HK1096588B - Therapeutic agent for mesothelioma - Google Patents

Description

Therapeutic agent for mesothelioma

Technical Field

The present invention relates to a novel mesothelioma therapeutic agent and a mesothelioma cell inhibitor.

Background

Mesothelioma is a tumor that occurs in the mesothelium covering the surface of the pleura, peritoneum, and pericardium that respectively surround the lung, heart, and other organs of the chest and the abdominal organs, such as the stomach, intestine, and liver. Diffuse pleural mesothelioma causes chest pain due to infiltration into intercostal nerves if on the pleural pleurodema side, and causes respiratory circulatory disturbance due to tumor proliferation and pleural fluid retention if on the visceral pleural pleurodema side (senior, medical progress (division 3 months) "respiratory disease" pp.469-472, 1999). In some cases, the adjacent mediastinal organs spread to directly infiltrate the heart and progress to the abdominal cavity through the diaphragm, while lymphatic or blood-borne metastasis increases and progresses toward the outside of the thoracic cavity (see above).

It has been reported that 3000 people in the united states suffered from diffuse pleural mesothelioma a year, which increased significantly from the 1980 s, mostly in men in the age of 60 years, with an incidence of about 5 times higher than in women (high wood, medical advance (3 months on minute manual) "respiratory disease" pp.469-472, 1999). Recent reports in europe and america indicate that the frequency of mesothelioma is rapidly increasing, and in british epidemiology statistics in 1995, it is expected that mesothelioma death will continue to increase in the next 25 years, and in the most serious cases, mesothelioma may account for 1% of the total deaths of men born in 1940 (midfield, respiration, volume 18, No. 9, pp.916-925, 1999).

Many different clinical disease classification methods are used for mesothelioma, and the disease classification method used in the conventional mesothelioma treatment reports is different, and therefore, this method has become a problem in comparison of therapeutic effects (Zhongye, respiration, volume 18, number 9, pp.916-925, 1999). Thus, by 1995, the International Mesothelia Interest Group (IMIG) proposed the International TNM classification scheme for malignant pleural Mesothelioma (Zhongye, respiration, Vol. 18, No. 9, pp.916-925, 1999).

In addition, the exposure of malignant mesothelioma to asbestos (asbestoss) has been causally related and has been demonstrated in animal experiments (field, medical progress (3 months of brochure) "respiratory disease" pp.406-408, 1999). Asbestos inhaled into the respiratory tract reaches just below the pleura, and at least through about 20 years of chronic stimulation, tumors spread superficially throughout the pleura (high wood, medical advance (3 months of brochure) "respiratory disease" pp.469-472, 1999). Thus, malignant mesotheliomas are classified as asbestos-related diseases, but not all malignant mesotheliomas are caused by asbestos, and it is confirmed that about half of patients have significant exposure (field, medical progress (3 months in minute manual) "respiratory disease" pp.406-408, 1999).

Malignant pleural mesothelioma is resistant, has a very poor prognosis and requires early countermeasures (Zhongye, respiration, volume 18, number 9, pp.916-925, 1999). For example: in chemotherapy for mesothelioma, Methotrexate (MTX), which is a folate antagonist, is highly effective, reaching 37%, when administered in combination with leucovin alone in ultra-large amounts, but chemotherapy for mesothelioma, which causes a large amount of pleural fluid retention, is technically difficult and not widespread (midfield, medical progress (3 months of manual) "respiratory disease" pp.570-573, 2003). In addition, diffuse pleural mesothelioma can be subjected to pleuropneumectomy or pleurectomy, but the disease is easy to recur after treatment, and particularly, the local recurrence rate after the operation is as high as 35-43% (alpine, medical progress (3 months in manual) "respiratory disease" pp.469-472, 1999).

It is known that various human mesothelioma cell lines and several mouse mesothelioma cell lines can express IL-6 in vitro, and it has been reported that IL-6 in serum is detected before Cancer cell proliferation and clinical symptoms, and changes in peripheral blood lymphoid tissues in mice transplanted with a mouse mesothelioma cell line AB22 highly expressing IL-6(Bielefeldt-Ohmann, Cancer Immunol Immunother 40: 241-K250, 1995). In addition, it is known that the level of IL-6 in the serum of a patient with malignant pleural mesothelioma is higher than that of a patient with lung adenocarcinoma accompanied by pleural effusion (pleural effusions), and that there is a significant correlation between the level of IL-6 in the serum and the number of platelets in a patient with malignant pleural mesothelioma, which is one of the clinical symptoms of malignant pleural mesothelioma (Nakano, British Journal of Cancer 77 (6): 907-912, 1998). In addition, it has been reported that IL-6 is highly expressed in tumor cells of a patient with peritoneal mesothelioma, and that the level of IL-6 in serum before death becomes high (Higashirara, Cancer October 15, 1992, volume 70, No.8, pp.2105-2108).

Bielefeldt-Ohmann et al reported that when rat anti-mouse IL-6 antibody (6B4) was administered to a mouse transplanted with AB22 2 times per week, the effect of significantly reducing the onset and progression of clinical symptoms was confirmed (Bielefeldt-Ohmann, Cancer Immunol Immunother 40: 241-ion 250, 1995). However, according to Bielefeldt, anti-IL-6 antibody has no direct proliferation inhibitory effect on AB22 in vitro, mice treated with anti-IL-6 antibody and untreated mice have no difference in appearance after death, and a considerable tumor mass is also seen in treated mice (Bielefeldt-Ohmann, Cancer Immunol Immunother 40: 241-ion 250, 1995). That is, inhibition of the proliferation of mesothelioma by an anti-IL-6 antibody has not been known either in vitro or in vivo.

Highwood, medical Advance (3 months of brochure) "respiratory disease" pp.469-472, 1999

Zhongye, respiration, volume 18, number 9, pp.916-925, 1999

Multidata, medical Advance (3 months of brochure) "respiratory disease" pp.406-408, 1999

Zhongye, medical Advance (3 months of brochure) "respiratory disease" pp.570-573, 2003

Bielefeldt-Ohmann，Cancer Immunol Immunother 40：241-250，1995

Higashihara，Cancer October 15，1992，volume 70，No.8，pp.2105-2108

Disclosure of Invention

It is not clear whether IL-6 antagonists act on mesothelioma to exhibit a proliferation-inhibiting effect. The purpose of the present invention is to provide a novel therapeutic agent for mesothelioma (an inhibitor of the proliferation of mesothelioma cells) which comprises an IL-6 antagonist as an active ingredient.

The present inventors have conducted various studies to develop a novel therapeutic agent for mesothelioma that inhibits the proliferation of mesothelioma cells, and as a result, have obtained a novel finding that the proliferation of mesothelioma cells can be inhibited by inhibiting or blocking signal transduction associated with IL-6, and have completed the present invention.

Accordingly, the present invention provides a therapeutic agent for mesothelioma, which comprises an interleukin-6 (IL-6) antagonist as an active ingredient.

The present invention also provides a mesothelioma cell growth inhibitor containing an interleukin-6 (IL-6) antagonist as an active ingredient.

The mesothelioma, such as pleural mesothelioma, more specifically, for example: malignant pleural mesothelioma. Among the malignant pleural mesotheliomas are diffuse pleural mesotheliomas.

Such IL-6 antagonists, for example, anti-IL-6 antibodies or anti-IL-6 receptor antibodies, preferably anti-IL-6 receptor monoclonal antibodies. The anti-IL-6 receptor antibody is particularly preferably a monoclonal antibody against human IL-6 receptor, for example: PM-1 antibody, or anti mouse IL-6 receptor monoclonal antibody, such as: MR16-1 antibody. The above antibody against the IL-6 receptor is preferably a recombinant antibody.

The anti-IL-6 receptor antibody may be a chimeric antibody, a humanized antibody or a human antibody. A particularly preferred antibody of the invention is a humanized PM-1 antibody.

The present invention may take the following forms.

(1) Use of an interleukin-6 (IL-6) antagonist in the manufacture of a therapeutic agent for mesothelioma.

(2) The use according to (1) above, wherein the mesothelioma is pleural mesothelioma.

(3) The use according to (2) above, wherein the pleural mesothelioma is malignant pleural mesothelioma.

(4) The use according to any one of (1) to (3) above, wherein the IL-6 antagonist is an anti-IL-6 receptor antibody.

(5) The use of (4) above, wherein the anti-IL-6 receptor antibody is an anti-IL-6 receptor monoclonal antibody.

(6) The use according to (4) above, wherein the anti-IL-6 receptor antibody is an anti-human IL-6 receptor monoclonal antibody.

(7) The use of (4) above, wherein the anti-IL-6 receptor antibody is a monoclonal antibody against a mouse IL-6 receptor.

(8) The use of any one of (4) to (7) above, wherein the antibody against IL-6 receptor is a recombinant antibody.

(9) The use according to (6) above, wherein the monoclonal antibody against a human IL-6 receptor is a PM-1 antibody.

(10) The use according to (7) above, wherein the monoclonal antibody against a mouse IL-6 receptor is an MR16-1 antibody.

(11) The use according to any one of (4) to (10) above, wherein the anti-IL-6 receptor antibody is a chimeric antibody, a humanized antibody or a human antibody against IL-6 receptor.

(12) The use of (11) above, wherein the humanized antibody against IL-6 receptor is a humanized PM-1 antibody.

(13) Use of an interleukin-6 (IL-6) antagonist in the manufacture of an inhibitor of mesothelioma cell proliferation.

(14) The use of (13) above, wherein the mesothelioma is pleural mesothelioma.

(15) The use of (14) above, wherein the pleural mesothelioma is malignant pleural mesothelioma.

(16) The use according to any one of (13) to (15) above, wherein the IL-6 antagonist is an anti-IL-6 receptor antibody.

(17) The use of (16) above, wherein the anti-IL-6 receptor antibody is an anti-IL-6 receptor monoclonal antibody.

(18) The use according to (16) above, wherein the anti-IL-6 receptor antibody is an anti-human IL-6 receptor monoclonal antibody.

(19) The use of (16) above, wherein the anti-IL-6 receptor antibody is a monoclonal antibody against a mouse IL-6 receptor.

(20) The use of any one of (16) to (19) above, wherein the antibody against the IL-6 receptor is a recombinant antibody.

(21) The use according to (18) above, wherein the monoclonal antibody against a human IL-6 receptor is a PM-1 antibody.

(22) The use of (19) above, wherein the monoclonal antibody against a mouse IL-6 receptor is an MR16-1 antibody.

(23) The use according to any one of (16) to (22) above, wherein the anti-IL-6 receptor antibody is a chimeric antibody, a humanized antibody or a human antibody against IL-6 receptor.

(24) The use of (23) above, wherein the humanized antibody against IL-6 receptor is a humanized PM-1 antibody.

In addition, the present invention may take the following forms.

(1) A method of treating mesothelioma, comprising administering an interleukin-6 (IL-6) antagonist to a subject in need of such treatment.

(2) The method of (1) above, wherein the mesothelioma is pleural mesothelioma.

(3) The method of (2) above, wherein the pleural mesothelioma is malignant pleural mesothelioma.

(4) The method of any one of (1) to (3) above, wherein the IL-6 antagonist is an anti-IL-6 receptor antibody.

(5) The method according to (4) above, wherein the anti-IL-6 receptor antibody is an anti-IL-6 receptor monoclonal antibody.

(6) The method according to (4) above, wherein the anti-IL-6 receptor antibody is a monoclonal antibody against human IL-6 receptor.

(7) The method of (4) above, wherein the anti-IL-6 receptor antibody is a monoclonal antibody against a mouse IL-6 receptor.

(8) The method of any one of (4) to (7) above, wherein the antibody against the IL-6 receptor is a recombinant antibody.

(9) The method according to (6) above, wherein the monoclonal antibody against a human IL-6 receptor is a PM-1 antibody.

(10) The method of (7) above, wherein the monoclonal antibody against a mouse IL-6 receptor is an MR16-1 antibody.

(11) The method of any one of (4) to (10) above, wherein the anti-IL-6 receptor antibody is a chimeric antibody, a humanized antibody or a human antibody against the IL-6 receptor.

(12) The method of (11) above, wherein the humanized antibody against IL-6 receptor is a humanized PM-1 antibody.

(13) A method of inhibiting mesothelioma cell proliferation, comprising administering an interleukin-6 (IL-6) antagonist to a subject in need of such inhibition.

(14) The method of (13) above, wherein the mesothelioma is pleural mesothelioma.

(15) The method of (14) above, wherein the pleural mesothelioma is malignant pleural mesothelioma.

(16) The method of any one of (13) to (15) above, wherein the IL-6 antagonist is an anti-IL-6 receptor antibody.

(17) The method of (16) above, wherein the anti-IL-6 receptor antibody is an anti-IL-6 receptor monoclonal antibody.

(18) The method according to (16) above, wherein the anti-IL-6 receptor antibody is a monoclonal antibody against human IL-6 receptor.

(19) The method of (16) above, wherein the anti-IL-6 receptor antibody is a monoclonal antibody against a mouse IL-6 receptor.

(20) The method of any one of (16) to (19) above, wherein the antibody against the IL-6 receptor is a recombinant antibody.

(21) The method according to (18) above, wherein the monoclonal antibody against a human IL-6 receptor is a PM-1 antibody.

(22) The method of (19) above, wherein the monoclonal antibody against a mouse IL-6 receptor is an MR16-1 antibody.

(23) The method of any one of (16) to (22) above, wherein the antibody against the IL-6 receptor is a chimeric antibody, a humanized antibody or a human antibody against the IL-6 receptor.

(24) The method of (23) above, wherein the humanized antibody against IL-6 receptor is a humanized PM-1 antibody.

Drawings

FIG. 1 shows the results of example 1, and shows the IL-6-producing ability of various malignant mesothelioma cell lines.

FIG. 2 shows the results of example 2, and shows the productivity (no production) of IL-6 receptor (IL-6R) of various malignant mesothelioma cell lines. The mRNA level of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) used as an internal standard is also shown.

FIG. 3 shows the results of example 3, and shows that the production of Vascular Endothelial Growth Factor (VEGF) by the malignant mesothelioma cell lines H2052 and H2452 is induced by IL-6 and IL-6R.

FIG. 4 shows the results of example 4, and shows the results of the same experiment as in example 3 with respect to malignant mesothelioma cell line H28, which produces Vascular Endothelial Growth Factor (VEGF) without induction by IL-6/IL-6R.

FIG. 5 shows the results of example 5, and shows that STAT3 phosphorylation was promoted by IL-6 stimulation in the IL-6-dependent VEGF-inducible strain H2025 cell line, whereas STAT3 phosphorylation was not promoted by IL-6 stimulation in the IL-6-independent VEGF-inducible strain H28 cell line.

FIG. 6 shows the results of example 6, showing that SOCS3 was inducible by IL-6 and IL-6R stimulation in IL-6-dependent VEGF-inducible strain H2025 cell line, whereas SOCS3 was non-inducibly expressed in IL-6-independent VEGF-inducible strain H28 cell line.

FIG. 7 shows the promoters of plasmids pGL3-VEGF and pGL3-VEGFmut used in example 7 and their neighboring structures.

FIG. 8 shows the results of example 7, and shows that IL-6-dependent activation of VEGF promoter was not observed by mutation of the p-STAT3 binding site in the VEGF promoter in a system in which the VEGF promoter was linked to a luciferase reporter gene.

FIG. 9 is a schematic diagram showing the mechanism of inducing a VEGF promoter (producing VEGF) by IL-6 stimulation in the H2052 cell line, which is estimated from the results of examples 5 to 7.

FIG. 10 shows the results of example 8, which shows that the proliferation of the H2052 cell line increased in the presence of IL-6R in a concentration-dependent manner with respect to IL-6.

FIG. 11 shows the results of example 9, and shows that the growth promotion of H2052 cell line by IL-6 and IL-6R was inhibited by MRA.

FIG. 12 shows the results of example 10, and shows that the proliferation of the H226 cell line increased in the presence of IL-6R in a concentration-dependent manner with respect to IL-6 and the inhibitory effect on MRA.

FIG. 13 shows the results of example 8, which shows that the proliferation of H226 cells is increased in the presence of IL-6R in a concentration-dependent manner with respect to IL-6.

FIG. 14 shows the results of example 11, and shows that the proliferation promoting effects of IL-6 and soluble IL-6 receptor-dependent malignant mesothelioma cell line H2052 cells and H226 are inhibited by the humanized PM-1 antibody.

FIG. 15 shows the results of example 12, and shows that the IL-6-stimulation-dependent VEGF induction production was inhibited by the humanized PM-1 antibody in malignant mesothelioma cell lines MSTO, H226, H2052 and H2452.

FIG. 16 shows the results of example 13, in which STAT3 phosphorylation induced by IL-6 and soluble IL-6 receptor stimulation was inhibited by the humanized PM-1 antibody in H2052 cell line and H2452 cell line.

FIG. 17 shows the results of example 14, which shows that the stimulation with IL-6 and a soluble IL-6 receptor did not inhibit the growth promotion of H2052 cell lines and H226 cell lines by an anti-VEGF antibody.

Detailed Description

IL-6 is a cytokine known as B cell stimulating factor 2(BSF2) or interferon beta 2.IL-6 was discovered as a differentiation factor involved in B lymphocyte activation (Hirano, T.et., Nature (1986)324, 73-76), and was later elucidated as a multifunctional cytokine affecting various cell functions (Akira, S.et al., adv.in Immunology (1993)54, 1-78). IL-6 has been reported to induce maturation of T lymphocytes (Lotz, M.et al., J.exp.Med. (1988)167, 1253-1258).

IL-6 through the cell on two proteins to transmit its biological activity. One is the ligand-binding protein IL-6 receptor (Taga, T.et., J.Exp.Med. (1987)166, 967-828, Yamasaki, K.et., Science (1987)241, 825-828) which binds IL-6 and has a molecular weight of about 80 kD. The IL-6 receptor is a membrane-bound type which penetrates a cell membrane and is expressed on the cell membrane, and is mainly present as a soluble IL-6 receptor composed of the extracellular region.

The other is a non-ligand-binding membrane protein gp130 with a molecular weight of about 130kD, which is involved in signal transduction. IL-6 forms an IL-6/IL-6 receptor complex with the IL-6 receptor, and then the biological activity of IL-6 is transmitted into cells by binding to gp130 (Taga, T.et., Cell (1989)58, 573-581).

IL-6 antagonists are substances that inhibit the transmission of IL-6 biological activity. Antibodies against IL-6 (anti-IL-6 antibodies), antibodies against IL-6 receptor (anti-IL-6 receptor antibodies), antibodies against gp130 (anti-gp 130 antibodies), IL-6 variants, IL-6 or partial peptides of IL-6 receptor, and the like have been known so far.

There have been several reports relating to anti-IL-6 receptor antibodies (Novick, D.et al., Hybridoma (1991)10, 137-35146, Huang, Y.W.et al., Hybridoma (1993)12, 621-630, International patent application publication No. WO 95-09873, French patent application publication No. FR 2694767, U.S. Pat. No. US 521628). Humanized PM-1 antibodies obtained by grafting the Complementarity Determining Regions (CDRs) of one of the mouse antibodies PM-1(Hirata, Y.et. al., J.Immunol. (1989)143, 2900-.

The IL-6 antagonist is preferably an anti-IL-6 receptor antibody, more preferably an anti-human IL-6 receptor monoclonal antibody or an anti-mouse IL-6 receptor monoclonal antibody. As the above-mentioned monoclonal antibody against the human IL-6 receptor, for example, PM-1 antibody, and further as the monoclonal antibody against the mouse IL-6 receptor, for example, MR16-1 antibody. The antibody is preferably a chimeric, humanized or human antibody, for example: humanized PM-1 antibodies.

The IL-6 antagonist used in the present invention can be used as an active ingredient of a therapeutic agent for mesothelioma, regardless of the origin, kind and shape thereof, as long as it inhibits the proliferation of mesothelioma cells.

IL-6 antagonists are substances that block IL-6 dependent signal transduction and inhibit the biological activity of IL-6. The IL-6 antagonist is preferably a substance having an inhibitory effect on the binding of any one of IL-6, IL-6 receptor and gp 130. As IL-6 antagonists, for example: anti IL-6 antibody, anti IL-6 receptor antibody, anti gp130 antibody, IL-6 variants, soluble IL-6 receptor variants or IL-6 receptor partial peptide, and show their same activity of low molecular substance.

The anti-IL-6 antibodies used in the present invention can be obtained in polyclonal or monoclonal form using well-known means. As the anti-IL-6 antibody used in the present invention, a monoclonal antibody derived from a mammal is particularly preferable. Examples of monoclonal antibodies derived from mammals include antibodies produced by hybridomas and antibodies produced by a host transformed with an expression vector containing an antibody gene by genetic engineering means. The antibody can inhibit the combination of IL-6 and IL-6 receptor by combining with IL-6, and block the transmission of IL-6 biological activity to cells.

As such antibodies, MH166(Matsuda, T.et al., Eur.J.Immunol. (1988)18, 951-956) and SK2 antibodies (Sato, K.et al., 21 st college of immunology, academic records (1991)21, 166) and the like can be mentioned.

Hybridomas that produce anti-IL-6 antibodies can be prepared essentially using well-known techniques, as follows. That is, IL-6 is used as a sensitizing antigen, immunization is carried out according to a usual immunization method, the obtained immunocytes are fused with well-known parent cells by a usual cell fusion method, and cells producing monoclonal antibodies are selected by a usual selection method.

Specifically, an anti-IL-6 antibody can be prepared as follows. For example: human IL-6 used as a sensitizing antigen for obtaining antibodies can be obtained by using the IL-6 gene/amino acid sequences disclosed in Eur.J.biochem (1987)168, 543-550, J.Immunol. (1988)140, 1534-1541, or Ag.biol.chem. (1990)54, 2685-2688.

The IL-6 gene sequence is inserted into a known expression vector system, and after transforming an appropriate host cell, the objective IL-6 protein is purified from the host cell or culture supernatant by a known method, and the purified IL-6 protein can be used as an antigen for sensitization. In addition, a fusion protein of IL-6 protein and another protein may be used as a sensitizing antigen.

The anti-IL-6 receptor antibody used in the present invention can be obtained in the form of a polyclonal or monoclonal antibody using well-known means. The anti-IL-6 receptor antibody used in the present invention is particularly preferably a monoclonal antibody derived from a mammal. Examples of monoclonal antibodies derived from mammals include antibodies produced by hybridomas and antibodies produced by hosts transformed with expression vectors containing antibody genes by genetic engineering techniques. The antibody inhibits the binding of IL-6 to IL-6 receptor by binding to IL-6 receptor, and blocks the transfer of the biological activity of IL-6 into cells.

As such antibodies, for example: 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 antibody or AUK146-15 antibody (International patent application publication No. WO92-19759), and the like. Of these, particularly preferred antibodies are PM-1 antibodies.

The hybridoma cell line producing the PM-1 antibody was collected as PM-1 at 7/12/3/year old and deposited with FERM BP-2998 under the Budapest treaty at the International patent organism depositary of the national institute of Industrial and technology (center 6, 1 st Carlo 1, 1 st Dida, Tokyo, prefecture). Furthermore, the hybridoma cell line producing the MR16-1 antibody was deposited as Rat-mousehybridoma MR16-1 in 9 years, 3 months, 13 days, and then deposited with FERM BP-5875 under the Budapest treaty at the International patent organism depositary of Integrated Industrial science and technology (center 6, 1 atm 1, 1 Carlo, Town, To.

Hybridomas producing anti-IL-6 receptor monoclonal antibodies can be prepared essentially using well-known techniques, as follows. That is, the monoclonal antibody can be prepared by immunizing according to a usual immunization method using an IL-6 receptor as a sensitizing antigen, fusing the obtained immunocytes with well-known parent cells by a usual cell fusion method, and screening cells producing the monoclonal antibody by a usual screening method.

Specifically, an anti-IL-6 receptor antibody can be prepared as follows. For example: the human IL-6 receptor used as a sensitizing antigen for obtaining an antibody can be obtained by using European patent application publication No. EP 325474, and the mouse IL-6 receptor can be obtained by using the gene/amino acid sequence of the IL-6 receptor disclosed in Japanese patent application publication No. JP-A3-155795.

The IL-6 receptor proteins are expressed on and detached from the cell membrane (soluble IL-6 receptor) (Yasukawa, K.et al, J.biochem. (1990)108, 673-676). Soluble IL-6 receptor antibodies are composed of a substantial extracellular domain of IL-6 receptor that binds to cell membranes, and soluble IL-6 receptors differ from membrane-bound IL-6 receptors by the absence of a cell membrane-penetrating domain or a combination of a cell membrane-penetrating domain and an intracellular domain. Any IL-6 receptor can be used as the IL-6 receptor protein as long as it can be used as a sensitizing antigen for producing an anti-IL-6 receptor antibody used in the present invention.

The IL-6 receptor gene sequence may be inserted into a known expression vector system, an appropriate host cell may be transformed, and the objective IL-6 receptor protein may be purified from the host cell or culture supernatant by a known method, and the purified IL-6 receptor protein may be used as a sensitizing antigen. In addition, IL-6 receptor-expressing cells or fusion proteins of IL-6 receptor proteins with other proteins can also be used as sensitizing antigens.

Coli (E.coli) containing plasmid pIBIBBSF 2R containing cDNA encoding the human IL-6 receptor was deposited under the Budapest treaty at the national institute of Integrated technology and Industrial science of independent administration patent depositary (center 6, 1, N.1, of Tokyo prefecture, Bobo, Tokyo prefecture) under HB 101-pIBBSF 2R and FERM BP-2232 at 1 month 9 in Yuanyuan (1989).

The anti-gp 130 antibody used in the present invention can be obtained in the form of a polyclonal or monoclonal antibody using well-known means. The anti-gp 130 antibody used in the present invention is particularly preferably a monoclonal antibody derived from a mammal. As monoclonal antibodies derived from mammals, there are antibodies produced by hybridomas and antibodies produced by hosts transformed by genetic engineering means with expression vectors containing antibody genes. The antibody inhibits the combination of IL-6/IL-6 receptor complex and gp130 by combining with gp130, and blocks the transmission of IL-6 biological activity to cells.

Examples of such antibodies include AM64 antibody (Japanese patent application laid-open No. Hei 3-219894), 4B11 antibody, 2H4 antibody (US 5571513), B-S12 antibody, and B-P8 antibody (Japanese patent application laid-open No. Hei 8-291199).

Hybridomas producing anti-gp 130 monoclonal antibodies can be prepared essentially as follows using well-known techniques. That is, gp130 was used as a sensitizing antigen, immunization was performed according to a usual immunization method, the obtained immunocytes were fused with well-known parental cells by a usual cell fusion method, and monoclonal antibody-producing cells were selected by a usual selection method.

Specifically, monoclonal antibodies can be prepared as follows. For example: gp130 used as a sensitizing antigen for obtaining an antibody can be obtained by using the gene/amino acid sequence of gp130 disclosed in european patent application publication No. EP 411946.

The gp130 gene sequence may be inserted into a known expression vector system, transformed into an appropriate host cell, and the gp130 protein of interest may be purified from the host cell or culture supernatant by a known method, and the purified gp130 receptor protein may be used as a sensitizing antigen. Alternatively, cells expressing gp130 or a fusion protein of gp130 protein and another protein may be used as a sensitizing antigen.

The mammal to be immunized with the sensitizing antigen is not particularly limited, and is preferably selected in consideration of suitability of the parent cell used for cell fusion, and a rodent such as: mouse, rat, hamster, and the like.

The sensitized antigen is immunized to the animal according to well-known methods. For example, as a general method, by injecting a sensitizing antigen into the abdominal cavity or subcutaneously of a mammal. Specifically, the sensitizing antigen is diluted to an appropriate amount with PBS (Phosphate-Buffered Saline) or physiological Saline, and the suspension solution may be mixed with a usual adjuvant such as: the Freund's complete adjuvant is mixed in appropriate amount, and preferably injected into mammal every 4-21 days after emulsification. In addition, in the immunization with a sensitizing antigen, an appropriate carrier can be used.

After the immunization was performed as described above and the increase in the desired antibody level in the serum was confirmed, the immune cells were taken out from the mammal and used for cell fusion. Spleen cells are particularly mentioned as ideal immune cells for cell fusion.

As the mammalian myeloma cells to be used as the other parent cell to be fused with the above-mentioned immunocytes, various cell lines known in the art are suitably used, such as: P3X63Ag8.653(Kearney, J.F.et. al.J.Immunol. (1979)123, 1548-1550), P3X63Ag8U.1(Current Topics in Microbiology and analysis (1978)81, 1-7), NS-1(Kohler.G.and Milstein, C.Eur.J.Immunol. (1976)6, 511-519), MPC-11 (Margulies.D.H.et., Cell (1976)8, 405-415), SP2/0(Shulman, M.et. Nature (1978)276, 269-270), FO (St.Groth, S.F.Immunol. (1980)35, 1-21), S.F.Immunol. (J.J.1980) 35, 1-21), S.194, Trdowni.J.313. Immunol. (1978, 121-11), and Nature (1976, 277-11, 1-11, 11-11, 11-11, 1-21, 11, 1, 11, 1.

Cell fusion of the above immunocytes with myeloma cells is carried out basically according to a well-known method, for example, the method of Milstein et al (Kohler.G. and Milstein, C., MethodsEnzymol. (1981)73, 3-46), etc.

More specifically, the cell fusion can be carried out in a conventional nutrient medium in the presence of, for example, a cell fusion promoter. As the fusion promoter, for example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like can be used, and if necessary, in order to further improve the fusion efficiency, can also be added and used dimethyl sulfoxide and other auxiliary agents.

The ratio of the immune cells to the myeloma cells to be used is, for example, preferably 1 to 10 times that of the immune cells to the myeloma cells. The culture medium used for the cell fusion may be, for example, an RPMI1640 culture medium suitable for the proliferation of the above myeloma cell line, an MEM culture medium, other conventional culture media used for the cell culture, or a serum supplement such as Fetal Calf Serum (FCS).

The cell fusion can be performed by mixing a predetermined amount of the immune cells and myeloma cells in the culture solution, and adding a PEG solution heated to about 37 ℃ in advance at a concentration of 30 to 60% (w/v), for example: PEG solutions having an average molecular weight of about 1000 to 6000 are mixed to form a desired fused cell (hybridoma). Then, appropriate culture medium is added one by one, and the operation of removing the supernatant by centrifugation is repeated, whereby a cell fusion agent or the like which affects the growth of a hybridoma can be removed.

The hybridoma is cultured and selected in a normal selection medium, for example, HAT medium (medium containing hypoxanthine, aminopterin, and thymidine). In order to provide sufficient time for complete death of cells (non-fused cells) other than the target hybridoma, the culture in the HAT culture solution is usually continued for several days to several weeks. Then, the hybridoma producing the target antibody is screened and cloned by a conventional limiting dilution method.

In addition to the above hybridomas obtained by immunizing an animal other than a human with an antigen, a human lymphocyte may be sensitized in vitro with a desired antigen protein or an antigen-expressing cell, and the sensitized B lymphocyte may be fused with a human myeloma cell, for example, U266, to obtain a desired human antibody having a binding activity to the desired antigen or antigen-expressing cell (see japanese patent publication No. 1-59878). Alternatively, desired human antibodies can be obtained by the above-described method by administering antigens or antigen-expressing cells to transgenic animals having a human antibody gene bank (reptotoire) (see international patent application publication nos. WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096, and WO 96/33735).

The monoclonal antibody-producing hybridomas thus prepared can be subcultured in a conventional culture medium, and can be stored in liquid nitrogen for a long period of time.

In order to obtain monoclonal antibodies from the hybridomas, the following methods can be used: the hybridoma is cultured according to a conventional method to obtain a culture supernatant thereof, or the following method: the hybridoma is administered to a mammal suitable for the hybridoma to proliferate and obtain ascites thereof, etc. The former method is suitable for obtaining an antibody of high purity, while the latter method is suitable for mass production of an antibody.

For example, a hybridoma producing an anti-IL-6 receptor antibody can be prepared according to the method disclosed in Japanese patent laid-open No. Hei 3-139293. PM-1 antibody-producing hybridomas deposited internationally under the terms of the Budapest treaty in the International patent organism depositary, national institute of advanced Industrial science and technology, national institute of technology and technology, national institute of ascites, national institute of technology and technology, center 6 of 1 st Care of 1T, 1 st Diway, Tokuwa prefecture, were injected into the abdominal cavity of BALB/c mice to obtain ascites fluid, and PM-1 antibody was purified from the ascites fluid, or the hybridomas were cultured in an appropriate medium, for example, RPMI1640 medium containing 10% fetal bovine serum and 5% BM-Condimed H1 (manufactured by Boehringer Mannheim), hybridoma SFM medium (manufactured by GIBCO-BRL), PFHM-II medium (manufactured by GIBCO-BRL), and PM-1 antibody was purified from the culture.

In the present invention, as the MONOCLONAL antibody, a recombinant antibody produced by cloning an antibody gene from a hybridoma, integrating the antibody gene into an appropriate vector, introducing the vector into a host, and using genetic recombination techniques (for example, see Borrebaeck C.A.K. and Larrick J.W.THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United kingdom by MACMILLIAN PUBLISHERS LTD, 1990) can be used.

Specifically, mRNA encoding the variable (V) region of an antibody is isolated from a cell, e.g., a hybridoma, that produces the antibody of interest. Isolation of mRNA Total RNA is prepared according to a well-known method, for example, guanidine ultracentrifugation (Chirgwin, J.M.et. al., Biochemistry (1979)18, 5294-. Alternatively, mRNA can be directly prepared by using QuickPrep mRNA purification kit (Pharmacia).

cDNA for the V region of the antibody can be synthesized from the resulting mRNA using reverse transcriptase. cDNA synthesis is performed using AMV reverse transcriptase strand 1 cDNA synthesis kit or the like. In addition, for the synthesis and amplification of cDNA, 5 '-Ampli FINDER RACE kit (manufactured by Clontech) and 5' -RACE method using PCR (Frohman, M.A.et. al., Proc.Natl.Acad.Sci.USA (1988)85, 8998-9002; Belyavsky, A.et. al., Nucleic Acids Res. (1989)17, 2919-2932) can be used. The target DNA fragment was purified from the obtained PCR product and ligated to the vector DNA. Then, a recombinant vector is prepared therefrom, and introduced into Escherichia coli or the like, colonies are selected, and a desired recombinant vector is prepared. By well-known methods, such as: the base sequence of the target DNA was confirmed by the deoxidation method.

If a DNA encoding the V region of the target antibody is obtained, it is ligated with the desired DNA encoding the constant region (C region) of the antibody, and incorporated into an expression vector. Alternatively, the DNA encoding the V region of the antibody may be incorporated into an expression vector containing the DNA of the C region of the antibody.

In order to produce the antibody used in the present invention, the antibody gene is incorporated into an expression vector which can be expressed under the control of an expression regulatory region, such as an enhancer or a promoter, as described below. Then, the host cell is transformed with the expression vector to express the antibody.

In the present invention, for the purpose of reducing human xenogeneic antigenicity, for example, a genetically modified antibody can be used, which is artificially modified, for example: chimeric (Chimeric) antibodies, Humanized (Humanized) antibodies, human (human) antibodies. These altered antibodies can be made using known methods.

The chimeric antibody can be produced by ligating a DNA encoding a V region of an antibody obtained as described above with a DNA encoding a C region of a human antibody, integrating the DNA into an expression vector, and introducing the vector into a host (see European patent application publication No. EP125023 and International patent application publication No. WO 92-19759). Using this known method, a chimeric antibody for use in the present invention can be obtained.

For example, plasmids containing DNA encoding the L chain and H chain V regions of the chimeric PM-1 antibody were designated as pPM-k3 and pPM-H1, respectively, and E.coli containing the plasmids were deposited under the Budapest treaty on the National Collections of Industrial and Marine bacteria Limited on 12.2.1991 (23 St Machar Drive, Aberdeen AB 21 RY Scotland, great british and North Ireland).

A humanized antibody is also called a reshaped (reshaped) human antibody, and is a product obtained by grafting a Complementarity Determining Region (CDR) of a non-human mammal, for example, a mouse antibody, to a complementarity determining region of a human antibody, and a general gene recombination method thereof is known (see european patent application publication No. EP125023 and international patent application publication No. WO 92-19759).

Specifically, a DNA sequence designed such that CDRs of a mouse antibody are connected to Framework Regions (FRs) of a human antibody is synthesized by PCR from a plurality of oligonucleotides having overlapping portions at their terminal portions. The obtained DNA is ligated with a DNA encoding a human antibody C region, and the ligated DNA is incorporated into an expression vector, which is introduced into a host to produce the vector (see European patent application publication No. EP 239400, International patent application publication No. WO 92-19759).

FR selection of a human antibody to which CDRs are ligated allows the CDRs to form a region of a good antigen-binding site. Amino acids in the framework regions of the antibody variable regions may also be substituted as necessary to form suitable antigen-binding sites for the complementarity determining regions of the reconstituted human antibodies (Sato, K.et al, Cancer Res. (1993)53, 851-.

For chimeric antibodies, humanized antibodies, human antibody C regions may be used. As the human antibody C region, such as C γ, for example: c γ 1, C γ 2, C γ 3, or C γ 4 may be used. In order to improve the stability of the antibody or its production, the C region of the human antibody may be modified.

Chimeric antibodies are composed of variable regions derived from antibodies derived from mammals other than humans and C regions derived from human antibodies, and humanized antibodies are composed of complementarity determining regions derived from antibodies derived from mammals other than humans, framework regions derived from human antibodies and C regions, and have low antigenicity in humans, and therefore can be used as antibodies in the present invention.

As a preferred specific example of the humanized antibody used in the present invention, a humanized PM-1 antibody (see International patent application publication No. WO92-19759) is exemplified.

As a method for obtaining a human antibody, in addition to the above-described methods, a technique for obtaining a human antibody by panning using a human antibody library is known. For example, the variable region of a human antibody may be displayed as a single chain antibody (scFv) on the surface of a phage by phage display, or a phage that binds to an antigen may be selected. If the genes of the selected phage are analyzed, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. If the DNA sequence of scFv that binds to an antigen is elucidated, an appropriate expression vector can be prepared using the sequence to obtain a human antibody. These processes are well known and reference may be made to WO 92/01047, WO 92/20791, WO93/06213, WO 93/11236, WO 93/19172, WO 95/01438, WO 95/15388.

The antibody gene constructed as described above can be obtained by expressing it by a known method. In the case of mammalian cells, the expression is carried out by using a commonly used and useful promoter, an expressed antibody gene, DNA to which a poly A signal peptide downstream of the 3' side is functionally linked, or a vector containing the DNA. For example: as the promoter/enhancer, human cytomegalovirus, i.e., early promoter/enhancer (human cytomegalovirus immediate early promoter/enhancer), is exemplified.

As another promoter/enhancer that can be used for the expression of the antibody of the present invention, a viral promoter/enhancer such as retrovirus, polyoma virus, adenovirus, simian virus 40(SV40), or a promoter/enhancer derived from mammalian cells such as human elongation factor 1 α (HEF1 α) can be used.

For example, when the SV40 promoter/enhancer is used, it can be easily carried out by the method of Mullingan et al (Mullingan, R.C. et al., Nature (1979)277, 108-114) and when the HEF 1. alpha. promoter/enhancer is used, the method of Mizushima et al (Mizushima, S.andNagata, S.nucleic Acids Res. (1990)18, 5322).

When Escherichia coli is used, a commonly used useful promoter, a signal peptide sequence for antibody secretion, and an expressed antibody gene can be functionally combined and expressed. For example, as a promoter, lacZ promoter, araB promoter can be mentioned. When the lacZ promoter is used, it can be performed according to the method of Ward et al (Ward, E.S.et al., Nature (1989)341, 544-242546; Ward, E.S.et al. FASEB J. (1992)6, 2422-2427), and when the araB promoter is used, it can be performed according to the method of Better et al (Better, M.et al. science (1988)240, 1041-1043).

As a signal peptide sequence for antibody secretion, a pelB signal peptide sequence can be used when an antibody is produced in the periplasm of E.coli (Lei, S.P.et al J.Bacteriol. (1987)169, 4379-4383). After the antibody produced in the periplasm is isolated, the structure of the antibody is appropriately refolded (refold) and used (see, for example, WO 96/30394).

As the replication origin, those derived from SV40, polyoma virus, adenovirus, Bovine Papilloma Virus (BPV) and the like can be used, and the expression vector may contain an Aminoglycoside Phosphotransferase (APH) gene, a thymidine deoxynucleoside kinase (TK) gene, an E.coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, a dihydrofolate reductase (dhfr) gene and the like as selection markers for gene copy number amplification in a host cell line.

For producing the antibody used in the present invention, any production system may be used. Production systems for antibody production have both in vitro and in vivo production systems. As production systems in vitro, there are, for example, those using eukaryotic cells and those using prokaryotic cells.

When eukaryotic cells are used, there are production systems using animal cells, plant cells, or fungal cells. As animal cells, (1) mammalian cells are known, for example: CHO, COS, myeloma, BHK (baby hamster kidney), HeLa, Vero, etc.; (2) amphibian cells, for example: xenopus oocytes, or (3) insect cells, e.g., sf9, sf21, Tn5, and the like. As plant cells, cells derived from tobacco (Nicotiana tabacum) are known, and callus culture can be performed on them. As fungal cells, yeasts are known, for example: yeasts (Saccharomyces) genus, such as Saccharomyces cerevisiae, filamentous fungi, such as: aspergillus (Aspergillus), for example: aspergillus niger (Aspergillus niger) and the like.

When prokaryotic cells are used, there are production systems using bacterial cells. Coli (e.coli) and bacillus subtilis are known as bacterial cells.

The antibody can be obtained by introducing a gene of the desired antibody into these cells by transformation, and culturing the transformed cells in vitro. The culture is carried out according to a well-known method. For example, DMEM, MEM, RPMI1640, IMDM may be used as the culture medium, or a serum additive such as Fetal Calf Serum (FCS) may be used in combination. Alternatively, the antibody can be produced in vivo by transferring cells into which the antibody gene has been introduced into the abdominal cavity of an animal, or the like.

In addition, as production systems in vivo, there are production systems using animals and production systems using plants. When animals are used, production systems for mammals and insects are used.

As the mammal, goat, pig, sheep, mouse, cow, etc. can be used (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). And silkworm can be used as the insect. When plants are used, tobacco can be used, for example.

The antibody gene is introduced into these animals or plants, and the antibody is produced and recovered in vivo in the animals or plants. For example: the antibody gene is inserted into the middle of a gene encoding a protein inherently produced in milk such as goat beta-casein, and a fusion gene is prepared. The DNA fragment containing the fusion gene into which the antibody gene has been inserted is injected into an embryo of a goat, and the embryo is introduced into a female goat. The desired antibody can be obtained from the milk produced by the transgenic goat born by the goat who received the embryo or by the offspring thereof. In order to increase the amount of milk containing the desired antibody produced from the transgenic goat, an appropriate hormone may be administered to the transgenic goat. (Ebert, K.M.et. al., Bio/Technology (1994)12, 699-.

In addition, when silkworms are used, the silkworms are infected with baculovirus into which a gene for the desired antibody is inserted, and the desired antibody is obtained from the body fluid of the silkworms (Maeda, S.et. al., Nature (1985)315, 592-594). In addition, when tobacco is used, the target antibody gene is inserted into a plant expression vector, for example, pMON530, and the vector is introduced into a bacterium such as Agrobacterium tumefaciens. Infection of tobacco with this bacterium, such as Nicotiana tabacum, can lead to the production of the desired antibody from the leaf of this tobacco (Julian, K. -C.Ma et al., Eur.J. Immunol. (1994)24, 131-.

When an antibody is produced by an in vitro or in vivo production system as described above, the host may be transformed by integrating DNA encoding the heavy chain (H chain) or the light chain (L chain) of the antibody into an expression vector, or by integrating DNA encoding the H chain and the L chain into one expression vector (see International patent application publication No. WO 94-11523).

The antibody that can be used in the present invention may be a fragment of the antibody or a modified product thereof, as long as it is suitable for use in the present invention. For example: as fragments of antibodies, for example, Fab, F (ab')₂Fv, or single chain Fv (scFv) in which the H chain of the Fv is linked to the L chain by a suitable linker.

Specifically, an enzyme for an antibody, for example: papain, pepsin, to produce antibody fragments, or to construct genes encoding these antibody fragments, which are expressed in appropriate host cells after introduction into an expression vector (see, 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, Pluuethun, A. & Skerra, A.methods in Enzymology (1989)178, 476-496, 476-thu, A.S. 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, 663-66, Bird, R.E. 137, ECH. 1991).

scFv can be obtained by linking the V region of the H chain of an antibody with the V region of the L chain. In this scFv, the V region of the H chain is linked to the V region of the L chain via a linker, preferably a peptide linker (Huston, J.S.et. al., Proc.Natl.Acad.Sci.U.S.A. (1988)85, 5879-5883). The V region of the H chain and the V region of the L chain in the ScFv may be derived from any of the antibodies described above. As the peptide linker for connecting the V region, for example, any single-chain peptide consisting of 12 to 19 amino acid residues can be used.

The DNA encoding scFv can be obtained by amplifying a DNA portion encoding an amino acid sequence expected from the above-mentioned sequences by PCR using a primer pair defining both ends of a DNA portion encoding an H chain or a V region of an H chain and a DNA encoding an L chain or a V region of an L chain as templates, and then combining a DNA encoding a peptide linker moiety and a primer pair defining both ends capable of linking to each of the H chain and the L chain, followed by amplification.

Once DNA encoding scFv is prepared, an expression vector containing the DNA and a host transformed with the expression vector can be obtained by a conventional method, and scFv can be obtained by a conventional method using the host.

These antibody fragments can be produced by obtaining the genes and expressing the genes in a host in the same manner as described above. Reference to "antibodies" in the present invention also includes fragments of such antibodies.

As a modified antibody, an antibody conjugated with various molecules such as polyethylene glycol (PEG) can be used. The "antibody" referred to in the present invention also includes these antibody modifications. In order to obtain such an antibody-modified product, the antibody obtained may be chemically modified. These methods are established in the art.

The antibody produced and expressed as described above can be isolated from the inside or outside of the cell and from the host, and purified to homogeneity. The antibody used in the present invention can be isolated and purified by affinity chromatography. As a column used in affinity chromatography, for example, a protein A column and a protein G column are used. Examples of the carrier used in the protein a column include Hyper D, POROS, Sepharose f.f. In addition, separation and purification methods generally used for proteins can be used without any limitation.

For example: the antibody used in the present invention can be isolated and purified by appropriately selecting and combining chromatography other than the above affinity chromatography, filtration, exclusion filtration, salting out, dialysis, and the like. Examples of chromatography include ion exchange chromatography, hydrophobic chromatography, and gel filtration. These chromatographies are suitable for HPLC (high Performance liquid chromatography). In addition, reverse phase hplc (reverse phase hplc) may also be used.

The concentration of the antibody obtained above can be measured by absorbance measurement, ELISA, or the like. That is, when the absorbance was measured, the sample was diluted with PBS (-) and then the absorbance at 280nm was measured, and 1mg/ml was calculated as 1.35 OD. When measured by ELISA, the measurement can be carried out as follows. That is, 100. mu.l of goat anti-human IgG (manufactured by TAG) diluted to 1. mu.g/ml with 0.1M bicarbonate buffer (pH9.6) was added to a 96-well plate (manufactured by Nunc), incubated overnight at 4 ℃ and the antibody was immobilized. After blocking, 100. mu.l of the antibody or antibody-containing sample used in the present invention or human IgG (manufactured by CAPPEL) as a standard was added thereto in an appropriate dilution, and the mixture was incubated at room temperature for 1 hour.

After washing, 100. mu.l of 5000-fold diluted alkaline phosphatase-labeled anti-human IgG (manufactured by BIOSOURCE) was added and incubated at room temperature for 1 hour. After washing, the substrate solution was added, and after incubation, absorbance at 405nm was measured using a MICROPLATE READER Model 3550 (manufactured by Bio-Rad) to calculate the concentration of the target antibody.

The IL-6 variants used in the present invention are substances which have binding activity to the IL-6 receptor and do not transmit the biological activity of IL-6. That is, IL-6 variants and IL-6 competitive binding to IL-6 receptor, because of not transmitting IL-6 biological activity, so can block dependent on IL-6 signal transduction.

IL-6 variants are prepared by introducing mutations by substitution of amino acid residues in the amino acid sequence of IL-6. IL-6 as an IL-6 variant body regardless of its origin, if considering antigenicity, etc., preferably human IL-6.

Specifically, the secondary structure of the amino acid sequence of IL-6 was predicted using a well-known molecular modeling program such as WHATIF (Vriend et al, J.mol.graphics (1990)8, 52-56), and the effect of the substituted amino acid residue on the whole was evaluated. After an appropriate substitution of amino acid residues is determined, a gene encoding an IL-6 variant can be obtained by introducing a mutation capable of substituting an amino acid into a vector containing a nucleotide sequence encoding a human IL-6 gene as a template by a commonly-performed PCR method. If necessary, the gene may be incorporated into an appropriate expression vector, and the IL-6 variant may be obtained by the above-described methods of expression, production and purification of the recombinant antibody.

As specific examples of IL-6 variants, mention may be made of the examples disclosed in Brakenhoff et al, J.biol.chem. (1994)269, 86-93 and Savino et al, EMBO J. (1994)13, 1357-.

The IL-6 partial peptide or IL-6 receptor partial peptide used in the present invention is a substance which has a binding activity to IL-6 receptor or IL-6, respectively, and does not transmit the biological activity of IL-6. That is, the IL-6 partial peptide or the IL-6 receptor partial peptide binds to the IL-6 receptor or IL-6, and by capturing them, the binding of IL-6 to the IL-6 receptor is specifically inhibited. As a result, IL-6-dependent signal transduction was blocked because the biological activity of IL-6 was not transmitted.

The IL-6 partial peptide or IL-6 receptor partial peptide is a peptide consisting of a part or all of the amino acid sequence of a region involved in the binding of IL-6 to an IL-6 receptor in the amino acid sequence of IL-6 or IL-6 receptor. Such peptides are generally composed of 10 to 80, preferably 20 to 50, more preferably 20 to 40 amino acid residues.

IL-6 partial peptides or IL-6 receptor partial peptides can be prepared as follows. The region involved in the binding of IL-6 to the IL-6 receptor is specifically designated in the amino acid sequence of IL-6 or IL-6 receptor, and a part or all of the amino acid sequence thereof is prepared by a conventionally known method, for example, genetic engineering means or peptide synthesis.

In order to prepare an IL-6 partial peptide or an IL-6 receptor partial peptide by genetic engineering, a DNA sequence encoding a desired peptide is incorporated into an expression vector, and the partial peptide can be obtained by the above-described methods of expression, production and purification of a recombinant antibody.

For the preparation of an IL-6 partial peptide or an IL-6 receptor partial peptide by peptide synthesis, a method generally used in peptide synthesis, for example, solid phase synthesis or liquid phase synthesis, can be used.

Specifically, the method can be carried out according to "development of pharmaceuticals" volume 14, "peptide synthesis", Tokyo island therapeutics, Guangchua bookshop, 1991. As the solid-phase synthesis method, for example, a method of binding a C-terminal amino acid corresponding to a peptide to be synthesized to a carrier insoluble in an organic solvent, and extending the peptide chain by alternately repeating a reaction of condensing amino acids in the order from the C-terminal to the N-terminal using amino acids in which an α -amino group and a side chain functional group are protected with an appropriate protecting group and a reaction of releasing the protecting group of the α -amino group of the amino acid or the peptide bound to a resin, one by one, is used. Solid-phase peptide synthesis methods are roughly classified into Boc method and Fmoc method depending on the kind of protecting group used.

After the target peptide is synthesized as described above, deprotection reaction and cleavage reaction of the peptide chain from the carrier are carried out. In the cleavage reaction for cleaving a peptide chain, hydrogen fluoride or trifluoromethanesulfonic acid is generally used if the Boc method is used, and TFA is generally used if the Fmoc method is used. If the protected peptide resin is treated by the Boc method, for example, in the presence of anisole in hydrogen fluoride. The protecting group is then deprotected and cleaved from the support, and the peptide recovered. The peptide was lyophilized to obtain a crude peptide. Further, when Fmoc method is used, deprotection reaction and cleavage reaction of the peptide chain from the carrier are carried out in the same manner as described above, for example, in TFA.

The obtained crude peptide can be separated and purified by HPLC. In elution, a water-acetonitrile solvent generally used for protein purification is used, and the elution is performed under optimum conditions. The resulting peak fractions corresponding to the entire chromatogram were collected and freeze-dried. The peptide fraction thus purified is identified by molecular weight analysis by mass spectrometry, amino acid composition analysis, amino acid sequence analysis, or the like.

Specific examples of the IL-6 partial peptide and the IL-6 receptor partial peptide include those disclosed in JP-A-2-188600, JP-A-7-324097, JP-A-8-311098 and U.S. Pat. No. 5,5210075.

The IL-6 signal transduction inhibitory activity of the IL-6 antagonist used in the present invention can be evaluated by a commonly used method. Specifically, IL-6-dependent cells were assayed for IL-6-dependent cells by culturing IL-6-dependent human myeloma cell line (S6B45, KPMM2), human Lennert T lymphoma cell line KT3, or IL-6-dependent cells MH60.BSF2, adding IL-6, and allowing IL-6 antagonist to coexist³Incorporation of H-thymidine.

In addition, by expressing the peptide as IL-6 receptorCulturing the cell in U266, and adding¹²⁵I-labeled IL-6, with addition of IL-6 antagonist, and determination of binding to IL-6 receptor expressing cells¹²⁵I labeled IL-6. In the above assay system, a negative control group containing no IL-6 antagonist is provided in addition to the group containing the IL-6 antagonist, and the IL-6 inhibitory activity of the IL-6 antagonist can be evaluated by comparing the results obtained from the two groups.

As shown in the examples below, since the effect of inhibiting the proliferation of mesothelioma cells by an anti-IL-6 receptor antibody was confirmed, it was revealed that IL-6 antagonists such as an anti-IL-6 receptor antibody can be used as a therapeutic agent for mesothelioma.

The subject of the invention is a mammal. The mammal to be treated is preferably a human.

The mesothelioma therapeutic agent or mesothelioma cell proliferation inhibitor of the present invention may be administered systemically or locally by oral or non-oral administration. For example, intravenous injection such as drip, intramuscular injection, intrathoracic injection, intraperitoneal injection, subcutaneous injection, suppository, enema, oral enteric agent, and the like can be selected, and an appropriate administration method can be selected according to the age and symptoms of the patient. The effective administration amount may be selected from the range of 0.01mg to 100mg per time, per 1kg body weight. Or the administration amount of 1 to 1000mg, preferably 5 to 50mg, can be selected for each patient.

The preferable dose and administration method are, for example, those of an anti-IL-6 receptor antibody, in which the amount of the antibody existing in blood is an effective dose, and specific examples thereof include 0.5 to 40mg, preferably 1 to 20mg, by intravenous injection such as intravenous drip, subcutaneous injection or the like, in accordance with an administration schedule such as 2 times/week, 1 time/2 week, 1 time/4 week or the like, for example, 1 time to several times within 1 month (4 weeks) per 1kg body weight. The administration schedule may be adjusted by extending the administration interval from 2 times/week or 1 time/week to 1 time/2 week, 1 time/3 week, 1 time/4 week, etc., while observing the trends in the disease conditions and blood test values.

The mesothelioma therapeutic agent or the mesothelioma cell growth inhibitor of the present invention may contain a pharmaceutically acceptable carrier or additive depending on the administration route. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, 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, pharmaceutically acceptable surfactants, and the like. The additives to be used may be selected from the above-mentioned compounds as appropriate or in combination according to the dosage form, but are not limited to these compounds.

Examples

The present invention will be specifically described below with reference to examples and reference examples, but the present invention is not limited to these examples.

Example 1 production of IL-6 by malignant mesothelioma cell lines

Cell concentration 5X 10 in 24-well plates supplemented with culture medium (RPMI supplemented with 10% Fetal Calf Serum (FCS))⁴The culture of malignant mesothelioma cell lines, MSTO, H2052, H28, H226 and H2452 was started per well, the cell culture solution was changed the next day, and then cultured for 3 more days, and the IL-6 concentration in the culture supernatant was measured by a full-automatic chemiluminescence enzyme immunoassay system (Fujirebio, Lumipulse), and the amount of the cell protein was used for supplementation and correction. The results are shown in FIG. 1. The experiment was repeated 3 times. The H28 cell line was a cell line that did not produce IL-6, and the other 4 lines were IL-6-producing lines. In particular, the H2052 cell line and the H226 cell line are considered to be IL-6-producing strains.

Example 2 expression of IL-6R in malignant mesothelioma cell lines

The expression level of IL-6 receptor was measured in 5 malignant mesotheliomas at the mRNA level. KT-3 cells were used as a positive control, and synovial cells (Synoviocytes) were used as a negative control. These cells were cultured in RPMI supplemented with 10% FCS for 48 hours, and the mRMA encoding IL-6 receptor (IL-6R) in the cells was measured by the reverse transcription-PCR (RTPCR) method of GeneAmp PCR System (Applied Biosystems) as a detection means. The results are shown in FIG. 2. In addition, the mRNA level of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) used as an internal standard control is shown in FIG. 2 (bottom).

From this result, it is considered that the malignant mesothelioma cells hardly express the IL-6 receptor. Pleural effusion in the case of malignant pleural mesothelioma is bloody pleural effusion, and many soluble IL-6 receptors are present, which are presumed to be involved in IL-6 stimulation transduction.

Example 3 stimulation with IL-6 induces VEGF production (1)

The malignant mesothelioma cell strains H2052 and H2452 are cultured in RPMI1640 medium to obtain the initial cell concentration of 5 × 10⁴Perwell, 3 sets of cultures were grown in 24-well plates. The next day the cell culture was changed and stimulation was started with (1) recombinant IL-6(10ng/ml), (2) recombinant IL-6(10ng/ml) + recombinant soluble IL-6R (100ng/ml) and (3) humanized PM-1 antibody as anti-IL-6 receptor antibody (WO 92/19759 reference) (25. mu.g/ml) (RPMI as media control). After 3 days of re-incubation, the cells were cultured by QuantikineHuman VEGF Immunoassay kit (R)&D Systems) the concentration of Vascular Endothelial Growth Factor (VEGF) in the culture supernatant was measured and corrected for the amount of cellular protein.

The results are shown in FIG. 3. As the figure shows, stimulation with IL-6 induced VEGF production in both H2052 and H2452 cell lines.

Example 4 stimulation with IL-6 induces VEGF production (2)

The experiment of example 3 was repeated using malignant mesothelioma cell line H28. The results are shown in FIG. 4. As the results indicate, the H28 cell line was a VEGF-producing strain and did not respond to IL-6 stimulation.

Example 5 phosphorylation of STAT3 by IL-6 stimulation

IL-6-dependent VEGF-inducible producer H2025 cell line and IL-6-independent VEGF-inducible producer H28 cell line were incubated with signal transducer and activator of transcription 3(STAT3) in the presence of recombinant IL-6(10ng/ml) and soluble recombinant IL-6 receptor (100ng/ml), and at 0, 0.5 and 1 hour of the incubation, analyses for phosphorylation from STAT3 to p-STAT3 were performed using Western blotting. The results are shown in FIG. 5.

As shown in fig. 5, STAT3 was demonstrated to be significantly phosphorylated in the H2052 cell line 30 minutes after IL-6 stimulation. In the control H28 cell line, only a small amount of STAT3 phosphorylation was seen on IL-6 stimulation. In addition, in fig. 5, p-STAT3 represents phosphorylated STAT3, and STAT3 represents the sum of phosphorylated and non-phosphorylated STAT 3.

Example 6 stimulation with IL-6 induces SOCS3

IL-6-dependent VEGF-inducible strain H2025 and IL-6-independent VEGF-inducible strain H28 were incubated in the presence of recombinant IL-6(10ng/ml) and soluble recombinant IL-6 receptor (100ng/ml), stimulated, and the expression levels of the induced supressor of cytokine 3(SOCS3) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were measured at 0, 2 and 4 hours of incubation, based on the measurement after the mRNA encoding them was amplified by RT-PCR. The results are shown in FIG. 6.

As shown in FIG. 6, in the H2052 cell line, induction of SOCS3mRNA was confirmed 2 hours after IL-6 stimulation. Constant high expression of SOCS3 was observed in H28 cell line.

Example 7 VEGF promoter analysis

A luciferase gene expression plasmid pGL3-VEGF controlled by the VEGF promoter, and a luciferase gene expression plasmid pGL3-VEGFmut controlled by a mutant VEGF promoter in which the phosphorylated STAT3 binding portion is disrupted were prepared (FIG. 7).

Transfection was performed at a rate of 1. mu.g pGL3-VEGF or pGL3-VEGFmut per 50000H 2052 cells, and transfected cells were stimulated with recombinant IL-6(10ng/ml) and recombinant soluble IL-6 receptor (100 ng/ml). After 2 days of stimulation, luciferase activities induced by the VEGF promoter and the mutant VEGF promoter were studied. The results are shown in FIG. 8.

As shown in fig. 8, no activation of the VEGF promoter due to IL-6 stimulation was found when phosphorylated STAT3 binding was deleted from the VEGF promoter. From the results of the above examples 5, 6 and 7, it is considered that the enhancement of VEGF production in the H2052 cell line due to IL-6 stimulation is mediated by the JAK-STAT system. The proposed system is shown in FIG. 9.

Example 8 promotion of H2052, H226 by addition of IL-6 and soluble IL-6 receptor Proliferation of

The H2052 cell line was inoculated into a 96-well plate containing 10% FCS-added RPMI medium at 500 cells/well, and cultured for 6 to 7 days in 5 groups in the presence or absence of 100ng/ml of recombinant soluble IL-6 and in the presence of IL-6 at various concentrations (0, 1, 10 or 100 ng/ml). The results are shown in FIGS. 10 and 13. As shown in these figures, the H2052 cell line and the H226 cell line proliferated in the presence of recombinant soluble IL-6 receptor (100ng/ml) in a concentration-dependent manner with IL-6.

Example 9 addition of IL-6 and IL-6R promoted proliferation of H2052 cells was addressed Antibody inhibition of IL-6R (anti-IL-6R antibody)

To see whether proliferation of H2052 cell lines promoted by IL-6 and IL-6 receptors was inhibited by anti-IL-6R antibody, H2052 cell lines were seeded at 500 cells/well in 96-well plates containing RPMI medium supplemented with 10% FCS, and cultured for 7 days in 3 groups in the presence of 10ng/ml of recombinant soluble IL-6 and 100ng/ml of recombinant soluble IL-6 receptor, and in the presence of humanized PM-1 antibody at various concentrations (0, 1. mu.g/ml, or 25. mu.g/ml). After the culture, the amount of proliferation (OD450) of the H2052 cell line was measured by MTS analysis. The results are shown in FIG. 11. From these results, it was found that the anti-IL-6R antibody inhibited proliferation in a concentration-dependent manner. In addition, as a control, human IgG1 was added at the same concentration instead of MRA, and no inhibitory effect was seen.

Example 10 proliferation of H226 by addition of IL-6 and soluble IL-6 receptor Promotion, and inhibition by anti-IL-6R antibodies

The H226 cell line was inoculated at 500 cells/well into 96-well plates containing 10% FCS-supplemented RPMI medium, and cultured for 7 days in 3 groups in the presence of 100ng/ml of recombinant soluble IL-6 receptor, and IL-6 at various concentrations (0, 1, 10 or 100ng/ml), or 25. mu.g/ml of MRA. The results are shown in FIG. 12. As shown in this figure, the IL-6-highly productive H226 cell line proliferated in a concentration-dependent manner with IL-6 in the presence of a recombinant soluble IL-6 receptor (100ng/ml) as in the IL-6-highly productive H2052 cell line, and the proliferation thereof was inhibited by an anti-IL-6R antibody.

Example 11H 2052 cells by addition of IL-6 and soluble IL-6 receptor Proliferation promotion of the strain or H226 cell line is inhibited by an anti-IL-6 receptor antibody

The H2052 cell line and the H226 cell line were inoculated at 200 cells/well into 96-well plates containing 10% FCS-supplemented RPMI medium, and cultured for 6 days in 5 groups in the presence of IL-6(10ng/ml) and soluble IL-6 receptor (100ng/ml) and in the presence of humanized PM-1 antibody at various concentrations (0, 1. mu.g/ml, 5. mu.g/ml). After the culture, the cell growth rates of the H2052 cell line and the H226 cell line were measured by MTS analysis. The cell proliferation rate is expressed as how many times the cells proliferated compared to the case where IL-6/sIL-6R was not added. The results are shown in FIG. 14. The results show that the anti-IL-6 receptor antibody completely inhibited the proliferation promoting effect of the H2052 cell line and the H226 cell line due to the addition of IL-6/sIL-6R. In addition, as a control, human IgG1(Sigma) was added at the same concentration in place of the anti-IL-6 receptor antibody, and no proliferation inhibitory effect was observed.

Example 12 inhibition of VEGF production by IL-6 stimulation with anti-IL-6 receptor antibodies Induction of

The inhibitory effect of anti-IL-6 receptor antibodies against IL-6-stimulated VEGF production by the malignant mesothelioma cell lines MSTO, H226, H2052 and H2452 was examined under the same conditions as in example 3, except for the concentrations of humanized PM-1 antibodies (0.1. mu.g/ml, 5. mu.g/ml). The results are shown in FIG. 15. This result indicates that anti-IL-6 receptor antibodies completely inhibit IL-6/sIL-6R stimulation-induced VEGF production. In addition, as a control, when human IgG1(Sigma) was added at the same concentration in place of the anti-IL-6 receptor antibody, little inhibition of induced VEGF production was observed.

Example 13 inhibition of STAT3 phosphorylation by anti-IL-6 receptor antibodies

It was examined whether STAT3 phosphorylation induced by stimulation with IL-6(10ng/ml) and soluble IL-6 receptor (100ng/ml) was inhibited by an anti-IL-6 receptor antibody at 5. mu.g/ml in the H2052 cell line and H2452 cell line which were VEGF-induced strains. The results are shown in FIG. 16. This result indicates that anti-IL-6 receptor antibodies significantly inhibit phosphorylated STAT3(p-STAT3) induced by IL-6/sIL-6R stimulation in malignant mesothelioma cells. In addition, as a control, human IgG1(Sigma) was added at the same concentration instead of the anti-IL-6 receptor antibody, and almost no inhibition of STAT3 phosphorylation was seen.

Example 14 Effect of anti-VEGF antibodies on malignant mesothelioma cell proliferation

The H2052 cell line and the H226 cell line were inoculated at 500 cells/well into a 96-well plate containing 10% FCS-supplemented RPMI medium, and cultured for 6 to 7 days in 5 groups in the presence of IL-6(10ng/ml) and recombinant soluble IL-6 receptor (100ng/ml) and in the presence of 1. mu.g/ml of anti-VEGF antibody. After the culture, the amount of proliferation was investigated by MTS analysis. As a control, human IgG1(Sigma) at the same concentration was used instead of the anti-VEGF antibody. The results are shown in FIG. 17. This result indicates that the anti-VEGF antibody does not inhibit cell proliferation caused by IL-6/sIL-6R stimulation. Therefore, it was found that the effect of IL-6/sIL-6R stimulation on malignant mesothelioma cell proliferation was not mediated by VEGF.

Reference example 1.Preparation of human soluble IL-6 receptor

Soluble IL-6 receptors were prepared by PCR using plasmid pBSF2R.236 containing cDNA encoding IL-6 receptors obtained according to Yamasaki et al (Yamasaki, K.et al, Science (1988)241, 825-828). The plasmid pBSF2R.236 was digested with the restriction enzyme Sph I to obtain an IL-6 receptor cDNA, which was inserted into mp18 (manufactured by Amersham). To introduce a stop codon into IL-6 receptor cDNA, a mutation was introduced into IL-6 receptor cDNA by PCR method using a designed synthetic oligonucleotide primer by an in vitro mutagenesis system (manufactured by Amersham). By this operation, a stop codon was introduced into the position of amino acid 345 to obtain cDNA encoding soluble IL-6 receptor.

In order to express the soluble IL-6 receptor cDNA in CHO cells, it was ligated with plasmid pSV (Pharmacia) to obtain plasmid pSVL 344. The soluble IL-6 receptor cDNA cut with Hind III-Sal I was inserted into plasmid pECEdhfr containing cDNA of dhfr to obtain CHO cell expression plasmid pECEdhfr 344.

The dhfr-CHO cell line DXB-11(Urlaub, G.et al, Proc.Natl.Acad.Sci.USA (1980)77, 4216-4220) was transfected with 10. mu.g of plasmid pECEdhfr344 using calcium phosphate precipitation (Chen, C.et al, mol.cell.biol. (1987)7, 2745-2751). The transfected CHO cells were cultured for 3 weeks in nucleotide-free α MEM selection medium containing 1mM glutamine, 10% dialysis FCS, 100U/ml penicillin and 100 μ g/ml streptomycin.

And (4) screening the selected CHO cells by using a limiting dilution method to obtain a single CHO cell clone. The CHO cell clone was amplified in methotrexate at a concentration of 20nM to 200nM to obtain a CHO cell line producing human soluble IL-6 receptor. The CHO cell line 5E27 was cultured in Iscove's modified Dulbecco's medium (IMDM, manufactured by Gibco) containing 5% FBS. Culture supernatants were recovered and the soluble IL-6 receptor concentration in the culture supernatants was determined by ELISA. This result confirmed the presence of soluble IL-6 receptor in the culture supernatant.

Reference example 2.Preparation of anti-human IL-6 antibody

Mu.g of recombinant IL-6(Hirano, T.et., Immunol.Lett. (1988)17, 41) was immunized with Freund's complete adjuvant in BALB/c mice, and immunization was continued every other week until anti-IL-6 antibodies could be detected in the serum. Immune cells were isolated from local lymph nodes and fused with myeloma cell line P3U1 using polyethylene glycol 1500. Hybridomas producing anti-human IL-6 antibodies were established by selecting them according to the method of Oi et al (Selective Methods in Cellular Immunology, W.H.Freeman and Co., San Francisco, 351, 1980) using HAT medium.

Hybridomas producing anti-human IL-6 antibodies can be assayed for IL-6 binding as described below. That is, a 96-well microplate made of soft polyethylene (Dynatech Laboratories, Inc., Alexandria, Va.) was coated with 100. mu.l of goat anti-mouse Ig (10. mu.l/ml, Cooper Biomedical, Inc., Malvern, Pa.) in 0.1M bicarbonate buffer (pH9.6) overnight at 4 ℃. The plates were then treated with 100. mu.l of 1% Bovine Serum Albumin (BSA) in PBS for 2 hours at room temperature.

After the plates were then washed with PBS, 100. mu.l of hybridoma culture supernatant was added to each well and incubated overnight at 4 ℃. Plates were washed and 125I-labeled recombinant IL-6 was added to each well as 2000cpm/0.5ng/well, after washing, the radioactivity in each well was measured using a Gamma counter (Beckman Gamma 9000, Beckman Instruments, Fullerton, Calif.). 32 out of 216 hybridoma clones were positive by IL-6 binding analysis. Stable mh166.bsf2 was finally obtained in these clones. The hybridoma produced anti-IL-6 antibody MH166 with the IgG1 kappa subtype.

Then using IL-6 dependenceMouse hybridoma clone mh60.bsf2, the neutralizing activity of MH166 antibody was studied in relation to hybridoma proliferation. MH60.BSF2 cells were plated at 1X 10⁴The mixture was dispensed at 200. mu.l/well, and then a sample containing MH166 antibody was added thereto, incubated for 48 hours, and added at 0.5. mu. Ci/well³After H-thymidine (New England Nuclear, Boston, Mass.), the culture was continued for another 6 hours. Cells were placed on glass filter paper and treated with an automatic collector (Labo Mash Science Co., Tokyo, Japan). Rabbit anti-IL-6 antibody was used as a control.

The results indicate that MH166 antibody potently inhibits MH60.BSF2 cells induced by IL-6³Incorporation of H thymidine. This indicates that MH166 antibody neutralizes the activity of IL-6.

Reference example 3.Preparation of anti-human IL-6 receptor antibody

IL-6 receptor was purified according to the attached recipe by binding anti-IL-6 receptor antibody MT18 prepared by the method of Hirata et al (Hirata, Y.et al.J.Immunol. (1989)143, 2900-2906) to CNBr-activated Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, NJ) (Yamasaki, K.et al., Science (1988)241, 825-828). Human myeloma cell line U266 was dissolved in 1mM p-aminophenylmethanesulfonyl fluoride hydrochloride (manufactured by Wako Chemicals) (digitonin buffer solution) containing 1% digitonin (manufactured by Wako Chemicals), 10mM triethanolamine (pH 7.8) and 0.15M NaCl, and mixed with MT18 antibody bound to agarose 4B particles. The particles were then washed 6 times with digitonin buffer as partially purified IL-6 receptor for immunization.

By a factor of 3X 10⁹The partially purified IL-6 receptor obtained from U266 cells was immunized into BALB/c mice 4 times every 10 days, and then made into hybridomas by a conventional method. The binding activity of hybridoma culture supernatants from growing positive wells to IL-6 receptor was investigated by the following method. Will be 5X 10⁷For U266 cells³⁵S-methionine (2.5mCi) was labeled and dissolved in the above-mentioned digitonin buffer. Dissolving U266 cells were mixed with 0.04ml volume of MT18 antibody bound to Sepharose 4B particles, then washed 6 times with digitonin buffer, and washed with 0.25ml of digitonin buffer (pH 3.4)³⁵The S-methionine-labeled IL-6 receptor was eluted and neutralized with 0.025ml of 1M Tris (pH 7.4).

0.05ml of hybridoma culture supernatant was mixed with 0.01ml of Protein G Sepharose (manufactured by Phramacia). After washing, the agarose was mixed with 0.005ml of the agarose prepared above³⁵S-labeled IL-6 receptor solution. The immunoprecipitated material was analyzed by SDS-PAGE and hybridoma culture supernatants that reacted with IL-6 receptor were investigated. This result established the positive hybridoma clone PM-1(FERM BP-2998). The antibody produced by hybridoma PM-1 has the IgG1 kappa subtype.

Human myeloma cell line U266 was used to study the inhibitory activity of the antibody produced by hybridoma PM-1 on the binding of IL-6 to the human IL-6 receptor. Preparation of human recombinant IL-6 from E.coli (Hirano, T.et al., Immunol.Lett. (1988)17, 41-45) by Bolton-Hunter reagent (New England Nuclear, Boston, Mass.)¹²⁵Marker I (Taga, t.et al, j.exp.med. (1987)166, 967-.

Will be 4X 10⁵Culture supernatant of U266 cells with 70% (v/v) hybridoma PM-1 and 14000cpm¹²⁵I labeled IL-6 was incubated for 1 hour. 70 μ l of the sample was spread on 400 μ l microfusion polyethylene tubes over 300 μ l FCS and after centrifugation, the radioactivity on the cells was measured.

This result indicates that the antibody produced by hybridoma PM-1 inhibits the binding of IL-6 to the IL-6 receptor.

Reference example 4.Preparation of anti-mouse IL-6 receptor antibody

Monoclonal antibodies against the mouse IL-6 receptor were prepared using the method described by Saito, T.et al, J.Immunol (1991)147, 168-173.

CHO cells producing a mouse soluble IL-6 receptor were cultured in IMDM medium containing 10% FCS, and the mouse soluble IL-6 receptor was purified using an affinity column (Saito, T.et al, supra) in which an anti-mouse IL-6 receptor antibody RS12 derived from the culture supernatant was fixed to Affigel 10 gel (manufactured by Biorad).

The resulting mouse soluble IL-6 receptor (50. mu.g) was mixed with Freund's complete adjuvant and injected into the abdomen of Wistar rats. After 2 weeks, additional immunization was performed with Freund's incomplete adjuvant. On day 45, rat spleen cells were harvested and 2X 10 cells were plated⁸Each cell is combined with 1X 10⁷Mouse myeloma cell P3U1 was subjected to cell fusion by a conventional method using 50% PEG1500 (manufactured by Boehringer Mannheim), and then hybridoma was selected by HAT medium.

The hybridoma culture supernatant was applied to a plate coated with a rabbit anti-rat IgG antibody (manufactured by Cappel) and reacted with a mouse soluble IL-6 receptor. Then, hybridomas producing mouse-soluble anti-IL-6 receptor antibodies were screened by ELISA using rabbit anti-mouse IL-6 receptor antibodies and alkaline phosphatase-labeled goat anti-rabbit IgG. Hybridoma clones confirmed to produce antibodies were sub-screened 2 times to obtain single hybridoma clones. This clone was named MR 16-1.

By using MH60.BSF2 cells (Matsuda, T.et al., J.Immunol. (1988)18, 951-956)³Incorporation of H-Thymidine the neutralizing activity of the antibody produced by this hybridoma in the transduction of IL-6 in mice was studied. According to 1 × 10⁴MH60.BSF2 cells were prepared in 96-well plates as per 200. mu.l/well. Adding 10pg/ml mouse IL-6 and MR16-1 antibody or RS12 antibody 12.3-1000 ng/ml to the plate, and reacting at 37 deg.C with 5% CO₂After culturing for 44 hours, 1. mu. Ci/well was added³H-thymidine. Measured after 4 hours³Incorporation of H-Thymidine. The results indicate that the MR16-1 antibody inhibits MH60.BSF2 cells³Incorporation of H thymidine.

Thus, it was revealed that the antibody produced by hybridoma MR16-1(FERM BP-5875) inhibited the binding of IL-6 to the IL-6 receptor.

Claims (22)

1. Use of an anti-IL-6 receptor antibody that inhibits the binding of interleukin-6 (IL-6) to an IL-6 receptor in the manufacture of a therapeutic agent for mesothelioma.

2. The use of claim 1, wherein the mesothelioma is pleural mesothelioma.

3. The use of claim 2, wherein the pleural mesothelioma is malignant pleural mesothelioma.

4. The use of claim 1, wherein the anti-IL-6 receptor antibody is a monoclonal anti-IL-6 receptor antibody.

5. The use of claim 1, wherein said anti-IL-6 receptor antibody is a monoclonal anti-human IL-6 receptor antibody.

6. The use of claim 1, wherein the anti-IL-6 receptor antibody is a monoclonal antibody against the mouse IL-6 receptor.

7. The use of claim 1, wherein the anti-IL-6 receptor antibody is a recombinant antibody.

8. The use of claim 5, wherein said monoclonal antibody against human IL-6 receptor is a PM-1 antibody.

9. The use of claim 6, wherein the monoclonal antibody against the mouse IL-6 receptor is the MR16-1 antibody.

10. The use of any one of claims 1 to 9, wherein the anti-IL-6 receptor antibody is a chimeric, humanized or human anti-IL-6 receptor antibody.

11. The use of claim 10, wherein the humanized antibody to the IL-6 receptor is a humanized PM-1 antibody.

12. Use of an anti-IL-6 receptor antibody that inhibits the binding of interleukin-6 (IL-6) to an IL-6 receptor for the preparation of a proliferation inhibitor for mesothelioma cells.

13. The use of claim 12, wherein the mesothelioma is pleural mesothelioma.

14. The use of claim 13, wherein the pleural mesothelioma is malignant pleural mesothelioma.

15. The use of claim 12, wherein the anti-IL-6 receptor antibody is a monoclonal anti-IL-6 receptor antibody.

16. The use of claim 12, wherein the anti-IL-6 receptor antibody is a monoclonal anti-human IL-6 receptor antibody.

17. The use of claim 12, wherein the anti-IL-6 receptor antibody is a monoclonal antibody against the mouse IL-6 receptor.

18. The use of claim 12, wherein the anti-IL-6 receptor antibody is a recombinant antibody.

19. The use of claim 16, wherein said anti-human IL-6 receptor monoclonal antibody is a PM-1 antibody.

20. The use of claim 17, wherein the monoclonal antibody against the mouse IL-6 receptor is the MR16-1 antibody.

21. The use of any one of claims 12 to 20, wherein the anti-IL-6 receptor antibody is a chimeric, humanized or human anti-IL-6 receptor antibody.

22. The use of claim 21, wherein the humanized antibody to the IL-6 receptor is a humanized PM-1 antibody.