HK1096859B - Methods for treating interleukin-6 related diseases - Google Patents

Description

Methods of treating interleukin-6 related diseases

Background

1. Field of the invention

The present invention relates to methods of treating interleukin-6 (IL-6) -associated diseases by combining an interleukin-6 antagonist (IL-6 antagonist), particularly an anti-interleukin-6 receptor (IL-6R) antibody (anti-IL-6R antibody), with an immunosuppressive agent, and administering the anti-IL-6R antibody at high doses.

2. Related Art

IL-6 is a cytokine, also known as B cell stimulating factor-2 (BSF2) or interferon beta 2. IL-6 has been found to be involved in the activation of cells of the B lymphocyte lineage as a differentiation factor (Hirano, T. et al, Nature, 324: 73-76, 1986), and IL-6 was later demonstrated to be a multifunctional cytokine affecting a variety of cell functions (Akira, S. et al, adv. in Immunology, 54: 1-78, 1993). IL-6 has been reported to induce cell maturation in T lymphocyte cell lines (Lotz, M. et al, J.Exp.Med., 167: 1253-one 1258, 1988).

IL-6 conducts its biological activity through two types of proteins on the cell. One is the IL-6 receptor, which is a ligand-binding protein with a molecular weight of approximately 80kD to which IL-6 binds (Taga, T. et al, J.Exp.Med., 166: 967-. In addition to the membrane-bound type that penetrates and is expressed on the cell membrane, the IL-6 receptor may also be a soluble form of the IL-6 receptor that contains primarily its extracellular region.

International publication WO92/19759 describes various types of anti-IL-6R antibodies, such as: humanized anti-IL-6R antibodies and chimeric anti-IL-6R antibodies. WO 96/11020 describes therapeutic agents for rheumatoid arthritis and synovial cell growth inhibitors whose main components are IL-6 antagonists such as anti-IL-6R antibodies. WO96/12503 describes the treatment of diseases caused by IL-6 production, examples of which are plasmacytosis, hyperimmunemia, anemia, nephritis, cachexia, rheumatoid arthritis, Castleman's disease and mesangial proliferative nephritis. WO 98/42377 describes diseases associated with sensitized T cells such as: a preventive/therapeutic agent for multiple sclerosis, uveitis, chronic thyroiditis, delayed hypersensitivity, contact dermatitis, and atopic dermatitis, wherein the active ingredient is an anti-IL-6R antibody.

WO 98/42377 describes therapeutic agents for systemic lupus erythematosus, wherein the active ingredient is an anti-IL-6R antibody. WO 99/47170 describes therapeutic agents for Crohn's disease, wherein the active ingredient is an anti-IL-6R antibody. WO 00/10607 describes a therapeutic agent for pancreatitis, wherein the active ingredient is an anti-IL-6R antibody. WO 02/3492 describes therapeutic agents for psoriasis wherein the active ingredient is an anti-IL-6R antibody. In addition, WO 02/080969 describes therapeutic agents for juvenile idiopathic arthritis in which the active ingredient is an anti-IL-6R antibody.

Summary of The Invention

As described above, its active ingredient is known as anti IL-6R antibody of various preventive or therapeutic agents. However, it is not known that an anti-IL-6R antibody in combination with an immunosuppressive agent, such as Methotrexate (MTX), can achieve a synergistic effect in the treatment of IL-6 related diseases, that an immunosuppressive agent, such as Methotrexate (MTX), can reduce or prevent allergy when used in combination with an anti-IL-6R antibody in the treatment of rheumatoid arthritis, and that high doses of anti-IL-6R antibody can reduce or prevent allergy when used in the treatment of rheumatoid arthritis with an anti-IL-6 antibody.

Accordingly, the present invention provides pharmaceutical compositions for treating IL-6 related disorders, comprising an interleukin-6 antagonist (IL-6 antagonist) and an immunosuppressive agent.

The invention also provides pharmaceutical compositions comprising an immunosuppressive agent to enhance the effect of treating IL-6 related disorders with an IL-6 antagonist.

The invention also provides pharmaceutical compositions comprising an immunosuppressive agent to prevent or reduce allergy when treating IL-6 related disorders with an IL-6 antagonist.

The invention further provides therapeutic agents for IL-6 related diseases for high dose administration, including IL-6 antagonists.

The invention further provides pharmaceutical compositions comprising high dose IL-6 antagonists to prevent or reduce allergy in the treatment of IL-6 related diseases.

The IL-6 antagonist is preferably an anti-IL-6R antibody. The anti-IL-6R antibody is preferably a monoclonal antibody against IL-6R. Preferably, the anti-IL-6R antibody is a monoclonal antibody against human IL-6R. Alternatively, preferably, the anti-IL-6R antibody is a monoclonal antibody against mouse IL-6R. Preferably, the anti-IL-6R antibody is a recombinant antibody. Preferably, the human anti-IL-6R monoclonal antibody is, for example, a PM-1 antibody. Preferably, the mouse anti-IL-6R monoclonal antibody is, for example, the MR16-1 antibody. Further, the antibody may be a chimeric, humanized or human antibody against IL-6R. It is particularly preferred that the anti-IL-6R antibody is, for example, a humanized PM-1 antibody.

When an IL-6 antagonist, in particular an anti-IL-6R antibody, is combined with an immunosuppressive agent, the dose of the IL-6 antagonist, in particular an anti-IL-6R antibody, for example, in the case of intravenous infusion, is a dose of 0.02 to 150mg/kg/4 weeks or a dose showing a blood anti-IL-6R antibody concentration equivalent thereto, preferably a dose of 0.5 to 30mg/kg/4 weeks or a dose showing a blood anti-IL-6R antibody concentration equivalent thereto, more preferably a dose of 2 to 8mg/kg/4 weeks or a dose showing a blood anti-IL-6R antibody concentration equivalent thereto.

When an IL-6 antagonist, particularly an anti-IL-6R antibody, is administered at a high dose, the dose of the IL-6 antagonist, particularly the anti-IL-6R antibody is, for example, a dose of not less than 4mg/kg/4 weeks or a dose showing a blood anti-IL-6R antibody concentration equivalent thereto, preferably a dose of 6 to 16mg/kg/4 weeks or a dose showing a blood anti-IL-6R antibody concentration equivalent thereto, more preferably a dose of 6 to 10mg/kg/4 weeks or a dose showing a blood anti-IL-6R antibody concentration equivalent thereto in the case of intravenous infusion.

When MTX is used as an immunosuppressive agent, the dose of MTX is, for example, a dose of 1 to 100 mg/body/week or a dose showing a blood MTX concentration equivalent thereto, preferably a dose of 4 to 50 mg/body/week or a dose showing a blood MTX concentration equivalent thereto, particularly preferably a dose of 10 to 25 mg/body/week or a dose showing a blood MTX concentration equivalent thereto.

The dose showing the concentration of the blood drug such as the anti-IL-6R antibody MTX means a dose giving an equivalent therapeutic effect, and even when the transition of the blood concentration varies depending on the administration method such as intravenous injection and subcutaneous injection, the dose is considered to be a dose showing the concentration of the blood drug such as the anti-IL-6R antibody or MTX as long as the therapeutic effect is equivalent.

Examples of IL-6-associated diseases include,

acute and chronic inflammatory and autoimmune diseases: nephritis, mesangial proliferative nephritis (mesangial proliferative nephritis), Crohn's disease, ulcerative colitis, pancreatitis, juvenile or systemic juvenile idiopathic arthritis, vasculitis, Kawasaki's disease, rheumatoid arthritis, systemic lupus erythematosus (systemic erythrodysus), psoriasis, Sjogren's syndrome, adult stills' disease;

tumor diseases: multiple myeloma, Castleman's disease, malignant lymphoma, renal cancer;

infectious diseases: HIV infection, EBV infection;

cachexia (cachexia): cachexia;

and (3) the other: plasmacytosis, hyperimmunemia, anemia and the like, preferably rheumatoid arthritis, plasmacytosis, hyperimmunemia, anemia, nephritis, cachexia, multiple myeloma, Castleman's disease, mesangial proliferative nephritis, systemic lupus erythematosus, Crohn's disease, pancreatitis, psoriasis, juvenile idiopathic arthritis, or systemic juvenile idiopathic arthritis.

The pharmaceutical compositions of the present invention may be administered orally or parenterally as well as systemically or topically. For example, intravenous injection, such as instillation, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, colonic injection, enteric coated oral drug, etc. may be selected. The administration method may be appropriately selected depending on the age and condition of the patient. The upper and lower limits of the actual dose are influenced by the frequency of administration, for example: the dose per dose increased when the dosing interval was long and the dose per dose decreased when the dosing interval was short.

For the preferred dosage and mode of administration of the anti-IL-6 receptor antibody, for example, the amount of free antibody present in the blood is an effective dose. As a specific example, there is a method of administering one to several times, for example, a method using intravenous injection such as drip and subcutaneous injection, according to the following administration schedule: 2 times/week, 1 time/2 week, 1 time/4 week, 1 time/6 week, 1 time/8 week, etc. The dosage regimen may be adjusted as the experimental data for observing disease status and blood changes, for example, by extending the dosage interval from 2/week or 1/week to 1/2 week, 1/3 week, 1/4 week, 1/6 week and 1/8 week.

When administered in combination with MTX, for example in the case of rheumatoid arthritis, typically the dose of anti-IL-6R antibody is higher than 0.5 mg/kg/week or a dose that shows equivalent or more antirheumatic effect. For example, when intravenous administration is carried out 1 time per 4 weeks, the dose is 0.02 to 150mg/kg, preferably 0.5 to 30mg/kg, more preferably 2 to 8 mg/kg.

The anti-IL-6R antibody and the immunosuppressive agent may be administered simultaneously or at intervals.

Immunosuppressants also include antirheumatics, adrenocortical hormones, and the like, and include, for example, the following:

A. immunosuppressant, antirheumatic, adrenocortical hormone agent

Immunosuppressant

Alkylating agent

Cyclophosphamide

Metabolic antagonists

Azathioprine, methotrexate, mizoribine

T cell activity inhibitor

Cyclosporin, tacrolimus

Antirheumatic agents:

hydroxychloroquine, sulfasalazine, leflunomide, etanercept, infliximab, adalimumab, D-penicillamine, orally available gold compounds, injectable gold compounds (intramuscular injection), minocycline, gold sodium thiomalate, auranofin, D-penicillamine, clobenzaprine, buclizine, acrilide;

adrenocortical hormone agents:

cortisones, hydrocortisone

Cortisone acetate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, fludrocortisone acetate

Prednisolone, prednisolone derivatives

Prednisolone, prednisolone sodium succinate, prednisolone sodium phosphate, haloprednisolone methylprednisolone acetate

Methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate triamcinolone

Triamcinolone, triamcinolone acetate, triamcinolone actide dexamethasone

Dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, dexamethasone palmitate betamethasone

Betamethasone (betamethasone sodium phosphate), betamethasone sodium phosphate and paramethasone

Paramethasone acetate

The dose of immunosuppressant, for example: in the treatment of rheumatoid arthritis in combination with MTX, for example in the case of oral administration, 1 to 100mg per subject per week, preferably 4 to 50mg, more preferably 7.5 to 25mg per subject.

In addition, a high dose of an anti-IL-6R antibody refers to a dose that prevents or reduces allergy, which is equal to or higher than the minimum effective dose for treating IL-6 related diseases. For example, in the case of treating rheumatoid arthritis with intravenous drip every 4 weeks, the dose includes 4mg/kg or more, preferably 6 to 16mg/kg, more preferably 6 to 10 mg/kg.

The above-described administration method, administration interval, and administration dose are examples of preferred examples. The method, interval and dose of administration showing similar therapeutic effects can be appropriately selected. For example, the administration method, interval and dose having similar effects to those of the above preferred examples can be selected by measuring the concentrations of various drugs in blood. The invention includes methods of administration, intervals and dosages that achieve equivalent blood concentrations to those of the above examples.

Detailed Description

The IL-6 antagonist used in the present invention does not need to be considered in terms of its origin, type, form, so long as it exhibits an effect of preventing or treating an IL-6-related disease.

IL-6 antagonists are substances that inhibit the biological activity of IL-6. The IL-6 antagonist is preferably a substance having an effect of inhibiting the binding to IL-6, IL-6R, or gp 130. IL-6 antagonists include anti-IL-6 antibodies, anti-IL-6R antibodies, anti-gp 130 antibodies, modified IL-6, modified soluble IL-6R, or IL-6R partial peptides, as well as low molecular weight substances that exhibit similar activity.

The anti-IL-6 antibody used in the present invention can be obtained in the form of a monoclonal or polyclonal antibody by a method known in the art. As the anti-IL-6 antibody used in the present invention, a monoclonal antibody derived from a mammal is preferred. Monoclonal antibodies from mammals include those produced by hybridomas and those produced by host cells transformed with expression vectors containing antibody genes by genetic engineering techniques. The antibody can inhibit the binding of IL-6 to IL-6 receptor by binding to IL-6, thereby blocking the transduction of IL-6 biologically active signals into cells.

Such antibodies include MH166(Matsuda, T.et al., Eur.J.Immunol., 18: 951-956, 1988) and SK2(Sato, K.et al., The 21st Proceedings of The Japanese society for Immunology, 21: 166, 1991).

Hybridomas that produce anti-IL-6 antibodies can be prepared essentially as follows using techniques known in the art. That is, hybridomas can be obtained by immunizing according to a standard immunization method using IL-6 as a sensitizing antigen, then fusing the resulting immune cells with parent cells known in the art by a standard cell fusion method, and screening monoclonal antibody-producing cells by a standard screening method.

Specifically, anti-IL-6 antibodies can be prepared as follows. For example, the methods can be implemented by the methods disclosed in eur.j.biochem., 168: 543, 550, 1987; j.immunol., 140: 1534-1541, 1988 or agr, biol, chem, 54: 2685 IL-6 Gene/amino acid sequence of 2688, 1990 to obtain human IL-6 which was used as a sensitizing antigen to obtain an antibody.

The IL-6 gene sequence is inserted into an expression vector known in the art, and then transformed into a suitable host cell, followed by purification of the objective IL-6 protein from the cell or culture supernatant by a method known in the art, and the purified IL-6 protein can be used as an sensitizing antigen. Similarly, fusion proteins of IL-6 with other proteins can also be used as sensitizing antigens.

The anti-IL-6 receptor antibody used in the present invention can be obtained in the form of a monoclonal or polyclonal antibody by a method known in the art. For the anti-IL-6 receptor antibody used in the present invention, a monoclonal antibody derived from a mammal is preferred. Monoclonal antibodies from mammals include antibodies produced by hybridomas and antibodies produced by transforming host cells with expression vectors containing antibody genes by genetic engineering techniques. The antibody can inhibit the binding of IL-6 to the IL-6 receptor by binding to the IL-6 receptor and block the transduction of IL-6 bioactive signals into cells.

Such antibodies include MR16-1 antibody (Tamura, T.et al., Proc.Nat l.Acad.Sci.USA, 90: 11924. Aca. 11928, 1993), PM-1 antibody (Hirata, Y.et al., J.Immunol., 143: 2900. 2906, 1989), AUK-12-20 antibody, AUK64-7 antibody or AUK146-15 antibody (International patent application publication No. WO 92-19759). Among them, particularly preferred antibodies include PM-1 antibodies.

A hybridoma cell line producing a PM-1 antibody as PM-1 has been internationally deposited at International patent organism depositary (AIST Tsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki Pref.) under the Budapest treaty with deposit number FERM BP-2998 on 12.7.1989. A hybridoma cell line producing the MR16-1 antibody as rat-mouse hybridoma MR16-1 has been deposited internationally under the Budapest treaty with the deposit number FERM BP-5875 on 13.3.1997 at the International patent organism depositary (AIST Tsukuba Central 6, 1-1, Higashi-chome, Tsukuba-shi, Ibarakiipref).

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

Specifically, an anti-IL-6 receptor antibody can be prepared as follows. For example, human IL-6 receptor used as ｃA sensitizing antigen to obtain an antibody can be obtained by the IL-6 receptor gene/amino acid sequences disclosed in European patent application publication Nos. EP325474 and JP-A-3-155795, respectively.

There are two types of IL-6 receptor proteins, namely, one expressed on cells and one separated from the cell membrane (soluble IL-6 receptor) (Yasukawa, K.et al., J.biochem., 108: 673-once 676). The soluble IL-6 receptor consists essentially of the extracellular domain of the IL-6 receptor, which is distinguished from membrane-bound IL-6 receptor by the absence of a transmembrane domain or the absence of a transmembrane domain and an intracellular domain. As long as it is used as a sensitizing antigen for producing an anti-IL-6 receptor antibody used in the present invention, both IL-6 receptor proteins can be used.

The gene sequence of IL-6 receptor is inserted into an expression vector known in the art, transformed into a suitable host cell, followed by purification of the target IL-6 receptor protein from the cells or culture supernatant by a method known in the art, and the purified IL-6 receptor protein can be used as an sensitizing antigen. Similarly, IL-6 receptor-expressing cells and IL-6 receptor fusion proteins with other proteins can also be used as sensitizing antigens.

The plasmid pIBIBBSF 2R comprises a cDNA encoding the human IL-6 receptor, E.coli HB 101-pIBBSF 2R containing this plasmid has been deposited internationally under the Budapest treaty with the accession number FERM BP-2232 at the International patent organism depositary (AIST Tsukuba Central 6, 1-1, Higashi-chome, Tsukuba-shi, Ibaraki Pref.) from 1/9 of 1989.

The anti-gp 130 antibody used in the present invention can be obtained as a monoclonal or polyclonal antibody by methods known in the art. For the anti-gp 130 antibody used in the present invention, a monoclonal antibody from a mammal is preferred. Monoclonal antibodies from mammals include antibodies produced by hybridomas and antibodies produced by transforming host cells with expression vectors containing antibody genes by genetic engineering techniques. The antibody can inhibit the binding of the IL-6/IL-6 receptor complex to gp130 by binding to gp130, thereby blocking the transduction of IL-6 bioactive signals into the cell.

Such antibodies include AM64 antibody (JP-A-3-219894), 4B11 antibody and 2H4 antibody (US5571513), B-S12 antibody and B-P8 antibody (JP-A-8-291199).

Hybridomas producing anti-gp 130 monoclonal antibodies can be prepared essentially by techniques known in the art as follows. That is, hybridomas can be obtained by immunizing with gp130 as a sensitizing antigen according to a standard immunization method, fusing the resulting immune cells with parent cells known in the art by a standard cell fusion method, and screening cells producing monoclonal antibodies by a standard screening method.

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

The gp130 gene sequence is inserted into an expression vector system known in the art, transformed into a suitable host cell, and then the target gp130 protein is purified from the cell or culture supernatant by a method known in the art, and the purified gp130 protein can be used as an sensitizing antigen. Similarly, cells expressing gp130 and fusion proteins of gp130 protein with other proteins can also be used as sensitizing antigens.

The mammal immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell for cell fusion. Generally, rodents such as mice, rats and hamsters are used.

Immunization of animals with a sensitizing antigen can be carried out by methods known in the art. As a general method, it is possible to inject the animal intraperitoneally or subcutaneously with a sensitizing antigen. In particular, it is preferable that the sensitizing antigen is appropriately diluted and suspended with PBS (phosphate buffered saline) or saline, mixed with an appropriate amount of a standard adjuvant such as freund's complete adjuvant, emulsified, and then administered to the mammal several times at intervals of 4 to 21 days. In addition, a suitable carrier may be used in immunization with a sensitizing antigen.

The animals were immunized as described above and then it was confirmed that the serum level of the desired antibody was increased. Next, immune cells are removed from the mammal for cell fusion. Preferably, the immune cells used for cell fusion include especially spleen cells.

As the mammalian myeloma cells as the partner parent cells to be fused with the above-mentioned immune cells, cell lines known in the art, for example: P3X63Ag8.653(Kearney, J.F.et., J.Immunol., 123: 1548-.

The cell fusion of the above immune cells with myeloma cells can be carried out essentially by methods known in the art, for example: the Milstein method (Kohler G.and Milstein C., Methods Enzymol., 73: 3-46, 1981).

More specifically, the above cell fusion can be carried out, for example, in a standard nutrient medium in the presence of a cell fusion promoter. As the cell fusion promoter, for example: polyethylene glycol (PEG), Sendai virus (HVJ) and the like can be used. Adjuvants may be further added/used as required, for example: dimethyl sulfoxide to improve the fusion efficiency.

The ratio of the immune cells to the myeloma cells to be used is preferably, for example, 1 to 10 times that of the immune cells to the myeloma cells. As a medium for the above cell fusion, RPMI 1640 medium, MEM medium suitable for growth of myeloma cell lines, and other standard media usable for this type of cell culture can be used, and further with serum supplements such as: fetal Calf Serum (FCS) combinations.

For cell fusion, the immune cells and myeloma cells can be thoroughly mixed in the above medium in a given amount, and a PEG solution, for example: PEG solution with average molecular weight of about 1000-6000 was added at standard concentration of 30-60% (w/v) preheated at 37 ℃ and mixed to form the desired fused cells (hybridomas). Subsequently, cell fusion agents and the like which are unfavorable for growth of hybridomas can be removed by repeating the operations of sequentially adding an appropriate medium and centrifuging to remove the supernatant.

Hybridomas can be selected by culturing in standard selection media, for example, HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). Typically, the culture in HAT medium is continued for several days to several weeks for a sufficient period of time until the cells other than the hybridoma of interest (non-fused cells) are dead. Hybridomas producing the desired antibody are then screened and cloned using standard limiting dilution methods.

Furthermore, in addition to the above hybridomas obtained by immunizing an animal other than a human with an antigen, a desired human antibody having a binding activity to a desired antigen or a cell expressing the antigen can also be obtained by sensitizing human lymphocytes with an antigen protein or a cell expressing an antigen in vitro, and then fusing the sensitized B lymphocytes with human myeloma cells such as U266 cells (see JP-B-1-59878). Furthermore, it is also possible to obtain a desired human antibody according to the above-mentioned method by administering an antigen or antigen-expressing cells to a transgenic animal containing all components of human antibody genes (see International patent application publication Nos. WO 93/12227, WO92/03918, WO 94/02602, WO 94/25585, WO 96/34096, WO 96/33735).

The hybridoma producing the monoclonal antibody prepared by the method can be cultured in a standard culture medium or stored in liquid nitrogen for a long time.

To obtain monoclonal antibodies from hybridomas, the following methods can be used: culturing the hybridoma by a standard method, and then obtaining the monoclonal antibody in the form of culture supernatant; alternatively, hybridomas are administered to compatible mammals for proliferation and monoclonal antibodies are obtained as ascites fluid. The former method is suitable for obtaining high purity antibody, and the latter method is suitable for large-scale production of antibody.

For example, ｃA hybridomｃA producing an anti-IL-6 receptor antibody can be prepared by the method disclosed in JP-A-3-139293. The following method is therefore carried out: a PM-1 antibody-producing hybridoma which is internationally deposited at International patent organism depositary (AIST Tsukuba Central 6, 1-1, Higashi-chome, Tsukuba-shi, Ibaraki Pref.) on the Budapest treaty under accession number FERM BP-2998 on 12.7.1989 is intraperitoneally injected into a BALB/c mouse to obtain ascites, and then the PM-1 antibody is purified from the ascites. Also, the following method may be implemented: the hybridoma is cultured in a suitable medium, such as: the PM-1 antibody was purified from its culture supernatant after culturing in RPMI 1640 medium, hybridoma SFM medium (supplied by GIBCO-BRL), or PFHM-II medium (supplied by GIBCO-BRL) containing 10% fetal bovine serum and 5% BM-conditioner H1 (supplied by Boehringer Mannheim).

In the present invention, as for the monoclonal antibody, a recombinant antibody prepared as follows can be used: antibody genes are cloned from hybridomas, inserted into an appropriate vector, introduced into host cells, and genetically engineered (see, e.g., Borrebaeck, C.A.K., and Larrick, J.M., Therapeutic Monoclonal antibodies, Macmillan Press, 1990).

Specifically, mRNA encoding the variable region (V) of an antibody can be isolated from a cell, such as a hybridoma, that produces the antibody of interest. For isolation of mRNA, total RNA can be prepared by methods known in the art, for example, guanidine ultracentrifugation (Chirgwin, J.M. et al, Biochemistry, 18: 5294-. Similarly, mRNA can also be prepared directly by the QuickPrepmRNA purification kit (Pharmacia).

cDNA for the V region of the antibody can be synthesized from the obtained mRNA by reverse transcriptase. cDNA can also be synthesized using AMV reverse transcriptase first strand cDNA synthesis kit or the like. Likewise, the 5 '-Ampli FINDER RACE kit (supplied by Clontech) and the 5' -RACE method using PCR (Frohman, M.A.et., Proc.Natl.Acad.Sci.USA, 85: 8998-. The DNA fragment of interest is purified from the obtained PCR product and then ligated to a vector DNA. Subsequently, the recombinant vector prepared in this manner is introduced into E.coli, and colonies are screened to prepare a desired recombinant vector. The base sequence of the DNA of interest can be confirmed by a method known in the art, for example, a deoxidation method.

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

To prepare the antibody for use in the present invention, the antibody gene may be incorporated into an expression vector for expression under the regulation of expression regulatory regions, such as enhancers and promoters described below. Subsequently, the expression vector is transformed into a host cell to express the antibody.

In the present invention, an artificially modified gene recombinant antibody, for example: chimeric, humanized and human antibodies to reduce alloantigenicity to humans. These modified antibodies can be prepared by known methods.

The chimeric antibody can be obtained by ligating the above-obtained DNA encoding the V region of the antibody with the DNA encoding the C region of the human antibody to be introduced into a host for production, and then incorporating into an expression vector (see European patent application publication No. EP 125023, International patent application publication No. WO92/19759). By these known methods, the chimeric antibody used in the present invention can be obtained.

For example, plasmids containing DNAs encoding L chain and H chain V regions of a PM-1 chimeric antibody were designated as pPM-k3 and pPM-H1, respectively, and E.coli containing these plasmids have been deposited internationally as NCIMB 40366 and NCIMB 40362 at national Industrial and Marine collections, Inc., 23 St.Machar Drive, Aberdeen AB 21 RY, Scotland, UK, respectively, on the basis of the Budapest treaty on 12/2.1991.

Humanized antibodies, also called reshaped human antibodies, which are antibodies in which Complementarity Determining Regions (CDRs) of a non-human mammal such as a mouse are grafted with human antibody complementarity determining regions, and conventional genetic recombination methods thereof are also known (see European patent application publication No. EP 125023, International patent application publication No. WO92/19759).

Specifically, a DNA sequence designed to link the CDRs of a mouse antibody with the Framework Regions (FRs) of a human antibody was synthesized by PCR from several oligonucleotides having overlapping portions at the ends. The obtained DNA is ligated with a DNA encoding a human antibody C region, and then incorporated into an expression vector, which is introduced into host cells to produce a humanized antibody (see European patent application publication EP 239400, International patent application publication WO 92/19759).

In the case of the FRs of the human antibody connected via the CDRs, the FRs are selected such that the complementarity determining regions form a good antigen binding site. Framework amino acids of the antibody variable region may be substituted, if desired, to allow the CDRs of the reshaped human antibody to form the correct antigen binding site (Sato, K.et al., Cancer Res., 53: 851-856, 1993).

Human antibody C regions can be used for chimeric and humanized antibodies. Human antibody C regions include C γ, for example, C γ 1, C γ 2, C γ 3 or C γ 4 can be used. The human antibody C region can be modified to improve antibody stability or its yield.

The chimeric antibody is composed of a variable region of an antibody derived from a non-human mammal and a C region derived from a human antibody. Humanized antibodies are composed of complementarity determining regions derived from non-human mammalian antibodies and framework regions and C regions derived from human antibodies. Therefore, these antibodies have reduced antigenicity in the human body and can be used as the antibody of the present invention.

Preferred specific examples of the humanized antibody used in the present invention include humanized PM-1 antibody (see International patent application publication No. WO 92-19759).

Similarly, as a method for obtaining a human antibody, in addition to the above-described method, a technique for obtaining a human antibody by panning using a human antibody library is also known. For example, the variable regions of human antibodies can be expressed on the phage surface as single chain antibodies (scFv) by phage display methods, and then the phage that bind the antigen are screened. The DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined by analyzing the genes of the selected phage. If the DNA sequence of an scFv that binds an antigen is elucidated, the sequence can be manipulated by a suitable expression vector to obtain a human antibody. These processes are already known and reference may be made to WO 92101047, WO92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438 and WO 95/15388.

The antibody gene constructed as described above can be expressed and obtained by a method known in the art. When mammalian cells are used, the antibody gene may be expressed from DNA functionally linked to a conventional promoter, the antibody gene to be expressed and a 3' downstream poly A signal, or may be expressed from a vector containing them. For example: the promoter/enhancer may include the human cytomegalovirus immediate early promoter/enhancer.

Similarly, as other promoters/enhancers that can be used for the expression of the antibody of the present invention, viral promoters/enhancers of retrovirus, polyoma virus, adenovirus, simian virus 40(SV40), and promoters/enhancers derived from mammalian cell human elongation factor 1 α (HEF1 α) can be used.

For example, expression can be carried out using the SV40 promoter/enhancer according to the method of Mulligan et al (Mulligan, R.C. et al., Nature, 277: 108-one 114, 1979) or expression can be carried out using the HEF1 α promoter/enhancer according to the method of Mizushima et al (Mizushima, S and Nagata, S., Nucleic Acids Res., 18: 5322, 1990).

For E.coli, the antibody gene can be expressed by functionally linking a conventional promoter, an antigen secretion signal sequence, and the antibody gene to be expressed. For example, the promoter may include lacZ promoter and araB promoter. In the case of using the lacZ promoter and the araB promoter, the method of Ward et al (Ward, E.S.et al, Nature, 341: 544. sup. 546, 1989; Ward, E.S.et al, FASEB J., 6: 2422. sup. 2427, 1992) and the method of Better et al (Better, M.et al, Science, 240: 1041. sup. 1043, 1988), respectively, can be employed.

As the antibody secretion signal sequence, in the case of production in the periplasm of E.coli, the pelB signal sequence can be used (Lei, S.P.et. al., Bacteriol., 169: 4379-A4383, 1987). Antibodies produced in the periplasm are isolated, followed by appropriate antibody structure refolding in order to use the antibody (see, e.g., WO 96/30394).

Origins of replication derived from SV40, polyoma virus, adenovirus, Bovine Papilloma Virus (BPV) may be used. In addition, in order to amplify the gene copy number in the host cell system, the expression vector may include an Aminoglycoside Phosphotransferase (APH) gene, a Thymidine Kinase (TK) gene, E.coli xanthine guanine phosphate glycosyltransferase (Ecogpt) gene, and dihydrofolate reductase (dhfr) gene, etc. as selection markers.

For the production of the antibody used in the present invention, an optional production system may be used. There are in vitro and in vivo production systems for the production of antibodies. In vitro production systems include those using eukaryotic cells and those using prokaryotic cells.

In the case of using eukaryotic cells, there are production systems using animal cells, plant cells or fungal cells. As the animal cell, (1) a mammalian cell is known, for example: CHO, COS, myeloma cells, BHK (baby hamster kidney cells), HeLa, Vero, etc., (2) amphibian cells, for example: xenopus oocytes, or (3) insect cells, such as: sf9, sf21, Tn5, and the like. As plant cells, cells derived from tobacco (Nicotiana tabacum) are known, and they can be cultured as callus. Yeasts, for example of the Saccharomyces genus (Saccharomyces), such as Saccharomyces cerevisiae, and filamentous fungi, for example of the Aspergillus genus (Aspergillus), such as Aspergillus niger, are known as fungal cells.

When prokaryotic cells are used, there are production systems that use bacterial cells. As bacterial cells, escherichia coli (e.coli) and Bacillus subtilis (Bacillus subtilis) are known.

The antibody can be obtained by introducing a target antibody gene into these cells through transformation, and then culturing the transformed cells in vitro. The cultivation may be performed according to methods known in the art. For example, DMEM, MEM, RPMI 1640 and IMDM can be used as the culture medium, but also with serum supplements such as: fetal Calf Serum (FCS) was used in combination. The antibody can be produced in vivo by transferring the antibody gene-introduced cells into the peritoneal cavity of the animal.

In another aspect, the in vivo production system includes a production system using animals and a production system using plant cells. In the case of using animal cells, there are production systems using mammals and insects.

As the mammal, goat, pig, sheep, mouse, cow and other animals can be used (Vicki Glaser, Spectrum Biotechnology Applications, 1993). Also, as the insect, silkworm can be used. In the case of using a plant, for example, tobacco can be used.

The antibody gene is introduced into these animals or plants, and the antibody is produced in the animals or plants and then collected. For example, the antibody gene may be prepared as a fusion gene by inserting the midstream of a gene encoding a protein naturally produced in milk, such as goat beta casein. The DNA fragment containing the fusion gene into which the antibody gene is inserted is injected into a goat embryo and then transferred to a female goat. The desired antibody can be obtained from the goat milk of a transgenic goat born from the goat receiving the embryo or a progeny goat thereof. To increase the amount of milk containing the desired antibodies, the transgenic goat may be given a suitable hormone (Ebert, K.M.et. al., Bio/Technology, 12: 699-702, 1994).

In the case of using silkworms, silkworms are infected with baculovirus into which a gene for a target antibody is inserted, and then the desired antibody is obtained from the silkworm body fluid (Maeda, S.et al., Nature, 315: 592-594, 1985). Further, in the case of using tobacco, a target antibody gene is inserted into a plant expression vector such as: pMON 530, and then introducing the vector into a bacterium, such as: agrobacterium tumefaciens. Infection of tobacco, such as Nicotiana tabacum, with these bacteria resulted in the desired antibody from the leaves of the tobacco (Julian, K.C., Ma, et al, Eur.J.Immunol., 24: 131-.

In the production of antibodies using the in vitro or in vivo production system as described above, the DNA encoding the heavy chain (H chain) or light chain (L chain) of an antibody may be incorporated separately into an expression vector, these vectors may be used to transform a host cell simultaneously, or the DNA encoding the H chain and L chain may be incorporated into a single expression vector, and a host may be transformed with the vector (see International patent application publication No. WO 94-11523).

The antibody used in the present invention may be an antibody fragment or a modification thereof, as long as the antibody is suitably used. For example, againstThe body segments include, for example: fab, F (ab')²Fv or single chain Fv (scFv), in which the H and L chains are joined by a suitable linker.

Specifically, enzymes may be used, for example: papain, pepsin treatment of antibodies to produce antibody fragments, or a gene encoding an antibody fragment can be constructed, introduced into an expression vector, and then expressed in a suitable host cell (see, e.g., Co, M.S. et al, J.Immunol., 152: 2968-2976, 1994; Better, M. & Horwitz, A.E., Methods in Enzymology, 178: 476-496, 1989; Pluuethun, A. & Skerra, A., Methods in Enzymology, 178: 476-496, 1989; Lamoyi, E., Methods in Enzymology, 121: 652-663, 1989; Rousseaux, J.et al, Methods in Enzymology, 121: 663-66, 1989; Bird, R.E.137, ECH., 1991).

scFv is obtained by linking the H chain V region with the L chain V region. In this scFv, the H chain V region and the L chain V region are preferably joined by a linker, particularly a peptide linker (Huston, J.S.et. al., Proc.Natl.Acad.Sci.USA, 85: 5879-. The H chain V region and the L chain V region in the scFv may be derived from any of the antibodies as described above. For the peptide linker connecting the V regions, a given single-chain peptide composed of, for example, 12 to 19 amino acid residues can be used.

Amplifying a DNA portion encoding a desired amino acid sequence in a template by a PCR method using DNAs encoding the H chain or the V region of the H chain and the L chain or the V region of the L chain of the antibody as templates and primers defining the ends of the DNA portion encoding the desired amino acid sequence; then further combining primers defining both ends of the peptide linker moiety to be linked to the H and L chains, and amplifying DNAs encoding the peptide linker moiety and both ends, thereby obtaining DNAs encoding scFv.

Further, once the DNA encoding scFv is obtained, an expression vector containing it and a host transformed with the expression vector can be obtained according to standard methods, and then scFv can be obtained using the host according to standard methods.

For these antibody fragments, their genes can be obtained as above, and expressed and produced by a host. The term "antibody" as used herein includes such antibody fragments.

As a modification to the antibody, an antibody conjugated with various molecules such as polyethylene glycol (PEG) may also be used. The term "antibody" as used herein includes such modified antibodies. Such modified antibody can be obtained by chemically modifying the obtained antibody. These methods are established in the art.

The antibody produced and expressed as described above can be isolated from the inside and outside of the cell and the inside and outside of the host, and purified to be homogeneous. The isolation and purification of the antibodies used in the present invention can be performed using affinity chromatography. Columns for affinity chromatography include, for example: protein a columns and protein G columns. Carriers for protein a columns include Hyper D, POROS, Sepharose f.f. Other separation/purification methods for conventional proteins may be used without limitation.

For example, chromatography other than the above-mentioned affinity chromatography, filtration, ultrafiltration, salting out, dialysis, and the like may be appropriately selected and combined to isolate and purify the antibody used in the present invention. Examples of chromatography include: ion exchange chromatography, hydrophobic chromatography, gel filtration, etc. Such chromatography can be applied to HPLC (high performance liquid chromatography). In addition, reverse phase HPLC may also be used.

The concentration of the antibody obtained above can be measured by measuring absorbance or by ELISA. That is, when the absorbance was measured, the antibody was appropriately diluted with PBS (-), followed by measuring the absorbance at 280mm, and the concentration was calculated as 1.35 OD-1 mg/ml. When ELISA is used, the measurement can be performed as follows. That is, goat anti-human IgG (supplied from TAG Co.) was diluted to 1. mu.g/ml with 0.1M bicarbonate buffer (pH 9.6), 100ml of this IgG was added to a 96-well plate (supplied from Nunc Co.), followed by 4 ℃ overnight incubation to immobilize the antibody. After blocking, 100. mu.l of an appropriately diluted antibody of the invention or an antibody-containing sample, or human IgG as standard (supplied by Cappel corporation) was added, followed by incubation at room temperature for 1 hour.

After washing, 100. mu.l of 5000-fold diluted alkaline phosphatase-labeled anti-human IgG (supplied by BioSource) was added, followed by incubation at room temperature for 1 hour. After washing, a substrate solution was added, incubated, and then absorbance at 405mm was measured with a Microplate Reader Model 3550 apparatus (provided by Bio-Rad) to calculate a target antibody concentration.

The modified IL-6 used in the present invention is a substance having IL-6 receptor binding activity but incapable of delivering IL-6 biological activity. That is, although the modified IL-6 and IL-6 competitive binding IL-6 receptor, but because it can not transmit IL-6 biological activity, so it can block IL-6 signal transduction.

Modified IL-6 can be obtained by introducing variations by substituting amino acid residues in the amino acid sequence of IL-6. IL-6 as a source of modified IL-6 may be obtained from any source, but human IL-6 is preferred in view of antigenicity.

In particular, IL-6 modification can be performed using molecular modeling programs known in the art, such as WHATIF (Vriend et al, mol. graphics, 8: 52-56, 1990) to predict IL-6 amino acid sequence secondary structure, and to further evaluate the effect of the amino acid residue to be substituted on the overall protein. After determining an appropriate amino acid residue to be substituted, a variant in which an amino acid is substituted is introduced by a conventional PCR method using a vector containing a base sequence encoding a human IL-6 gene as a template to obtain a modified IL-6-encoding gene. Modified IL-6 can be obtained by incorporating the coding gene into a suitable desired vector, followed by recombinant antibody expression, production and purification methods as described above.

Specific examples of modified IL-6 are disclosed in Brakenhoff et al, J.biol.chem., 269: 86-93, 1994 and Savino et al, EMBO j., 13: 1357, 1367, 1994, WO 96/18648 and WO 96/17869.

The partial peptide of IL-6 or the partial peptide of IL-6 receptor used in the present invention has an activity of binding to IL-6 receptor or IL-6, respectively, and is a substance that does not transfer the biological activity of IL-6. That is, the IL-6 partial peptide or the IL-6 receptor partial peptide specifically inhibits the binding of IL-6 to the IL-6 receptor by binding and capturing the IL-6 receptor or IL-6, respectively. As a result, since they do not transmit IL-6 bioactivity, they can block signaling by IL-6.

The partial peptide of IL-6 or the partial peptide of IL-6 receptor is a peptide consisting of part or all of the amino acid sequence of IL-6 or IL-6 receptor involved in the binding of IL-6 to the IL-6 receptor. Such peptides typically consist of 10-80 amino acid residues, preferably 20-50, more preferably 20-40. Partial peptides of IL-6 or partial peptides of the IL-6 receptor can be obtained by determining the region of the amino acid sequence of IL-6 or IL-6 receptor which is involved in binding to IL-6 or IL-6 receptor, and then using known conventional methods, for example: the amino acid sequence of the polypeptide is partially or completely synthesized by genetic engineering technology or peptide synthesis.

In order to obtain a partial peptide of IL-6 or a partial peptide of IL-6 receptor by genetic engineering techniques, a DNA sequence encoding a desired peptide may be inserted into an expression vector, followed by the methods of recombinant antibody expression, production and purification described above.

For the preparation of a partial peptide of IL-6 or a partial peptide of IL-6 receptor by peptide synthesis, a typical method of peptide synthesis may be used, for example: solid phase synthesis or liquid phase synthesis.

Specifically, the synthesis can be carried out according to the method described in Zoku Iyakuhin, Kaihatu Vol.14 Peptide Gosei, (Ed., Yajima, H., Hirokawa Shoten, 1991). For the solid phase synthesis method, for example, the following methods can be used: the peptide chain is extended by binding an amino acid corresponding to the C-terminal end of the peptide to be synthesized to a support insoluble in an organic solvent, and then alternately repeating a reaction of sequentially condensing one (the. alpha. -amino group and the side chain functional group are protected with an appropriate protecting group in the amino acid) in the direction from the C-terminal end to the N-terminal end of the amino acid, and a reaction of eliminating the protecting group of the. alpha. -amino group of the amino acid or the peptide attached to the resin. Solid phase synthesis methods can be roughly classified into Boc method and Fmoc method according to the type of protecting group used.

After the target peptide is synthesized in this manner, deprotection reaction and cleavage reaction of the peptide chain from the support are carried out. In the cleavage reaction of peptide chain, hydrogen fluoride or trifluoromethanesulfonic acid and TFA are typically used for anisole in Boc method and Fmoc method, respectively. In the Boc method, the above protected peptide-resin is treated with hydrogen fluoride in the presence of anisole, followed by removal of the protecting group and cleavage separation of the peptide from the support to recover the peptide. The peptide was freeze-dried to yield the crude peptide. On the other hand, in the Fmoc method, for example: in TFA, the deprotection reaction and the cleavage reaction of the peptide chain from the support were carried out in the same manner as described above.

The crude peptide obtained was isolated and purified by HPLC. Eluting under optimal conditions with water-acetonitrile solvents typically used for protein purification. Fractions corresponding to peaks in the obtained chromatogram were collected and freeze-dried. The peptide fraction purified by the above method can be identified by mass spectrometry molecular weight analysis, amino acid composition analysis or amino acid sequence analysis.

Specific examples of IL-6 and partial peptides of IL-6 receptor are disclosed in JP-A-2-188600, 7-324097 and 8-311098, and U.S. Pat. No. 5210075.

The pharmaceutical composition of the present invention may contain pharmaceutically acceptable carriers and additives depending on its administration mode. Examples of carriers and additives include: water, a pharmaceutically acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium arginine, water-soluble dextran, sodium carboxymethyl starch, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum arabic, casein, gelatin, agarose, diglycerin, propylene glycol, polyethylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, Human Serum Albumin (HSA), mannitol, sorbitol, lactose, an acceptable surfactant as a pharmaceutical additive, and the like. The additives used may be selected from the above as appropriate or in appropriate combination depending on the dosage form, but are not limited thereto.

Examples

The present invention is specifically described below by way of examples and reference examples, but the present invention is not limited thereto.

Example 1:

MRA is a recombinant humanized monoclonal antibody of the IgG1 subclass anti-human interleukin-6 receptor, which inhibits the function of the cytokine interleukin-6 (IL-6). In early studies in japan and europe, MRA showed promise for the treatment of rheumatoid arthritis and was well tolerated.

This example is a phase II pilot trial to determine the optimal dose of MRA for treatment of rheumatoid arthritis when administered alone and in combination with methotrexate. The potential efficacy of repeated intravenous administration of MRA (both alone and in combination with methotrexate) was evaluated in patients with active rheumatoid arthritis despite treatment with methotrexate for a defined period of time and compared to methotrexate alone. The effectiveness, safety and tolerability of MRA was assessed.

The method comprises the following steps:

study subjects: patients with rheumatoid arthritis who were diagnosed based on the 1987 disease classification of the American College of Rheumatology (ACR) for a period of at least 6 months were enrolled. Patients must have active disease and they do not respond adequately or have a burst of disease to MTX administered at a dose of at least 12.5 mg/week or 10 mg/week (in the case of intolerance) for at least 6 months.

Research and design: double-blind, randomized, parallel group studies by central randomization

Dose and mode of administration: seven groups are as follows: 0mg/kg (placebo) + MTX, 2mg/kg MRA + MTX, 4mg/kg MRA + MTX, 8mg/kg MRA + MTX, 2mg/kg MRA + MTX placebo, 4mg/kg MRA + MTX placebo and 8mg/kg MRA + MTX placebo. MRA or placebo was administered by intravenous infusion at 4 week intervals. MTX or MTX placebo is administered orally 1 time per week at 10-25 mg/week.

The research method comprises the following steps: the indicated doses were co-administered 4 times by intravenous infusion at 4 week intervals, with efficacy and safety assessed every 2 weeks up to 16 weeks and observed at week 20 follow-up. The primary endpoint (primary endpoint) of the efficacy study was the ACR20 rate at week 16 (4 weeks after the last dose). The secondary endpoint (secondary endpoint) included the ratio of ACR50 and ACR70 at week 16 (4 weeks after the last dose).

ACR improvement criteria: when in the following 7 items, the swollen joint number and painful joint number were improved by 20% or more and an improvement of 20% or more was observed in 3 of the remaining 5 items, this case was determined as an ACR improvement criterion of 20% or more. In addition, 50% and 70% improved cases refer to cases in which the above-mentioned 20% improved fraction was improved by 50% and 70%, respectively.

(1) Swollen joint number

(2) Number of tender joints

(3) Patient assessment of pain

(4) General assessment of patient activity

(5) General assessment of disease activity by physicians

(6) Patient assessment of physiological function

(7) CRP or ESR

TABLE 1

	2mg/kg MRA	4mg/kg MRA	8mg/kg MRA	MTX
					ACR 20	30.8％	61.1％	62.7％	40.8％

ACR 50	5.8％	27.8％	41.2％	28.6％
					ACR 70	1.9％	5.6％	15.7％	16.3％
	2mg/kg MRA+MTX	4mg/kg MRA+MTX	8mg/kg MRA+MTX
					ACR 20	64.0％	63.3％	73.5％
ACR 50	32.0％	36.7％	53.1％
					ACR 70	14.0％	12.2％	36.7％

A statistically significantly higher improvement rate of ACR20 was observed in all groups except the 2mg/kg group where MRA was administered alone, compared to the control group. In the MRA 8mg/kg + MTX group, the improvement rates of ACR50 and 70 were 53.1% and 36.7%, respectively, which were statistically significantly higher than those of the control group, which were 28.6% and 16.3%, respectively. In the MRA single-dose group, a statistically significant dose dependence was observed in the ACR20 improvement rate. In addition, statistically significant dose-dependent responses were observed in both the MRA alone and the MTX combined group for the improvement rates of ACR50 and ACR 70.

Swelling joint count reduction (Table 2)

The mean swollen joint count was similar at baseline in all treatment groups.

As the duration of drug exposure increased for all seven treatment groups, the average swollen joint count decreased cumulatively from baseline. The mean decrease in swollen joint count in the MRA 8mg/kg group was statistically significant compared to the decrease in the MTX group (p ═ 0.010). At week 16, the mean difference (95% CI) between the MRA 8mg/kg group and the MTX group was-2.31 (-4.07, -0.55). There was a statistically significant linear dose relationship (p < 0.001) between the MRA treatment alone groups. The mean decrease in swollen joint counts for the MRA 8mg/kg + MTX group was statistically significant (p < 0.001) compared to the decrease for the MTX group. The mean difference (95% CI) between the MRA 8mg/kg + MTX group and the MTX group was-3.62 (-5.39, -1.84). There was a statistically significant linear dose relationship between the MRA combination treatment groups (p ═ 0.004).

TABLE 2

	2mg/kgMRA	4mg/kgMRA	8mg/kgMRA	MTX
					Baseline: average value of N. + -. SD	5211.6±4.6	5411.1±4.4	5112.2±5.2	4912.7±4.2
Change from baseline: mean N. + -. SD at week 16	42-4.5±5.7	43-5.8±4.1	43-8.4±4.6	39-5.7±6.1

	2mg/kgMRA+MTX	4mg/kgMRA+MTX	8mg/kgMRA+MTX
					Baseline: average value of N. + -. SD	5011.9±4.3	4911.9±3.9	4911.8±3.9
Change from baseline: average value of N at week 16±SD	46-6.2±4.6	42-6.8±5.4	44-9.4±4.0

Among 359 patients enrolled, the safety assessment group, the complete analysis group, and PPS (per protocol set) were 359, 354, and 307, respectively. A total of 359 patients were enrolled, 299 completed the study, and 60 withdrawals. Of the patients who were withdrawn, 33 were adverse, 1 was concurrent with other diseases, 7 were adverse, 7 were prohibited from concomitant use, 5 were withdrawn with informed consent (informad present), 1 was missed, and 22 were absent (including multiple causes).

Among the serious adverse reactions that could not deny the causal relationship, 5 infections were reported. That is, it was reported that 1 patient had foot abscess and osteomyelitis in the 2mg/kg MRA group, 1 patient had chest infection and pleuritis in the 8mg/kg MRA + MTX group, and 1 patient had septicemia, 1 patient had septicemia in the 8mg/kg MRA + MTX group, and 1 patient had joint infection in the 8mg/kg MRA + MTX group. In addition, there were 5 reported allergies that were serious adverse reactions that could not deny the causal relationship, i.e., 4 patients in the 2mg/kg MRA group and 1 in the 4mg/kg MRA group were reported to be allergic. All these allergic events occurred after either the 3 rd or 4 th MRA administration without combining with MTX.

With respect to the liver function laboratory data values, although elevated ALT and AST levels were observed as a result of MRA administration, these elevations were comparable to those observed in other rheumatoid arthritis patients. An increase in lipid (total cholesterol, HDL cholesterol and triglycerides) -related laboratory data was observed in the MRA group. However, the actuated pulse hardening Index (Atherogenic Index) was not changed overall.

A slight temporary decrease in neutrophil count occurred in some patients. Clinically significant changes in the disease activity parameters, i.e. reduction in CRP and ESR and elevation in hemoglobin, were observed in a dose-dependent manner.

Infusion reaction

Infusion reactions were defined as adverse reactions that occurred within 24 hours of administration of the study drug. The number of patients presenting with infusion response in each treatment group suggests a possible counter-dose response (invertedose-response) of the MRA.

anti-MRA antibodies

The production of anti-MRA antibodies was detected. There was no antibody production in the 8mg/kg treatment group (either treatment alone or in combination with MTX). In the 2 or 4mg/kg treatment groups, the MTX combination treatment group showed fewer antibody producing events than the MRA treatment group alone.

Results

Significant dose responses were observed in both MA alone and MRA in combination with methotrexate. The effectiveness of MRA in treating rheumatoid arthritis patients was demonstrated in both MRA alone and in combination with methotrexate. In addition, the safety of MRA was also demonstrated in MRA alone and in combination with methotrexate.

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

The soluble IL-6 receptor was prepared by PCR using the plasmid pBSF2R.236 containing cDNA encoding the IL-6 receptor obtained according to the method of Yamasaki et al (Yamasaki, K.et. al., Science, 241: 825-828, 1988). The plasmid pBSF2R.236 was digested with the restriction enzyme Sph1 to obtain IL-6 receptor cDNA, which was then inserted into mp18 (supplied by Amersham). Mutations were introduced into IL-6 receptor cDNA by PCR using synthetic oligomeric primers designed to introduce stop codons into the IL-6 receptor cDNA using an in vitro mutagenesis system (supplied by Amersham). This procedure introduces a stop codon at amino acid 345 to obtain a cDNA encoding a soluble IL-6 receptor.

The soluble IL-6 receptor cDNA was ligated with plasmid pSV (supplied by Pharmacia) to obtain plasmid pSVL344 for expression of the cDNA in CHO cells. The HindIII-SalI-digested soluble IL-6 receptor cDNA was inserted into a plasmid pECEdhfr containing a cDNA for dhfr to obtain a CHO cell expression plasmid pECEdhfr 344.

Mu.g of pECEdhfr344 plasmid was transfected into the dhfr-CHO cell line DXB-11(Urlaub, G.et al, Proc.Natl.Acad.Sci.USA, 77: 4216-. Transfected CHO cells were cultured for 3 weeks in α MEM selection medium containing 1mM glutamine, 10% dialyzed FCS, 100U/ml penicillin, and 100 μ g/ml streptomycin and no nucleosides.

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

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

BALB/c mice were immunized with 10. mu.g of recombinant IL-6(Hirano, T.et., Immunol.Lett., 17: 41, 1988) together with Freund's complete adjuvant, and immunization was continued every other week until anti-IL-6 antibodies were detected in the serum. Immune cells were obtained from regional lymph nodes and fused with the myeloma cell line P3U1 using polyethylene glycol 1500. Hybridomas producing anti-human IL-6 antibodies were established by screening hybridomas using HAT medium according to the method of Oi et al (Selective methods in Cellular Immunology, W.H.Freeman and Co., San Francisco, 351, 1980).

IL-6 binding assays were performed on hybridomas producing anti-human IL-6 antibodies as follows. That is, flexible polyethylene 96-well microtiter plates (Dynatech Laboratories, Inc., available from Alexandra, Va.) were coated with 100. mu.l goat anti-mouse Ig (10. mu.l/ml; available from Cooper biomedicalcal Inc., Malvern, Pa.) in 0.1M bicarbonate-carbonate buffer (pH 9.6) overnight at 4 ℃. Next, the plates were treated with 100. mu.l of 1% Bovine Serum Albumin (BSA) in PBS for 2 hours at room temperature.

The plate was washed with PBS, then 100. mu.l hybridoma culture supernatant was added to each well and incubated overnight at 4 ℃. Washing the plate, then¹²⁵I-labeled recombinant IL-6 was added to 2000cpm/0.5 ng/well, the plates were washed and the radioactivity in each well was measured using a Gamma counter (Beckman Gamma 9000, Beckman instruments, Fullerton, Calif.). As a result, 32 out of 216 hybridoma clones were positive in the IL-6 binding assay. Among these clones, the stable clone MH166.BSF2 was finally obtained. The anti-IL-6 antibody MH166 produced by this hybridoma was the IgG1 kappa subtype.

Next, neutralizing activity of MH166 antibody was tested against hybridoma growth with IL-6 dependent mouse hybridoma clone mh60.bsf 2. MH60.BSF2 cells at 1X 10⁴The sample containing MH166 antibody was added to the sample distributed at 200. mu.l/well, followed by incubation for 48 hours, and then 0.5. mu. Ci/well was added³H thymidine (New England Nuclear, Bos ton, MA). After another 6 hours of incubation, the cells were placed on glass filter paper and treated with an automatic collector (Labo mass Science co., Tokyo, Japan). Rabbit anti-IL-6 antibody was used as a control.

As a result, MH166 antibody inhibited MH60 BSF2 cell pairs induced by IL-6 in a dose-dependent manner³Uptake of H thymidine. This indicates that MH166 antibody neutralizes the activity of IL-6.

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

IL-6 receptor was purified by combining the anti-IL-6 receptor antibody MT18 prepared according to the method of Hirano et al (Hirano, Y.et al., J.Immunol., 143: 2900-2906, 1989) with Sepharose 4B (supplied by Pharmacia Fine Chemicals; Piscataway, NJ) activated with CNBr according to the protocol attached (Yamasaki, K.et al., Science, 241: 825-828, 1988). Human myeloma cell line U266 was lysed with 1mM 1mM p-aminophenylmethanesulfonyl fluoride hydrochloride (supplied by Wako Chemicals) containing 1% digitonin (supplied by Wako Chemicals), 10mM ethanolamine (pH 7.8) and 1.5M NaCl (supplied by Wako Chemicals) (digitonin buffer), and mixed with MT18 antibody bound to agarose 4B beads. The beads were then washed 6 times with digitonin buffer and used as partially purified IL-6 receptor for immunization.

By a factor of 3X 10⁹BALB/c mice were immunized 4 times every 10 days with the above partially purified IL-6 receptor obtained from each U266 cell, followed by preparation of hybridomas using standard methods. The following method was used to examine the IL-6 receptor binding activity in hybridoma supernatants from growth positive wells. 5X 10⁷For U266 cells³⁵S-methionine (2.5mCi) was labeled and dissolved in the above-mentioned digitonin buffer. A volume of 0.04ml of MT18 antibody bound to Sepharose 4B beads was mixed with lysed U266 cells, followed by 6 washes with digitonin buffer and elution with 0.25ml of digitonin buffer (pH 3.4)³⁵The S-methionine-labeled IL-6 receptor was neutralized with 0.025ml of 1M Tris (pH 7.4).

Hybridoma culture supernatant (0.05ml) was mixed with 0.01ml protein G Sepharose (supplied by Pharmacia). After washing, the agarose was mixed with 0.005ml of the one prepared above³⁵S-methionine-labeled IL-6 receptor solution. The immunoprecipitates were analyzed by SDS-PAGE and hybridoma culture supernatants reactive with IL-6 receptor were detected. Thus, a positive-reacting hybridoma clone PM-1(FERM BP-2998) was established. The antibody produced by hybridoma PM-1 is of the IgG kappa subtype.

The inhibitory activity of the antibody produced by hybridoma Pm-1 on the binding of IL-6 to the human IL-6 receptor was examined using the human myeloma cell line U266. Preparation of human recombinant IL-6 from E.coli (Hirano, T.et al., Immunol.Lett., 17: 4)1-45, 1988) and performed with Bolton-Hunter reagent (New England Nuclear, Boston, Mass.)¹²⁵And I, marking.

U266 cells were plated at 4X 10⁵With 70% (v/v) hybridoma PM-1 culture supernatant and 14000cpm¹²⁵I labeled IL-6 was incubated together. Samples (70. mu.l) were plated on 300. mu.l FCS in 400. mu.l microcentrifuge polyethylene tubes, centrifuged, and the cell radioactivity measured.

As a result, antibodies produced by hybridoma PM-1 were shown to inhibit the binding of IL-6 to the IL-6 receptor.

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

The methods described by Saito, t.et al, j.immunol., 147: 168-173, 1991, and a monoclonal antibody against the mouse IL-6 receptor.

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 from the culture supernatant using an affinity column on Affigel 10 gel (supplied by Biorad) to which an anti-mouse IL-6 receptor antibody RS12 (see Saito, T.et al, supra) was immobilized.

The resulting mouse soluble IL-6 receptor (50. mu.g) was mixed with Freund's complete adjuvant and injected into the abdominal cavity of Wistar rats. Two weeks later, re-immunization was started with Freund's incomplete adjuvant. On day 45, splenocytes from rats were harvested and treated with 2X 10 PEG1500 (supplied by Boehringer Mannheim) according to standard procedures⁸Each cell is combined with 1X 10⁷Mouse myeloma P3U1 cells were fused, followed by selection of hybridomas in HAT medium.

Hybridoma culture supernatants were added to plates coated with rabbit anti-rat IgG antibody (supplied by Cappel) and reacted with mouse soluble IL-6 receptor. Subsequently, hybridomas producing antibodies against the mouse soluble IL-6 receptor were screened by ELISA using rabbit anti-mouse IL-6 receptor antibody and alkaline phosphatase-labeled sheep anti-rabbit IgG. The hybridoma clones confirmed to produce the antibody were subcloned twice to obtain hybridoma monoclonals. This clone was named MR 16-1.

By using MH60.BSF2 cell (Matsuda, T.et al., J.Immunol., 18: 951-956, 1988) pairs³Uptake of H thymidine and detection of neutralizing activity of the antibody produced by the hybridoma on mouse IL-6 signaling. MH60.BSF2 cells were prepared as 1X 10 in 96-well plates⁴Cells/200. mu.l/well. Add 10pg/ml mouse IL-6 and 12.3-1000ng/ml MR16-1 antibody or RS12 antibody to the plate, followed by 5% CO₂Incubated at 37 ℃ for 44 hours, followed by addition of 1. mu. Ci/well³H thymidine. After 4 hours, measure³Uptake of H thymidine. As a result, the MR16-1 antibody inhibited MH60 BSF2 cell pairs³Uptake of H thymidine.

Thus, the antibody produced by hybridoma MR16-1(FERM BP-5875) was shown to inhibit the binding of IL-6 to the IL-6 receptor.

Claims (18)

1. A pharmaceutical composition for treating rheumatoid arthritis comprising MRA and methotrexate.

2. The pharmaceutical composition of claim 1, wherein the dose of MRA is 0.02 to 150mg/kg/4 weeks or a dose that shows equivalent blood MRA concentration thereto.

3. The pharmaceutical composition of claim 2, wherein the dose of MRA is 0.5-30mg/kg/4 weeks or a dose that shows equivalent blood MRA concentration thereto.

4. The pharmaceutical composition of claim 3, wherein the MRA dose is a dose of 2 to 8mg/kg/4 weeks or a dose that exhibits a blood MRA concentration equivalent thereto.

5. The pharmaceutical composition of claim 1, wherein the dose of methotrexate is 1-100 mg/subject/week.

6. The pharmaceutical composition of claim 5, wherein the dose of methotrexate is 4-50 mg/subject/week.

7. The pharmaceutical composition of claim 6, wherein the dose of methotrexate is 7.5-25 mg/subject/week.

8. The pharmaceutical composition of any one of claims 1-7, prepared as a formulation for simultaneous administration of MRA and methotrexate.

9. The pharmaceutical composition of any one of claims 1-7, prepared as a formulation for administration of MRA and methotrexate at intervals in time.

Use of MRA and methotrexate in the preparation of a pharmaceutical composition for the treatment of rheumatoid arthritis.

11. The use of claim 10, wherein the MRA dose is a dose of 0.02 to 150mg/kg/4 weeks or a dose that exhibits a blood MRA concentration equivalent thereto.

12. The use of claim 30, wherein the MRA dose is a dose of 0.5 to 30mg/kg/4 weeks or a dose that exhibits a blood MRA concentration equivalent thereto.

13. The use of claim 31, wherein the MRA dose is a dose of 2 to 8mg/kg/4 weeks or a dose which exhibits a blood MRA concentration equivalent thereto.

14. The use of claim 10, wherein the dose of methotrexate is 1-100 mg/subject/week.

15. The use of claim 14, wherein the dose of methotrexate is 4-50 mg/subject/week.

16. The use of claim 15, wherein the dose of methotrexate is 7.5-25 mg/subject/week.

17. The use according to any one of claims 10 to 16 for the simultaneous administration of MRA and methotrexate.

18. The use according to any one of claims 10 to 16 for the administration of MRA and methotrexate at spaced intervals in time.