Use of a pm-1 antibody or of a mh 166 antibody for enhancing the anti-tumor effect of cisplatin or carboplatin

Technical Field

0001The present invention relates to an antitumor agent effect enhancer comprising an interleukin-6 antagonist, which assists and enhances the effects of antitumor agents in the treatment of tumors.

Background Art

0002Various drugs such as alkylating agents, metabolism antagonists, antitumor antibiotics and platinum compounds have been used in the past for chemotherapy on human tumors. In the case where remarkable therapeutic effects are not observed when these antitumor agents are used alone, therapeutic methods have been devised in which a plurality of antitumor agents are used concomitantly (<nplcit id="ncit0001" npl-type="s"><text>Frei, E. III, Cancer Res., 32, 2593-2607, 1992</text></nplcit>). Tumor cells exhibit various sensitivities to antitumor agents, and certain cells are known to exhibit resistance to therapy by antitumor agents (Magrath, I., New Directions in Cancer Treatment, 1989, Springer-Verlag). Acquisition of resistance to therapy by tumor cells is said to be caused by, for example, the occurrence of multi-drug resistance (MDR) (<nplcit id="ncit0002" npl-type="s"><text>Tsuruo, T. et al., Cancer Res., 42, 4730-4733, 1982</text></nplcit>), reduction in cell uptake of antitumor agent (Sherman, S.E. et al., Science, 230, 412, 1985), increased DNA repair activity (Borch, R.F., Metabolism and Action of Anticancer Drugs, Powis, G. &amp; Prough, R. ed., Taylor &amp; Francis, London, 1987, 163-193), or promotion of inactivation of antitumor agent in the cells (<nplcit id="ncit0003" npl-type="s"><text>Teicher, B.A. et al., Cancer Res., 46, 4379, 1986</text></nplcit>). In such cases, there are many cases in which the expected therapeutic effects are not observed simply by administering antitumor agent.

0003Renal cell carcinoma exhibits resistance to therapy to antitumor agents such as cisplatin, adriamycin and vinblastine (<nplcit id="ncit0004" npl-type="s"><text>Kakehi, Y. et al., J. Urol., 139, 862-864, 1988</text></nplcit>; <nplcit id="ncit0005" npl-type="s"><text>Kanamaru, H. et al., J. Natl. Cancer Inst., 81, 844-847, 1989</text></nplcit>; <nplcit id="ncit0006" npl-type="s"><text>Teicher, B.A. et al., Cancer Res., 47, 388-393, 1987</text></nplcit>). Platinum compounds such as cisplatin that possess antitumor effects bond with DNA and inhibit DNA synthesis and cell division (<nplcit id="ncit0007" npl-type="s"><text>Pinto, A.L. et al., Biochica et Biophysica Acta, 780, 167-180, 1985</text></nplcit>).

0004The expression of glutathione-S transferase-π (GST-π), inhibition of the effects of cisplatin caused by an increase in intracellular levels of substances containing sulfidryl groups, increased DNA repair activities, or activation of oncogenes such as c-myc is considered to be involved in the resistance to therapy of renal cell carcinoma to cisplatin (<nplcit id="ncit0008" npl-type="s"><text>Sklar, M.D. et al., Cancer Res., 51, 2118-2123, 1991</text></nplcit>; <nplcit id="ncit0009" npl-type="s"><text>Mizutani, Y. et al., Cancer in press, 1994</text></nplcit>; <nplcit id="ncit0010" npl-type="s"><text>Nakagawa, K. et al., Japan J. Cancer Res., 79, 301-305, 1988</text></nplcit>).

0005In addition, changes in membrane permeability and transport capabilities in tumor cells are said to cause a decrease in cisplatin uptake within the cells, thus increasing resistance to therapy to cisplatin (<nplcit id="ncit0011" npl-type="s"><text>Richon, V. et al., Cancer Res., 47, 2056-2061, 1987</text></nplcit>; <nplcit id="ncit0012" npl-type="s"><text>Waud, W.R. et al., Cancer Res., 47, 6549-6555, 1987</text></nplcit>). Glutathione, an example of a substance containing sulfidryl groups that is present in the largest amounts in mammalian animal cells, is reported to inactivate cisplatin in cells, and certain types of tumors have been shown to have higher intracellular glutathione and metallothionein levels (<nplcit id="ncit0013" npl-type="s"><text>Hromas, R.A. et al., Cancer LETT., 34, 9-13, 1987</text></nplcit>; <nplcit id="ncit0014" npl-type="s"><text>Taylor, D.M. et al., Eur. J. Cancer, 12, 249-254, 1976</text></nplcit>).

0006Glutathione is a tripeptide thiol. It plays an important role in the inactivation of DNA-bonding substances such as alkylating agents and cisplatin and in the repair of cell damage caused by these. One of the effects of GST-π is that it promotes inactivation of antitumor agents by causing antitumor agents like those described above to bond to glutathione.

0007Since renal cell carcinoma produces interleukin-6 (IL-6) and expresses IL-6 receptor (IL-6R), it has been suggested that IL-6 plays some type of role in the growth activity of renal cell carcinoma (<nplcit id="ncit0015" npl-type="s"><text>Miki, S. et al. FEBS Lett., 250, 607-610, 1989</text></nplcit>; <nplcit id="ncit0016" npl-type="s"><text>Takenawa, J. et al., J. Natl. Cancer. Inst., 83, 1668-1672, 1991</text></nplcit>). Moreover, it has been reported that the level of IL-6 in the serum increases when the prognosis for treatment of renal cell carcinoma patients is poor (<nplcit id="ncit0017" npl-type="s"><text>Blay, J. et al., Cancer Res., 52, 3317-3322, 1992</text></nplcit>; <nplcit id="ncit0018" npl-type="s"><text>Tsukamoto, T. et al., J. Urol., 148, 1778-1782, 1992</text></nplcit>). However, a clear correlation between IL-6 and the resistance to therapy of renal cell carcinoma to antitumor agents has not yet been found, and is still unknown.

0008IL-6 is a multi-functional cytokine referred to as B cell stimulating factor-2 or Interferon β2. IL-6 was discovered as a differentiation factor involved in the activation of B lymphoid cells (<nplcit id="ncit0019" npl-type="s"><text>Hirano, T. et al., Nature, 324, 73-76, 1986</text></nplcit>). Later, it was clearly shown to be a multi-functional cytokine that affects the functions of various cells (<nplcit id="ncit0020" npl-type="s"><text>Akira, S. et al., Adv. in Immunology, 54, 1-78, 1993</text></nplcit>). IL-6 transmits its biological activity by means of two proteins present on cells.

0009One of them is IL-6R, a ligand bonding protein having a molecular weight of approximately 80 KD to which IL-6 bonds. In addition to the cell bonding form which is expressed on the cell membrane by passing through the cell membrane, it is also present as soluble IL-6R (sIL-6R), which is mainly composed of its extracellular region. Another protein is gp130 having a molecular weight of approximately 130 KD that is involved in signal transmission of non-ligand bonding. IL-6 and IL-6R form an IL-6/IL-6R complex. As a result of then bonding with the other membrane protein gp130, the biological activity of IL-6 is transmitted to the cell (Taga, et al., J. Exp. Med., 196, 967, 1987).

0010Although platinum compounds like cisplatin and antitumor agents such as mitomycin C induce apoptosis in tumor cells, IL-6 has been reported to inhibit apoptosis induced by antitumor agents (<nplcit id="ncit0021" npl-type="s"><text>Kerr, J. et al., Cancer, 73, 2013-2026, 1994</text></nplcit>; <nplcit id="ncit0022" npl-type="s"><text>Sachs, L. et al., Blood, 82, 15-21, 1993</text></nplcit>). In addition, antitumor agents like cisplatin and mitomycin C have cytotoxic effects on tumor cells as a result of producing free radicals (<nplcit id="ncit0023" npl-type="s"><text>Oyanagi, Y. et al., Biochem. Pharmacol., 26, 473-476,1997</text></nplcit>; <nplcit id="ncit0024" npl-type="s"><text>Nakano, H. et al., Biochem. Biophys. Acta., 796, 285-293,1984</text></nplcit>). IL-6, however, is known to promote the expression of manganase superoxide dismutase (MnSOD), which has the effect of decomposing free radicals, and IL-6 antibody inhibits the expression of this promoted MnSOD (<nplcit id="ncit0025" npl-type="s"><text>Ono, M. et al., Biochem. Biophys. Res. Commun., 182, 1100-1107, 1992</text></nplcit>; <nplcit id="ncit0026" npl-type="s"><text>Dougall, W.C. et al., Endocrinology, 129, 2376-2384, 1991</text></nplcit>).

0011However, none of these reports state that the effects of antitumor agents are enhanced by blocking the biological activity of IL-6. In addition, there are no reports of actually attempting to use an IL-6 antagonist as an effect enhancer of antitumor agents.

0012Although antitumor agents have been used in the treatment of tumors in the past, since large doses of these drugs produce adverse side effects such as nausea, vomiting, kidney and liver function disorders and inhibition of bone marrow function, there have been cases in which it was dangerous to administer the required dose of antitumor agent for adequately demonstrating antitumor effects. In addition, since there are tumors having resistance to therapy in which chemotherapy using ordinary antitumor agents is not effective, there is a need for an effect enhancer that increases the sensitivity of these tumors to antitumor agents.

0013The object of the present invention is to provide a novel antitumor agent effect enhancer that assists and enhances the effects of antitumor agents and increases the sensitivity of therapy-resistant tumor cells to antitumor agents. The present invention is thus directed to the use of an IL-6 antagonist for the preparation of a medicament for enhancing the effect of cisplatin or carboplatin in the treatment of a tumor wherein the IL-6 antagonist is the PM-1 or MH166 antibody and said tumor is renal cell carcinoma.

Disclosure of the Invention

0014As a result of various studies on the effects of IL-6 antagonists on changes in sensitivity of tumor cells to antitumor agents, the inventors of the present invention found that IL-6 antagonists such as IL-6 antibody or IL-6R antibody increase the sensitivity of tumor cells to antitumor agents, that therapeutic effects are observed with lower doses of antitumor agents, and that therapeutic effects appear by concomitant use of ordinary antitumor agents with an effect enhancer comprising an IL-6 antagonist against tumors exhibiting resistance to therapy.

0015Namely, the present invention relates to an antitumor agent effect enhancer containing an IL-6 antagonist. More specifically, the present invention refers to the use of an IL-6 antagonist for the preparation of a medicament for enhancing the effect of an antitumor agent.

0016Specific examples PM-1 antibody are humanized PM-1 antibody. Antitumor agents used in combination with IL-6 antagonists are cisplatin and carboplatin.

Brief Description of the Drawings

0017<figref idref="f0001">Figs. 1A and 1B</figref> indicate the cytotoxicity against renal cell carcinoma line Caki-1 in the presence of cisplatin at a concentration of 0, 0.1, 1 or 10 µg/ml and IL-6 antibody (<figref idref="f0001">Fig. 1A</figref>) or IL-6R antibody (<figref idref="f0001">Fig. 1B</figref>). Diamonds indicate the cytotoxicity (%) in the presence of cisplatin only, squares that in the presence of cisplatin and 0.1 µg/ml I L-6 antibody or IL-6R antibody, triangles that in the presence of cisplatin and 1 µg/ml IL-6 antibody or IL-6R antibody, and circles that in the presence of cisplatin and 10 µg/ml IL-6 antibody or IL-6R antibody.

0018<figref idref="f0002">Figs. 2A and 2B</figref> indicate the cytotoxicity against renal cell carcinoma line Caki-1 in the presence of mytomycin C at a concentration of 0,0.1, 1 or 10 µg/ml and IL-6 antibody (<figref idref="f0002">Fig. 2A</figref>) or IL-6R antibody (<figref idref="f0002">Fig. 2B</figref>). Diamonds indicate the cytotoxicity (%) in the presence of mitomycin C only, squares that in the presence of mitomycin C and 0.1 µg/ml IL-6 antibody or IL-6R antibody, triangles that in the presence of mitomycin C and 1 µg/ml IL-6 antibody or IL-6R antibody, and circles that in the presence of mitomycin C and 10 µg/ml IL-6 antibody or IL-6R antibody.

0019<figref idref="f0003">Figs. 3A and 3B</figref> indicate the cytotoxicity against renal cell carcinoma line Caki-1/DDP in the presence of cisplatin at a concentration of 0, 0.1, 1 or 10 µg/ml and IL-6 antibody (<figref idref="f0003">Fig. 3A</figref>) or IL-6R antibody (<figref idref="f0003">Fig. 3B</figref>). Diamonds indicate the cytotoxicity (%) in the presence of cisplatin only, squares that in the presence of cisplatin and 0.1 µg/ml IL-6 antibody or IL-6R antibody, triangles that in the presence of cisplatin and 1 µg/ml IL-6 antibody or IL-6R antibody, and circles that in the presence of cisplatin and 10 µg/ml IL-6 antibody or IL-6R antibody.

0020<figref idref="f0004">Figs. 4A and 4B</figref> indicate the cytotoxicity against renal cell carcinoma line ACHN in the presence of cisplatin at a concentration of 0, 0.1, 1 or 10 µg/ml and IL-6 antibody (<figref idref="f0004">Fig. 4A</figref>) or IL-6R antibody (<figref idref="f0004">Fig. 4B</figref>). Diamonds indicate the cytotoxicity (%) in the presence of cisplatin only, squares that in the presence of cisplatin and 0.1 µg/ml IL-6 antibody or IL-6R antibody, triangles that in the presence of cisplatin and 1 µg/ml IL-6 antibody or IL-6R antibody, and circles that in the presence of cisplatin and 10 µg/ml IL-6 antibody or IL-6R antibody.

0021<figref idref="f0005">Figs. 5A and 5B</figref> indicate the cytotoxicity against renal cell carcinoma line A704 in the presence of cisplatin at a concentration of 0, 0.1, 1 or 10 µg/ml and IL-6 antibody (<figref idref="f0005">Fig. 5A</figref>) or IL-6R antibody (<figref idref="f0005">Fig. 5B</figref>). Diamonds indicate the cytotoxicity (%) in the presence of cisplatin only, squares that in the presence of cisplatin and 0.1 µg/ml IL-6 antibody or IL-6R antibody, triangles that in the presence of cisplatin and 1 µg/ml IL-6 antibody or IL-6R antibody, and circles that in the presence of cisplatin and 10 µg/ml IL-6 antibody or IL-6R antibody.

0022<figref idref="f0006">Figs. 6A and 6B</figref> indicate the cytotoxicity against fresh renal cell carcinoma obtained from Patent 1 in the presence of cisplatin at a concentration of 0, 0.1, 1 or 10 µg/ml and IL-6 antibody (<figref idref="f0006">Fig. 6A</figref>) or IL-6R antibody (<figref idref="f0006">Fig. 6B</figref>). Diamonds indicate the cytotoxicity (%) in the presence of cisplatin only, squares that in the presence of cisplatin and 0.1 µg/ml IL-6 antibody or IL-6R antibody, triangles that in the presence of cisplatin and 1 µg/ml IL-6 antibody or IL-6R antibody, and circles that in the presence of cisplatin and 10 µg/ml IL-6 antibody or IL-6R antibody.

0023<figref idref="f0007">Figs. 7A and 7B</figref> indicate the cytotoxicity against fresh renal cell carcinoma obtained from Patent 2 in the presence of cisplatin at a concentration of 0, 0.1, 1 or 10 µg/ml and IL-6 antibody (<figref idref="f0007">Fig. 7A</figref>) or IL-6R antibody (<figref idref="f0007">Fig. 7B</figref>). Diamonds indicate the cytotoxicity (%) in the presence of cisplatin only, squares that in the presence of cisplatin and 0.1 µg/ml IL-6 antibody or IL-6R antibody, triangles that in the presence of cisplatin and 1 µg/ml IL-6 antibody or IL-6R antibody, and circles that in the presence of cisplatin and 10 µg/ml IL-6 antibody or IL-6R antibody.

0024<figref idref="f0008">Figs. 8A and 8B</figref> indicate the cytotoxicity against fresh renal cell carcinoma obtained from Patent 3 in the presence of cisplatin at a concentration of 0, 0.1, 1 or 10 µg/ml and IL-6 antibody (<figref idref="f0006">Fig. 6A</figref>) or IL-6R antibody (<figref idref="f0006">Fig. 6B</figref>). Diamonds indicate the cytotoxicity (%) in the presence of cisplatin only, squares that in the presence of cisplatin and 0.1 µg/ml IL-6 antibody or IL-6R antibody, triangles that in the presence of cisplatin and 1 µg/ml IL-6 antibody or IL-6R antibody, and circles that in the presence of cisplatin and 10 µg/ml IL-6 antibody or IL-6R antibody.

0025<figref idref="f0009">Figs. 9A and 9B</figref> indicate the cytotoxicity against renal cell carcinoma line Caki-1 in the presence of carboplatin at a concentration of 0, 1, 10 or 100 µg/ml and IL-6 antibody (<figref idref="f0009">Fig. 9A</figref>) or IL-6R antibody (<figref idref="f0009">Fig. 9B</figref>). Diamonds indicate the cytotoxicity (%) in the presence of carboplatin only, squares that in the presence of carboplatin and 0.1 µg/ml IL-6 antibody or IL-6R antibody, triangles that in the presence of carboplatin and 1 µg/ml IL-6 antibody or IL-6R antibody, and circles that in the presence of carboplatin and 10 µg/ml IL-6 antibody or IL-6R antibody.

0026A in <figref idref="f0010">Fig. 10</figref> is a diagram showing the results of Northern blotting of the total RNA of Caki-1 using a GST-π cDNA probe in order to investigate the expression of GST-π mRNA in renal cell carcinoma line Caki-1 treated with a culture medium (control), cisplatin at 10 µg/ml or IL-6 antibody or IL-6R antibody at 10 µg/ml. Lane 1 shows Caki-1 treated with culture medium only, lane 2 that treated with cisplatin, lane 3 that treated with IL-6 antibody, and lane 4 that treated with IL-6R antibody. B in <figref idref="f0010">Fig. 10</figref> is a diagram of a gel used for Northern blotting stained with ethidium bromide. The diagram shows that RNA is present in each lane.

Detailed Description

0027The antitumor agent effect enhancer comprising an IL-6 antagonist of the present invention enhances antitumor effects by being used in combination with antitumor agents during treatment of tumors. In addition, it enables the required dose of antitumor agent to be decreased since it has the effect of increasing the sensitivity to antitumor agents of therapy-resistant tumors for which therapeutic effects are not observed with ordinary chemotherapy.

0028Antitumor agents for which antitumor effects are enhanced by the effect enhancer of the present invention are chemotherapy drugs having tumor therapeutic effects that inhibit the development and growth of tumor cells by acting on tumor cells. These chemotherapy drugs are platinum compounds; selected from cisplatin and carboplatin. Platinum compounds have a platinum atom that forms complexes with other atoms. These compounds exhibit antitumor effects by bonding with DNA, inhibiting DNA synthesis of tumor cells and inhibiting division of tumor cells. Known examples of platinum compounds having antitumor effects that have been used so far include cisplatin (cis-diamine dichloroplatinum (II) having the structural formula shown below): <chemistry id="chem0001" num="0001"><img file="EP0800829B2_D0001.tif" /></chemistry> carboplatin (cis-diamine (1,1-cyclobutanedicarboxylato)-platinum (II) having the structural formula shown below): <chemistry id="chem0002" num="0002"><img file="EP0800829B2_D0002.tif" /></chemistry>

0029These antitumor agents can be prepared using routine methods. For example, platinum compounds may be used orally or parenterally, and preferably as an injection formulation, by mixing with a pharmaceutical carrier and an excipient, as necessary. For preparing an injective formulation, it may be mixed with distilled water, a salt solution such as sodium chloride or potassium chloride, glucose solution or physiological saline. The amount of antitumor agent in these formulations is preferably a unit dose that is convenient for use according to the patient's age, symptoms and so forth. For example, in using for tumor treatment in an adult, it is daily administered at 10 to 2000 mg/m² (body surface area), administered continuously for 5 days according to the dose, or a recovery period of 1 to 4 weeks may be provided between administrations.

0030Tumor cells in which antitumor effects are observed due to the effect enhancer of the present invention are tumors that have IL-6R and exhibit growth and/or resistance to therapy provided by IL-6 as one of their physiologically active substances. According to the present invention the tumor is renal cell carcinoma (<nplcit id="ncit0027" npl-type="s"><text>Miki, S. et al., FEBS Letter, 250, 607-610, 1989</text></nplcit>).

0031The IL-6 antagonist used in the present invention is an IL-6 antibody or an IL-6R antibody.

0032There are no particular limitation on the animal species of monoclonal antibody-producing cells provided it is mammalian, and they may originate in human antibody or non-human mammalian antibody. Monoclonal antibodies of rabbit or rodent origin are preferable as monoclonal antibodies of non-human mammalian origin due to the ease of preparation. Although there are no particular limitations on rodent antibodies, preferable examples include mouse, rat and hamster antibodies.

0033The IL-6 antibody according to the present invention is the MH166 antibody (<nplcit id="ncit0028" npl-type="s"><text>Matsuda, et al., Eur. J. Immunol., 18, 951-956, 1988</text></nplcit>). The IL-6R antibody according to the present invention is the PM-1 antibody (<nplcit id="ncit0029" npl-type="s"><text>Hirata, et al., J. Immunol., 143, 2900-2906, 1989</text></nplcit>).

0034Of these antibodies, IL-6R antibodies are particularly preferable, that is the above-mentioned PM-1 antibody.

0035Monoclonal antibodies can be prepared in the following manner basically using known technology. Namely, using IL-6, or IL-6R for the sensitizing antigen, a host is immunized in accordance with a routine immunization method, the resulting immunized cells are fused with known parent cells by routine cell fusion, and a monoclonal antibody-producing cell is then selected by routine screening methods.

0036More specifically, the following procedure may be performed to prepare monoclonal antibody. For example, an antigen of human origin is preferable for the above-mentioned sensitizing antigen, and in the case of human IL-6, antigen is obtained by using the gene sequence of human IL-6 disclosed in Hirano, et al., Nature, 324, 73, 1986. After inserting the gene sequence of human IL-6 into a known expression vector system and transforming suitable host cells, the target IL-6 protein is purified from the host cells or culture supernatant, and the purified IL-6 protein may then be used as the sensitizing antigen.

0037In the case of human IL-6R, IL-6R protein can be obtained by following a method similar to that of the above-mentioned human IL-6 by using the gene sequence disclosed in European Patent Publication No. <patcit id="pcit0001" dnum="EP325474A"><text>EP325474</text></patcit>. There are two types of IL-6R, namely that which is expressed on the cell membrane, and a soluble type which is liberated from the cell membrane (sIL-6R) (<nplcit id="ncit0030" npl-type="s"><text>Yasukawa, et al., J. Biochem., 108, 673-676, 1990</text></nplcit>). sIL-6R is mainly composed of the extracellular region of IL-6R bonded to the cell membrane, and is different from membrane-bound IL-6R in that-it lacks a cell membrane permeating region or cell membrane permeating region and intracellular region.

0038Although there are no particular limitations, mammals that are immunized with sensitizing antigen are preferably selected in consideration of compatibility with the parent cells used in cell fusion, and mice, rats, hamsters or rabbits are commonly used.

0039Immunization of an animal with sensitizing antigen is performed in accordance with known methods. As a typical example, immunization is performed by intraperitoneally or subcutaneously injecting a sensitizing antigen into an animal. More specifically, after diluting and suspending the sensitizing antigen in a suitable amount of phosphate-buffered saline (PBS) or physiological saline, the resulting suspension is mixed with a suitable amount of an ordinary adjuvant, such as Freund's complete adjuvant, as desired followed by emulsification. The emulsion is then preferably administered to the mammal in several doses every 4 to 21 days. In addition, a suitable carrier can also be used during immunization with sensitizing antigen.

0040Thus, after immunizing the mammal and confirming that the desired antibody level has risen in the serum, the immunized cells are extracted from the mammal and used in cell fusion. A particularly preferable example of immunized cells are spleen cells.

0041Various known cell lines are preferably used for the mammalian myeloma cells that serve as the parent cells that are fused with the above-mentioned immunized cells, examples of which include P3 (P3x63Ag8.653) (J. Immunol., 123, 1548, 1978), p3-U1 (<nplcit id="ncit0031" npl-type="s"><text>Current Topics in Microbiology and Immunology, 81, 1-7, 1978</text></nplcit>), NS-1 (<nplcit id="ncit0032" npl-type="s"><text>Eur. J. Immunol., 6, 511-519, 1976</text></nplcit>), MPC-11 (<nplcit id="ncit0033" npl-type="s"><text>Cell, 8, 405-415, 1976</text></nplcit>), SP2/0 (<nplcit id="ncit0034" npl-type="s"><text>Nature, 276, 269-270, 1978</text></nplcit>), F0 (<nplcit id="ncit0035" npl-type="s"><text>J. Immunol. Meth., 35, 1-21, 1980</text></nplcit>), S194 (<nplcit id="ncit0036" npl-type="s"><text>J. Exp. Med., 148, 313-323, 1978</text></nplcit>), and R210 (<nplcit id="ncit0037" npl-type="s"><text>Nature, 277, 131-133, 1979</text></nplcit>).

0042Cell fusion of the above-mentioned immunized cells and myeloma cells can basically be carried out in accordance with known methods, an example of which is the method of Milstein, et al. (<nplcit id="ncit0038" npl-type="s"><text>Milstein, et al., Methods Enzymol., 73, 3-46, 1981</text></nplcit>).

0043More specifically, the above-mentioned cell fusion is, for example, performed in an ordinary nutrient culture liquid in the presence of cell fusion promoter. Examples of fusion promoters that are used include polyethylene glycol (PEG) and Sendai virus (HVJ). Moreover, a fusion aid such as dimethylsulfoxide can also be added to increase fusion efficiency as desired.

0044The ratio of immunized cells and myeloma cells used is preferably, for example, 1 to 10 times more immunized cells than myeloma cells. RPMI 1640 culture medium, MEM culture medium or other conventional culture media used in this type of cell culturing can be used for the culture liquid used for the above-mentioned cell fusion. Moreover, these culture media can be used in combination with a serum supplement such as fetal calf serum (FCS).

0045For cell fusion, prescribed amounts of the above-mentioned immunized cells and myeloma cells are mixed well in the above-mentioned culture medium followed by the addition of PEG solution, such as PEG solution having a mean molecular weight of 1000 to 6000, warmed in advance to about 37°C normally at a concentration of 30 to 60% (w/v) and mixing to form the target fused cells (hybridoma). Next, by repeating a procedure consisting of sequentially adding a suitable culture medium, centrifuging and removing the supernatant, cell fusion agents and so forth unsuitable for hybridoma growth can be removed.

0046Said hybridoma is selected by culturing in a conventional selective culture medium such as HAT culture medium (culture medium containing hypoxanthine, aminopterin and thymidine). Culturing in said HAT culture medium is normally continued for several days to several weeks to allow adequate time for cells other than the target hybridoma cells (non-fusion cells) to die. Next, a conventional limited dilution method is performed followed by screening and single-cloning of hybridoma that produces the target antibody.

0047Hybridoma thus prepared that produces monoclonal antibody can be subcultured in an ordinary culture medium, and can be stored for a long time in liquid nitrogen.

0048In order to acquire monoclonal antibody from said hybridoma, said hybridoma is cultured in accordance with routine methods, and obtained in the form of culture supernatant. Alternatively, the hybridoma is grown by administering to a mammal with which it has compatibility and obtaining the monoclonal antibody in its ascites. The former method is suited for obtaining highly pure antibody, while the later method is suited for large-volume production of antibody.

0049In addition, monoclonal antibody can not only be obtained from hybridoma formed by fusing antibody-producing cells obtained by immunizing with antigen, but monoclonal antibody can also be used that is produced using recombinant gene technology by cloning antibody gene, incorporating it in a suitable vector and introducing the vector into a known cell line such as COS or CHO (see, for example, <nplcit id="ncit0039" npl-type="s"><text>Vandamme, A-M. et al., Eur. J. Biochem., 192, 767-775, 1990</text></nplcit>).

0050Moreover, monoclonal antibody obtained by the above-mentioned method can be purified to high purity using routine purification means such as salting out, gel filtration or affinity chromatography.

0051Monoclonal antibody prepared in this manner is able to recognize antigen with high sensitivity and high accuracy by routine immunological means such as radioimmunoassay (RIA), enzyme immunoassay (EIA, ELISA) and immunofluorescence analysis.

0052The monoclonal antibody used in the present invention is not limited to monoclonal antibody produced by hybridoma. Monoclonal antibody which has been artificially altered for the purpose of lowering heteroantigenicity to humans and so forth is more preferable. For example, chimeric antibody composed of the variable region of a mammalian monoclonal antibody other than human monoclonal antibody, for example mouse monoclonal antibody, and the constant region of human antibody can be used. This type of chimeric antibody can be produced using known chimeric antibody production methods, and particularly recombinant gene technology.

0053Moreover, reshaped human antibody can also be used in the present invention. In this type of antibody, the complementarity determining region of non-human mammalian antibody, such as mouse antibody, is grafted to the complementarity determining region of human antibody, by known general gene recombination technique. A reshaped human antibody that is useful in the present invention can be obtained using this known technique, a preferable example of which is reshaped PM-1 antibody (see, for example, <patcit id="pcit0002" dnum="WO9219759A"><text>International Patent Publication No. WO92-19759</text></patcit>).

0054Furthermore, the amino acids of the framework (FR) region of the variable region of an antibody may be substituted so as that the complementarity determining region of reshaped human antibody forms a suitable antibody binding site (<nplcit id="ncit0040" npl-type="s"><text>Sato, et al., Cancer Res., 53, 1-6, 1993</text></nplcit>). Moreover, a gene that code for antibody fragment such as F(ab')₂, Fab, Fv, single chain Fv(scFv) linking Fv of H chain and L chain with a suitable linker can be contructed, and the antibody fragment can then be expressed in a suitable host cell and can be used for the above-mentioned purpose provided it binds with antigen and inhibit IL-6 activity (see, for example, <nplcit id="ncit0041" npl-type="s"><text>Bird, et al., TIBTECH, 9, 132-137, 1991</text></nplcit>).

0055An scFv is composed by linking the H chain V region and L chain V.region of an antibody. In this scFv, the H chain V region and L chain V region are linked by means of a linker, and preferably by a peptide linker (<nplcit id="ncit0042" npl-type="s"><text>Huston, J.S. et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883, 1988</text></nplcit>). The H chain V region and L chain V region in the scFv may be derived from any of the above-mentioned antibodies. These V regions are preferably linked by a peptide linker. Examples of peptide linkers that are used include any single chain peptide comprising 12 to 19 amino acid residues.

0056DNA coding for scFv is obtained by using DNA that codes for the H chain or H chain V region of the above-mentioned antibody and DNA coding for the L chain or L chain V region as templates, amplifying the DNA portion of their sequences that codes for the desired amino acid sequence using a primer pair that defines both of its ends, and combining with a primer pair that defines the DNA that codes for the peptide linker portion as well as both ends so as to link with the H chain and the L chain.

0057In addition, once a DNA coding for scFv has been prepared, an expression vector that contains it and a host transformed by said expression vector can be obtained in accordance with routine methods. In addition, scFv can be obtained in accordance with routine methods by using that host. Since scFv has greater ability to migrate into tissue than antibody molecule, it is expected to be used as substance that has similar functions as reshaped human antibody.

0058The antitumor agent effect enhancer comprising IL-6 antagonist of the present invention can be effectively used in the treatment of any tumors having IL-6R, growing by using IL-6 as one of its physiological active substances, and/or exhibiting resistance to therapy, provided it blocks IL-6 signal transmission and assists and enhances the effect of the antitumor agent.

0059The antitumor agent effect enhancer comprising IL-6 antagonist of the present invention can be administered systemically or locally, and preferably parenterally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection or subcutaneous injection. Moreover, it can be used in the form of a pharmaceutical composition or kit with at least one pharmaceutical carrier or diluent.

0060The dose in humans of the antitumor agent effect enhancer comprising IL-6 antagonist of the present invention varies according to the condition and age of the patient or according to the administration method. However, it is necessary to select a suitable dose. For example, in the case of IL-6R antibody, a divided dose of four administrations or less within a range of approximately 1 to 1000 mg/patient can be selected. In addition, it can also be administered at a dose of 1 to 10 mg/kg/week. However, the antitumor agent effect enhancer comprising IL-6 antagonist of the present invention is not limited to these doses.

0061The antitumor agent effect enhancer comprising IL-6 antagonist of the present invention can be prepared in accordance with routine methods (Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S.A.). For example, preparations for injection may be prepared by dissolving purified IL-6 antagonist in a solvent such as physiological saline, buffer or glucose solution and adding an adsorption prevention agent such as Tween80, gelatin or human serum albumin (HSA). Alternatively, it can also be prepared by freeze-drying for reconstitution prior to use. Examples of vehicles that can be used for freeze-drying include sugar alcohols and sugars such as mannitol and glucose.

Examples

0062Although the following provides a detailed explanation of the present invention using examples, reference examples and experiments, the present invention is not limited to them.

Example 1. Effect of IL-6 Antibody or IL-6R Antibody on Sensitivity of Tumor Cells to Antitumor Agents

0063The effect of IL-6 antibody or IL-6R antibody on the sensitivity of tumor cells to various antitumor agents was investigated.

(1) Preparation of Human Renal Cell Carcinoma

0064Human renal cell carcinoma line Caki-1, cisplatin-resistant strain Caki-1/DDP, a subline of Caki-1, human renal cell carcinoma ACHN and human renal cell carcinoma A704 (<nplcit id="ncit0043" npl-type="s"><text>Giard, D.J. et al., J. Natl. Cancer Inst., 51, 1417-1423, 1973</text></nplcit>) were cultured so as to form a single layer in plastic dishes in RPMI 1640 culture medium containing 25 mM HEPES, 2 mM L-glutamine, 1% non-essential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin and 10% heat-inactivated fetal bovine serum (FBS) (all manufactured by Gibco) (to be referred to as complete culture medium).

0065On the other hand, fresh tumor cells were obtained from renal cell carcinoma patients in accordance with the method of <nplcit id="ncit0044" npl-type="s"><text>Mizutani, Y. et al. (Cancer, 69, 537-545, 1992</text></nplcit>). Renal cell carcinoma tumor tissue was obtained during surgical procedures on three renal cell carcinoma patients. After confirming the tissue to be renal cell carcinoma by histological classification, the tumor tissue was finely decomposed by 3 mg/ml of collagenase (Sigma Chemical Co.) to prepare a tumor cell suspension. After washing three times with RPMI 1640 culture medium, the cell suspension was layered over a non-continuous gradient composed of 2 ml each of 100%, 80% and 50% Ficol-Hypaque in 15 ml volumetric plastic tubes followed by centrifugation for 30 minutes at 400 xg. The lymphocyte-rich monocyte layer contained in the above-mentioned 100% layer was removed, and tumor cells and mesothelial cells were obtained from the 80% layer.

0066To prevent contamination by other cells, the cell suspension rich in tumor cells was layered over a non-continuous gradient composed of 3 ml each of 25%, 15% and 10% Percoll in complete culture medium contained in 15 ml plastic tubes followed by centrifugation for 7 minutes at 25 xg and room temperature. After washing the resulting tumor cells and suspending in complete culture medium, the presence of tumor cells was confirmed by trypan blue staining. The tumor cells prepared in this manner were used in the following experiment.

(2) Confirmation of IL-6 Production of Renal Cell Carcinoma by ELISA

0067The presence of IL-6 in the culture supernatant of renal cell carcinoma line Caki-1, Caki-1/DDP, ACHN, A704 and fresh tumor cells derived from renal cell carcinoma patients (Nos. 1-3) was investigated by ELISA (enzyme-linked immunosorbent assay).

0068100 µl of IL-6 antibody was added to 96-well ELISA plates to coat the ELISA plates with IL-6 antibody by allowing to stand at least overnight. These plates were stored for a maximum of 4 weeks at 4°C until the time of use. The plates coated with IL-6 antibody were washed three times and blocked for 1 hour with ELISA PBS containing 1% PBS (bovine serum albumin). After washing twice, 100 µl of tumor cell culture supernatant or <u>E.</u><u>coli</u> recombinant IL-6 as the control (Yasukawa, et al., Biotechnol. Lett., 12, 419, 1990) were added to each well.

0069The plates were incubated for 1 hour and washed three times followed by the addition of 100 µl of anti-IL-6 polyclonal antibody (<nplcit id="ncit0045" npl-type="s"><text>Matsuda, T. et al., Eur. J. Immunol., 18, 951-956, 1988</text></nplcit>) to each well. The plates were then incubated for 1 hour followed by the addition of alkaline phosphate-bound goat anti-rabbit IgG to each well and incubating for an additional hour. The plates were washed and incubated with alkaline phosphate substrate (Sigma 104, Sigma Chemical Co.). Two hours later, the absorbance at 405 nm was measured with an ELISA READER (Immunoreader, Japan Intermed Co., Ltd.). According to those results, the renal cell carcinoma were all clearly shown to produce IL-6 (see Table 1). <tables id="tabl0001" num="0001"><table frame="all"><title>Table 1</title><tgroup cols="2" rowsep="0"><colspec colnum="1" colname="col1" colwidth="48mm" /><colspec colnum="2" colname="col2" colwidth="78mm" /><thead valign="top"><row rowsep="1"><entry namest="col1" nameend="col2" align="center">Concentration of IL-6 in Supernatant</entry></row><row rowsep="1"><entry align="center">RCC Cells</entry><entry align="center">IL-6 Concentration (pg/ml; mean ± standard deviation</entry></row></thead><tbody><row><entry align="center">Caki-1</entry><entry align="center">1337±35</entry></row><row><entry align="center">Caki-1/DDP</entry><entry align="center">3900±325</entry></row><row><entry align="center">A704</entry><entry align="center">1290±141</entry></row><row><entry align="center">ACHN</entry><entry align="center">1282±106</entry></row><row><entry align="center">Fresh RCC cells (Patient No. 1)</entry><entry align="center">1236±71</entry></row><row><entry align="center">Fresh RCC cells (Patient No. 2)</entry><entry align="center">42±4</entry></row><row rowsep="1"><entry align="center">Fresh RCC cells (Patient No. 3)</entry><entry align="center">2579±219</entry></row></tbody></tgroup></table></tables>

(3) Effect of IL-6 Antibody or IL-6R Antibody on the Cytotoxicity of Antitumor agents

0070The MTT method (<nplcit id="ncit0046" npl-type="s"><text>Mizutani, Y. et al., Cancer, 73, 730-737, 1994</text></nplcit>) was used to investigate the effect of IL-6 antibody or IL-6R antibody on the sensitivity of various renal cell carcinomas, namely Caki-1, Caki-1/DDP, ACHN, A704 and fresh tumor cells derived from renal cell carcinoma patients (Nos. 1 to 3), to various concentrations of antitumor agents, namely cisplatin (cis-diaminedichloroplatinum (II)), mitomycin C (MMC), adriamycin (ADR), vinblastine (VBL) and 5-fluorouracil (5-FU).

0071100 µl of the above-mentioned renal cell carcinoma tumor cell suspensions (2 x 10⁴ cells) were added to a 96-well bottom-surface microtiter plate (Corning Glass Works, Corning). The plate was incubated in a humid environment at 37°C in the presence of 5% CO₂ and the tumor cells were cultured for 24 hours. The cell culture supernatant was aspirated and the tumor cells were washed three times with RPMI 1640 culture medium. 200 µl of a solution containing each antitumor agent or control in the form of complete culture solution were added to each well in the presence of IL-6 antibody (<nplcit id="ncit0047" npl-type="s"><text>Matsuda, T. et al., Eur. J. Immunol., 18, 951-956, 1988</text></nplcit>) or IL-6R antibody (<nplcit id="ncit0048" npl-type="s"><text>Hirata, Y. et al., J. Immunol., 143, 2900-2906, 1989</text></nplcit>) followed by culturing for 24 hours at 37°C. 20 µl of MTT solution (5 mg/ml; Sigma Chemical Co.) were added to each well after which culturing was continued for 4 hours in a humid environment at 37°C and in the presence of 5% CO₂. The culture medium was then removed from each well and replaced with isopropanol containing 0.05 N HCl (Sigma Chemical Co.).

0072The absorbance of the solution in each well at 540 nm was measured with a microculture plate reader (Immunoreader, Japan Intermed Co., Ltd.). The rate of cytotoxicity was calculated with the following formula.<maths id="math0001"><math display="block"><mi>Cytotoxicity</mi><mfenced><mo>%</mo></mfenced><mo>=</mo><mfenced open="[" close="]"><mn>1</mn><mo>-</mo><mfenced><mi>absorbance of experimental group</mi><mo>/</mo><mi>absorbance of control group</mi></mfenced></mfenced><mo>⁢</mo><mi>x</mi><mspace width="1em" /><mn>100.</mn></math><img file="EP0800829B2_D0003.tif" /></maths>

0073As a result, cytotoxicity caused by cisplatin (see <figref idref="f0001">Figs. 1A and 1B</figref>) or MMC (see <figref idref="f0002">Figs. 2A and 2B</figref>) in Caki-1 cells clearly increased in the presence of antibody. In the experiment group to which control antibody MOPC3 (J. Natl. Cancer Inst. (Bethesda), 41, 1083, 1968) was added, there were no effects on sensitivity to cisplatin or MMC observed. In comparison with the case of adding antitumor agent alone, the amount of antitumor agent required to elicit cytotoxic effects equivalent to those in the presence of antitumor agent and IL-6 antibody or IL-6R antibody was 1/10 to 1/100. On the other hand, there was no change in the sensitivity of tumor cells to antitumor agents in the presence of IL-6 antibody or IL-6R antibody and ADR, VBL or 5-FU.

0074The resistance of Caki-1/DDP cells to cisplatin was overcome by having IL-6 antibody or IL-6R antibody present with the cisplatin (see <figref idref="f0003">Figs. 3A and 3B</figref>). The combined effects of IL-6 antibody or IL-6R antibody and cisplatin were similarly observed for the other renal cell carcinoma lines ACHN (see <figref idref="f0004">Figs. 4A and 4B</figref>) and A704 (see <figref idref="f0005">Figs. 5A and 5B</figref>) as well as for the fresh tumor cells obtained from the three renal cell carcinoma patients (see <figref idref="f0006">Figs. 6A, 6B</figref>, <figref idref="f0007">7A, 7B</figref>, <figref idref="f0008">8A and 8B</figref>).

Example 2. Effect of IL-6 Antibody or IL-6R Antibody on the Sensitivity of Renal Cell Carcinoma to Carboplatin

0075The cytotoxicity when IL-6 antibody or IL-6R antibody was present with carboplatin prepared at various concentrations was examined in the same manner as Example 1 using renal cell carcinoma line Caki-1. As a result, the sensitivity of Caki-1 cells to carboplatin was enhanced (see <figref idref="f0009">Figs. 9A and 9B</figref>).

Example 3. Effect of IL-6 Antibody or IL-6R Antibody on Intracellular Accumulation of Antitumor Agents

0076Caki-1 cells were cultured for 24 hours in the presence of combination of 10 µg/ml of cisplatin or 100 µg/ml of 5-FU or culture medium as control, and 10 µg/ml of control antibody MOPC3/C, or 10 µg/ml of IL-6 antibody or IL-6R antibody.

0077Next, the culture medium was removed and the cells were washed three times with RPMI 1640 culture liquid. The intracellular accumulation of cisplatin was measured by flameless atomic absorption spectrometry (<nplcit id="ncit0049" npl-type="s"><text>Daley-Tates, P.T. et al., Biochem. Pharmacol., 34, 2263-2369</text></nplcit>; <nplcit id="ncit0050" npl-type="s"><text>Riley, C.M. et al., Analytical Biochem., 124, 167-179, 1982</text></nplcit>). The Zeeman Z-8000 (Zeeman Z-8000 Spectrophotometer, Hitachi Co., Ltd.) was used for the measuring instrument. In addition, the intracellular accumulation of 5-FU was measured by gas chromatography and mass spectrometry (<nplcit id="ncit0051" npl-type="s"><text>Marunaka, T. et al., J. Pharm. Sci., 69, 1296-1300, 1980</text></nplcit>). The JMS-D300 mass spectrometer equipped with the JGC-20KP gas chromatograph (JOEL) was used for the measuring instrument. Those results are shown in Table 2. The results clearly showed that IL-6 antibody or IL-6R antibody do not have any effect whatsoever on the accumulation of cisplatin and 5-FU within tumor cells. <tables id="tabl0002" num="0002"><table frame="all"><title>Table 2</title><tgroup cols="5"><colspec colnum="1" colname="col1" colwidth="15mm" /><colspec colnum="2" colname="col2" colwidth="39mm" /><colspec colnum="3" colname="col3" colwidth="21mm" /><colspec colnum="4" colname="col4" colwidth="25mm" /><colspec colnum="5" colname="col5" colwidth="26mm" /><thead valign="top"><row><entry namest="col1" nameend="col5" align="center">Intracellular Accumulation of CDDP or 5-FU (ng/10⁷ cells) Treatment</entry></row><row><entry align="center">Drug</entry><entry align="center">Control (culture medium)</entry><entry align="center">Control Ab</entry><entry align="center">Anti-IL-6 mAb</entry><entry align="center">Anti-IL-6R mAb</entry></row></thead><tbody><row rowsep="0"><entry align="center">CDDP</entry><entry align="center">0.28±0.05</entry><entry align="center">0.27±0.02</entry><entry align="center">0.26±0.05</entry><entry align="center">0.27±0.02</entry></row><row><entry align="center">5-FU</entry><entry align="center">1.48±0.32</entry><entry align="center">1.57±0.45</entry><entry align="center">1.50±0.19</entry><entry align="center">1.63±0.31</entry></row></tbody></tgroup></table></tables>

0078Figures in the table were calculated from the data obtained from three experiments (mean ± standard deviation).

Example 4. Effect of Cisplatin, IL-6 Antibody or IL-6R Antibody on Expression of Glutathione S-transferase-π (GST-π)

0079Caki-1 cells were cultured for 4 hours with control culture medium, 10 µg/ml of cisplatin or 10 µg/ml of IL-6 antibody or IL-6R antibody. Next, the total RNA of the cells was prepared according to the method of <nplcit id="ncit0052" npl-type="s"><text>Mizutani, Y. et al. (Cancer, 73, 730-737, 1994</text></nplcit>) followed by electrophoresis in 1.2% agarose-2.2 M HCHO gel in 1 x MOPS buffer containing 200 mM MOPS (3-[N-morpholino]propane sulfonate), 50 mM sodium acetate and 10 mM sodium EDTA so as to result in 10 µg of RNA per lane. Next, the RNA was transcribed to a Biodyne A membrane (Poll) in 20 x SSC solution containing 3 M NaCl and 0.3 M sodium citrate (pH 7.0). A 50 to 100 ng GST-π cDNA probe (<nplcit id="ncit0053" npl-type="s"><text>Nakagawa, K. et al., J. Biol. Chem., 265, 4296-4301, 1990</text></nplcit>) was labeled by random oligoprimer elongation using a³²P-dCTP (NEN). The above-mentioned nylon membrane to which the RNA was transcribed was cross-linked with ultraviolet rays and hybridized with the above-mentioned probe. Those results are shown in <figref idref="f0010">Fig. 10</figref>.

0080The expression of GST-π mRNA of Caki-1 cells was not affected at all by cisplatin. However, when IL-6 antibody or IL-6R antibody was added, expression of GST-π mRNA decreased. Based on these findings, it was suggested that the decrease in the level of expression of GST-π mRNA is involved in the increase in sensitivity of renal cell carcinoma to cisplatin caused by IL-6 antibody or IL-6R antibody.

Industrial Applicability

0081The sensitivity of tumor cells to antitumor agents was observed at lower doses as a result of combining IL-6 antagonists such as IL-6 antibody or IL-6R antibody with antitumor agents, thereby confirming that combined effects are demonstrated by IL-6 antagonists and antitumor agents. Moreover, tumor cells exhibiting resistance to therapy to antitumor agents were proven to be treatable as a result of IL-6 antagonists enhancing their sensitivity to antitumor agents.

0082In addition, the antitumor agent effect enhancer comprising IL-6 antagonist of the present invention is able to reduce the cytotoxic effects of antitumor agents on tissue by allowing the required dose of antitumor agent to be lowered, thus giving it considerable value in use as an enhancer of antitumor agent effects.