Can resolve the structural instability of IgG4 (Angal, S. et al., Mol. Immunol. (1993) 30, 105-
108).

A chimeric antibody is composed of the V region of a non-human mammal-derived antibody and a C region of a human antibody. The humanized antibody is composed of the CDRs of a non-human mammal-derived antibody and the FRs and C regions of a human antibody. Since the amino acid sequences derived from mammals other than humans are minimally reduced, the antigenicity in the human body is reduced, which is useful as an active ingredient of the therapeutic agent of the present invention. Preferred specific examples of the humanized antibody that can be used in the present invention include a humanized WS-4 antibody (see International Patent Application Publication No. WO 96/02576). Humanization
For the WS-4 antibody, the CDR of the mouse-derived WS-4 antibody is used, the FR of the human antibody REI for the L chain, and the human antibody VD for the H chain.
It is obtained by linking to FR1-3 of H26 and FR4 of human antibody 4B4, and partially substituting amino acid residues of FR so as to have antigen-binding activity.

Escherichia coli having a plasmid containing the L-chain or H-chain of the humanized WS-4 antibody was isolated from Escherichia
coli DH5α (HEF-RVLa-gκ) and Escherichia coli J
As M109 (HEF-RVHg-gγ1), the Institute of Biotechnology and Industrial Technology (I 1-3, Higashi 1-3-1, Tsukuba, Ibaraki Prefecture)
On July 12, 1994, FERM BP-4738 and FERM BP-4738 respectively
It has been deposited internationally under the Budapest Treaty as 4741.

5. Modified Antibody The antibody used in the present invention may be a fragment of an antibody or a modified product thereof, as long as it binds to IL-8 and inhibits the activity of IL-8. For example, antibody fragments include Fab, F (ab ') 2, Fv
Alternatively, a single chain Fv (scFv) in which Fvs of an H chain and an L chain are linked by an appropriate linker can be used. In particular,
The antibody is treated with an enzyme such as papain or pepsin to generate an antibody fragment, or a gene encoding these antibody fragment is constructed, introduced into an expression vector, and then expressed in an appropriate host cell (for example, , Co, MS e
t al., J. Immunol. (1994) 152, 2968-2976; Better,
M. and Horwitz, AH, Methods Enzymol. (1989) 17
8, 476-496; Pluckthun, A. and Skerra, A., Methods
Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods.
Enzymol. (1986) 121, 652-663; Rousseaux, J. et a
l., Methods Enzymol. (1986) 121, 663-669; Bird,
RE and Walker, BW, Trends Biotechnol. (1991)
9, 132-137).

The scFv can be obtained by linking the H chain V region and L chain V region of the antibody. In this scFv, the H chain
The V region and the L chain V region are connected via a linker, preferably a peptide linker (Huston, JS et al.,
Proc. Natl. Acad. Sci. USA (1988) 85, 5879-588
3). The H chain V region and L chain V region in the scFv may be derived from any of the antibodies described above. As the peptide linker connecting the V regions, for example, any single-chain peptide consisting of 12 to 19 amino acid residues is used.

The DNA encoding the scFv is obtained by using, as templates, DNA encoding the H chain or H chain V region and DNA encoding the L chain or L chain V region of the antibody. The DNA portion encoding the amino acid sequence is amplified by PCR using primer pairs defining both ends thereof, and then the DNA encoding the peptide linker portion and both ends thereof are ligated to H and L chains, respectively. By combining and amplifying primer pairs defined as described above. In addition, once the DNA encoding scFv
Is prepared, an expression vector containing them and a host transformed with the expression vector can be obtained according to a conventional method, and scFv can be obtained using the host according to a conventional method.

[0036] The fragments of these antibodies can be produced in the same manner as described above, by obtaining the gene, expressing the gene, and using a host. “Antibodies” in the claims of the present application also include fragments of these antibodies. As a modified antibody, an anti-IL-8 antibody bound to various molecules such as polyethylene glycol (PEG) can also be used. The “antibody” referred to in the claims of the present application also includes these modified antibodies. Such a modified antibody can be obtained by subjecting the obtained antibody to chemical modification. These methods are already established in this field.

6. Expression and Production of Recombinant Antibody or Modified Antibody The antibody gene constructed as described above can be expressed and obtained by a known method. For mammalian cells,
Expression can be carried out using an expression vector containing a useful promoter commonly used, an antibody gene to be expressed, and a DNA having a polyA signal operably linked to the 3 ′ downstream thereof. For example, as a promoter / enhancer, human cytomegalovirus immediate early promoter / e
nhancer).

Other promoters / enhancers that can be used for expression of the antibodies used in the present invention include retroviruses, polyomaviruses, adenoviruses, and the like.
Viral promoter / enhancer such as Simian virus 40 (SV40) and human elongation factor 1
A promoter / enhancer derived from a mammalian cell such as α (HEF1α) may be used. For example, when the SV40 promoter / enhancer is used, the method of Mulligan, RC et al. (Nature (1979) 277, 108-114) and HEF1α
Mizushim if using promoter / enhancer
a, S et al. (Nucleic Acids Res. (1990) 18, 5322)
Can be easily implemented.

In the case of Escherichia coli, expression can be performed by operably linking a useful promoter which is commonly used, a signal sequence for antibody secretion, and an antibody gene to be expressed.
For example, as the promoter, lacz promoter, araB
Promoters can be mentioned. When using the lacz promoter, the method of Ward, ES et al. (Nature (198
9) 341, 544-546; FASEB J. (1992) 6, 2422-2427)
In addition, when using the araB promoter, Better,
The method of M. et al. (Science (1988) 240, 1041-1043) may be used.

As a signal sequence for antibody secretion,
When produced in the periplasm of E. coli, the pelB signal sequence (Lei, SP et al., J. Bacteriol. (1987) 169,
4379-4383). After separating the antibody produced in the periplasm, the structure of the antibody is appropriately refolded and used (for example, International Patent Application Publication No. WO
96/30394). As the replication origin, those derived from SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like can be used. Furthermore, in order to amplify the gene copy number in a host cell system, an expression vector is used as a selection marker. , Aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase (dhfr) gene and the like.

For the production of the antibodies used in the present invention,
Any production system can be used, and production systems for producing antibodies include in vitro and in vivo production systems. in
Examples of the in vitro production system include a production system using eukaryotic cells and a production system using prokaryotic cells. When eukaryotic cells are used, there are production systems using animal cells, plant cells, and fungal cells. Examples of animal cells include (1) mammalian cells such as CHO, COS, myeloma, BHK (baby hamster k
idney), HeLa, Vero, (2) amphibian cells, such as Xenopus oocytes, or (3) insect cells,
For example, sf9, sf21, and Tn5 are known. Examples of plant cells include the genus Nicotiana, specifically, Nicotiana tabacum.
Originating cells are known and may be callus cultured.

As fungal cells, (1) yeast, for example,
Saccharomyces genus, specifically, Saccharomyces cerevisiae,
Or (2) a filamentous fungus, for example, Aspergillus (Aspe
rgillus), specifically, Aspergillus niger (As
pergillus niger) is known. When using prokaryotic cells, there is a production system using bacterial cells. As bacterial cells, Escherichia coli and Bacillus subtilis are known.

The desired antibody gene is introduced into these cells by transformation, and the transformed cells are transformed in vitro.
An antibody can be obtained by culturing in. Culture is performed according to a known method. For example, DMEM, MEM, RPMI1640, IMDM, and the like can be used as a culture solution for mammalian cells, and a serum replacement solution such as fetal calf serum (FCS) can 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 peritoneal cavity of animals. Further in vivo production systems include production systems using animals and plants. When using animals, there are production systems using mammals and insects.

As mammals, goats, pigs, sheep, mice and cattle can be used (Glaser, V.,
SPECTRUM Biotechnology Applications, 1993). Silkworms can be used as insects. When using a plant, for example, tobacco can be used.
An antibody gene is introduced into these animals or plants, and antibodies are produced in the animals or plants and collected. For example,
An antibody gene is prepared as a fusion gene by inserting the antibody gene in the middle of a gene encoding a protein uniquely produced in milk, such as goat β-casein. A DNA fragment containing the fusion gene into which the antibody gene has been inserted is injected into a goat embryo, and the embryo is introduced into a female goat.

A desired antibody is obtained from milk produced by a transgenic goat born from a goat that has received an embryo or a descendant thereof. Hormones may optionally be used in the transgenic goat to increase the amount of milk containing the desired antibody produced from the transgenic goat. (Eber
t, KM et al., Bio / Technology (1994) 12, 699-702
).

When using a silkworm, the baculovirus into which the antibody gene of interest is inserted is infected to the silkworm,
The desired antibody is obtained from the body fluid of the silkworm (Maeda, S. et.
al., Nature (1985) 315, 592-594). Furthermore, when tobacco is used, the desired antibody gene is inserted into a plant expression vector, for example, pMON530, and this vector is inserted into Agrobacterium tumefaciens.
mefaciens). The bacteria are transferred to tobacco, such as Nicotiana Tabacam (Nico
tiana tabacum) to obtain the desired antibody from the leaves of this tobacco (Ma, JK et al., Eur. J. Immunol. (199
4) 24, 131-138).

As described above, when producing an antibody in an in vitro or in vivo production system, the host may be co-transformed by separately incorporating DNAs encoding the H or L chain of the antibody into an expression vector. Alternatively, the DNAs encoding the H and L chains are integrated into a single expression vector,
The host may be transformed (International Patent Application Publication No. WO
94/11523).

7. Separation and Purification of Antibody The antibody expressed and produced as described above can be separated from the host inside and outside the cell and from the host and purified to homogeneity. The antibody used in the present invention can be separated and purified by affinity chromatography. Columns used for affinity chromatography include, for example, a protein A column and a protein G column.
For example, as a column using a protein A column, Hy
per D, POROS, Sepharose FF (Pharmacia) and the like. In addition, the separation and purification methods used for ordinary proteins may be used, and are not limited at all.

For example, chromatography columns other than the above-mentioned affinity chromatography, filters,
If appropriate selection and combination of ultrafiltration, salting out, dialysis, etc.,
Antibodies can be separated and purified (Antibodies: A La
boratory Manual.Ed Harlowand David Lane, Cold Spr
ing Harbor Laboratory, 1988). Examples of chromatography other than affinity chromatography include ion exchange chromatography, hydrophobic chromatography, gel filtration and the like (Strategies for P
rotein Purification and Characterization: A Labora
tory Course Manual. Ed Daniel R. Marshak et al., C
old Spring Harbor Laboratory Press, 1996).

8. Measurement of Antibody Concentration The concentration of the antibody obtained in step 7 was measured by measuring absorbance or enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay).
say; ELISA). That is,
When the absorbance is measured, the obtained antibody is appropriately diluted with PBS, and then the absorbance at 280 nm is measured.The extinction coefficient differs depending on the species and subclass.
Calculate mg / ml as 1.4 OD. In addition, in the case of using ELISA, it can be measured as follows.

That is, 0.1 M bicarbonate buffer (pH 9.6)
100 μl of goat anti-human IgG antibody diluted to 1 μg / ml with
Add to 96-well plate (Nunc) and incubate overnight at 4 ° C to immobilize antibody. After blocking, appropriately diluted antibody or a sample containing the antibody used in the present invention, or human IgG1 at a known concentration as a concentration standard
Add 00 μl and incubate at room temperature for 1 hour. After washing, 100 μl of an alkaline phosphatase-labeled anti-human IgG antibody diluted 5000 times is added, and the mixture is incubated at room temperature for 1 hour. After washing, adding the substrate solution, and incubating, the MICROPLATE READER Model 3550 (Bio-Ra
The absorbance at 405 nm is measured using d), and the concentration of the target antibody is calculated from the absorbance of the concentration standard human IgG.

9. Confirmation of Antibody Activity The antigen-binding activity (Antibodies:
A Laboratory Manual.Ed Harlow and David Lane, Cold
Spring Harbor Laboratory, 1988), ligand receptor binding inhibitory activity (Harada, A. et al., Int. Immunol.
(1993) 5, 681-690), known methods can be used.

As a method for measuring the antigen-binding activity of the anti-IL-8 antibody used in the present invention, ELISA, EIA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay) or a fluorescent antibody method can be used. . For example, when using ELISA, IL
IL-8 is added to a 96-well plate on which a polyclonal antibody against -8 is immobilized, and then a sample containing the desired anti-IL-8 antibody, for example, a culture supernatant or purified antibody of an anti-IL-8 antibody-producing cell is added. Add. Add a secondary antibody that recognizes the target anti-IL-8 antibody labeled with an enzyme such as alkaline phosphatase, incubate and wash the plate, then add an enzyme substrate such as p-nitrophenyl phosphate and measure the absorbance. Can be used to evaluate the antigen binding activity.

As a method for measuring the ligand receptor binding inhibitory activity of the anti-IL-8 antibody used in the present invention, an ordinary Cell ELISA or a ligand receptor binding assay can be used. For example, in the case of the Cell ELISA method, blood cells or cancer cells expressing IL-8 receptor, for example, neutrophils are cultured and adhered to a 96-well plate, and immobilized with paraformaldehyde or the like. Alternatively, a membrane fraction of cells expressing the IL-8 receptor is prepared to prepare a solid-phased 96-well plate.

A sample containing an anti-IL-8 antibody of interest, such as a culture supernatant of an anti-IL-8 antibody-producing cell or a purified antibody, and IL-8 labeled with a radioisotope such as ¹²⁵ I After incubating and washing the plate, the amount of IL-8 bound to the IL-8 receptor can be measured by measuring the radioactivity, and the ligand receptor binding inhibitory activity of the anti-IL-8 antibody can be evaluated. it can. For example, in an assay for inhibiting the binding of IL-8 to the IL-8 receptor on cells, a blood cell or a cancer cell expressing the IL-8 receptor, such as neutrophils, is separated by a method such as centrifugation and then suspended in a cell suspension. Prepare as a suspension. A radioisotope, for example, an IL-8 solution labeled with ¹²⁵ I or a mixed solution of unlabeled IL-8 and labeled IL-8, and a solution containing a concentration-adjusted anti-IL-8 antibody are used as a cell suspension. To be added. After a certain time, the cells may be separated and the radioactivity of the labeled IL-8 bound on the cells may be measured.

As a method for measuring the ability of the anti-IL-8 antibody used in the present invention to inhibit neutrophil migration (chemotaxis), a well-known method known in the art,
For example, the method of Grob, PM et al. (J. Biol. Chem. (199
0) 265, 8311-8316) can be used. Specifically, using a commercially available chemotaxis chamber,
The IL-8 antibody is cultured in a culture solution such as RPMI 1640, DMEM, MEM,
After dilution with IMDM or the like, IL-8 is added, and this is dispensed to the lower layer of the chamber partitioned by a filter. Next, the prepared cell suspension, for example, a neutrophil suspension, is added to the upper layer of the chamber, and left for a certain time. Since the migrating cells adhere to the lower surface of the filter attached to the chamber, the number of the cells may be measured by a method using a staining solution or a fluorescent antibody. In addition, it is also possible to make a judgment with the naked eye under a microscope or perform an automatic measurement using a measuring instrument.

10. Administration Method and Preparations The diagnostic findings for rheumatoid arthritis include morning stiffness,
Joint swelling of the hand region, symmetric joint swelling, multiple joint swelling, etc. are used (Mimori, T. et al., Diagnosis and Therapy Vol. 83, No.
7 1995 (65) 1187-1190, Arnett, FC et al., Arthri
tis Rheum. (1988) 31, 315-324). Thus, animals with artificially produced joint swelling are useful as experimental animal systems for testing the therapeutic effects of rheumatoid arthritis.
The therapeutic agent containing the anti-IL-8 antibody of the present invention as an active ingredient is systemically administered parenterally, for example, by intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, intraarticular injection and the like. Alternatively, it can be administered locally. The administration method can be appropriately selected depending on the age and symptoms of the patient.

The therapeutic agent containing the anti-IL-8 antibody of the present invention as an active ingredient can be used to cure or at least partially prevent the symptoms of the disease and its complications in patients already suffering from the disease. It is administered in a sufficient amount. For example, the effective dose is selected in the range of 0.01 mg / kg to 1000 mg / kg of body weight at a time. Or 5-20 per patient
A dose of 00mg / body can be chosen. However, the therapeutic agent containing the anti-IL-8 antibody of the present invention is not limited to these doses.

The administration may be carried out after the diagnosis of rheumatoid arthritis is confirmed, or may be administered when rheumatoid arthritis is suspected from the symptoms. The therapeutic agent containing the anti-IL-8 antibody of the present invention as an active ingredient can be formulated according to a conventional method (Re
mington's Pharmaceutical Science, latest edition, M
ark Publishing Company, Easton, USA), together with pharmaceutically acceptable carriers and additives.

Examples of such carriers and pharmaceutical additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, Water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA ) Mannitol, sorbitol, lactose,
Surfactants and the like that are acceptable as pharmaceutical additives.

The actual additive is appropriately or in combination selected from the above depending on the dosage form of the therapeutic agent of the present invention, but is not limited to these. For example,
When used as an injection, a purified anti-IL-8 antibody is dissolved in a solvent such as physiological saline, a buffer, a glucose solution and the like, and an anti-adsorption agent such as Tween 80, Tween is added thereto.
n20, gelatin, human serum albumin and the like can be used. Alternatively, it may be freeze-dried for reconstitution before use, and as an excipient for freeze-drying, for example, sugar alcohols and sugars such as mannitol and glucose can be used.

[0062]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to Reference Examples and Examples, but the present invention is not limited thereto. Reference Example 1. Production of monoclonal antibody against human IL-8
Preparation of Ibridomas BALB / c mice were immunized with human IL-8 by a conventional method, and spleen cells were collected from the mice in which immunization was established. The spleen cells were fused with mouse myeloma cells P3X63Ag8.653 by a conventional method using polyethylene glycol to prepare a hybridoma producing a mouse monoclonal antibody against human IL-8. As a result of screening using the binding activity to human IL-8 as an index, a hybridoma cell line WS-4 was obtained.
The antibody produced by hybridoma WS-4 is human IL-8
Inhibited the binding to neutrophils and had a neutralizing activity. (K
o, Y. et al., J. Immunol. Methods (1992) 149, 227
-235).

The H and L chain isotypes of the antibody produced by the hybridoma WS-4 were examined using a mouse monoclonal antibody isotyping kit. as a result,
It was revealed that the antibody produced by hybridoma WS-4 had a mouse κ type L chain and a mouse γ1 type H chain. The hybridoma cell line WS-4 is a Mouse hybrid
As oma WS-4, the Institute of Biotechnology, Institute of Industrial Science and Technology (1-1-3 Higashi 1-3, Tsukuba, Ibaraki Prefecture)
On July 7, it was deposited internationally under the Budapest Treaty as FERM BP-5507.

Reference Example 2 Humanized Antibody Against Human IL-8
Preparation of Humanized WS-4 Antibody by International Patent Application Publication No. WO 96-02576
Was prepared by the method described in (1). From the hybridoma WS-4 prepared in Reference Example 1, total RNA was prepared by a conventional method, and a single-stranded cDNA was synthesized therefrom. Mouse WS by PCR
DNA encoding the V region of the H chain and L chain of the -4 antibody was amplified. The primers used for the PCR method were Jones, S.
T. and Bendig, MM, Bio / Technology (1991) 9, 88-
The primers described in 89 were used. The DNA fragment amplified by the PCR method is purified, and the DNA fragment containing the gene encoding the mouse WS-4 antibody L chain V region and the mouse WS-4 antibody H
A DNA fragment containing the gene encoding the chain V region was isolated. Each of these DNA fragments was ligated to a plasmid pUC-type cloning vector, and introduced into E. coli competent cells to obtain E. coli transformants.

The transformant was cultured by a conventional method, and a plasmid containing the above DNA fragment was purified from the obtained cells. The nucleotide sequence of the DNA encoding the V region in the plasmid is determined by a conventional method, and each V
Region CDRs were identified. In order to prepare a vector for expressing the chimeric WS-4 antibody, cDNA encoding the L region and the V region of the H chain of the mouse WS-4 antibody is converted to a HEF vector in which DNA encoding the human C region has been linked in advance. Each was inserted separately. In order to produce humanized WS-4 antibody, the genetic modification of CDR-4
Region CDRs were implanted into human antibodies. In order to form an appropriate antigen-binding site, substitution of a DNA sequence to partially substitute FR amino acids in the V region of the CDR-grafted antibody was performed.

In order to express the V regions of the L chain and H chain of the humanized WS-4 antibody thus produced in mammalian cells as antibodies, the DNAs encoding each are separately inserted into a HEF vector. Then, a vector for expressing the L chain or H chain of the humanized WS-4 antibody was prepared. By inserting these two expression vectors into COS cells at the same time,
A cell line producing the WS-4 antibody was established. The humanized WS-4 antibody obtained by culturing this cell line was tested for its ability to bind to IL-8 and neutralize IL-8 by ELISA and IL-8 / neutrophil binding inhibition test, respectively. Was. As a result, it was found that the humanized WS-4 antibody binds to human IL-8 and inhibits the binding of IL-8 to neutrophils to the same extent as the mouse WS-4 antibody.

Escherichia coli having a plasmid containing the L chain and the H chain of the humanized WS-4 antibody was isolated from Escherichia
coli DH5α (HEF-RVLa-gκ) and Escherichia coli J
As M109 (HEF-RVHg-gγ1), the Institute of Biotechnology and Industrial Technology (I 1-3, Higashi 1-3-1, Tsukuba, Ibaraki Prefecture)
On July 12, 1994, FERM BP-4738 and FERM BP-4738 respectively
Deposited internationally under the Budapest Treaty as 4741.

Example 1. Phosphate buffered saline (PBS:
Bovine serum albumin (BSA: Nacalai Tesque) solution dissolved in Nissui Pharmaceutical at a concentration of 10 mg / ml
It was mixed with d's complete adjuvant (Sigma) and emulsified. This emulsion was used for Japanese white rabbits (n = 6, male, 3
(2 months old, Gokita Laboratory) Each animal was sensitized by injection with 2 ml (0.4 ml for each subcutaneous back, 0.5 ml for each of the right and left thigh muscles, and 0.1 ml each for the right and left hind footpads). 21 days after immunization, a 10 mg / ml BSA solution (solvent PBS) and Freund's
0.4 ml of an emulsified solution obtained by mixing and emulsifying an equal amount of incomplete adjuvant (Sigma) was injected subcutaneously into the back of a BSA-immunized rabbit.

Further, an intradermal reaction was performed 7 days after (28 days after immunization), and it was confirmed that sensitization to BSA was established. That is, 10 μg of BSA was dissolved in 0.1 ml of PBS and injected into the inner skin of the right ear. Three days later, redness of 10 mm or more in diameter was evaluated as positive. After confirming the sensitization to BSA (31 days after immunization), the sensitized rabbit was anesthetized with Nembutal (Dainippon Pharmaceutical, dose 0.4 mg / kg body weight), and
10 mg of mouse WS-4 antibody against IL-8 or 10 mg of mouse P3.6.2.8.1 antibody as a control antibody was diluted with physiological saline (Otsuka Pharmaceutical Co., Ltd.), and 3 ml of the solution was injected into the ear vein.

Immediately after administration of the antibody, an antigen solution prepared by diluting 0.5 ml of a 10 mg / ml BSA solution (solvent PBS) with 1.5 ml of physiological saline was injected into the left knee joint cavity using a 23G needle.
24 hours later, Nembutal over anesthesia (dose 2.0m
g / kg of body weight), and the diameters of the left and right knee joints were measured with a caliper at the knee joint space to calculate the difference between the left knee joint diameter that caused joint swelling and the normal right knee joint diameter. Joint swelling was noted. Table 1 shows the results.

[0071]

[Table 1]

The mean difference in the diameter of the left and right knee joints in the control antibody-administered group was 4.2 ± 1.2 mm, whereas that in the WS-4 antibody-administered group was 2.7 ± 0.8 mm. Statistical analysis was performed between the two groups regarding the difference between the left and right knee joint diameters, and a significant difference was observed. That is, in this example, it was revealed that the WS-4 antibody significantly suppressed joint swelling.
It has never been reported that an anti-IL-8 antibody suppresses joint swelling which is a diagnostic criterion for rheumatoid arthritis and an indicator of a therapeutic effect, and this is the first fact shown by the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location C12R 1:91)

Claims (7)

[Claims] 1. A therapeutic agent for rheumatoid arthritis comprising an anti-IL-8 antibody as an active ingredient. 2. The therapeutic agent according to claim 1, wherein the anti-IL-8 antibody is a monoclonal antibody. 3. The therapeutic agent according to claim 1, wherein the anti-IL-8 antibody is an antibody against mammalian IL-8. 4. The therapeutic agent according to claim 3, wherein the anti-IL-8 antibody is an antibody against human IL-8. 5. The therapeutic agent according to claim 2, wherein the anti-IL-8 antibody is a WS-4 antibody. 6. The therapeutic agent according to claim 1, wherein the anti-IL-8 antibody is a humanized or chimeric antibody. 7. The therapeutic agent according to claim 2, wherein the anti-IL-8 antibody is humanized WS-4.