WO1997040215A1 - Medicament contre l'infarctus du myocarde contenant comme principe actif un anticorps anti-il-8 - Google Patents

Medicament contre l'infarctus du myocarde contenant comme principe actif un anticorps anti-il-8 Download PDF

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Publication number
WO1997040215A1
WO1997040215A1 PCT/JP1997/001405 JP9701405W WO9740215A1 WO 1997040215 A1 WO1997040215 A1 WO 1997040215A1 JP 9701405 W JP9701405 W JP 9701405W WO 9740215 A1 WO9740215 A1 WO 9740215A1
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Prior art keywords
antibody
therapeutic agent
cells
active ingredient
myocardial infarction
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PCT/JP1997/001405
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English (en)
Japanese (ja)
Inventor
Akira Matsumori
Kouji Matsushima
Takaki Koga
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Chugai Seiyaku Kabushiki Kaisha
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Priority to AU24051/97A priority Critical patent/AU2405197A/en
Publication of WO1997040215A1 publication Critical patent/WO1997040215A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a therapeutic agent for myocardial infarction comprising an anti-interleukin-8 (IL-8) antibody as an active ingredient.
  • the present invention also relates to a therapeutic agent for unstable angina pectoris, comprising an anti-IL-8 antibody as an active ingredient.
  • the present invention relates to a therapeutic agent for myocardial ischemia-reperfusion injury containing an anti-1L-8 antibody as an active ingredient.
  • IL-8 is a protein belonging to the CXC chemokine subfamily, and was formerly a monocyte-derived neutrophil chemotactic factor, a neutrophil activator protein-1 (neutrophil attra ctant / act i vat on protein-1) and neutrophil a ctivating factor.
  • IL-8 is a factor that activates neutrophils and causes neutrophils to acquire migratory ability. Inflammatory cytokines such as IL-1 / 3 and TNF- ⁇ (Koch, AE et al., J Investig. Med.
  • Ischemic heart disease such as myocardial infarction or angina, or cardiac-lung bypass surgery, surgery to perform artificial cardiac arrest, or decreased oxygen supply due to reduced coronary blood flow or disruption during heart transplantation, Inhibits cardiomyocyte energy production, reduces cardiac function, and leads to irreversible cytotoxicity of cardiomyocytes.
  • Reperfusion therapy such as coronary thrombolysis, percutaneous coronary angioplasty (PTCA), or coronary artery bypass grafting (CABG) has been widely used to restore blood flow to patients with ischemic heart disease.
  • PTCA percutaneous coronary angioplasty
  • CABG coronary artery bypass grafting
  • nitrites, calcium antagonists, beta receptor antagonists, etc., and antiplatelet drugs have been used for the prevention of recurrence for myocardial infarction for the purpose of coronary vasodilation and reduction of cardiac load.
  • thrombolytic therapy and reperfusion therapy such as PTCA and CABG have recently been common.
  • no drug has been reported to have a direct ameliorating effect on myocardial injury associated with reperfusion therapy.
  • the present inventors have conducted intensive studies to provide such a therapeutic agent, and
  • the present invention provides a therapeutic agent for myocardial infarction containing an anti-IL-8 antibody as an active ingredient.
  • the present invention also provides a therapeutic agent for acute myocardial infarction containing an anti-1L-8 antibody as an active ingredient.
  • the present invention also provides a therapeutic agent for unstable angina pectoris, comprising an anti-IL-8 antibody as an active ingredient.
  • the present invention also provides a therapeutic agent for myocardial ischemia-reperfusion injury, comprising an anti-1L-8 antibody as an active ingredient.
  • the present invention also provides a therapeutic agent for myocardial ischemia-reperfusion injury accompanying cardiac-lung bypass surgery, which contains an anti-1L-8 antibody as an active ingredient.
  • the present invention also provides a therapeutic agent for myocardial ischemia-reperfusion injury accompanying surgery for performing artificial cardiac arrest, which comprises an anti-IL-8 antibody as an active ingredient.
  • the present invention also provides a therapeutic agent for myocardial ischemia-reperfusion injury associated with heart transplantation, which contains an anti-1 antibody as an active ingredient.
  • the present invention also provides a therapeutic agent for myocardial infarction, a therapeutic agent for unstable angina, or a therapeutic agent for myocardial ischemia-reperfusion injury, comprising an anti-IL-8 monoclonal antibody as an active ingredient.
  • the present invention also relates to a therapeutic agent for myocardial infarction, a therapeutic agent for unstable angina pectoris, or a myocardial ischemia-reperfusion, which comprises an antibody against mammal 1 as an active ingredient.
  • a therapeutic agent for disorders which comprises an antibody against mammal 1 as an active ingredient.
  • the present invention also provides a therapeutic agent for myocardial infarction, a therapeutic agent for unstable angina, or a therapeutic agent for myocardial ischemia-reperfusion injury, comprising an antibody against human 1L-8 as an active ingredient.
  • the present invention also provides a therapeutic agent for myocardial infarction, a therapeutic agent for unstable angina, or a therapeutic agent for myocardial ischemia-reperfusion injury, comprising a WS-4 antibody as an active ingredient.
  • the present invention also relates to a therapeutic agent for myocardial infarction, a therapeutic agent for unstable angina pectoris, or a therapeutic agent for myocardial ischemia-reperfusion injury, which comprises a humanized or chimerized anti-IL-8 antibody as an active ingredient.
  • a therapeutic agent for myocardial infarction a therapeutic agent for unstable angina pectoris, or a therapeutic agent for myocardial ischemia-reperfusion injury, which comprises a humanized or chimerized anti-IL-8 antibody as an active ingredient.
  • the present invention further provides a therapeutic agent for cardiac myocardial infarction, a therapeutic agent for unstable angina, or a therapeutic agent for myocardial ischemia-reperfusion injury, comprising a humanized WS-4 antibody as an active ingredient.
  • FIG. 1 is a graph in which CK activity in plasma was measured over time in a heron myocardial infarction model, and the average value of the WS-4 antibody-administered group was compared with the average value of the solvent-administered group as a negative control.
  • FIG. 2 is a graph in which the LDH activity in plasma was measured over time in a heron myocardial infarction model, and the average value of the WS-4 antibody-administered group was compared with the average value of the solvent-administered group as a negative control.
  • the anti-1L-8 antibody used in the present invention may be of any origin and type (monoclonal, monoclonal, Polyclonal) and any shape
  • the anti-1 antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using known means.
  • a mammalian monoclonal antibody is particularly preferable.
  • Monoclonal antibodies derived from mammals include those produced by hybridomas and those produced by a host transformed with an expression vector containing the antibody gene by genetic engineering techniques. By binding to 1L-8, this antibody inhibits the binding to IL-8 receptor expressed on neutrophils, blocks 1L-8 signal transduction, and activates 1L-8 biological activity. Is an antibody that inhibits
  • Such antibodies include the WS-4 antibody (Ko, Y. et al., J. Immunol 1. Methods (1992) 149, 227-235) and the DM / C7 antibody (Mulligan, M. S. et. al., J. Immunol. (1993) 150, 5585-5595), Pep-1 antibody and Pep-3 antibody (International Patent Application Publication No.WO 92/04372) or 6G4.2.5 antibody and A5.12.14 antibody (International Patent Application Publication No. W095 / 23865; Boylan, AM et al., J. Clin. Invest. (1992) 89, 1257-1267).
  • 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 (Ichiki, Tsukuba, Higashi 1-3-1, Ibaraki Prefecture) in 1996. On April 17, it was internationally deposited under the Budapest Treaty as FERM BP-5507.
  • a hybridoma producing a monoclonal antibody can be basically produced using a known technique as follows. That is, IL-8 is used as a sensitizing antigen, and immunized according to a usual immunization method, and the obtained immune cells are compared with known parent cells by a normal cell fusion method. It can be produced by fusing and screening monoclonal antibody-producing cells by a usual screening method.
  • a monoclonal antibody may be prepared as follows.
  • human IL-8 is described in Matsushima, K. et al., J. Exp. Med. (1988)
  • Human 1 has been reported to be produced in a variety of cells and undergo different processing at the N-terminus (Leonard, B.J. et al.,
  • IL-8 having amino acid residues of 79, 77, 72, 71, 70 and 69 has been known, but it has been known to obtain the anti-1L-8 antibody used in the present invention.
  • the number of amino acid residues is not limited as long as it can be used as an antigen of the present invention.
  • the target IL-8 protein 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 is known from the host cell or culture supernatant.
  • the purified 1-8 protein may be used as a sensitizing antigen.
  • the mammal to be immunized with the sensitizing antigen is not particularly limited, but is selected in consideration of compatibility with the parent cell used for cell fusion.
  • rodent animals for example, mice, rats, hamsters, etc., are used.
  • Immunization of an animal with a sensitizing antigen is performed according to a known method.
  • the sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal.
  • sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal.
  • PBS Phosphate-Buffered Saline
  • physiological saline physiological saline
  • an appropriate carrier can be used at the time of immunization with a sensitizing antigen.
  • the immune cells are removed from the mammal and subjected to cell fusion. Examples include spleen cells.
  • mammalian cell lines already known as mammalian myeloma cells as the other parent cells to be fused with the immune cells include, for example, P3 (P3x63Ag8.653) (Kearney, JF et al. , J. Immnol. (1979) 123, 1548-1550), P3x63Ag8U.1 (Yel ton, DE et al., Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 ( Kohler, G. and Milstein, C., Eur. J. Immunol.
  • the cell fusion between the immune cells and myeloma cells is basically known. Method, for example, the method of Milstein et al. (Gaifre, G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46). .
  • the cell fusion is performed, for example, in a normal nutrient culture in the presence of a cell fusion promoter.
  • a cell fusion promoter for example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like are used, and if necessary, an auxiliary such as dimethyl sulfoxide for enhancing the fusion efficiency.
  • An agent can be added and used.
  • the ratio of the use of the immune cells to the myeloma cells is preferably, for example, 10 times the number of the immune cells to the myeloma cells.
  • the culture medium used for the cell fusion for example, an RPM 640 culture medium, a MEM culture medium suitable for the growth of the myeloma cell line, and other ordinary culture mediums used for this kind of cell culture can be used.
  • serum replacement such as fetal calf serum (FCS) can be used in combination.
  • a predetermined amount of the immune cells and myeloma cells are mixed well in the culture medium, and the mixture is preliminarily heated to about 37 ° C., for example, a PEG solution having an average molecular weight of about 1000 to 6000.
  • the desired fused cells are formed by adding and mixing the cells at a concentration of 30-60% (w / v). Subsequently, by repeatedly adding an appropriate culture solution, and centrifuging to remove the supernatant, a cell fusion agent or the like that is not preferable for the growth of the hybridoma 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, aminobuterin and thymidine). Culture in the HAT culture medium is continued for a period of time sufficient to kill cells other than the target hybridoma (non-fused cells), usually several days to several weeks. Then, a conventional limiting dilution method is performed to screen the hybridomas that produce the desired antibody. And single cloning.
  • a HAT culture solution a culture solution containing hypoxanthine, aminobuterin and thymidine.
  • sensitizing human lymphocytes to -8 in vitro and sensitizing sensitized lymphocytes to human-derived permanent divisions It can also be fused with a functional myeloma cell, for example, U266, to obtain a desired human antibody having an activity of binding to IL-8 (see Japanese Patent Publication No. 1-59878).
  • a transgenic animal having a human antibody gene liver immunity is immunized with IL-8 as an antigen to obtain anti-IL-8 antibody-producing cells, and the immortalized cells are obtained. May be used to obtain a human antibody to I8 (International Patent Application Publication Nos. W092 / 03918, W093 / 12227, W094 / 02602, W094 / 25585, W096 / 33735 and W096 / 34096).
  • Hybridomas that produce monoclonal antibodies produced in this way can be subcultured in ordinary culture media and can be stored for a long period in liquid nitrogen. It is.
  • a method of culturing the hybridoma according to an ordinary method and obtaining the culture supernatant, or transferring the hybridoma to a mammal compatible therewith A method of transplanting, growing, and obtaining ascites is used.
  • the former method is suitable for obtaining high-purity antibodies, while the latter method is suitable for mass production of antibodies.
  • a recombinant antibody produced by cloning an antibody gene from a hybridoma, incorporating the antibody gene into an appropriate vector, introducing the antibody into a host, and producing the recombinant antibody using a gene recombination technique is described in the present invention.
  • a gene recombination technique See, for example, Borrebaeck, CK and Larrick, JW, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by ACMILLAN PUBLISHERS LTD, 1990).
  • 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 isolation can be performed by known methods, for example, guanidine ultracentrifugation (Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-5299), AGPC method (Chom czynski, P. and Sacchi, Prepare total RNA using N., Anal. Biochem. (1987) 162, 156-159) and purify mRNA from total RNA using mRNA Purification Kit (Pharmacia).
  • mRNA can be directly prepared by using the QuickPrep mRNA Purification Kit (Pharmacia).
  • cDNA for the antibody V region is synthesized using reverse transcriptase.
  • Synthesis of cDNA can also be performed using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku).
  • AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku).
  • PCR polymerase chain reaction
  • a recombinant vector is prepared from this, introduced into Escherichia coli or the like, and a colony is selected to prepare a desired recombinant vector.
  • the nucleotide sequence of the target DNA is confirmed by a known method, for example, the dideoxynucleotide titanium emission method.
  • 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, insert the DNA encoding the V region of the antibody into an expression vector that already contains the DNA of the C region of the antibody. Is also good.
  • 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.
  • an expression control region for example, an enhancer or a promoter.
  • host cells are transformed with the expression vector to express the antibody.
  • Expression of the antibody gene can be performed by co-transforming host cells by separately incorporating DNA encoding the heavy chain (H chain) or light chain (chain) of the antibody into an expression vector, or by transforming the host cell.
  • the host cell may be transformed by incorporating DNA encoding the L and L chains into a single expression vector (see International Patent Application Publication No. WO 94/11523).
  • a recombinant antibody artificially modified for the purpose of, for example, reducing the antigenicity to humans such as a chimeric antibody or a humanized antibody.
  • These modified antibodies can be produced using known methods.
  • the chimeric antibody is obtained by ligating the DNA encoding the antibody V region other than the human antibody obtained as described above to the DNA encoding the human antibody C region, and using this in the expression vector. It is obtained by incorporation, introduction into a host, and production (see European Patent Application Publication No. EP 125023, 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 H chain of Chimera WS-4 antibody was Escherichia coli DH5 (HEF-chWS4L-g c) and Escherichia coli JM109 (HEF-chWS4H, respectively).
  • -g r 1 at the Institute of Biotechnology and Industrial Technology (1-3 1-3, Higashi, Tsukuba, Ibaraki, Japan) on July 12, 1994, as FERM BP-4739 and FERM BP, respectively.
  • -4740 deposited internationally under the Budapest Treaty.
  • the humanized antibody is also referred to as a reshaped human antibody, and the complementarity determining region (CDR) of a mammal other than human, for example, a mouse antibody, is complemented with the human antibody. It has been transplanted into the sex-determining region, and its general genetic recombination technique is also known (see European Patent Application Publication No. EP 125023, International Patent Application Publication No. WO 96/02576).
  • a DNA sequence designed to link the mouse antibody CDR and the human antibody framework region (FR) is composed of several DNA sequences with overlapping portions at the ends.
  • the DNA is synthesized by splitting it into two oligonucleotides, and synthesized into a single DNA by PCR.
  • the obtained DNA is ligated to DNA encoding the human antibody C region, then incorporated into an expression vector, and then introduced into a host to produce the same (European Patent Application Publication No. EP 239400, See International Patent Application Publication No. W096 / 02576).
  • the human antibody FR linked via the CDR is selected so that the CDR forms a favorable antigen-binding site. If necessary, the amino acid of the PR in the V region of the antibody may be substituted so that the complementarity determining region of the humanized antibody forms an appropriate antigen-binding site (Sato, K. et al. , Cancer Res. (1993) 53, 851-856).
  • a human antibody C region is used depending on the purpose, and, for example, Ca1, Cr2, Cr3, and C4 can be used.
  • the human antibody C region may be modified in order to improve the stability of the antibody or its production.
  • the antibody subclass is selected to be G4
  • the amino acid sequence CPSCP of a part of the lgG4 hinge region is converted to the amino acid sequence CPPCP of the IgGl hinge region. It can eliminate the structural instability of IgG4 (Angal, S. et al., Mo 1. Immunol. (1993) 30, 105-108).
  • Chimeric antibodies consist of the V region of an antibody derived from a mammal other than a human and the C region derived from a human antibody.
  • the humanized antibody comprises the CDRs of an antibody derived from a mammal other than the human and the FRs derived from the human antibody.
  • amino acid sequences derived from mammals other than human are minimally reduced, resulting in a decrease in antigenicity in the human body, and as an active ingredient of the therapeutic agent of the present invention. Useful.
  • humanized WS-4 antibody the CDR of mouse-derived WS-4 antibody was used.
  • FR of human antibody RE1 was used.
  • FR3 of human antibody VDH26 and human antibody 4B4 were used. The amino acid residue of FR is partially substituted 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 Escherichia coli DH5a (HBF-RVLa- / c) and Escherichia coli J 109 (HEF, respectively).
  • the antibody used in the present invention may be an antibody fragment or a modified product thereof, as long as it binds to 1-8 and inhibits the activity of IL-8.
  • antibody fragments include Fab, F (ab ') 2, Fv, or single chain Fv (scFv) in which Hv and Lv Fv are linked by an appropriate linker.
  • an antibody is treated with an enzyme such as papine or pepsin to generate an antibody fragment, or a gene encoding these antibody fragments is constructed and introduced into an expression vector. And expressed in a suitable host cell (eg, Co, MS et al., J. Immunol. (1994) 152 , 968-2976; Better, M. and Horwitz, AH, Methods Enzymol.
  • a suitable host cell eg, Co, MS et al., J. Immunol. (1994) 152 , 968-2976; Better, M. and Horwitz, AH, Methods Enzymol.
  • scFv can be obtained by linking the V region of the H chain to the V region of the antibody.
  • the H chain V region and the L chain V region are linked via a linker, preferably a peptide linker (Huston, J.S. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883).
  • the H chain V region and L chain V region in the scFv may be derived from any of those described as the above antibodies.
  • the peptide linker that connects the V regions for example, an arbitrary single-chain peptide consisting of amino acid residues 12 to 19 is used.
  • the scFv-encoding DNA is a DNA encoding the H chain or the H chain V region of the antibody, and a DNA encoding the L chain or the L chain V region of the antibody.
  • a portion of the DNA encoding the desired amino acid sequence is amplified by PCR using a pair of primers that define both ends of the DNA, and then a DNase encoding a portion of the peptide linker is further amplified. It can be obtained by combining and amplifying a pair of primers that define A and both ends thereof to be linked to an H chain and an L chain, respectively.
  • expression vectors containing them and a host transformed with the expression vectors can be obtained according to a conventional method. Can be used to obtain scFv in accordance with a conventional method.
  • antibody fragments can be obtained and expressed in the same manner as described above, and produced by a host.
  • antibody also includes fragments of these antibodies.
  • an anti-R8 antibody conjugated to various molecules such as polyethylene glycol (PEG) can also be used.
  • PEG polyethylene glycol
  • the “antibody” referred to in the claims of the present application also includes these modified antibodies.
  • Such a modified antibody can be obtained by chemically modifying the obtained antibody. These methods are already established in this field.
  • the antibody gene constructed as described above can be expressed and obtained by a known method.
  • expression is carried out in an expression vector containing a commonly used useful promoter, an antibody gene to be expressed, and a DNA to which a polyA signal is functionally linked downstream of the 3 'end.
  • the promoter Z enhancer includes human cytomegalovirus early promoter / enhancer (human cy to megalovirus immediate ear promoter / enhancer).
  • SV40 immunoreactor 40
  • HEF1a hypertrophy function ia
  • the method of Mu 11 igan, RC et al. (Nature (1979) 277, 108-114), or when using the HEFla promoter-Z enhancer, It can be easily carried out according to the method of Mizushima, S et al. (Nucleic Acids Res. (1990) 18, 5322).
  • Escherichia coli it can be expressed by functionally linking a useful promoter commonly used, a signal sequence for antibody secretion, and a gene to be expressed.
  • examples of the promoter include a lacz promoter and an araB promoter. When using the lacz promoter, the method of Ward, ES et al.
  • the pelB signal sequence (Lei, SP et al., J. Bacteriol. (1987) 169, 4379-4383) may be used as a signal sequence for antibody secretion when producing E. coli periplasm. I just need. After isolating the antibody produced in the periplasm, the antibody structure is appropriately refolded and used (see, for example, International Patent Application Publication No. WO 96/30394).
  • the expression vector is used as a selectable marker as an aminoglycoside trans- ferase (APH) gene, a thymidine kinase (TK) gene, or Escherichia coli xanthine guanine phosphonate. It can contain a ribosyl transferase (Ecogpt) gene, a dihydrofolate reductase (dhfr) gene, and the like.
  • APH aminoglycoside trans- ferase
  • TK thymidine kinase
  • Escherichia coli xanthine guanine phosphonate Escherichia coli xanthine guanine phosphonate. It can contain a ribosyl transferase (Ecogpt) gene, a dihydrofolate reductase (dhfr) gene, and the like.
  • any production system can be used. Production systems for producing antibodies include in Vro
  • Examples of the in vitro production system include a production system using eukaryotic cells and a production system using prokaryotic cells.
  • animal cells When using eukaryotic cells, use animal cells, plant cells, and fungal cells. Production systems.
  • animal cells include (1) mammalian cells, for example, CH0, COS, myeloma, BH (baby hamster kidney), HeLa, Vero, (2) amphibian cells, for example, African toad frog oocytes, or (3) Insect cells such as sf9, sf21, and Tn5 are known.
  • plant cells for example, cells derived from the genus Nicotiana, more specifically, cells derived from Nicotiana tabacum, are known to be callus-cultured.
  • fungal cells examples include (1) yeast, for example, genus Saccharomyces, more specifically, Saccharomyces cerevisiae, or (2) filamentous fungi, for example, The genus Aspergillus, specifically, Aspergillus niger, is known.
  • Escherichia coli Escherichia coli
  • Bacillus subtilis are known as bacterial cells.
  • the desired antibody gene is introduced into these cells by transformation.
  • Antibodies can be obtained by culturing the transformed cells in vitro. Culture is performed according to a known method. For example, DMEM, MEM, RPMI 1640. IMDM, or the like can be used as a culture solution for mammalian cells, and a serum collection solution such as fetal calf serum (FCS) can be used in combination. Alternatively, the antibody may be produced in vivo by implanting the cells into which the antibody gene has been introduced into the peritoneal cavity of the animal.
  • FCS fetal calf serum
  • Further in vivo production systems include production systems using animals and plants. When using animals, there are production systems using mammals and insects.
  • mice As mammals, goats, pigs, sheep, mice, and mice can be used (Glaser, V., SPECTRUM Biotechnology Appli cat i ons, 1993). When a plant that can use silkworm is used as an insect, for example, tobacco can be used.
  • an antibody gene is introduced into these animals or plants, and antibodies are produced and recovered in the animals or plants.
  • an antibody gene is inserted into a gene encoding a protein uniquely produced in milk, such as goat casein, to prepare a fusion gene.
  • a DNA fragment containing the fusion gene into which the antibody gene has been introduced is injected into a goat embryo, and this embryo is introduced into a female goat.
  • the desired antibody is obtained from the milk produced by the transgenic X nicka or the progeny of the goat that has received the embryo.
  • formone may be appropriately used in the transgenic kid. (Ebert, K. M. et al., Bio / Technology (1994) 12, 699-702).
  • the baculovirus into which the target antibody gene is inserted is infected to the silkworms, and a desired antibody is obtained from the body fluid of the silkworms (Maeda, S. et al., Nature (1985)). 315, 592-594).
  • the antibody gene of interest is introduced into a vector for plant expression, for example, pMON530, and this vector is used to transform Agrobacterium tumefaciens into Agrobacterium tumefaciens. Introduce them into the territory.
  • Nicotiana tabacum Naturala tabacum
  • Nicotiana tabacum Naturala tabacum
  • the DNA encoding the H or L chain of the antibody is separately incorporated into an expression vector, and the host is co-transformed.
  • H chain And the DNA encoding the L chain can be incorporated into a single expression vector to transform the host (see International Patent Application Publication No. WO 94/11523).
  • 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. Separation and purification of the antibody used in the present invention can be performed by affinity chromatography.
  • Columns used for affinity chromatography include, for example, protein A column and protein G column.
  • Hyper D, P0R0S, Sepharose FF (Pharmacia) and the like can be mentioned.
  • any other separation and purification methods used for ordinary proteins may be used, and there is no limitation.
  • antibodies can be separated by appropriately selecting and combining chromatographic single columns other than the above-mentioned affinity chromatographs, final letters, ultrafiltration, salting out, dialysis, etc.
  • Chromatography other than affinity chromatography includes, for example, ion-exchange chromatography, hydrophobic chromatography, gel filtration, and the like.
  • the concentration of the antibody obtained in step 7 can be measured by absorbance measurement or enzyme-linked immunosorbent assay (ELISA) or the like.
  • ELISA enzyme-linked immunosorbent assay
  • the absorbance at 280 nm is measured, and the extinction coefficient varies depending on the species and subclass.
  • 1 mg / ml is calculated as 1.40D.
  • the measurement can be performed as follows. That is, goat anti-human IgG antibody 1001 diluted to 1 g / ml with 0.1 M bicarbonate buffer (pH 9.6) was added to a 96-well plate (Nunc) and incubated at 4 ° C for 10 min.
  • the antigen-binding activity of the antibody used in the present invention (Antibodies: A Labora tory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), the ligand receptor binding inhibitory activity (Harada, A. et al., Known methods can be used for the measurement of Int. Immunol. (1993) 5, 681-690).
  • EL1SA As a method for measuring the antigen binding activity of the anti-IL-8 antibody used in the present invention, EL1SA, EIA (enzyme immunoassay), RIA (radioimmunoassay) or a fluorescent antibody method can be used.
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • fluorescent antibody method a fluorescent antibody method.
  • Ile8 is added to a 96-well plate on which a polyclonal antibody against 1L-8 is immobilized, and then a sample containing the desired pile-8 antibody, for example, -Add culture supernatant of 8 antibody-producing cells and purified antibody.
  • the antigen binding activity can be evaluated by adding an enzyme substrate such as P-diphenylphosphoric acid and measuring the absorbance.
  • IL-8 receptor such as neutrophils
  • a membrane fraction of cells expressing the 1L-8 receptor is prepared, and a solid-phased 96-well plate is prepared.
  • a sample containing anti-1 Le 8 antibody for example, a culture supernatant or purified antibody anti IL- 8 antibody-producing cells, radioisotopes, For example, labeled with 1 25 1 etc. - adding 8
  • the amount of IL-8 bound to the 1L-8 receptor can be measured by measuring the radioactivity, and the inhibitory activity of the anti-IL-8 antibody on ligand receptor 1 binding Can be evaluated.
  • an assay for inhibiting the binding of I-8 to the 1L-8 receptor on cells includes blood cells or cancer cells that express the IL-8 receptor, such as neutrophils, separated by centrifugation or other means. Then, it is prepared as a cell suspension.
  • Cell suspension of a solution containing a radioactive isotope, for example, 12 -labeled 1-8, or a mixed solution of unlabeled -8 and labeled 1-8, and a concentration-adjusted solution containing anti-8-antibody Add to the suspension. After a certain period of time, the cells may be separated and the radioactivity of labeled Ile8 bound on the cells may be measured.
  • chemotaxis As a method for measuring the ability of the anti-1R8 antibody used in the present invention to inhibit neutrophil migration (chemotaxis), a known method, for example, Grob, PM These methods (J. Biol. Chem. (1990) 265, 8311-8316) can be used. Specifically, using a commercially available chemotaxis chamber,
  • the prepared cell suspension for example, a neutrophil suspension
  • the number of the cells may be measured by a method using a staining solution or a fluorescent antibody.
  • 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, etc. Can be administered in a controlled manner.
  • the administration method can be appropriately selected depending on the age and symptoms of the patient.
  • the therapeutic agent containing the anti-1L-8 antibody of the present invention as an active ingredient can cure or at least partially prevent the symptoms of the disease and its complications in patients already suffering from the disease. Is administered in a sufficient amount.
  • the effective dose is selected from the range of 0.1 Olmg to 100000 / kg body weight at a time.
  • a dose of 5-2000 mg / body per patient can be chosen.
  • the therapeutic agent containing the anti-8 antibody of the present invention is not limited to these doses.
  • the administration may be performed after myocardial infarction, unstable angina pectoris or myocardial ischemia-reperfusion injury occurs, or reperfusion after temporarily blocking blood flow It may be given at the time of administration, when a thrombolytic agent is used, or when reperfusion is predicted after PTCA.
  • the therapeutic agent containing the anti-1L-8 antibody of the present invention as an active ingredient can be formulated according to a conventional method (Remington's Pharmaceutical S.A.). cience, latest edition, Mark Publishing Company, Easton, USA), and pharmaceutically acceptable carriers and additives.
  • Examples of such carriers and excipients include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, vinyloxyvinylpolymer, and vinyloxamine.
  • Methyl cell mouth sodium, sodium polyacrylate, sodium alginate, water-soluble dextran, carboxymethyl starch sodium, zinc, Methylcellulose, ethylcellulose, xanthan gum, arabic gum, casein, agar, polyethylene glycol, diglycellin, glycerin, propylene glycol, vase Lin, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, Surfactants and the like that are acceptable as pharmaceutical additives.
  • HSA human serum albumin
  • the actual additive is selected appropriately or in combination from the above according to the dosage form of the therapeutic agent of the present invention, but is not limited thereto.
  • the purified anti-8 antibody when used as an injection, 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 20, gelatin, human serum albumin and the like can be used.
  • a solvent for example, a physiological saline solution, a buffer solution, a glucose solution, and the like
  • an anti-adsorption agent for example, Tween 80, Tween 20, gelatin, human serum albumin and the like
  • the lyophilization vehicle include sugar alcohols and sugars such as mannitol and glucose. Can be used.
  • a therapeutic agent containing the anti-1L-8 antibody or a fragment thereof of the present invention as an active ingredient is useful as a therapeutic agent for myocardial infarction, unstable angina and myocardial ischemia-reperfusion injury. .
  • BALB / c mice were immunized with human IL-8 by a conventional method, and spleen cells were collected from the immunized mice.
  • 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. Screening was performed using the binding activity to human 1L-8 as an index, and as a result, a hybridoma cell line WS-4 was obtained.
  • the antibody produced by the hybridoma WS-4 inhibited the binding of human IL-8 to neutrophils and had a neutralizing activity.
  • H- and L-chain isotypes of antibody produced by Hypri-Doma WS-4 were analyzed using mouse monoclonal antibody isotyping kit. I checked. As a result, it was revealed that the antibody produced by the hybridoma WS-4 had a mouse c-type chain and a mouse type 1 H chain o
  • Hypri-doma cell line WS-4 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 Prefecture) on April 17, 1996. On the day, it was deposited internationally under the Budapest Treaty as FERM BP-5507.
  • Humanized WS-4 antibody was prepared by the method described in International Patent Application Publication No. WO 96-02576.
  • RNAs were prepared by a conventional method, and single-stranded cDNA was synthesized therefrom.
  • the DNA encoding the V region of the H and L chains of the mouse WS-4 antibody was amplified by PCR. Primers used in the PCR method were primers described in Jones, ST and Bendig, M.M., Bio / Technology (1991) 9, 88-89.
  • 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 gene encoding the mouse WS-4 antibody H chain V region are purified. DNA fragment was isolated.
  • Each of these DNA fragments was ligated to a plasmid pUC-based cloning vector and introduced into E. coli competent cells to obtain an E. coli transformant.
  • 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 was determined by a conventional method, and the CDR of each V region was identified from the amino acid sequence.
  • a cDNA encoding the V region of the L chain and H chain of the mouse WS-4 antibody is previously coded for the human C region. Separately insert each DNA into HEF vector Entered.
  • the V region CDR of the mouse WS-4 antibody was transplanted to a human antibody using a genetic technique based on CDR transplantation.
  • substitution of a DNA sequence was performed to partially replace the amino acid of FR in the V region of the CDR-grafted antibody.
  • the DNAs encoding the respective regions are added to a HEF vector. These were separately inserted to prepare vectors expressing the L chain or H chain of the humanized WS-4 antibody.
  • a cell line producing a humanized WS-4 antibody was established.
  • the ability of humanized WS-4 antibody obtained by culturing this cell line to bind to IL-8 and to neutralize IL-8 was determined by ELISA and IL-8 / neutrophil binding inhibition test, respectively. Examined. As a result, it was found that the humanized WS-4 antibody binds to human IL-8 and inhibits the binding of 1L-8 to neutrophils to the same extent as the mouse WS-4 antibody. did.
  • Escherichia coli having a plasmid containing the L chain and the H chain of the humanized WS-4 antibody were Escherichia coli DH5a (HEF-VLa-g) and Escherichia coli JM109 (HBF- RVHg-g 1) was submitted to the Institute of Biotechnology, Institute of Industrial Science and Technology (I 1-3 1-3 Higashi, Tsukuba, Ibaraki Prefecture) on July 12, 1994, as FERM BP-4738 and FERM BP, respectively. -Deposited 4741 internationally under the Budapest Treaty.
  • Nembutal dynabot, dose 25-30 mg / kg body weight
  • the trachea was exposed by longitudinal anterior neck incision, an endotracheal intubation tube was introduced by tracheostomy, and the chest was opened using an electric massager and thoracotomy device under artificial respiration. At that time, mixed gas (95% oxygen, carbon dioxide, etc.) was added to the inhaled air to maintain the blood oxygen partial pressure at 130-140 mmHg. After exposing the heart and pericardiotomy, a thread was threaded on the left circumflex of the coronary artery. Electrodes of an electrocardiograph (Nihon Kohden) were set on the limbs and allowed to stand for 1 hour.
  • CK activity and LDH activity For the measurement of CK activity and LDH activity, 0.5 ml of blood was collected from the femoral vein by heparin (shimizu) blood collection, and the same treatment was performed over time.
  • the left circumflex branch of the coronary artery was ligated using a polyethylene tube (hibiki size6) and a modified arterial clamp (jujube), and discoloration of the ischemic area and elevation of the ST segment on the electrocardiogram were confirmed.
  • a polyethylene tube hibiki size6
  • a modified arterial clamp jujube
  • a mouse WS-4 antibody against human IL-8 at a concentration of 5 mg / ml (solvent: Na-Na phosphate buffer PH6.0) was administered over a period of 1 minute (dose of 5 mg / kg body weight, lml / kg body weight) or a solvent (lml / kg body weight) was administered from the femoral vein as a negative control.
  • a solvent lml / kg body weight
  • the value of the WS-4 antibody-administered group increased sharply due to reperfusion, but was lower than the average value of the solvent-administered group, and thereafter remained constant and suppressed the increase. This indicated that administration of the WS-4 antibody suppressed an increase in blood CK activity, which is an indicator of cardiomyocyte damage (FIG. 1).
  • the mean LDH activity in the blood in the vehicle-administered group increased sharply by reperfusion, and then gradually decreased over time.
  • the value of the WS-4 antibody-administered group increased sharply by reperfusion, the value was lower than that of the solvent-administered group, and the degree of the subsequent decrease was stronger than that of the solvent-administered group. This indicated that administration of the WS-4 antibody suppressed an increase in blood LDH activity, which is an indicator of cardiomyocyte damage (Fig. 2).
  • WS-4 antibody suppressed the release of CK and LDH into the blood, which are caused by damage to cardiomyocytes due to ischemia reperfusion. It means that myocardial cells were protected against perfusion injury. Therefore, it was shown to be useful as a therapeutic agent for myocardial ischemia-reperfusion injury including myocardial infarction.
  • the infarct lesion can be quantified by performing the following processing.
  • the blood was removed by injecting 10 ml of physiological saline (Otsuka Pharmaceutical) retrograde from the aorta of the isolated heart, followed by religating the thread used for ischemia left in the left circumflex and re-ligating the aorta.
  • the isolated heart was separated into a normal perfused area stained with Evans blue and an ischemic area not stained with Evans blue.
  • the ischemic area was in a solution of triphenyl nitrazolium chloride (Sigma, TTC) at 37 ° C.
  • the infarct lesion can be quantified by measuring the tissue weight of each of the normal perfusion site, the ischemic region non-necrotic region, and the ischemic region necrotic region.
  • anti-1-8 antibody reduced blood levels of CK activity and LDH activity. This fact indicates that the anti-IL-8 antibody is useful as a therapeutic agent for myocardial infarction, unstable angina and myocardial ischemia-reperfusion injury.

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Abstract

Médicament contre l'infarctus du myocarde, l'angor instable et les troubles de la reperfusion ischémique myocardique, contenant comme principe actif un anticorps anti-interleukine 8.
PCT/JP1997/001405 1996-04-23 1997-04-23 Medicament contre l'infarctus du myocarde contenant comme principe actif un anticorps anti-il-8 WO1997040215A1 (fr)

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AU24051/97A AU2405197A (en) 1996-04-23 1997-04-23 Myocardial infarction remedy containing anti-il-8 antibody as active ingredient

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JP13735896 1996-04-23
JP8/137358 1996-04-23

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6458355B1 (en) 1998-01-22 2002-10-01 Genentech, Inc. Methods of treating inflammatory disease with anti-IL-8 antibody fragment-polymer conjugates
US6468532B1 (en) 1998-01-22 2002-10-22 Genentech, Inc. Methods of treating inflammatory diseases with anti-IL-8 antibody fragment-polymer conjugates
US6870033B1 (en) 1997-02-21 2005-03-22 Genentech, Inc. Antibody fragment-polymer conjugates and humanized anti-IL-8 monoclonal antibodies
US7214776B2 (en) 1999-01-21 2007-05-08 Genentech, Inc. Antibody fragment-polymer conjugates and uses of same

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996002576A1 (fr) * 1994-07-13 1996-02-01 Chugai Seiyaku Kabushiki Kaisha Anticorps humain reconstitue contre l'interleukine-8 humaine

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996002576A1 (fr) * 1994-07-13 1996-02-01 Chugai Seiyaku Kabushiki Kaisha Anticorps humain reconstitue contre l'interleukine-8 humaine

Non-Patent Citations (3)

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Title
CARDIOVASCULAR RESEARCH, 1995, Vol. 29, TAKAHASHI MASAFUMI et al., "Effects of Endotherial Interleukin-8 on Neutrophil Migration Across an Endothelial monolayer", pages 670-675. *
CHEMICAL ABSTRACTS, 1994, Vol. 121, Abstract No. 131719, TADASHI MUKODA et al., "Interleukin-8 and Reflow Disorder (in Japanese)"; & JIKKEN IGAKU, 1994, Vol. 12, No. 10, pages 65 to 67, Particularly page 67, "In Conclusion". *
NATURE, 1993, Vol. 365, SEKIDO NOBUAKI et al., "Prevention of Lung Reperfusion Injury in Rabbits by a Monoclonal Antibody Against Interleukin-8", pages 654-657. *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6870033B1 (en) 1997-02-21 2005-03-22 Genentech, Inc. Antibody fragment-polymer conjugates and humanized anti-IL-8 monoclonal antibodies
US6458355B1 (en) 1998-01-22 2002-10-01 Genentech, Inc. Methods of treating inflammatory disease with anti-IL-8 antibody fragment-polymer conjugates
US6468532B1 (en) 1998-01-22 2002-10-22 Genentech, Inc. Methods of treating inflammatory diseases with anti-IL-8 antibody fragment-polymer conjugates
US7214776B2 (en) 1999-01-21 2007-05-08 Genentech, Inc. Antibody fragment-polymer conjugates and uses of same
US7507405B2 (en) 1999-01-21 2009-03-24 Genentech, Inc. Antibody fragment-polymer conjugates and uses of same
US7842789B2 (en) 1999-01-21 2010-11-30 Genentech, Inc. Antibody fragment-polymer conjugates and uses of same
US8147830B2 (en) 1999-01-21 2012-04-03 Genentech, Inc. Antibody fragment-polymer conjugates and uses of same
US8652468B2 (en) 1999-01-21 2014-02-18 Genentech, Inc. Methods of binding TNF-α using anti-TNF-α antibody fragment-polymer conjugates

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