JPH10182488A - Septicemia-treating agent containing anti-interleukin-8 antibody as active ingredient - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the new subject treating agent useful for treating septicemia or septic shock by making the agent include an anti-interleukin-8 antibody as an active ingredient. SOLUTION: This septicemia-treating agent includes anti-interleukin-8 antibody (anti-IL-8 antibody) as an active ingredient. The anti-IL-8 antibody is preferably a monoclonal antibody derived from a mammal, especially WS-4 antibody. The monoclonal antibody is obtained by immunizing an animal by using IL-8 as a sensitizing antibody, performing cell-fusion of the obtained immunized cells with myeloma cells as parent cells, further screening and cloning the obtained hybridoma and culturing the screened and cloned hybridoma. The WS-4 antibody is obtained from Mouse hybridoma WS-4 (FERM BP-5507). The objective treating agent can be orally administered and the effective divided dose thereof is, for example, 0.001-1,000mg/kg.

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 sepsis and septic shock containing an anti-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).

[0003] IL-8 is a factor that induces the activation and migration of neutrophils, and inflammatory cytokines such as IL-1β and TNF-α (Koch, AE et al., J. Investig. Med. 1995) 43,
28-38; Larsen, CG et al., Immunology (1989) 6
8, 31-36) and mitogens such as PMA and LPS (Yoshimura,
T. et al., Proc. Natl. Acad. Sci. USA (1987) 8
4, 9233-9237) and heavy metals such as cadmium (Hori
guchi, H. et al., Lymphokine Cytokine Res. (1993)
12, 421-428) and other stimuli. It is also known that human umbilical vein endothelial cells exposed to hypoxia express IL-8 (Karakurum,
M. et al., J. Clin. Invest. (1994) 93,1564-1570
).

In order for IL-8 to exert its biological activity, IL-8
Must bind to the IL-8 receptor and stimulate cells expressing the IL-8 receptor. The IL-8 receptor to which IL-8 binds and transmits a signal into cells has already been cloned, and its amino acid sequence has been elucidated. Human IL-8 receptors include IL-8 receptor A (IL-8 receptor α, IL-8 receptor 2 or CXCR1) and IL-8 receptor B (IL-8 receptor β, IL-8 There is a receptor called receptor 1 or CXCR2 (Murphy, PM and Tiffany, H. et al.
L., Science (1991) 253, 1280-1283; Holmes, WE
et al., Science (1991) 253, 1278-1280).

[0005] It is assumed that both have a structure that penetrates the cell membrane seven times, and both are associated with GTP-binding proteins in cytoplasmic domains (Horuk, R., Trends Pharmaceuticals).
col.Sci. (1994) 15, 159-165), and transmits IL-8 signals into cells. Therefore, by inhibiting the binding between IL-8 and the IL-8 receptor, it becomes possible to inhibit the biological activity of IL-8.

[0006] In 1991, the Society of Intensive Care
Critical Care Medicine) and American Society of Chest Disease (Ameri
A consensus conference was held jointly by the Can College of Chest Physicians, and the systemic inflammatory response syndrome (S
A concept of disease called ystemic Inflammatory Response Syndrome (SIRS) was proposed. That is, the following diagnosis is based on the biological response to invasion such as trauma, burns, severe pancreatitis, and infection.
Diagnosis is a condition with clinical findings of any two or more of the four items (Bone, RC et al., Chest (1992) 10
1, 1644-1655).

(1) Body temperature is higher than 38 ° C. or lower than 36 ° C. (2) Heart rate is higher than 90 times / minute (3) Respiratory rate is higher than 20 times / minute or PaCO 2 (arterial blood carbon dioxide partial pressure) ) Is less than 32 torr (4) White blood cell count is more than 12000 / μl or 4000 / μ
<l or immature leukocytes> 10%

[0008] Sepsis is a disease that has clinical findings of any two or more of the four diagnostic items of SIRS and is caused by infection. There may or may not be evidence of the pathogen causing the infection. Trauma, burns, and severe pancreatitis are distinguished from sepsis by the fact that the direct cause is not infection. Septic shock is a disease associated with abnormal perfusion such as hypotension despite maintaining a sufficient circulating fluid volume in sepsis. If sepsis progresses, it becomes septic shock within a few hours, and causes a decrease in total peripheral vascular resistance, a decrease in myocardial contractility, a peripheral circulatory failure, a decrease in blood pressure, and the like.

[0009] Serum or plasma of sepsis patients contains inflammatory cytokines such as IL-1β, IL-6, IL-8 and TNF-α (Thijs, LG and Hack, CE, I
ntensive Care Med. (1995) 21 Suppl 2, 258-263,),
Furthermore, it has been reported that the production of chemokines such as MCP-1, MCP-2 and MIP-1α in addition to IL-8 is increased (Bos
sink, AW et al., Blood (1995) 86, 3841-3847: Fu
jishima, S. et al., Intensive Care Med. (1996) 22,
1169-1175).

In addition to these cytokines, leukotriene B4, thromboxane B2 and prostaglandin are higher than normal values as eicosanoids.
It has been reported that the complement system is also activated (Takak
uwa, T. et al., Res Commun. Chem. Pathol. Pharmaco
l. (1994) 84, 291-300). As described above, it is assumed that a variety of factors are involved as septic aggressive factors and determine the pathology of sepsis in a complicated manner. Therefore, anti
IL-8 antibody was not known to have any therapeutic effect on sepsis or septic shock.

[0011]

At present, for sepsis, the presence of an infectious foci in the body is searched, and surgical drainage / resection or antibiotic therapy is performed. In addition, vasopressors and steroids are used for septic shock (Illustrated Pathology Internal Medicine Course [Vol. 17] Infectious Diseases, Medical View, pp. 96-97). However, the overall mortality rate of patients with sepsis is still 25-90% (Merck Manual, 1st edition, Medical View, p. 73). This indicates that the efficacy of these treatments and therapeutic agents is limited. Therefore, development of an effective therapeutic agent has been demanded.

It is an object of the present invention to provide new therapeutic agents for such diseases.

[0013]

Means for Solving the Problems The present inventors have conducted intensive studies to provide such a therapeutic agent, and as a result, have found that the intended purpose can be achieved by using an anti-IL-8 antibody. Was completed. That is, the present invention provides a therapeutic agent for sepsis, comprising an anti-IL-8 antibody as an active ingredient. The present invention also provides a therapeutic agent for septic shock, comprising an anti-IL-8 antibody as an active ingredient.

The present invention also provides a therapeutic agent for sepsis or septic shock, comprising an anti-IL-8 monoclonal antibody as an active ingredient. The present invention also provides a therapeutic agent for sepsis or septic shock, comprising as an active ingredient an antibody against IL-8 of a mammal. The invention also relates to humans
Provided is a therapeutic agent for sepsis or septic shock, which comprises an antibody against IL-8 as an active ingredient.

The present invention also provides a therapeutic agent for sepsis or septic shock, comprising a WS-4 antibody as an active ingredient. The present invention also relates to an anti-IL-8 having a human antibody constant region.
Provided is a therapeutic agent for sepsis or septic shock, comprising an antibody as an active ingredient. The present invention also provides a therapeutic agent for sepsis or septic shock, comprising a humanized or chimeric anti-IL-8 antibody as an active ingredient.

The present invention also provides a therapeutic agent for sepsis or septic shock, comprising a humanized WS-4 antibody as an active ingredient. The present invention also provides an agent for improving arterial blood pressure lowering in septic shock, comprising an anti-IL-8 antibody as an active ingredient. The present invention further provides an agent for reducing respiratory rate increase in septic shock, comprising an anti-IL-8 antibody as an active ingredient.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION

1. Anti-IL-8 Antibody The anti-IL-8 antibody used in the present invention is not limited in its origin, type (monoclonal or polyclonal) and shape as long as it has a therapeutic effect on sepsis and septic shock.

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 antibodies produced by hybridomas and recombinant antibodies produced by hosts transformed with expression vectors containing antibody genes. The anti-IL-8 antibody used in the present invention is IL
IL expressed by neutrophils by binding to -8
An antibody that blocks IL-8 signaling by inhibiting binding to the -8 receptor.

As such an antibody, 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 designated as Mouse hybridoma WS-4 by the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology (1-1-3 Higashi, Tsukuba, Ibaraki, Japan).
On April 17, 1996, it was deposited internationally under the Budapest Treaty as FERM BP-5507.

2. Antibodies produced by hybridomas Monoclonal antibodies are basically prepared using known techniques,
A hybridoma can be prepared and obtained as follows. That is, using IL-8 as a sensitizing antigen, immunizing it according to a normal immunization method, fusing the obtained immune cells with a known parent cell by a normal cell fusion method, and performing a normal screening method, It can be prepared by screening monoclonal antibody-producing cells.

Specifically, a monoclonal antibody can be prepared as follows.

For example, IL-8 used as a sensitizing antigen for obtaining antibodies is described in Matsushima, K. et.
al., J. Exp. Med. (1988) 167, 1883-1893
For IL-8, see Harada, A. et al., Int.
93) 5, 681-690; for dog IL-8, Ishikawa, J. e.
t al., Gene (1993) 131, 305-306, and for sheep IL-8, Seow, HF et al., Immunol. Cell Biol. (199
4) 72, 398-405 and Monkey IL-8 by Villinger, F.
et al., J. Immunol. (1995) 155, 3946-3954, and for guinea pig IL-8, Yoshimura, T. and Johnson,
DG, J. Immunol. (1993) 151, 6225-6236, Pig I
Goodman, RB et al., Biochemistry (1
992) 31, 10483-10490, each IL-8
It is obtained by using the gene / amino acid sequence.

It has been reported that human IL-8 is produced in various cells and undergoes different processing at the N-terminus (Leonard, EJ et al., Am. J. Respir. Cel).
l. Mol. Biol. (1990) 2, 479-486). So far, 7
Human IL-8 having 9, 77, 72, 71, 70 and 69 amino acid residues is known, but the anti-IL-8 used in the present invention is
The number of amino acid residues is not limited as long as it can be used as an antigen for obtaining an antibody.

After transforming an appropriate host cell by inserting the gene sequence of IL-8 into a known expression vector system, the desired IL-8 protein is known from the host cell or the culture supernatant. The purified IL-8 protein may be used as a sensitizing antigen.

As a mammal immunized with a sensitizing antigen,
Although not particularly limited, selection is preferably made in consideration of compatibility with a parent cell used for cell fusion. In general, rodents, lagomorphs, and primates are used. As rodent animals, for example, mice, rats, hamsters and the like are used. As an animal of the order Lagomorpha, for example, a rabbit is used. As a primate animal, for example, a monkey is used. As the monkeys, for example, monkeys of the lower nose (old world monkeys), more specifically, cynomolgus monkeys, rhesus monkeys, baboons, chimpanzees 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 desired antibody level is increased in the serum. Then, immune cells such as lymph node cells or spleen cells are removed from the mammal and subjected to cell fusion. However, preferred immune cells include spleen cells in particular.

As mammalian myeloma cells as the other parent cells fused with the immune cells, various cell lines already known, 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) and the like 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 and the myeloma cells is preferably, for example, 1 to 10 times that of 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 cloning of a hybridoma producing the desired antibody are performed.

Further, in addition to obtaining the above-mentioned hybridoma by immunizing an animal other than human with an antigen, human
Sensitized to IL-8, and fused the sensitized lymphocytes with a myeloma cell having permanent dividing ability derived from a human, for example, U266.
A hybridoma that produces a desired human antibody having an activity of binding to A can also be obtained (see Japanese Patent Publication No. 1-59878).
Furthermore, transgenic animals having a repertoire of human antibody genes were immunized with IL-8 as an antigen to obtain anti-IL-8 antibody-producing cells, which were then fused with myeloma cells to obtain IL-8. Human antibodies may be obtained (International Patent Application Publication Nos. WO 92/03918, WO 9
3/12227, WO 94/02602, WO 94/25585, WO 96/33735
And WO 96/34096).

The hybridoma producing the monoclonal antibody thus produced can be subcultured in a usual 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.

In addition to producing antibodies using hybridomas, cells in which immune cells such as sensitized lymphocytes producing antibodies are immortalized by oncogenes may be used.

3. Recombinant Antibody Monoclonal antibodies can also be obtained as recombinant antibodies produced using genetic recombination techniques. For example, a recombinant antibody is produced by cloning the antibody gene from an immune cell such as a hybridoma or a sensitized lymphocyte producing the antibody, inserting the clone into an appropriate vector, and introducing this into a host. In the present invention, this recombinant antibody can be used (for example, Borrebaeck, CAK and
Larrick, JW, THERAPEUTIC MONOCLONAL ANTIBODIE
S, Published in the United Kingdom by MACMILLAN PU
BLISHERS 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 was 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 also 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.

A target DNA fragment is purified from the obtained PCR product and ligated with 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.

When the DNA encoding the V region of the desired anti-IL-8 antibody is obtained, it is ligated to the DNA encoding the constant region (C region) of the desired antibody, and this is inserted into an expression vector. Alternatively, the DNA encoding the antibody V region may be incorporated into an expression vector that already contains the antibody C region DNA. As the antibody C region, an antibody C region derived from the same animal species as the V region may be used, or an antibody C region derived from an animal species different from the V region may be used.

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 As the recombinant antibodies used in the present invention, modified antibodies prepared by using genetic engineering techniques for the purpose of, for example, reducing heterologous antigenicity to humans can be used.
The modified antibody has a human antibody C region and, for example, a chimera (Ch
imeric) antibodies and humanized antibodies. These modified antibodies can be produced using known methods.

The chimeric antibody is obtained by combining the DNA encoding the antibody V region other than the human antibody obtained as described above with the human antibody C.
It is obtained by ligating to a DNA encoding the region, inserting the DNA into an expression vector, introducing into a host, and producing (see European Patent Application Publication No. EP 125023, International Patent Application Publication No. WO96 / 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 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, respectively, FERM BP-4739 and FERM B
Deposited internationally under the Budapest Treaty as P-4740.

The humanized antibody is also called a reshaped human antibody, and is used to determine the complementarity determining region of a non-human mammal, for example, a mouse antibody.
gion (CDR) is transplanted to the CDR of a human antibody.
The general recombination technique is also known (European Patent Application Publication No. EP 125023, International Patent Application Publication No. WO96)
/ 02576).

More 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).

The FR of a human antibody linked via CDRs is
Those whose CDRs form a good antigen-binding site are selected. If necessary, the amino acids of FR in the antibody V region may be substituted such that the CDRs of the humanized antibody form an appropriate antigen-binding site (Sato, K. et al., Cancer Res. (1993) 53 ,
851-856). Preferred specific examples of the humanized antibody 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 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-47, respectively.
41, deposited internationally under the Budapest Treaty.

To produce the anti-IL-8 antibody used in the present invention, the antibody gene is incorporated into an expression vector so as to be 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.

The expression of the antibody gene is determined by determining the antibody heavy chain (H chain).
Alternatively, host cells may be co-transformed by separately incorporating DNAs encoding the light chain (L chain) into an expression vector, or DNA encoding the H chain and the L chain may be integrated into a single expression vector. Alternatively, the host cell may be transformed (see International Patent Application Publication No. WO 94/11523). A chimeric antibody comprises a V region derived from a non-human mammalian antibody and a C region derived from a human antibody.A humanized antibody comprises CDRs derived from a non-human mammal antibody and FRs and C regions derived from a human antibody. Since the amino acid sequences derived from mammals other than those of the present invention are minimally reduced, the antigenicity in the human body is reduced, and this is useful as an active ingredient of the therapeutic agent of the present invention.

As the human antibody C region to be used, for example, Cγ1, Cγ2, Cγ3 and Cγ4 can be used. In addition, the human antibody C region may be modified to improve the stability of the antibody or its production. For example, when the antibody subclass is selected as IgG4, the amino acid sequence Cys-Pro-Ser-Cys-Pro of the hinge region of IgG4 is converted into the amino acid sequence Cys-Pro-Pro-Cys-Pro of the hinge region of IgG1. Can eliminate the structural instability of IgG4 (An
gal, S. et al., Mol. Immunol. (1993) 30, 105-10
8).

5. Antibody Fragments and Modified Antibodies The antibodies used in the present invention may be antibody fragments or modified antibodies as long as they bind to IL-8 and inhibit the activity of IL-8. For example, antibody fragments include Fab, F (ab ') 2, Fv, or a single chain Fv (scFv) in which Fvs of an H chain and an L chain are linked by an appropriate linker.

Specifically, 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 and introduced into an expression vector. (Eg, Co, MS et al., J. Immunol. (1994)
152, 2968-2976; Better, M. and Horwitz, AH, Met
hods Enzymol. (1989) 178, 476-496; Pluckthun, A.
and Skerra, A., Methods Enzymol. (1989) 178, 497-5
15; Lamoyi, E., Methods Enzymol. (1986) 121,652-66.
3; Rousseaux, J. et al., Methods Enzymol. (1986)
121, 663-669; Bird, RE and Walker, BW, Tren
ds 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 the DNA encoding the H chain or the V region of the H chain and the DNA encoding the L chain or the V region of the L chain of the antibody as templates. 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.

Further, once DNAs encoding scFv are prepared, an expression vector containing them and a host transformed with the expression vector can be obtained in accordance with a conventional method. According to the usual method, sc
You can get Fv.

[0058] These antibody fragments can be produced in the same manner as described above, by obtaining the gene, expressing it, and producing it by a host. The “antibody” in the claims of the present application also includes these antibody fragments. Anti-IL-8 conjugated with various molecules such as polyethylene glycol (PEG) as a modified antibody
Antibodies 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. Recombinant antibody, modified antibody, or antibody
Expression and Production of Fragment 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 commonly used useful promoter / enhancer, 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, a human cytomegalovirus immediate early promoter / enhancer (human cytomegalovirus immediate early)
promoter / enhancer).

Other promoters / enhancers that can be used for the 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 using the SV40 promoter / enhancer, the method of Mulligan, RC et al. (Nature
(1979) 277, 108-114) and HEF1α promoter /
When an enhancer is used, it can be easily carried out according to the method of Mizushima, S. et al. (Nucleic Acids Res. (1990) 18, 5322).

In the case of Escherichia coli, expression can be carried out by operably linking a useful promoter commonly used, a signal sequence for antibody secretion, and an antibody gene to be expressed.
For example, promoters include 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)
And when using the araB promoter, see Better, M.
These methods may be followed (Science (1988) 240, 1041-1043).

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).

The origin of replication includes SV40, polyoma virus, adenovirus, bovine papilloma virus (BP
V) or other origins of replication.
In order to amplify the gene copy number in the host cell system, the expression vector is selected as an aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthine guanine phosphoribosyl transferase (Ecogpt) gene, dihydrofolate reductase (dhfr) as a selection marker. It can contain genes 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. Animal cells include (1) mammalian cells such as CHO, COS, myeloma, BHK (baby hamster kidney), HeLa, Vero,
(2) amphibian cells, eg, Xenopus oocytes, or (3) insect cells, eg, sf9, sf21, Tn
Five are known. Known plant cells include, for example, cells derived from the genus Nicotiana, more specifically, Nicotiana tabacum, which may be callus-cultured. As fungal cells,
(1) Yeast, for example, Saccharomyces
) Genus, specifically, Saccharomyces cerevisiae (Sacch
aromyces cerevisiae) or (2) filamentous fungi, for example, the genus Aspergillus, more specifically Aspergillus niger.

When using prokaryotic cells, there is a production system using bacterial cells. As bacterial cells, Escheric (Escheric
hia coli) and Bacillus subtilis are known. In these cells,
An antibody can be obtained by introducing a target antibody gene by transformation and culturing the transformed cells in vitro. Culture is performed according to a known method. For example, as a culture solution for mammalian cells, DMEM, MEM, RPMI1640, IMDM
Etc. can be used. At that time, fetal calf serum (FCS
) And the like, or serum-free culture may be used. 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.

Examples of the in vivo production system include a production system using animals and a production system using plants. When using animals, there are production systems using mammals and insects.

As mammals, goats, pigs, sheep, mice, and cows can be used (Glaser, V.,
SPECTRUM Biotechnology Applications, 1993). When a mammal is used, a transgenic animal can be used. For example, the antibody gene is prepared as a fusion gene by inserting the antibody gene into a gene such as goat β casein, which encodes a protein uniquely produced in milk.
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 progeny thereof. Hormones may optionally be used in transgenic goats to increase the amount of milk containing the desired antibody produced from the transgenic goats (Ebert, KM et al., Bio /
Technology (1994) 12, 699-702).

Also, silkworms can be used as insects. When a silkworm is used, the silkworm is infected with a baculovirus into which an antibody gene of interest has been inserted, and a desired antibody is obtained from the body fluid of the silkworm (Maeda, S. et al.,
ture (1985) 315, 592-594). Furthermore, when using a plant, for example, tobacco can be used. 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.
ns). The bacteria are transferred to tobacco, such as Nicotiana Tabacam
tabacum) and obtain the desired antibody from the leaves of this tobacco (Ma, JK et al., Eur. J. Immunol. (1994) 24,
131-138).

The antibody gene is introduced into these animals or plants as described above, and the antibodies are produced in the animals or plants and collected. In vitro or in vivo as described above
When an antibody is produced in the production system of (1), DNAs encoding the antibody H chain or L chain may be separately incorporated into an expression vector to co-transform the host. Or H chain and L
The DNA encoding the strand may be incorporated into a single expression vector to transform the host (see International Patent Application Publication No. WO 94/11523).

7. Separation and Purification of Antibodies The antibodies expressed and produced as described above can be separated from the inside and outside of cells and from the host and purified to homogeneity. The separation and purification of the antibody used in the present invention may be carried out by using the separation and purification methods used for ordinary proteins, and is not limited at all. For example, an antibody can be separated and purified by appropriately selecting and combining a chromatography column such as affinity chromatography, a filter, ultrafiltration, salting out, dialysis and the like (Antibodies:
A Laboratory Manual. Ed Harlow and David Lane, Col
d Spring Harbor Laboratory, 1988). Columns used for affinity chromatography include a protein A column and a protein G column.

For example, as a column using a protein A column, Hyper D, POROS, Sepharose FF (Pharma
cia) and the like. Examples of chromatography other than affinity chromatography include ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein P).
urification and Characterization: A Laboratory Cou
rse Manual. Ed Daniel R. Marshak et al., Cold Spri
ng Harbor Laboratory Press, 1996). Furthermore, these chromatographys can be performed using liquid phase chromatography such as HPLC and FPLC.

8. Measurement of Antibody Concentration The concentration of the antibody obtained above was measured by measuring absorbance or enzyme-linked immunosorbent assay.
assay; ELISA) or the like. That is, when the absorbance is measured, the obtained antibody is
After appropriate dilution, absorbance at 280 nm is measured, and the extinction coefficient varies depending on the species and subclass. In addition, in the case of using ELISA, it can be measured as follows. That is, 100 μl of a goat anti-human IgG antibody diluted to 1 μg / ml with 0.1 M bicarbonate buffer (pH 9.6) was added to a 96-well plate (Nunc) and incubated at 4 ° C. overnight to immobilize the antibody. I do.

After blocking, 100 μl of an appropriately diluted antibody used in the present invention or a sample containing the antibody, or human IgG having a known concentration as a concentration standard is added.
Incubate for 1 hour at room temperature. 5000 after washing
Add 100 μl of a 1: 2 diluted alkaline phosphatase-labeled anti-human IgG antibody and incubate at room temperature for 1 hour. After washing, add the substrate solution, and incubate,
40 using MICROPLATE READER Model 3550 (Bio-Rad)
The absorbance at 5 nm is measured, and the concentration of the target antibody is calculated from the absorbance of the concentration standard human IgG.

For measuring the antibody concentration, BIAcore (Phar
macia) can be used.

9. Confirmation of Antibody Activity Antigen binding activity of the antibody used in the present invention (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 ELISA is used, a polyclonal antibody against IL-8 is immobilized on a 96-well plate on which IL-8 is immobilized.
Then, a sample containing the desired anti-IL-8 antibody, for example, a culture supernatant of anti-IL-8 antibody-producing cells or a purified antibody is added. Add a secondary antibody that recognizes the anti-IL-8 antibody of interest, labeled with an enzyme such as alkaline phosphatase, incubate and wash the plate, add an enzyme substrate such as p-nitrophenyl phosphate, and measure the absorbance. Thus, the antigen binding activity can be evaluated. BIAcore (Pharmacia) can be used to measure the antigen-binding activity of the antibody.

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 the desired anti-IL-8 antibody,
For example, a culture supernatant or a purified antibody of an anti-IL-8 antibody-producing cell,
After adding a radioisotope, for example, IL-8 labeled with ¹²⁵ I, incubating and washing the plate, the amount of IL-8 bound to the IL-8 receptor can be measured by measuring the radioactivity. The ligand receptor binding inhibitory activity of the IL-8 antibody can be evaluated.

For example, for IL-8 receptors on cells
In the IL-8 binding inhibition assay, blood cells or cancer cells expressing the IL-8 receptor, such as neutrophils, are separated by means such as centrifugation and then prepared as a cell suspension. Cell suspension of a solution of IL-8 labeled with a radioisotope, e.g., 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 Add to the solution. After a period of time, the cells were separated and labeled IL-8 bound on the cells.
May be measured.

As a method for measuring the ability of the anti-IL-8 antibody to be used in the present invention to inhibit neutrophil migration (chemotaxis), a 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, an anti-IL-8 antibody was cultured in a culture solution such as RPMI.
After dilution with 1640, DMEM, MEM, IMDM, etc., add IL-8,
This is dispensed into the lower layer of the chamber partitioned by the 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 Preparation The therapeutic agent containing the anti-IL-8 antibody of the present invention as an active ingredient is parenterally administered, for example, by intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection It can be administered systemically or locally by injection or the like. 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 is administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially prevent the symptoms of the disease. Is administered. For example, an effective dose is
It is selected from the range of 0.01mg to 1000mg per kg of body weight at a time. Alternatively, a dose of 5-2000 mg / body per patient 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 sepsis or septic shock has occurred, or may be administered when sepsis or septic shock is predicted to occur. The administration period 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 formulated according to a conventional method (Remington's Pharmaceutical).
Science, latest edition, Mark Publishing Company,
Easton, U.S.A.), 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, acacia, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA ), Mannitol, sorbitol, lactose, surfactants acceptable as pharmaceutical additives, and the like.

The actual additives are appropriately or in combination selected from the above depending on the dosage form of the therapeutic agent of the present invention, but are not limited thereto. For example,
When used as an injection, a purified anti-IL-8 antibody is dissolved in a solvent, for example, a physiological saline solution, a buffer solution, a glucose solution, and the like, and an anti-adsorption agent, for example, Tween 80, Tween
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.

Sepsis is a disease that has clinical findings of any two or more of the four diagnostic items of SIRS and is caused by infection. There may or may not be evidence of the pathogen causing the infection. Trauma, burns, and severe pancreatitis are distinguished from sepsis by the fact that the direct cause is not infection. Septic shock refers to sepsis accompanied by abnormal perfusion such as hypotension despite maintaining a sufficient amount of circulating fluid. If sepsis progresses, it becomes septic shock within a few hours, and causes a decrease in total peripheral vascular resistance, a decrease in myocardial contractility, a peripheral circulatory failure, a decrease in blood pressure, and the like.

As shown in the examples below, the anti-
The therapeutic agent containing an IL-8 antibody as an active ingredient suppresses a decrease in arterial blood pressure, an increase in respiratory rate and a change in body temperature during administration of endotoxin to a rabbit known as an experimental system for the above-mentioned diseases, and the survival of an endotoxin-treated rabbit. Improved the rate. Therefore, the therapeutic agent containing the anti-IL-8 antibody of the present invention as an active ingredient is useful as a therapeutic agent for sepsis or septic shock. Further, the therapeutic agent containing the anti-IL-8 antibody of the present invention as an active ingredient is useful for improving the decrease in arterial blood pressure in septic shock and reducing the increase in respiratory rate in septic shock.

[0092]

EXAMPLES The present invention will now be described specifically with reference to Examples and Reference Examples, but the present invention is not limited to these Examples. 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 isotype of the H chain and L chain of the antibody produced by the hybridoma WS-4 was 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 a humanized WS-4 antibody, the CDRs
The V region CDR of the mouse WS-4 antibody was transplanted into a human antibody using a genetic engineering technique by a transplantation method. In order to form an appropriate antigen-binding site, FRs in the V region of the CDR-grafted antibody
The amino acid sequence was partially substituted for the DNA sequence.

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 the respective regions were 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.

E. 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-47, respectively.
41 was deposited internationally under the Budapest Treaty.

Example 1 A New Zealand white rabbit (female, n = 5 per group, weight: 2.8 to 3.2 kg) was intramuscularly administered with 0.5 mg / kg body weight of diazepam and 35 mg / kg body weight of pentobarbital. Anesthetized. After standing for 30 minutes,
A 24G Terumo catheter was inserted, and 5 mg / kg body weight ketamine was administered from the intravenous catheter to anesthetize. Then, a 22G Terumo catheter was inserted into the auricular artery.

Thereafter, the following treatment was performed until the end of anesthesia.
(i) 20 mg / kg of ketamine per hour from intravenous catheter
Additional injections were made at the body weight ratio. (ii) From an intravenous catheter,
Physiological saline was infused at a rate of 5 ml / kg body weight per hour. (i
ii) Arterial blood pressure was continuously measured using an arterial catheter. (iv) Blood was collected periodically from the arterial catheter.
(v) To prevent clogging of the catheter, 2.5 IU / ml of heparin was injected at a rate of 1 ml / kg body weight per hour from an arterial catheter. (vi) Respiratory rate and rectal temperature were measured periodically.

After completion of the catheter insertion, the catheter was allowed to stand for 45 minutes, and the baseline arterial blood pressure, respiratory rate, and rectal temperature were measured. Immediately thereafter, 3 mg / kg of mouse WS-4 antibody against human IL-8
Body weight or mouse P3.6.2.8.1 antibody as control antibody
3 mg / kg body weight or physiological saline was administered at 1.8 ml / kg body weight via an intravenous catheter. Five minutes later, over 20 minutes, 0.5 mg / kg body weight of lipopolysaccharide (LPS, Esche
richia coli O127: B8, Sigma) or 2 ml / kg body weight saline was administered via an intravenous catheter.

The experimental group was divided into four groups: an anti-IL-8 antibody administration group, a control antibody administration group, an LPS group and a normal group. In the group to which the anti-IL-8 antibody was administered, mouse WS-4 antibody was administered at 0 minutes, and LPS was administered at 5 to 25 minutes. In the control antibody administration group, mouse P
3.6.2.8.1 antibody was administered and LPS was administered 5-25 minutes. L
In the PS group, only saline was administered at 0 minutes, and L was administered at 5 to 25 minutes.
PS was administered. The normal group received only saline at 0 min and 5-25 min.

4 hours after LPS administration, anesthesia and measurement of each parameter were terminated, and each was returned to the same cage as before the experiment. Thereafter, the survival rate was evaluated until 7 days later. Changes over time in arterial blood pressure, respiratory rate and rectal temperature are shown in FIGS. 1, 2 and 3, respectively. FIG. 4 shows the change over time in the survival rate.

(1) Arterial Blood Pressure In each group to which LPS was administered (anti-IL-8 antibody administration group, control antibody administration group and LPS group), arterial blood pressure was significantly (p <0.05) lower than in the normal group. On the other hand, shock symptoms in which arterial blood pressure decreased due to LPS administration were shown. However, the anti-IL-8 antibody administration group was significantly (p <
0.05) reduced arterial blood pressure. No significant difference was observed in arterial blood pressure between the control antibody administration group and the LPS group (see FIG. 1). These results indicate that anti-IL-8 antibody reduces blood pressure drop, which is one of the symptoms of sepsis and septic shock.

(2) Respiratory rate In each group to which LPS was administered (anti-IL-8 antibody-administered group, control antibody-administered group and LPS group), the respiratory rate increased significantly (p <0.05) compared to the normal group. (However, excluding the control antibody administration group at 165 minutes) showed an increase in respiratory rate, which is one of the diagnostic items for SIRS. However, in the group treated with the anti-IL-8 antibody, the increase in respiratory rate tended to be reduced as compared with the group treated with the control antibody and the LPS group.
0.05) reduction in respiratory rate increase was observed. No significant difference was observed in respiratory rate between the control antibody administration group and the LPS group (see FIG. 2). These results indicate that anti-IL-8 antibody reduces the increase in respiratory rate, one of the symptoms of sepsis and septic shock.

(3) Rectal Body Temperature In each group to which LPS was administered (anti-IL-8 antibody-administered group, control antibody-administered group and LPS group), there was no statistically significant difference, but the rectum was higher than that in the normal group. Body temperature tended to decrease.
At that time, in the anti-IL-8 antibody administration group, the control antibody administration group and LP
The body temperature drop tended to be reduced as compared with the S group (see FIG. 3). This suggests that anti-IL-8 antibody reduces changes in body temperature, which is one of the diagnostic items for sepsis and septic shock.

(4) Survival Rate In the LPS group, all 5 cases died by 48 hours, and in the control antibody administration group, 2 out of 5 cases survived 7 days later.
In the anti-IL-8 antibody administration group, 4 out of 5 cases survived 7 days later (see FIG. 4). This indicated that the anti-IL-8 antibody rescued lethality due to LPS administration. In the normal group without LPS administration, all cases survived.

As described in (1) to (4) above,
The IL-8 antibody alleviated hypotension, increased respiratory rate and changes in body temperature, symptoms of sepsis including septic shock. Furthermore, lethality due to endotoxin administration was rescued.

Anti-IL-8 antibodies reduce the decrease in blood pressure, increase in respiratory rate and changes in body temperature caused by bacterial toxins, and rescue bacterial toxins. This fact indicates that the anti-IL-8 antibody is useful as a therapeutic agent for sepsis and septic shock, an agent for improving arterial blood pressure lowering, and an agent for reducing respiratory rate increase.

[Brief description of the drawings]

FIG. 1 shows the time course of arterial blood pressure from 0 to 240 minutes when an antibody or saline was administered at 0 minutes and LPS or saline was administered at 5 to 25 minutes. FIG. During the period indicated by the line a, the arterial blood pressure of each of the anti-IL-8 antibody-administered group, control antibody-administered group and LPS group was significantly (p <0.05) lower than that of the normal group. In the period shown by the line b, the group treated with the anti-IL-8 antibody was significantly more significant than the LPS group (p <0.0
5) The decrease in arterial blood pressure was reduced. The period shown by the line c,
The group treated with anti-IL-8 antibody was significantly (p <0.0
5) The decrease in arterial blood pressure was reduced.

FIG. 2 shows the time course of respiratory rate from 0 to 240 minutes when an antibody or saline was administered at 0 minutes and LPS or saline was administered at 5 to 25 minutes. FIG. d period, the anti-IL-8 antibody administration group,
Each of the control antibody administration group and the LPS group significantly (p <0.05) increased the respiratory rate compared to the normal group (except for the control antibody administration group at 165 minutes). The period indicated by the line e
The -8 antibody administration group significantly (p <0.05) reduced respiratory rate increase compared to the LPS group.

FIG. 3 shows the time course of rectal body temperature from 0 to 240 minutes when an antibody or saline was administered at 0 minutes and LPS or saline was administered at 5 to 25 minutes. FIG.

FIG. 4 is a graph showing the change over time in the survival rate up to 7 days later.

Claims (11)

[Claims] 1. A therapeutic agent for sepsis, comprising an anti-IL-8 antibody as an active ingredient. 2. The therapeutic agent according to claim 1, wherein the sepsis is septic shock. 3. The therapeutic agent according to claim 1, wherein the anti-IL-8 antibody is a monoclonal antibody. 4. The therapeutic agent according to claim 1, wherein the anti-IL-8 antibody is an antibody against mammalian IL-8. 5. The therapeutic agent according to claim 1, wherein the anti-IL-8 antibody is an antibody against human IL-8. 6. The therapeutic agent according to claim 1, wherein the anti-IL-8 antibody is a WS-4 antibody. 7. The therapeutic agent according to claim 1, wherein the anti-IL-8 antibody has a human antibody constant region. 8. The therapeutic agent according to any one of claims 1 to 7, wherein the anti-IL-8 antibody is a humanized or chimerized antibody. 9. The therapeutic agent according to any one of claims 1 to 8, wherein the anti-IL-8 antibody is a humanized WS-4 antibody. 10. An agent for improving arterial blood pressure lowering in septic shock, comprising an anti-IL-8 antibody as an active ingredient. 11. An agent for reducing respiratory rate increase in septic shock, comprising an anti-IL-8 antibody as an active ingredient.