US20070134242A1 - Mesothelioma therapeutic agent - Google Patents

Mesothelioma therapeutic agent Download PDF

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US20070134242A1
US20070134242A1 US10/575,455 US57545504A US2007134242A1 US 20070134242 A1 US20070134242 A1 US 20070134242A1 US 57545504 A US57545504 A US 57545504A US 2007134242 A1 US2007134242 A1 US 2007134242A1
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antibody
receptor
mesothelioma
cells
human
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Norihiro Nishimoto
Tadamitsu Kishimoto
Yasuo Adachi
Koichi Takayama
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Chugai Pharmaceutical Co Ltd
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Chugai Pharmaceutical Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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]
    • C07K16/248—IL-6
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to a novel mesothelioma therapeutic agent and mesothelioma cell inhibitor.
  • Mesothelioma is a tumor that occurs in the mesothelium that covers the surface of the pleura, peritoneum and pericardium that respectively envelop the organs of the chest cavity such as the lungs and heart, and abdominal organs such as the digestive tract and liver.
  • chest pain is caused by invasion of the intercostals nerves on the side of the chest wall pleura, and respiratory and circulatory disorders may occur due to tumor growth and accumulation of pleural fluid in the pleura on the organ side (Takagi, Journal of Clinical and Experimental Medicine, (March Supplement), “Respiratory Diseases”, pp. 469-472, 1999).
  • malignant mesothelioma has a causative relationship with exposure to asbestos, and this has also been demonstrated in animal experiments (Tada, Journal of Clinical and Experimental Medicine (March Supplement), “Respiratory Diseases”, pp. 406-408, 1999). Asbestos that has been inhaled into the respiratory tract reaches a location directly beneath the pleura where a tumor eventually develops due to chronic irritation for at least about 20 years, and this tumor spreads in a thin layer over the entire surface of the pleura (Takagi, Journal of Clinical and Experimental Medicine (March Supplement), “Respiratory Diseases”, pp. 469-472, 1999).
  • pleuropulmonary excision and pleurectomy are performed for diffuse pleural mesothelioma
  • susceptibility to relapse following treatment there is increased susceptibility to relapse following treatment, and the post-surgical local relapse rate in particular is high at 35-43% (Takagi, Journal of Clinical and Experimental Medicine (March Supplement), “Respiratory Diseases”, pp. 469-472, 1999).
  • serum IL-6 levels in patients with malignant pleural mesothelioma are higher in comparison with pulmonary adenoma patients complicated with pleural effusion, and with respect to thrombocytosis, which is one of the clinical symptoms of malignant pleural mesothelioma, there is known to be a remarkable correlation between serum IL-6 levels and platelet counts (Nakano, British Journal of Cancer 77(6): 907-912, 1998).
  • the tumor cells of pleural mesothelioma patients express high levels of IL-6, and IL-6 levels in the serum have been reported to increase prior to death (Higashihara, Cancer, Oct. 15, 1992, Vol. 70, No. 8, pp. 2105-2108).
  • Bielefeldt-Ohmann, et al. reported that effects were observed that considerably diminished the onset and progression of clinical symptoms (Bielefeldt-Ohmann, Cancer Immunol. Immunother. 40: 241-250, 1995).
  • anti-IL-6 antibody does not have a direct growth inhibitory effect on AB22 in vitro, there were no differences observed in the postmortem appearances of mice treated with anti-IL-6 antibody and those not treated with said antibody, and tumor masses of considerable size were observed even in the treated mice (Bielefeldt-Ohmann, Cancer Immunol. Immunother. 40: 241-250, 1995). Namely, growth inhibition of mesothelioma by anti-IL-6 antibody has not been known both in vitro and in vivo.
  • An object of the present invention is to provide a novel mesothelioma therapeutic agent (mesothelioma cell growth inhibitor) that contains an IL-6 antagonist as its active ingredient.
  • the inventor of the present invention obtained the novel finding that the growth of mesothelioma cells can be inhibited by inhibiting or interrupting signal transmission relating to IL-6, thereby leading to completion of the present invention.
  • the present invention provides a mesothelioma therapeutic agent that contains an interleukin-6 (IL-6) antagonist as its active ingredient.
  • IL-6 interleukin-6
  • the present invention also provides a growth inhibitor against mesothelioma cells that contains an interleukin-6 (IL-6) antagonist as its active ingredient.
  • IL-6 interleukin-6
  • the aforementioned mesothelioma is, for example, pleural mesothelioma, and more specifically, malignant pleural mesothelioma. Diffuse pleural mesothelioma is included in malignant pleural mesothelioma.
  • the aforementioned IL-6 antagonist is, for example, an antibody to IL-6 or an antibody to IL-6 receptor, and preferably a monoclonal antibody to IL-6 receptor.
  • the aforementioned antibody to IL-6 receptor is particularly preferably a monoclonal antibody to human IL-6 receptor such as PM-1 antibody, or a monoclonal antibody to mouse IL-6 receptor such as MR16-1 antibody.
  • the aforementioned antibody to IL-6 receptor is preferably a recombinant antibody.
  • the aforementioned antibody to IL-6 receptor may be a chimeric antibody, humanized antibody or human antibody.
  • a particularly preferable antibody is humanized PM-1 antibody.
  • the present invention can also be in the forms indicated below.
  • IL-6 interleukin-6
  • the present invention can also adopt the forms indicated below.
  • a treatment method for mesothelioma comprising: administering an interleukin-6 (IL-6) antagonist to a subject requiring that treatment.
  • IL-6 interleukin-6
  • a method for inhibiting the growth of mesothelioma cells comprising: administering an interleukin-6 (IL-6) antagonist to a subject requiring that inhibition.
  • IL-6 interleukin-6
  • FIG. 1 is a graph that shows the results of Example 1 by indicating the productivities of IL-6 by various malignant mesothelioma cell lines.
  • FIG. 2 is a graph that shows the results of Example 2 by indicating the productivities (lack thereof) of IL-6 receptor (IL-6R) by various malignant mesothelioma cell lines. Furthermore, GAPDH indicates the amount of mRNA of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) used as an internal control.
  • FIG. 3 is a graph that shows the results of Example 3, wherein the production of vascular endothelial growth factor (VEGF) by malignant mesothelioma cell lines H2052 and H2452 is induced by IL-6 and IL-6R.
  • VEGF vascular endothelial growth factor
  • FIG. 4 is a graph that shows the results of Example 4 in which a similar experiment to Example 3 was conducted for malignant mesothelioma cell line H28 by indicating that this cell line produces vascular endothelial growth factor (VEGF) without requiring induction by IL-6/IL-6R.
  • VEGF vascular endothelial growth factor
  • FIG. 5 is a graph that shows the results of Example 5, wherein in contrast to phosphorylation of STAT3 due to stimulation by IL-6 being promoted in cell line H2025, in which production of VEGF is induced by IL-6, phosphorylation of STAT3 due to stimulation by IL-6 is not promoted in cell line H28, in which production of VEGF is not induced by IL-6.
  • FIG. 6 is a graph that shows the results of Example 6, wherein in contrast to expression of SOCS3 due to stimulation by IL-6 and IL-6R being induced in cell line H2025, in which production of VEGF is induced by IL-6, SOCS3 is non-inductively expressed in cell line H28, in which production of VEGF is not induced by IL-6.
  • FIG. 7 is a drawing showing the structure of a promoter of plasmids pGL3-VEGF and pGL3-VEGFmut along with its vicinity used in Example 7.
  • FIG. 8 is a graph that shows the results of Example 7, wherein in a system in which VEGF promoter is coupled to luciferase reporter gene, in the case of altering the p-STAT3 binding site within the VEGF promoter, activation of the VEGF promoter by IL-6 does not occur.
  • FIG. 9 is a schematic drawing showing the mechanism of induction of VEGF promoter (production of VEGF) due to stimulation by IL-6 in cell line H2052 as predicted from the results of Examples 5 through 7.
  • FIG. 10 is a graph that shows the results of Example 8, wherein the growth of cell line H2052 increases IL-6 concentration-dependently in the presence of IL-6R.
  • FIG. 11 is a graph that shows the results of Example 9, wherein promotion of the growth of cell line H2052 by IL-6 and IL-6R is inhibited by MRA.
  • FIG. 12 is a graph that shows the results of Example 10, wherein the growth of cell line H226 increases IL-6 concentration-dependently in the presence of IL-6R, and that MRA demonstrates inhibitory effects on that growth.
  • FIG. 13 is a graph that shows the results of Experiment 8 by indicating that the growth of cell line H226 increases IL-6 concentration-dependently in the presence of IL-6R.
  • FIG. 14 is a graph that shows the results of Example 11, wherein growth promotion of malignant mesothelioma cell lines H2052 and H226 by IL-6 and soluble IL-6 receptor is inhibited by humanized PM-1 antibody.
  • FIG. 15 is a graph that shows the results of Example 12, wherein induction of VEGF production by IL-6 stimulation in the malignant mesothelioma cell lines MSTO, H226, H2052 and H2452 is inhibited by humanized PM-1 antibody.
  • FIG. 16 is a graph that shows the results of Example 13, wherein phosphorylation of STAT3 induced by IL-6/soluble IL-6R stimulation in the cell lines H2052 and H2452 is inhibited by humanized PM-1 antibody.
  • FIG. 17 is a graph that shows the results of Example 14, wherein promotion of the growth of the cell lines H2052 and H226 by IL-6 and soluble IL-6R stimulation is not inhibited by anti-VEGF antibody.
  • IL-6 is a cytokine also referred to as B cell stimulating factor 2 (BSF2) or interferon ⁇ 2.
  • BSF2 B cell stimulating factor 2
  • IL-6 was initially discovered to be differentiation factor involved in activation of B lymphocytic cells (Hirano, T. et al., Nature (1986) 324, 73-76), after which it was determined to be a multifunctional cytokine having effects on the functions of various cells (Akira, S. et al., Adv. In Immunology (1993) 54, 1-78). IL-6 has also been reported to induce maturation of T lymphocytic cells (Lotz, M. et al., J. Exp. Med. (1988) 167, 1253-1258).
  • IL-6 transmits its biological activity by means of various proteins on cells.
  • One of these is the ligand-binding protein of IL-6 receptors having a molecular weight of about 80 kD (Taga, T. et al., J. Exp. Med. (1987) 166, 967-981; Yamasaki, K. et al., Science (1987) 241, 825-828).
  • IL-6 receptors In addition to existing in a membrane-bound form that is expressed on the cell membrane by penetrating the cell membrane, IL-6 receptors also exist as soluble IL-6 receptors mainly composed of an extracellular region.
  • gp130 Another of these a membrane protein gp130 having a molecular weight of about 130 kD that is involved in the signal transmission of non-ligand binding.
  • IL-6 and IL-6 receptors form an IL-6/IL-6 receptor complex, and as a result of subsequently binding with gp130, the biological activity of IL-6 is transmitted into cells (Taga, T. et al., Cell (1989) 58, 573-581).
  • IL-6 antagonists are substances that inhibit the transmission of the biological activity of IL-6.
  • IL-6 antagonists include antibody to IL-6 (anti-IL-6 antibody), antibody to IL-6 receptors (anti-IL-6 receptor antibody), antibody to gp130 (anti-gp130 antibody), IL-6 variant, and IL-6 or IL-6 receptor partial peptide.
  • the aforementioned IL-6 antagonist is preferably an antibody to IL-6 receptor, and more preferably a monoclonal antibody to human IL-6 receptor or monoclonal antibody to mouse IL-6 receptor.
  • An example of the aforementioned monoclonal antibody to human IL-6 receptor is PM-1 antibody, while an example of monoclonal antibody to mouse IL-6 receptor is MR16-1 antibody.
  • the aforementioned antibody is preferably a chimeric antibody, humanized antibody or human antibody, an example of which is humanized PM-1 antibody.
  • IL-6 antagonist used in the present invention provided it inhibits the growth of mesothelioma cells and is useful as an active ingredient of a mesothelioma therapeutic agent.
  • IL-6 antagonists are substances that inhibit the biological activity of IL-6 by interrupting signal transmission by IL-6.
  • IL-6 antagonists are preferably substances that have inhibitory action against the binding of IL-6, IL-6 receptor and gp130.
  • Examples of IL-6 antagonists include anti-IL-6 antibody, anti-IL-6 receptor antibody, anti-gp130 antibody, IL-6 variants, soluble IL-6 receptor variants, IL-6 receptor partial peptides and low molecular weight substances demonstrating similar activity.
  • Anti-IL-6 antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using known methods.
  • Monoclonal antibody of mammalian origin is particularly preferable for the anti-IL-6 antibody used in the present invention.
  • monoclonal antibodies of mammalian origin include those produced by hybridomas and those produced by hosts transformed with an expression vector containing antibody gene using genetic engineering techniques. This antibody interrupts the transmission of the biological activity of IL-6 to cells as a result of inhibiting binding of IL-6 to IL-6 receptors by binding with IL-6.
  • Anti-IL-6 antibody-producing hybridoma can basically be produced in the manner described below using known technology. Namely, this antibody can be produced by using IL-6 as a sensitizing antigen, immunizing with this in accordance with ordinary immunization methods, fusing the resulting immunocytes with known host cells according to ordinary cell fusion methods, and then screening for cells that produce monoclonal antibody according to ordinary screening methods.
  • IL-6 antibody production of IL-6 antibody should be carried out in the manner described below.
  • Human IL-6 used as sensitizing antigen for acquiring antibody is obtained by using the IL-6 gene/amino acid sequence disclosed in Eur. J. Biochem. (1987) 168, 543-550; J. Immunol. (1988) 140, 1534-1541; or, Agr. Biol. Chem. (1990) 54, 2685-2688.
  • the target IL-6 protein is purified by known methods from the host cells or culture supernatant, followed by using this purified IL-6 protein as a sensitizing antigen.
  • a fused protein consisting of IL-6 protein and another protein may also be used as sensitizing antigen.
  • the anti-IL-6 receptor antibody used in the present invention can be obtained in the form of a polyclonal antibody or monoclonal antibody using known means.
  • Monoclonal antibody of mammalian origin is particularly preferable for the anti-IL-6 receptor antibody used in the present invention.
  • Examples of monoclonal antibody originating in mammalian cells include those produced by hybridomas and those produced in a host transformed with an expression vector that contains antigen gene by genetic engineering techniques. As a result of this antibody binding to IL-6 receptors, binding of IL-6 to IL-6 receptors is inhibited, thereby interrupting the transmission of the biological activity of IL-6 to cells.
  • Examples of these antibodies include MR16-1 antibody (Tamura, T. et al., Proc. Natl. Acad. Sci. USA (1993) 90, 11924-11928), PM-1 antibody (Hirata, Y. et al., J. Immunol. (1989) 143, 2900-2906), AUK12-20 antibody, AUK64-7 and AUK146-15 antibody (International Unexamined Patent Publication No. WO 92-19759).
  • PM-1 antibody is particularly preferable for use as the antibody.
  • a PM-1 antibody-producing hybridoma cell line has been internationally deposited based on the Budapest Treaty under the designation FERM BP-2998 on Jul. 12, 1989 at the International Patent Organism Depository of the National Institute of Advanced Industrial Science and Technology (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan) as PM-1.
  • an MR16-1 antibody-producing hybridoma cell line has been internationally deposited based on the Budapest Treaty under the designation FERM BP-5875 on Mar. 13, 1997 at the International Patent Organism Depository of the National Institute of Advanced Industrial Science and Technology (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan) as Rat-mouse hybridoma MR16-1.
  • Anti-IL-6 receptor monoclonal antibody-producing hybridoma can basically be produced in the manner described below using known technology. Namely, this hybridoma can be produced by using IL-6 receptor as sensitizing antigen, immunizing with this in accordance with ordinary immunization methods, fusing the resulting immunocytes with known host cells according to ordinary cell fusion methods, and screening for cells that produce monoclonal antibody according to known screening methods.
  • anti-IL-6 receptor antibody should be produced in the manner described below.
  • human IL-6 receptor used as sensitizing antigen for acquisition of antibody is obtained by using the IL-6 receptor gene/amino acid sequence disclosed in European Unexamined Patent Publication No. EP 325474, while mouse IL-6 receptor is obtained by using the IL-6 receptor gene/amino acid sequence disclosed in Japanese Unexamined Patent Publication No. 3-155795.
  • IL-6 receptor protein There are two types of IL-6 receptor protein consisting of that which is expressed on the cell membrane and that which is released from the cell membrane (soluble IL-6 receptor) (Yasukawa, K. et al., J. Biochem. (1990) 108, 673-676).
  • Soluble IL-6 receptor antibody is substantially composed of the extracellular region of IL-6 receptor that binds to the cell membrane, and differs from membrane-bound IL-6 receptor in that it is missing a cell membrane-penetrating region or cell membrane-penetrating region and intracellular region.
  • IL-6 receptor protein may use either IL-6 receptor provided it can be used as sensitizing antigen for producing the anti-IL-6 receptor antibody used in the present invention.
  • the target IL-6 receptor After inserting the gene sequence of IL-6 receptor into a known expression vector system to transform suitable host cells, the target IL-6 receptor is purified by known methods from the host cells or culture supernatant followed by using the purified IL-6 receptor protein as sensitizing antigen.
  • a fused protein consisting of cells that express IL-6 receptor or IL-6 receptor protein and another protein may also be used as sensitizing antigen.
  • Escherichia coli E. coli containing plasmid pIBIBSF2R that contains cDNA encoding human IL-6 receptor has been internationally deposited based on the Budapest Treaty under the deposit number FERM BP-2232 on Jan. 9, 1989 at the International Patent Organism Depository of the National Institute of Advanced Industrial Science and Technology (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan) as HB101-pIBIBSF2R.
  • Anti-gp130 antibody used in the present invention can be obtained in the form of polyclonal antibody or monoclonal antibody using known means.
  • Monoclonal antibody of mammalian origin is particularly preferable for the anti-gp130 antibody used in the present invention.
  • Monoclonal antibodies of mammalian origin include those produced by hybridomas and those produced in a host transformed with an expression vector that contains antibody gene by genetic engineering techniques. This antibody inhibits binding of IL-6/IL-6 receptor complex to gp130 and interrupts the transmission of the biological activity of IL-6 to cells by binding with gp130.
  • AM64 antibody Japanese Unexamined Patent Publication No. 3-219894
  • 4B11 antibody 2H4 antibody
  • 2H4 antibody U.S. Pat. No. 5,571,513
  • B-P8 antibody Japanese Unexamined Patent Publication No. 8-291199
  • Anti-gp130 monoclonal antibody-producing hybridoma can basically be produced in the manner described below using known technology. Namely, this hybridoma can be produced by using gp130 as a sensitizing antigen, immunizing with this in accordance with ordinary immunization methods, fusing the resulting immunocytes with known host cells according to ordinary cell fusion methods, and screening for cells that produce monoclonal antibody according to ordinary screening methods.
  • gp130 used as sensitizing antigen for acquiring antibody is obtained by using the gp130 gene/amino acid sequence disclosed in European Unexamined Patent Publication No. EP 411946.
  • the target gp130 protein After inserting the gene sequence of gp130 into a known expression vector system to transform suitable host cells, the target gp130 protein is purified from the host cells or culture supernatant by known methods, followed by using the purified gp130 receptor protein as sensitizing antigen.
  • a fused protein consisting of cells expressing gp130 or gp130 protein and another protein may also be used as sensitizing antigen.
  • the mammal immunized with sensitizing antigen is preferably selected in consideration of compatibility with the host cells used for cell fusion, typical examples of which include rodents such as mice, rats and hamsters.
  • Immunization of the animal with sensitizing antigen is carried out in accordance with known methods.
  • immunization is preferably carried out by injecting sensitizing antigen into the abdominal cavity or beneath the skin of a mammal. More specifically, a suspension of sensitizing antigen diluted to a suitable amount with phosphate-buffered saline (PSB) or physiological saline is mixed with a suitable amount of an ordinary adjuvant such as Freund's complete adjuvant as desired followed by emulsifying and administering several times to the mammal every 4 to 21 days.
  • a suitable carrier can be used when immunizing with the sensitizing antigen.
  • immunocytes are removed from the mammal and used for cell fusion.
  • Spleen cells are a particularly preferable example of immunocytes used for cell fusion.
  • Fusion of the aforementioned immunocytes and myeloma cells can basically be carried out in compliance with known methods such as the method of Milstein, et al. (Kohler, G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46).
  • the aforementioned cell fusion is carried out, for example, in an ordinary nutritive culture liquid in the presence of a cell fusion promoter.
  • fusion promoters include polyethylene glycol (PEG) and Sendai virus (HVJ), and an assistant such as dimethylsulfoxide can also be added to enhance fusion efficiency as desired.
  • the ratio of immunocytes and myeloma cells used is preferably 1 to 10 times more immunocytes than myeloma cells.
  • culture liquids that can be used for the aforementioned cell fusion include RPMI1640 culture liquid preferable for growth of the aforementioned myeloma cells, MEM culture liquid and other ordinary culture liquids used for this type of cell culturing.
  • a serum supplement such as fetal calf serum (FCS) can also be used in combination with the aforementioned culture liquid.
  • Cell fusion is carried out by thoroughly mixing predetermined amounts of the aforementioned immunocytes and myeloma cells in the aforementioned culture liquid, adding PEG solution, such as PEG solution having an average molecular weight of about 1000 to 6000 and pre-warmed to 37° C., normally at a concentration of 30 to 60% (w/v), and then mixing to form the target fused cells (hybridoma).
  • PEG solution such as PEG solution having an average molecular weight of about 1000 to 6000 and pre-warmed to 37° C., normally at a concentration of 30 to 60% (w/v)
  • cell fusion agents and so forth that are detrimental to hybridoma development can be removed by repeating the procedure consisting of sequentially adding suitable culture liquid and then centrifuging to remove the supernatant.
  • the hybridoma is selected by culturing in an ordinary selective culture liquid such as HAT culture liquid (culture liquid containing hypoxanthine, aminopterin and thymidine). Culturing in said HAT culture liquid is continued for a duration, which is normally from several days to several weeks, that is sufficient for destroying those cells other than the target hybridoma (non-fused cells). Next, hybridoma that produces the target antibody is then screened and cloned by carrying out ordinary limiting dilution methods.
  • HAT culture liquid culture liquid containing hypoxanthine, aminopterin and thymidine
  • a desired human antibody having binding activity with a desired antigen or antigen-expressing cells can be obtained by sensitizing human lymphocytes with a desired antigen protein or antigen-expressing cells in vitro, and then fusing the sensitized B lymphocytes with myeloma cells such as U266 (refer to Japanese Examined Patent Publication No. 1-59878).
  • a desired human antibody may also be acquired in accordance with the aforementioned method by administering antigen or antigen-expressing cells into a transgenic animal having a repertoire of human antibody genes (refer to International Unexamined Patent Publications Nos. WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096 or WO 96/33735).
  • Hybridoma that produces monoclonal antibody produced in this manner can be sub-cultured in ordinary culture liquid and stored in liquid nitrogen for a long period of time.
  • a method in which the hybridoma is cultured in accordance with ordinary methods and obtained in the form of the culture supernatant, or a method in which the hybridoma is grown by administering to a mammal that is compatible therewith followed by obtaining the form of ascites, can be employed to acquire monoclonal antibody from the hybridoma.
  • the former method is suitable for obtaining highly pure antibody, while the latter method is suitable for producing antibody in large volume.
  • production of anti-IL-6 receptor antibody-producing hybridoma can be carried out according to the method disclosed in Japanese Unexamined Patent Publication No. 3-139293. This can be carried out using a method in which the PM-1-producing hybridoma internationally deposited based on the Budapest Treaty under the designation FERM BP-2998 on Jul.
  • Recombinant antibody produced using gene recombination technology by cloning antibody gene from a hybridoma, incorporating in a suitable vector and then inserting into a host can be used as monoclonal antibody in the present invention (refer to, for example, Borrebaeck, C. A. K. and Larrick, J. W., Therapeutic Monoclonal Antibodies, publishing in the United Kingdom by MacMillan Publishers Ltd., 1990).
  • mRNA that encodes antibody variable (V) region is isolated from cells such as a hybridoma that produce the target antibody. Isolation of mRNA is carried out by preparing total RNA according to a known method such as guanidine centrifugation (Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-5299) or AGPC (Chomczynski, P. et al., Anal. Biochem. (1987) 162, 156-159), and the preparing mRNA using an mRNA Purification Kit (Pharmacia) and so forth. In addition, mRNA can be prepared directly by using the QuickPrep mRNA Purification Kit (Pharmacia).
  • cDNA of the antibody V region is synthesized from the resulting mRNA using reverse transcriptase.
  • Synthesis of cDNA can be carried out using, for example, the AMV Reverse Transcriptase First-Strand cDNA Synthesis Kit.
  • the 5′-Ampli Finder Race Kit (Clontech) and 5′-RACE method using PCR can be used to synthesize and amplify the cDNA.
  • the target DNA fragment is purified from the resulting PCR product and coupled with vector DNA.
  • a recombinant vector is then produced from this and inserted into E. coli and so forth followed by selecting a colony to prepare the desired recombinant vector.
  • the base sequence of the target DNA is then confirmed by a known method such as the deoxy method.
  • DNA that encodes the V region of the target antibody is then coupled with DNA that encodes a desired antibody constant region (C region) followed by incorporation into an expression vector.
  • DNA encoding the V region of the antibody may be incorporated into an expression vector that contains DNA of the antibody C region.
  • an antibody gene as will be described later is incorporated into an expression vector that expressed under the control of, for example, an enhancer or a promoter.
  • host cells are transformed with this expression vector to allow expression of the antibody.
  • a gene recombinant antibody that has been artificially altered for the purpose of lowering interspecies antigenicity to humans can be used, examples of which include chimeric antibody, humanized antibody and human antibody. These altered antibodies can be produced using known methods.
  • Chimeric antibodies are obtained by coupling DNA encoding antibody V region obtained in the manner described above with DNA encoding human antibody C region followed by incorporating the coupled product into an expression vector and producing by inserting into a host (refer to European Unexamined Patent Publication No. EP 125023 or International Unexamined Patent Publication No. WO 92-19759). Chimeric antibody that is useful in the present invention can be obtained by using this known method.
  • plasmids containing DNA that encode the V regions of the L chain and H chain of chimeric PM-1 antibody have been named pPM-k3 and pPM-h1, respectively, and Escherichia coli retaining these plasmids have been internationally deposited based on the Budapest Treaty under the designation NCIMB40366 and NCIMB40362, respectively, on Feb. 12, 1991 at the National Collections of Industrial and Marine Bacteria Limited (23 St Machar Drive, Aberdeen AB2 1RY, Scotland, Commonwealth of Great Britain and Northern Ireland).
  • Humanized antibodies which are also referred to as reshaped antibodies, are obtained by transplanting the complementarity determining region (CDR) of mammals other than humans such as mice to the complementarity determining region of human antibody, and their typical gene recombination techniques are known (refer to European Unexamined Patent Publication No. EP 125023 or International Unexamined Patent Publication No. WO 92-19759).
  • CDR complementarity determining region
  • a DNA sequence designed so as to couple CDR of mouse antibody with the framework (FR) region of human antibody is synthesized by PCR from a plurality of oligonucleotides produced so as to have overlapping portions on their ends.
  • the resulting DNA is then coupled with DNA that encodes human antibody C region followed by incorporation into an expression vector and insertion into a host to produce the DNA in that host (refer to European Unexamined Patent Publication No. EU 239400 or International Unexamined Patent Publication No. WO 92-19759).
  • An FR for which the complementarity determining region forms a satisfactory antigen binding site is selected for the FR of the human antibody coupled by means of CDR.
  • the amino acids of the framework region of the antibody variable region may be substituted so that the complementary determining region of the reconfigured human antibody forms a suitable antigen binding site (Sato, K. et al., Cancer Res. (1993) 53, 851-856).
  • Human antibody C region is used for chimeric antibody and humanized antibody.
  • An example of a human antibody C region is C ⁇ , and for example, C ⁇ 1, C ⁇ 2, C ⁇ 3 or C ⁇ 4 can be used.
  • the human antibody C region may be modified to improve stability of the antibody or its production.
  • Chimeric antibodies are composed of a variable region of an antibody of mammalian origin other than humans and a C region originating in human antibody, while humanized antibodies are composed of a complementarity determining region of an antibody of mammalian origin other than humans and a framework region and C region originating in human antibody, and since their antigenicity in humans is decreased, they are useful as antibodies used in the present invention.
  • a preferable specific example of a humanized antibody used in the present invention is humanized PM-1 antibody (refer to International Unexamined Patent Publication No. WO 92-19759).
  • a phage that binds to an antigen can also be selected by using a variable region of human antibody as a single chain antibody (scfv) and expressing on the surface of a phage using the phage display method.
  • the DNA sequence that encodes the human antibody variable region that binds to the antigen can then be determined by analyzing the genes of the selected phage. Once the DNA sequence of the scfv that binds to the antigen has been identified, an expression vector that is equivalent to that sequence can then be produced to acquire the human antibody.
  • Antibody gene constructed in the manner described above can be expressed and acquired by known methods.
  • antibody gene can be expressed with DNA or vector that contains said DNA in which a commonly used useful promoter, antibody gene to be expressed, and poly A signal downstream from the 3′ side are functionally bound.
  • a promoter/enhancer is human cytomegalovirus immediately early promoter/enhancer.
  • virus promoters/enhancers such as those of retrovirus, polyoma virus, adenovirus or Simeon virus 40 (SV40), as well as promoters/enhancers originating in mammalian cells such as human elongation factor 1 ⁇ (HEF1 ⁇ ).
  • antibody in the case of using SV40 promoter/enhancer, antibody can be easily expressed in accordance with the method of Mulligan et al. (Mulligan, R. C. et al., Nature (1979) 277, 108-114) or in the case of HEF1 ⁇ promoter/enhancer, antibody can be easily expressed in accordance with the method of Mizushima et al. (Mizushima, S. and Nagata, S., Nucleic Acids Res. (1990) 18, 5322).
  • antibody can be expressed by functionally binding a commonly used useful promoter, signal sequence for secreting antibody and antibody gene to be expressed.
  • promoters include lacZ promoter and araB promoter.
  • lacZ promoter antibody should be expressed in accordance with the method of Ward et al. (Ward, E. S. et al., Nature (1989) 341, 544-546; Ward, E. S. et al., FASEB J. (1992) 6, 2422-2427), or in the case of using araB promoter, antibody should be expressed in accordance with the method of Better et al. (Better, M. et al., Science (1988) 240, 1041-1043).
  • the pelB signal sequence (Lei, S. P. et al., J. Bacteriol. (1987) 169, 4379-4383) should be used for the signal sequence for antibody secretion in the case of producing in the periplasm of E. coli . After separating the antibody produced in the periplasm, the antibody is used after suitably refolding the antibody structure (refer to, for example, WO 96/30394).
  • replication sources examples include those originating in SV40, polyoma virus, adenovirus or bovine papilloma virus (BPV), and in order to increase the number of gene copies in the host cell system, the expression vector can contain a selection marker such as aminoglycoside phosphotransferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene or dihydrofolate reductase (dhfr) gene.
  • APH aminoglycoside phosphotransferase
  • TK thymidine kinase
  • Ecogpt E. coli xanthine-guanine phosphoribosyl transferase
  • dhfr dihydrofolate reductase
  • the production system for antibody production may be an in vitro system or an in vivo system.
  • in vitro production systems include production systems that use eukaryotic cells and a production systems that use prokaryotic cells.
  • the production system may use animal cells, plant cells or fungal cells.
  • animal cells include: (1) mammalian cells such as CHO, COS, myeloma, BHK (baby hamster kidney), HeLa and Vero cells, (2) amphibian cells such as African tree frog follicular cells, and (3) insect cells such as sf9, sf21 and Tn5 cells.
  • plant cells include cells originating in Nicotiana tabacum , and they should be cultured in calluses.
  • fungal cells include yeasts cells such as Saccharomyces species including Saccharomyces cerevisiae , and molds such as Aspergillus species including Aspergillus niger.
  • bacterial cells In the case of using prokaryotic cells, a production system that uses bacterial cells is used.
  • bacterial cells include E. coli and Bacillus subtilis.
  • Antibody is obtained by inserting the target antibody gene into these cells by transformation, and then culturing the transformed cells in vitro. Culturing is carried out in accordance with known methods. For example, DMEM, MEM, RPMI1640 or IMDM can be used for the culture liquid, and a serum supplement such as fetal calf serum (FCS) can also be used in combination. In addition, antibody may also be produced in vivo by transferring cells into which the antibody gene has been inserted to the abdominal cavity and so forth of an animal.
  • FCS fetal calf serum
  • examples of in vivo production systems include production systems that use animals and production systems that use plants.
  • examples of production systems include those that use mammals or insects.
  • Examples of mammals that can be used include goats, pigs, sheep, mice and rabbits (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993).
  • Silkworms can be used for the insects.
  • tobacco plants can be used, for example.
  • Antibody gene is inserted into these animals or plants followed by production and recovery of the antibody within the bodies of the animals or plants.
  • an antibody gene is inserted at an intermediate location in a gene that encodes a protein uniquely produced in milk such as goat ⁇ casein to prepare in the form of a fused protein.
  • a DNA fragment that contains the fused protein into which the antibody gene has been inserted is then injected into a goat embryo and this embryo is then introduced into a female goat.
  • the desired antibody is then obtained from the milk produced by a transgenic goat or its progeny that is born from the goat that has received the embryo.
  • a suitable hormone may be used in the transgenic goat to increase the amount of milk containing the desired antibody produced from the transgenic goat (Ebert, K. M. et al., Bio/Technology (1994) 12, 699-702).
  • a silkworm is infected with baculovirus into which the target antibody gene has been inserted followed by obtaining the desired antibody from the silkworm body fluid (Maeda, S. et al., Nature (1985) 315, 592-594).
  • the target antibody gene is inserted into a plant expression vector such as pMON 530, after which this vector is inserted into bacteria such as Agrobacterium tumefaciens . This bacteria is then used to infect a tobacco plant such as Nicotiana tabacum to obtain the desired antibody from the tobacco leaves (Jilian, K. -C. Ma, et al., Eur. J. Immunol. (1994) 24, 131-138).
  • a host in the case of producing antibody with an in vitro or in vivo production system, a host may be simultaneously transfected by separately incorporating DNA encoding antibody heavy chain (H chain) or light chain (L chain) in separate expression vectors, or transforming a host by incorporating DNA encoding H chain and L chain in a single expression vector (refer to International Unexamined Patent Publication No. WO 94-11523).
  • Antibody used in the present invention may be an antibody fragment or modified product thereof provided is can be preferably used in the present invention.
  • antibody fragments include Fab, F(ab′)2, Fv or H chain and single chain Fv (scfv) in which Fv or Fv or H chain and L chain are coupled with a suitable linker.
  • an antibody fragment by treating an antibody with an enzyme such as papain or pepsin, or constructing a gene that encodes these antibody fragments and inserting it into an expression vector, it is expressed in suitable host cells (refer to, for example, Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976; Better, M. & Horwitz, A. H., Methods in Enzymology (1989) 178, 476-496; Plueckthun, A. & Skerra, A., Methods in Enzymology (1989) 178, 476-496; Lamoyi, E., Methods in Enzymology (1989) 121, 652-663; Rousseaux, J. et al., Methods in Enzymology (1989) 121, 663-666; Bird, R. E. et al., TIBTECH (1991) 9, 132-137).
  • suitable host cells herein, Co, M. S. et al., J. Immun
  • the aforementioned scFv is obtained by coupling antibody H chain V region with L chain V region.
  • the H chain V region and L chain V region are coupled with a linker and preferably a peptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879-5883).
  • the H chain V region and L chain V region in scFv may have for their origins any of the origins described for the aforementioned antibodies.
  • An example of a peptide linker used to couple the V regions is an arbitrary single chain peptide composed of 12-19 amino acid residues.
  • DNA that encodes scFv is obtained by using DNA that encodes the aforementioned antibody H chain or H chain V region and DNA that encodes L chain or L chain V region as templates, amplifying a DNA portion that encodes a desired amino acid sequence among these sequences by PCR using a primer pair that defines both ends, and then amplifying by combining with a prime pair that defines the DNA that encodes the peptide linker portion as well as both of its ends so that each H chain and L chain is linked.
  • expression vectors that contain them as well as hosts that have been transformed by said expression vectors can be obtained in accordance with ordinary methods.
  • scFv can be obtained in accordance with ordinary methods by using those hosts.
  • antibody fragments can be produced from a host by acquiring and expressing their genes in the same manner as previously described.
  • the “antibody” referred to in the present invention includes these antibody fragments.
  • Antibodies bound with various types of molecules such as polyethylene glycol (PEG) can be used for antibody modified products.
  • the “antibody” referred to in the present invention includes these antibody modified products. These antibody modified products can be obtained by carrying out chemical modification on a resulting antibody. These methods have already been established in this field.
  • expressed antibody can be separated from inside or outside cells or from a host and purified to uniformity. Separation and purification of antibody used in the present invention can be carried out by affinity chromatography. Examples of columns used for affinity chromatography include a protein A column and protein G column. Examples of carriers used for a protein A column include Hyper D, POROS and Sepharose F.F. Other separation and purification methods used with ordinary proteins may also be used and there are no limitations thereon.
  • Antibody used in the present invention can be separated and purified by suitably selecting and combining, for example, chromatography other than the aforementioned affinity chromatography, filtration, ultrafiltration, salting out or dialysis.
  • chromatography other than the aforementioned affinity chromatography, filtration, ultrafiltration, salting out or dialysis.
  • examples of other types of chromatography include ion exchange chromatography, hydrophobic chromatography and gel filtration. These types of chromatography can be applied to high-performance liquid chromatography (HPLC). In addition, reverse phase HPLC may also be used.
  • Concentration of antibody obtained as described above can be measured by measurement of optical absorbance, ELISA and so forth. Namely, in the case of measuring optical absorbance, after suitably diluting with PBS( ⁇ ), optical absorbance at 280 nm is measured followed by calculating concentration based on 1.35 OD representing 1 mg/ml.
  • concentration by ELISA can be carried out in the manner described below. Namely, 100 ⁇ l of goat anti-human IgG (TAG) diluted to 1 ⁇ g/ml with 0.1 M bicarbonate buffer (pH 9.6) are added to a 96-well plate (Nunc) followed by incubating overnight at 4° C. to immobilize the antibody on the plate. After blocking, 100 ⁇ l of suitably diluted antibody used in the present invention, sample containing antibody or human IgG standard (Cappel) are added followed by incubating for 1 hour at room temperature.
  • TAG goat anti-human IgG
  • Cappel human IgG standard
  • IL-6 variant used in the present invention is a substance that has binding activity with IL-6 receptor but does not transmit the biological activity of IL-6. Namely, although IL-6 variant competes with IL-6 for binding with IL-6 receptor, since it does not transmit the biological activity of IL-6, signal transmission by IL-6 is interrupted.
  • IL-6 variant is produced by introducing a mutation by substituting an amino acid residue in the amino acid sequence of IL-6.
  • human IL-6 is preferable in consideration of antigenicity and so forth.
  • this is carried out by predicting the secondary structure of the amino acid sequence of IL-6 using a known molecular modeling program such as WHATIF (Vriend, et al., J. Mol. Graphics (1990) 8, 52-56), and then evaluating the effects on all of the amino acid residues to be substituted.
  • a suitable substituted amino acid residue by then using a vector containing a base sequence that encodes human IL-6 gene as a template and introducing a mutation such that the amino acid is substituted by PCR carried out in the normal manner, a gene that encodes IL-6 variant is obtained. This can then be incorporated in a suitable expression vector as necessary to obtain IL-6 variant in compliance with the aforementioned recombinant antibody expression, production and purification methods.
  • IL-6 variants are disclosed in Brakenhoff et al., J. Biol. Chem. (1994) 269, 86-93, Savino et al., EMBO J. (1994) 13, 1357-1367, WO 96-18648 and WO 96-17869.
  • IL-6 partial peptide or IL-6 receptor partial peptide used in the present invention is a substance that has binding activity with IL-6 receptor or IL-6, respectively, and does not transmit the biological activity of IL-6. Namely, IL-6 partial peptide or IL-6 receptor partial peptide specifically inhibit binding of IL-6 to IL-6 receptor by capturing IL-6 receptor or IL-6 by binding thereto. As a result, signal transmission by IL-6 is interrupted since the biological activity of IL-6 is not transmitted.
  • IL-6 partial peptide or IL-6 receptor partial peptide is a peptide that is composed of an amino acid sequence of a portion or entirety of the region involved in binding between IL-6 and IL-6 receptor in the amino acid sequence of IL-6 or IL-6 receptor.
  • Such a peptide is normally composed of 10 to 80, preferably 20 to 50 and more preferably 20 to 40 amino acid residues.
  • IL-6 partial peptide of IL-6 receptor partial peptide can be produced by a method in which the region involved in binding between IL-6 and IL-6 receptor is identified in the amino acid sequence of IL-6 or IL-6 receptor, and a portion or all of that amino acid sequence is normally known, examples of which include genetic engineering techniques and peptide synthesis methods.
  • IL-6 partial peptide or IL-6 receptor partial peptide can be produced using genetic engineering techniques by incorporating DNA that encodes a desired peptide in an expression vector and obtaining the desired peptide in compliance with the aforementioned recombinant antibody expression, production and purification methods.
  • IL-6 partial peptide or IL-6 receptor partial peptide can be produced using peptide synthesis methods by using a method normally used in peptide synthesis, such as a solid phase synthesis method or liquid phase synthesis method.
  • Solid phase synthesis a method is used in which, for example, a peptide chain is elongated by alternately repeating a reaction in which an amino acid corresponding to the C terminal of the peptide to be synthesized is bound to a support that is insoluble in organic solvent, and amino acids in which the ⁇ -amino groups and side chain functional groups are protected with suitable protecting groups are sequentially bound in order from the C terminal to the N terminal, and a reaction in which said protecting groups of ⁇ -amino groups of amino acids or peptide bound to the resin are eliminated.
  • Solid phase peptide synthesis methods are broadly divided into the Boc method and Fmoc method depending on the type of protecting groups used.
  • a de-protecting reaction and reaction for severing the peptide chain from the support are carried out.
  • the reaction for severing the peptide chain hydrogen fluoride or trifluoromethane sulfonic acid are normally used in the Boc method, while TFA is normally used in the Fmoc method.
  • Boc method for example, the aforementioned protected peptide resin is treated in hydrogen fluoride in the presence of anisole.
  • the protecting groups are eliminated and the peptide is severed from the support followed by recovery of the peptide.
  • a crude peptide is then obtained by freeze-drying the product.
  • a de-protecting reaction and reaction for severing the peptide chain from the support can be carried out using the same procedure as described above in TFA.
  • the resulting crude peptide can be separated and purified by applying to HPLC. Elution should be carried out under the optimum conditions using a water-acetonitrile-based solvent normally used for purification of protein. The fraction corresponding to the peak of the resulting chromatography profile is then separated and freeze-dried. The peptide fraction purified in this manner is then identified by molecular weight analysis using mass spectrometry, amino acid composition analysis or amino acid sequence analysis.
  • IL-6 partial peptides and IL-6 receptor partial peptides are disclosed in Japanese Unexamined Patent Publication No. 2-188600, Japanese Unexamined Patent Publication No. 7-324097, Japanese Unexamined Patent Publication No. 8-311098 and U.S. Pat. No. 5,210,075.
  • IL-6 signal transmission inhibitory activity of IL-6 antagonist used in the present invention can be evaluated by normally used methods. More specifically, IL-6-dependent human myeloma line (S6B45, KPMM2), human Lennert's T lymphoma cell line KT3 or IL-6-dependent cell line MH60 or BSF2 is cultured followed by the addition of IL-6 while simultaneously in the presence of IL-6 antagonist and measurement of the uptake of 3 H-thymidine by IL-6-dependent cells.
  • S6B45, KPMM2 human myeloma line
  • KT3 human Lennert's T lymphoma cell line KT3
  • IL-6-dependent cell line MH60 or BSF2 is cultured followed by the addition of IL-6 while simultaneously in the presence of IL-6 antagonist and measurement of the uptake of 3 H-thymidine by IL-6-dependent cells.
  • 125 I-labeled IL-6 bound to IL-6 receptor expressing cells is measured by culturing IL-6 receptor expressing cells in the form of U266 cells followed by the addition of 125 I-labeled IL-6 and the simultaneous addition of IL-6 antagonist.
  • a negative control group that does not contain IL-6 antagonist is provided in addition to the group in which IL-6 antagonist is present, and comparison of the results obtained from the two groups makes it possible to evaluate the IL-6 inhibitory activity of the IL-6 antagonist.
  • anti-IL-6 receptor antibody As will be indicated in the examples to be described later, since growth inhibitory effects on mesothelioma cells have been observed for anti-IL-6 receptor antibody, anti-IL-6 receptor antibody and other IL-6 antagonists were suggested as being useful as therapeutic agents for mesothelioma.
  • the treatment target in the present invention is a mammal.
  • the mammal of the treatment target is preferably a human.
  • the mesothelioma therapeutic agent or mesothelioma cell growth inhibitor of the present invention can be administered systemically or locally either orally or parenterally.
  • intravenous infusion or other form of intravenous injection, intramuscular injection, intrathoracic injection, intraperitoneal injection, subcutaneous injection, suppositories, enema or oral enteric-coated pills and so forth can be selected, and the administration method can be suitably selected according to the age and symptoms of the patient.
  • the effective dose is selected within the range of 0.01 mg to 100 mg per kilogram of body weight per administration.
  • a dose of 1 to 1000 mg, and preferably 5 to 50 mg can be selected per patient.
  • the preferable dose and administration method in the case of anti-IL-6 receptor antibody is such that the amount of free antibody present in the blood is the effective dose, a specific example of which is a method by which it is administered using a method such as intravenous drip or other form of intravenous injection or subcutaneous injection and so forth according to an administration schedule such as twice a week, once a week, once every two weeks or once every four weeks in a single administration or divided among several administrations at a dose of 0.5 mg to 40 mg, and preferably 1 mg to 20 mg, per month (4 weeks) per 1 kilogram of body weight.
  • the administration schedule can be adjusted such as by lengthening the administration interval from twice per week or once per week to once every two weeks, once every three weeks or once every four weeks while observing the patient's condition and trends in blood test values.
  • the mesothelioma therapeutic agent or mesothelioma cell growth inhibitor of the present invention may contain a pharmaceutically acceptable carrier or additive depending on the administration route.
  • a pharmaceutically acceptable carrier or additive include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, sodium polyacrylate, sodium arginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum Arabic, casein, gelatin, agar, diglycerin, propylene glycol, polyethylene glycol, Vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose and surfactants acceptable for use as pharmaceutical additives.
  • additives used are selected are suitably selected or combined from the aforementioned additives according to the drug form
  • IL-6 receptor The expression levels of IL-6 receptor were measured for five malignant mesothelioma cell lines at the mRNA level.
  • KT-3 cells were used for the positive control, and synoviocytes were used for the negative control. These cells were cultured for 48 hours in RPMI containing 10% FCS, and mRNA that encodes IL-6 receptor IL-6R) in the cells was measured by reverse-transcribed PCR (RTPCR) using the GeneAmp PCR System (Applied Biosystems) for the detection device.
  • RTPCR reverse-transcribed PCR
  • FIG. 2 (bottom) indicates the amount of mRNA of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) used for the internal control.
  • malignant mesothelioma cells are believed to express hardly any IL-6 receptor. Since the pleural fluid in cases of malignant pleural mesothelioma consists of bloody pleural fluid, soluble IL-6 receptor is surmised to be present in large amounts, and this is believed to be involved in transmission of IL-6 irritation.
  • Tumor cells of malignant mesothelioma cell lines H2052 and H2452 were cultured in three series in 24-well plates at an initial cell concentration of 5 ⁇ 10 4 /well in RPMI1640 medium.
  • the cell culture liquid was replaced followed by commencement of stimulation by (1) recombinant IL-6 (10 ng/ml), (2) recombinant IL-6 (10 ng/ml)+recombinant soluble IL-6R (100 ng/ml), and (3) inhibition by the anti-IL-6 receptor antibody, humanized PM-1 antibody (refer to WO 92/19759) (25 ⁇ g/ml) (RPMI was used for the medium control).
  • VEGF vascular endothelial growth factor
  • Example 3 The experiment of Example 3 was repeated using malignant mesothelioma cell line H28. Those results are shown in FIG. 4 . As is clear from these results, although cell line H28 produces high levels of VEGF, it did not respond to stimulation by IL-6.
  • Cell line H2025 in which production of VEGF is induced by IL-6
  • cell line H28 in which production of VEGF is not induced by IL-6
  • STAT3 signal transducer and activator of transcription 3
  • FIG. 5 prominent phosphorylation of STAT3 was observed for cell line H2052 at 30 minutes after IL-6 stimulation. In contrast, only slight phosphorylation of STAT3 was observed in response to IL-6 stimulation for cell line H28. Furthermore, p-STAT3 in FIG. 5 indicates phosphorylated STAT3, while STAT3 indicates the sum of phosphorylated and non-phosphorylated STAT3.
  • Cell line H2025 in which production of VEGF is induced by IL-6
  • cell line H28 in which production of VEGF is not induced by IL-6
  • recombinant IL-6 10 ng/ml
  • soluble recombinant IL-6 receptor 100 ng/ml
  • SOCS3 induced suppressor of cytokine signaling 3
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • 50,000H2052 cells each were transfected with 1 ⁇ g of pGL3-VEGF or pGL3-VEGFmut, and the transfected cells were stimulated with recombinant IL-6 (10 ng/ml) and recombinant soluble IL-6 receptor (100 ng/ml). After stimulating for 2 days, the luciferase activity induced from the VEGF promoter and mutated VEGF promoter was examined. Those results are shown in FIG. 8 .
  • Cell line H2052 cells were disseminated in a 96-well plate containing RPMI medium containing 10% FCS at 500 cells/well followed by culturing in five series for 6 to 7 days in the presence or absence of recombinant soluble IL-6 receptor at 100 ng/ml and in the presence of various concentrations (0, 1, 10 or 100 ng/ml) of IL-6. Those results are shown in FIG. 10 and FIG. 13 . As is clear from these graphs, cell line H2052 and cell line H226 grow IL-6 concentration-dependently in the presence of recombinant soluble IL-6 receptor (100 ng/ml).
  • H2052 cells were disseminated in a 96-well plate containing RPMI medium containing 10% FCS at 500 cells/well followed by culturing in three series for 7 days in the presence of recombinant soluble IL-6 at 10 ng/ml and recombinant soluble IL-6 receptor at 100 ng/ml and in the presence of various concentrations (0.1 or 25 ⁇ g/ml) of humanized PM-1 antibody. Following culturing, the amount of growth of H2052 cells (OD 450) was measured by MTS assay. Those results are shown in FIG. 11 . As a result, anti-IL-6 antibody was determined to inhibit growth concentration-dependently. On the other hand, inhibitory effects were not observed in the case of adding human IgG1 instead of MRA at the same concentration as a control.
  • Cell line H226 cells were disseminated in a 96-well plate containing RPMI medium containing 10% FCS at a concentration of 500 cells/well followed by culturing in three series for 7 days in the presence of recombinant soluble IL-6 receptor at 100 ng/ml and in the presence of various concentrations (0, 1, 10 or 100 ng/ml) of IL-6. Those results are shown in FIG. 12 . As is clear from the graph, H226 cells that produce high levels of IL-6 grew IL-6 concentration-dependently in the presence of recombinant soluble IL-6 receptor (100 ng/ml) while their growth was inhibited by anti-IL-6R antibody in the same manner as H2052 cells producing high levels of IL-6.
  • Cells of cell line H2052 and cell line H225 were disseminated in a 96-well plate at 200 cells/well in RPMI medium containing 10% FCS followed by culturing in five series for 6 days in the presence of IL-6 (10 ng/ml) and soluble IL-6 receptor (100 ng/ml) and in the presence of various concentrations (0, 1 ⁇ g/ml, 5 ⁇ g/ml) of humanized PM-1 antibody. After culturing, the cell growth rates of cell line H2052 and cell line H226 were measured by MTS assay. The cell growth rates indicated how many times cell growth increased as compared with the absence of addition of IL-6/sIL-6R. Those results are shown in FIG. 14 .
  • anti-IL-6 receptor antibody completely inhibited the growth promoting action induced by addition of IL-6/sIL-6R in cell line H2052 and cell line H226.
  • growth inhibitory effects were not observed in the case of adding the same concentrations of human IgG1 (Sigma) as a control instead of anti-IL-6 receptor antibody.
  • Cells of cell line H2052 and cell line H226 were disseminated in a 96-well plate at 500 cells/plate in RPMI medium containing 10% FCS, followed by culturing in five series for 6-7 days in the presence of IL-6 (10 ng/ml) and recombinant soluble IL-6 receptor (100 ng/ml) and in the presence of 1 ⁇ g/ml of anti-VEGF antibody. After culturing, the amounts of growth were examined by MTS assay. The same concentration of human IgG1 (Sigma) was used for the control instead of anti-VEGF antibody. Those results are shown in FIG. 17 .
  • anti-VEGF antibody did not inhibit cell growth induced by IL-6/sIL-6R stimulation.
  • growth action on malignant mesothelioma cells induced by IL-6/sIL-6R stimulation was clearly determined to not be mediated by VEGF.
  • Soluble IL-6 receptor was produced by PCR using a plasmid pBSF2R.236 that contains DNA encoding IL-6 receptor obtained according to the method of Yamasaki, et al. (Yamasaki, K. et al., Science (1988) 241, 825-828). Plasmid pBSF2R.236 was then digested with restrictase SphI to obtain IL-6 receptor cDNA which was then inserted into mp18 (Amersham). A mutation was introduced into the IL-6 receptor cDNA by PCR with the In Vitro Mutagenesis System (Amersham) using a synthetic oligoprimer designed so as to insert a stop codon into the IL-6 receptor cDNA. As a result of this procedure, a stop codon was inserted at the location of amino acid 345, and cDNA was obtained that encodes soluble IL-6 receptor.
  • plasmid pSVL344 Soluble IL-6 receptor cDNA severed with HindIII-SalI was then inserted into plasmid pECEdhfr containing dhfr cDNA to obtain CHO cell expression plasmid pECEdhfr344.
  • plasmid pECEdhfr344 10 ⁇ g of plasmid pECEdhfr344 were then used to transfect dhfr-CHO cell line DXB-11 (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA (1980) 77, 4216-4220) according to the calcium phosphate precipitation method (Chen, C. et al., Mol. Cell. Biol. (1987) 7, 2745-2751). The transfected CHO cells were then cultured for 3 weeks in nucleoside-free ⁇ MEM selective culture liquid containing 1 mM glutamine, 10% dialyzed FCS, 100 U/ml of penicillin and 100 ⁇ g/ml of streptomycin.
  • the selected CHO cells were then screened by a limiting dilution method to obtain a single CHO cell clone.
  • This CHO cell clone was amplified with methotrexate at a concentration from 20 nM to 200 nM to obtain human soluble IL-6 receptor-producing CHO cell line 5E27.
  • CHO cell line 5E27 was cultured in Iscove's Modified Dulbecco's Medium (IMDM, Gibco) containing 5% FBS. After recovering the culture supernatant, the concentration of soluble IL-6 receptor in the culture supernatant was measured by ELISA. As a result, soluble IL-6 receptor was confirmed to be present in the culture supernatant.
  • a BALB/c mouse was immunized with 10 ⁇ g of recombinant IL-6 (Hirano, T. et al., Immunol. Lett. (1988) 17, 41) together with Freund's complete adjuvant, and this was continued once a week until anti-IL-6 antibody was able to be detected in the serum.
  • Immunocytes were excised from local lymph nodes and fused with myeloma cell line P3U1 using polyethylene glycol 1500.
  • a hybridoma was selected according to the method of Oi, et al. using HAT culture liquid (Selective Methods in Cellular Immunology, W. H. Freeman and Co., San Francisco, 351, 1980) to establish a hybridoma that produced anti-human IL-6 antibody.
  • IL-6 binding assay was performed in the manner described below on the hybridoma that produced anti-human IL-6 antibody. Namely, a flexible polyvinyl 96-well microplate (Dynatech Laboratories, Alexandria, Va.) was coated overnight at 4° C. with 100 ⁇ l of goat anti-mouse Ig (10 ⁇ g/ml, Cooper Biomedical, Malvern, Pa.) in 0.1 M carbonate-hydrogen carbonate buffer (pH 9.6). Next, the plate was treated for 2 hours at room temperature with 100 ⁇ l of PBS containing 1% bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • hybridoma culture supernatant After washing the plate with PBS, 100 ⁇ l of hybridoma culture supernatant were added to each well followed by incubating overnight at 4° C. The plate was washed and 125 I-labeled recombinant IL-6 was added to each well at 2000 cpm/0.5 ng/well followed by measuring the radioactivity of each well after washing using a gamma counter (Beckman Gamma 9000, Beckman Instruments, Fullerton, Calif.). 32 of 216 hybridoma clones were positive according to the IL-6 binding assay. Stable MH166.BSF2 was ultimately obtained from these clones. The anti-IL-6 antibody MH166 produced by said hybridoma had IgG1 ⁇ subtype.
  • MH60.BSF2 cells were divided among the wells of a microplate at 1 ⁇ 10 4 /200 ⁇ l/well followed by the addition of sample containing MH166 antibody and culturing for 48 hours, and then additionally culturing for 6 hours after adding 3 H-thymidine (New England Nuclear, Boston, Mass.) at 0.5 ⁇ Ci/well. The cells were then placed on glass filter paper and treated with an automatic harvester (Labo Mash Science, Tokyo, Japan). Rabbit anti-IL-6 antibody was used for the control.
  • MH166 antibody was clearly demonstrated to neutralize IL-6 activity.
  • Anti-IL-6 receptor antibody MT18 produced according to the method of Hirata, et al. (Hirata, Y. et al., J. Immunol. (1989) 143, 2900-2906) was bound to activated Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, N.J.) according to attached protocol to purify IL-6 receptor (Yamasaki, K. et al., Science (1988) 241, 825-828).
  • Human myeloma cell line U266 was solubilized with p-paraaminophenyl methane sulfonyl fluoride hydrochloride (Wako Chemicals) containing 1% digitonin (Wako Chemicals), 10 mM triethanol amine (pH 7.8) and 0.15 M NaCl (digitonin buffer), and then mixed with MT18 antibody bound to Sepharose 4B beads. Subsequently, the beads were washed six times with digitonin buffer to obtain partially purified IL-6 receptor for immunization.
  • Wako Chemicals p-paraaminophenyl methane sulfonyl fluoride hydrochloride
  • digitonin 10 mM triethanol amine
  • 0.15 M NaCl digitonin buffer
  • a BALB/c mouse was immunized four times every 10 days with the aforementioned partially purified IL-6 receptor obtained from 3 ⁇ 10 9 U266 cells followed by production of hybridoma in accordance with ordinary methods.
  • the binding activity of hybridoma culture supernatant from positive growth wells to IL-6 receptor was investigated according to the method described below. 5 ⁇ 10 7 U266 cells were labeled with 35 S-methionine (2.5 mCi) and solubilized with the aforementioned digitonin buffer.
  • solubilized U266 cells were then mixed with MT18 antibody bound to Sepharose 4B beads having a volume of 0.04 ml followed by washing six times with digitonin buffer, eluting the 35 S-methionine-labeled IL-6 receptor with 0.25 ml of digitonin buffer (pH 3.4) and neutralizing with 0.025 ml of 1 M Tris (pH 7.4).
  • hybridoma culture supernatant 0.05 ml of hybridoma culture supernatant were mixed with 0.01 ml of Protein G Sepharose (Pharmacia). After washing, the Sepharose was incubated with 0.005 ml of 35 S-methionine-labeled IL-6 receptor solution prepared as described above. The immunoprecipitate was then analyzed with SDS-PAGE and hybridoma culture supernatant that reacts with IL-6 receptor was investigated. As a result, reaction-positive hybridoma clone PM-1 (FERM BP-2998) was established. Antibody produced from hybridoma PM-1 have the IgG1 ⁇ subtype.
  • IL-6 binding inhibitory activity of antibody produced by hybridoma PM-1 on human IL-6 receptor was investigated using human myeloma cell line U266.
  • Human recombinant IL-6 was prepared from E. coli (Hirano, T. et al., Immunol. Lett. (1988) 17, 41-45) and labeled with 125 I using Bolton-Hunter reagent (New England Nuclear, Boston, Mass.) (Taga, T. et al., J. Exp. Med. (1987) 166, 967-981).
  • Monoclonal antibody to mouse IL-6 receptor was prepared according to the method described in Saito, T. et al., J. Immunol. (1991) 147, 168-173.
  • CHO cells producing mouse soluble IL-6 receptor were cultured in IMDM culture liquid containing 10% FCS followed by purification of mouse soluble IL-6 receptor from the culture supernatant using an affinity column in which anti-mouse IL-6 receptor antibody RS12 (refer to the aforementioned Saito, T. et al.) was immobilized in Affigel 10 gel (BioRad).
  • mice soluble IL-6 receptor 50 ⁇ g of the resulting mouse soluble IL-6 receptor were mixed with Freund's complete adjuvant and injected into the abdomen of a Wistar rat. The rat was additionally immunized with Freund's incomplete adjuvant starting two weeks later. Rat spleen cells were then harvested on day 45, and after fusing 2 ⁇ 10 8 cells with 1 ⁇ 10 7 cells of mouse myeloma cell line P3U1 using 50% PEG1500 (Boehringer-Mannheim) in accordance with ordinary methods, the fused cells were screened for hybridoma with HAT medium.
  • hybridoma culture supernatant After adding hybridoma culture supernatant to a plate coated with rabbit anti-rat IgG antibody (Cappel), the mouse soluble IL-6 receptor was allowed to react.
  • hybridoma that produced antibody to mouse soluble IL-6 receptor was screened by ELISA using rabbit anti-mouse IL-6 receptor antibody alkaline phosphatase-labeled sheep anti-rabbit IgG.
  • the hybridoma clones that were confirmed to produce antibody were sub-screened twice to obtain a single hybridoma clone. That clone was named MR16-1.
  • the neutralizing activity during information transmission by mouse IL-6 of this hybridoma-producing antibody was investigated according to the uptake of 3 H-thymidine using MH60.BSF2 cells (Matsuda, T. et al., J. Immunol. (1988) 18, 951-956).
  • 1 ⁇ 10 4 cells/200 ⁇ l/well of MH60.BSF2 cells were prepared in a 96-well plate.
  • 10 pg/ml of mouse IL-6 and 12.3 to 1000 ng/ml of MR16-1 antibody or RS12 antibody were added to this plate and cultured for 44 hours at 37° C. and 5% CO 2 followed by the addition of 1 ⁇ Ci/well of 3 H-thymidine.
  • the uptake of 3H-thymidine was then measured 4 hours later.
  • MR16-1 antibody inhibited the uptake of 3 H-thymidine by MH60.BSF2 cells.
  • hybridoma MR16-1 (FERM BP-5875) was clearly determined to inhibit binding of IL-6 to IL-6 receptor.

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