Therapeutic agents against chronic rheumatoid arthritis, containing antagonist of il-6 as an effective component

 

(57) Abstract:

The invention relates to medicine, in particular to pharmaceutical compositions for the treatment of chronic rheumatoid arthritis and inhibitor of the growth of synovial cells. The composition contains as an effective component an antibody against the receptor of interleukin-6, and the inhibitor contains an antagonist of interleukin-6, selected from the group consisting of antibodies, interleukin-6, antibodies to the receptor of Il-6, gp130 antibodies, modified interleukin-6, and receptor antisense interleukin-6, a partial peptide of interleukin 6 and a partial peptide of the receptor of interleukin-6. The invention provides the possibility of suppressing the abnormal growth of synovial cells in patients with rheumatoid arthritis. 2 c. and 4 C.p. f-crystals, 1 tab., 6 Il.

The present invention relates to a therapeutic agent against chronic rheumatoid arthritis or inhibitor of the growth of synovial cells containing antagonist of interleukin-6 as an effective component.

Background of the invention

Rheumatoid arthritis is a systemic chronic inflammatory disease, which is an abnormal growth in sousta patients with rheumatoid arthritis there is a noticeable growth of synovial cells, the formation of the multilayer structure due to the abnormal growth of synovial cells (formation of pannus), invasion of the synovial cells in cartilage and bone tissue, vascularization in the direction of the synovial tissue and infiltration of inflammatory cells such as lymphocytes and macrophages. It is reported that the mechanisms of occurrence of rheumatoid arthritis is based on factors such as heredity, bacterial infection and promoting his various cytokines and growth factors, but in General, the mechanism of occurrence of the disease remains unclear.

In recent years, in the synovial membrane and synovial fluid of patients with rheumatoid arthritis detected cytokines and growth factors, including interleukin-1 (IL-1), interleukin-8 (IL-8), tumor necrosis factor (TNF), transforming - growth factor (TGF ), a growth factor, fibroblast (FGF) and platelet-derived growth factor (PDGF) (Nouri et al., Clin. Exp. Immunol. 55; 295 to 302, 1984; Thornton et al., Clin. Exp. Immunol. 86: 79-86, 1991; Saxne, et al. Arthritis Rheum. 31:1041-1045, 1988; Seitz et al., J. Clin. Invest. 87: 463-469, 1991; Lafyatis et al.,J. lmmunol. 143:1142-1148, 1989; Melnyk et al., Arthritis Rheum. 33:493-500, 1990).

It is believed that IL-1, TNF and PDGF are especially powerful factors in the growth of synovial cells (Thornton et al., Clin. Exp. lmnunol. 86:79-86, 1991; Lafyatis et al., J. lmmunol. the production of interleukin-6 (IL-6) synovial cells (lto et al. Arthritis Rheum. 35:1197-1201, 1992).

IL-6 is a cytokine that is known as the factor 2, stimulation of b-cells or interferon 2 . IL-6 is described as a factor of differentiation, contributes to the activation of b-lymphocytes (Hira-no,T., et al. Nature 324, 73-76, 1986), and, as was discovered later, is a multifunctional cytokine that affects the functioning of several cell types (Akira, S. et al., Adv.in Immunology, 54, 1-78, 1993). For the induction activity of IL-6 required two functionally distinct membrane molecules. One of them is the IL-6 receptor (IL-6R) with a molecular weight of approximately 80 KD, which specifically binds to IL-6.

IL-6R exists in membranifolia form, which is expressed on the cell membrane and penetrates the cell membrane, as well as in the form of soluble IL-6R (sIL-6R), which is mainly composed of the extracellular domain. Another protein is gp130 with a molecular mass of approximately 130 KD, which is not a ligand-fixing, but rather functions as a mediating signal transduction. IL-6 and IL-6R form a complex IL-6/IL-6R, which in turn communicates with another membrane protein gpl30 inducyruya biological activity of IL-6 in relation to the cell (Taga et al., J. Exp. Med.196:967, 1 is it excessive interleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6R) (Houssiau et al. Arthritis Rheum. 31: 784-788, 1988; Hira-no et al., Eur. J. lmmunol. 18:1797 - 1801, 1988; Yoshioka et al., Japn. J. Rheumatol., in press), and because similar results were also obtained in animal models of reumatoidea arthritis (Takai et al. Arthritis Rheum. 32: 594-600, 1989; Leisten et al., Clin. Immunol. Immunopathol. 56: 108-115, 1990), it is assumed that IL-6 is somehow plays a role in rheumatoid arthritis.

However, the publication did not pass the examination, the Japan patent N 4-89433 noted that peptides that strongly promotirovat production of IL-6, are effective as therapeutic agents against rheumatoid arthritis.

In addition, Hiqaki with TCS. suggested that synovial cells of patients with rheumatoid arthritis have the reaction of "weak" versus IL-6, and IL-6 thus performs the function of the inhibitor against the growth of synovial cells (Clinical Immunology, 22:880-887, 1990). Thus, there are contradictory messages, considering the ratio of IL-6 to rheumatoid arthritis, and this relationship is still unclear.

Recently Wendling with TCS. reported that antibodies against IL-6 in patients with rheumatoid arthritis temporarily facilitates clinical and biological symptoms, also increasing the level of IL-6 in serum (J. Rheumato cells in rheumatoid arthritis or he has inhibitory action, and thus it is still unknown, or has not IL-6 direct effect on synovial cells of patients with rheumatoid arthritis.

Disclosure of the invention

Anti-inflammatory steroid agents such as corticosteroids, have been used as medicines for rheumatoid arthritis, but their prolonged use causes undesirable side effects such as skin lesions and inhibition of the function of the cortical substance of the adrenal gland, have been searching drugs with fewer side effects.

The aim of the present invention is to provide a new therapeutic agents for the treatment of rheumatoid arthritis, devoid of the above disadvantages. More specifically, the present invention provides a pharmaceutical composition for inhibiting abnormal growth of synovial cells in rheumatoid arthritis, an effective component of which is an antagonist of interleukin - 6, and a pharmaceutical composition for the treatment of rheumatoid arthritis, which has the same effect.

The authors of the present invention conducted diligent research on the role of IL-6 for synovial cells PR is navalnyj cells with one IL-6, and therefore, investigated the factors other than IL-6, which has resulted in the creation of the present invention based on the discovery that although one IL-6 almost does not detect effects on growth of synovial cells, a strong impact on the growth of synovial cells takes place in the presence of both IL-6 and soluble IL-6R, and, moreover, that this effect on the growth of synovial cells is suppressed by adding antagonist, which inhibits the activity of IL-6, such as IL-6 antibody or IL-6R antibody.

In other words, the present invention relates to pharmaceutical compositions for the treatment of chronic rheumatoid arthritis, containing antagonist of IL-6 as an effective component. More specifically the present invention relates to pharmaceutical compositions for the treatment of rheumatoid arthritis, containing antagonist of IL-6 as an effective component and suppressing the abnormal growth of synovial cells. The present invention also relates to an inhibitor of the growth of synovial cells, the effective component of which is an antagonist of IL-6.

Brief description of drawings

Fig. 1 is a graph showing absorption3H-thymidine in synovial cells in pressui action of antibodies to IL-6 or antibodies to IL-6R (antibodies to IL-6R) on the absorption of3H-thymidine in synovial cells in the presence of both IL-6 and sIL-6R.

Fig. 3 is a graph showing the effect of antibody to IL-6 or antibodies to IL-6R on the absorption of3H-thymidine in synovial cells in the presence of both IL-6 and slL-6R.

In Fig. 4 is a graph showing the inhibitory effect of the antibody to IL-6R at the beginning of the disease in the mouse model of collagen-induced arthritis.

In Fig. 5 is a graph showing the antibody levels of collagen in the serum of mice with induced arthritis.

Fig. 6 is a photographic image obtained by histopathological analysis of the joint of the hind paws of mice with arthritis induced by collagen. Photograph (a) refers to a mouse of the group, which was administered antibody IL-6 recipes, a photograph (b) refers to the mouse of the group, which was administered to the control antibody. In the group which was administered antibody IL-6-receptor, invasion of granulation tissue in cartilage and bone (chronic proliferative synovitis) is clearly suppressed.

Detailed description of the invention

Pharmaceutical composition for the treatment of rheumatoid arthritis according to the invention is a medicinal product, which gives a soothing and therapeutic effect on the symptoms of the disease.

The antagonist of IL-6, used according to the invention, can be obtained from any source, if it is a substance that blocks the signaling of IL-6 and inhibits the biological activity of IL-6. Antagonists of IL-6 include antibody IL-6, antibody to IL-6R, the antibody gpl30, modified IL-6, antisense IL-6R and partial peptides of IL-6R or IL-6R.

The antibody used as an antagonist according to the invention, such as antibody-IL-6 antibody IL-6R or gp130 antibody may be of any origin or type (monoclonal, polyclonal), but especially preferred are monoclonal antibodies derived from mammals. Such antibodies bind to IL-6, IL-6R or gp130, inhibiting the binding between IL-6 and IL-6R or IL - 6R and gp130, and thus block the signal transduction of IL - 6 by inhibiting the biological activity of IL-6.

Species of animal for producing monoclonal antibodies of the cells is not particularly limited, while the animal is a mammal, and can use human antibodies or antibodies derived from another mammal, not a person. Monoclonal antibody derived from a mammal, not a person, Preste to get. There is no special restrictions on the type of rodents, but preferred examples are mice, rats and hamsters.

Examples of such antibodies, which are antibodies to IL-6, include MN (Matasuda et al., Eur J. Immunol. 18:951-956, 1988) and SK2 antibody (Sato et al. . Journal for the 21st General Meeting of the Japan Immunology Association, 21:116, 1991). Examples of antibodies to IL-6R include antibody PM-1 (Hirata et al. , J. lmmunol. 143:2900-2906, 1989), AUK12-20, AUK64-7 and AUK146-15 (which has not passed the examination of the application for international patent N WAIS-19759). Example antibodies to gp130 antibody is A (Japanese not passed examination patent publication N 3-219894).

Among them, preferred is an antibody PM-1.

Monoclonal antibodies can be obtained by the following method, which is based on known techniques. Namely, the use of IL-6, IL-6R or gp130 as a sensitizing antigen for immunization according to the conventional method, and the resulting immunocytes then merge with known parent cells by a conventional method, merge cells, and producing monoclonal antibody cell sorting usual way of screening, receiving antibodies.

Specifically monoclonal antibodies can be obtained in the following way. For example, if the CE is a major human IL-6, described Harada with TCS. Nature, 324:73, 1986. The genetic sequence of human IL-6 is inserted into a known expression vector system and used to transform appropriate host cells, after which the desired protein IL-6 purified from host cells or from the culture supernatant and purified protein IL-6 is then used as a sensitizing antigen.

In the case of human IL-6R protein IL-6R can be obtained in the same manner described above to obtain human IL-6, using the gene sequence described as the application for the European patent N EP 325474. There are two types of IL-6R, one of which is expressed on the cell surface and a soluble form (sIL - 6R), which is separated from the cell membrane. sIL-6R consists essentially of the extracellular domain of IL-6R, which is attached to the cell membrane, and is different from the membrane-bound IL-6R that lacks the transmembrane domain or the transmembrane domain and intracellular domain.

In the case of human gp130 this protein can be obtained in the same manner as described above for human IL-6, using the gene sequence described in the application for the European patent N EP 411946.

MLC is playing, considering their compatibility with the parent cells used to merge cells, and, as a rule, you can use mice, rats, hamsters and rabbits.

Immunization of animals sensitizing antigen can be implemented widely known method. For example, a common method is intraperitoneally or subcutaneous injection mammals sensitizing antigen. In particular, sensitizing antigen is diluted preferably equivalent to the number SFR (phosphate buffered saline) or fisiologicas solution, suspended and used in conjunction with a suitable number of conventional adjuvant, such as complete beta-blockers, if this is desirable, and then enter the mammal multiple times every 4-21 days. For immunization sensitizing antigen is also possible to use appropriate media.

After such immunization and confirm the increased serum level of desired antibodies in mammals take immunocytes and used to merge cells, and particularly preferred immunocytes are splenic cells.

The parent cells used for fusion with the above immuno who's cell strains including P3 (P3x6Aq8.653) (J. Immunol. 123:1548, 1978), p3-UI (Current Topics in Microbiology and lmmunology 81: 1-7, 1978), NS-1 (Eur. J. lmmunol. 6:511-519, 1976), MPC-11 (Cell, 8:405-415, 1976), SP2/0 (Nature, 276:269- 270, 1978), OF (J. Immunol. Meth. 35:1-21, 1980), S194 (J. Exp. Med. 148:313-323, 1978), R210 (Nature, 277:131-133,1979). The fusion of immune cells with myeloma cells may be based on a well-known method such as the method of Milstein with TCS. (Milstein et al. Methods Enzymol. 73:3-46, 1981).

More specifically the above-mentioned cell fusion is carried out in conventional nutrient culture in the presence of a promoter merge cells. Used the promoter of the merger may constitute, for example, polyethylene glycol (PEG) or Sendai virus (HVJ), and, if desirable, can also be added such auxiliary means as dimethylsulfoxide, to increase the effectiveness of the merger.

The ratio used immunocytes and myeloma cells is preferably 1-10 times the number of immune cells against myeloma cells. Used culture medium for merging cells may represent, for example, culture medium RPM11640 or culture medium MEM (minimal supportive environment), which are suitable for the growth of strains of myeloma cells, or other normal culture medium, primeton, such as fetal calf serum (FCS).

Merging cells is implemented by mixing the above-mentioned quantities of immunocytes and myeloma cells in the above-described culture medium, with the addition of PEG solution, preheated to 37oC, for example, by adding to the culture medium PEG with an average molecular weight of 1000-6000 mm., usually at a concentration of 30-60% (m/o) and subsequent mixing with the formation of the desired fused cells (hybridomas). Then repeat the procedure of gradual addition of a suitable culture medium and centrifugation to remove the supernatant, and carry out the removal tool to merge cells, etc., that is unfavorable for growing hybrid.

Suitable hybridoma selected by cultivation in normal selective culture medium, such as cultural GAT-medium (containing gipoksantin, aminopterin and thymine) Growing in culture GAT environment continue for a specified time, usually within a few days or several weeks for loss of other non hybridomas cells (nesmith cells). Then carry out a normal limited breeding, and hybridoma producing is received in this way, you can get subculture in normal solution culture, and they can also be placed in liquid nitrogen for long term storage.

To obtain monoclonal antibodies from hybridomas last cultivated in the usual way, then remove supertint culture or use another method, in which hybridoma injected a compatible mammal, grow and get ascitic fluid. The first method is suitable for obtaining high-purity antibody, while the latter method is suitable for mass production of antibodies.

Monoclonal antibodies obtained by these methods, then you can clean to a high degree, using traditional cleaning methods such as salting out, gel filtration, affinity chromatography or similar cleaning methods.

Monoclonal antibodies obtained in this way, you can then check on the degree of sensitivity and purity recognition of antigen conventional immunological methods, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (EA, L1SA), the method of fluorescent antibodies (immunofluorescence assay), and similar methods.

Monoclonal antibodies used in the present izobretali monoclonal antibodies which artificially modified in order to reduce heterogeneity against people. For example, you can use chimeric antibody, which comprises variable regions of the monoclonal antibody of another mammal, not a person, such as a mouse, and a constant region of a human antibody, and this chimeric antibody can be obtained by known methods of obtaining chimeric antibodies, in particular by genetic recombination.

Reconstructed human antibodies can also be used according to the present invention. Get them using a complementarity determining region of murine antibodies or antibodies of another mammal, not a man, to replace the complementarity determining region of a human antibody, and traditional methods of genetic recombination for this purpose are well known. One of such known methods can be used to obtain a reconstructed human antibodies, which are useful according to the invention. A preferred example of such a reconstructed human antibodies is hPM-1 (see not passed the examination of the application for international patent N WO 92-19759).

< / the way that complementarity determining region reconstructed human antibody forms an appropriate binding site of antibodies (Sato et al. Cancer Res.53:851-856, 1993). In addition, the above objective may also be achieved by constructing a gene encoding the antibody fragment, which binds to the antigen for inhibiting the activity of IL-6, such as Fab or Fv or single-chain Fv(scFv), FvH - and L-chains are linked via an appropriate linker, and use it for expression in the appropriate cell hosts (see , for example, Bird et al., TIBTECH, 9:132-137,1991; Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883, 1988).

Modified IL-6 used in the present invention, can be represented as IL-6, described in Brakenhoff et al., J. Biol. Chem. 269: 86-93, 1994, or in Savino et al., EMBO J. 13:1357-1367, 1994.

Used a modified IL-6 can be obtained by introducing a mutation such as substitution, deletion or insertion in the amino acid sequence of IL-6, to retain the binding activity of IL-6R, aluminiowa at the same time, the signal transfer function of IL-6. The source of IL-6 may occur from an animal of any kind, if it has the above properties, but from the point of view of antigenicity preferably ispolzovovanie IL-6 can be predicted using well-known molecular modeling program, such as WHATIF (Vreind et al., J. Mol.Graphics, 8:52-56, 1990), with which it is also possible to evaluate the influence of the mutated amino acid residues to the entire structure. After determining the corresponding mutated amino acid residues of the vector containing the nucleotide sequence encoding the gene for human IL-6, used as a matrix for the introduction of mutations by means of the traditionally used method of PCR (polymerase chain reaction), and receive the gene encoding the modified IL-6. Then it is introduced into a suitable expression vector, if necessary, and Express in cells of E. coli or in mammalian cells and then use either entirely in the culture supernatant, or after isolation and purification by conventional means, and evaluate the activity of binding IL-6R and the signal transfer activity neutralized IL-6.

A partial peptide of IL-6 or a partial peptide of IL-6R used in the present invention, can have any sequence, as they are associated with IL-6R or IL-6, respectively, and do not have activity transfer function of IL - 6. Partial peptides of IL-6 and partial peptides of IL-6R is described in published U.S. patent N 5210075. Antisense oligonucleotide IL-6 as described in the application on patentkontor which is the antagonist of IL-6, the present invention is effective for the treatment of rheumatoid arthritis, if it blocks the signal transduction of IL-6 and inhibits the abnormal growth of synovial cells induced IL-6, which are implicated in the disease. Example 1 demonstrates in vitro, the vast growth effect on synovial cells taken from a patient with rheumatism. In example 2, the injected antibody IL-6-receptor in a murine model of arthritis immunized with collagen type 11, and the relevant data show (1) suppressed the development of arthritis is based on the arthritic index (Fig. 4), (2) suppress the production of antibodies against collagen type 11 in the blood of immunized collagen mice (Fig.5), and (3) suppression of invasion of granulation tissue in cartilage and bone (proliferative synovitis) in the joints of the hind legs of mice used as models of arthritis, which was administered antibody of receptor of IL-6 (Fig.6).

Taking into account the above-mentioned data (1) and (2) the results confirm the inhibitory effect of the antibody of receptor of IL-6, especially in the beginning on the development of arthritis model mice. Results (3) show that the invasion of granulation tissue in cartilage and bone tissue is suppressed, and this is confirmed by the results obtained in example 1 (ing farmacevticheskaja composition of the present invention for the treatment of rheumatoid arthritis has excellent initial effect on rheumatoid arthritis.

The pharmaceutical composition of the present invention for the treatment of rheumatoid arthritis preferably injected parenterally, for example, by intravenous, intramuscular, intraperitoneally or subcutaneous injection, either systemically or topically. In addition, the composition may be in the form of a set of drugs together, at least one type of carrier or diluent for the drug.

The dosage of the pharmaceutical compositions of the present invention for the treatment of rheumatoid arthritis when administered to humans varies depending on the pathological condition and age of the patient, and the method of administration, and appropriate and appropriate dose should be selected taking into account these circumstances. As an example, you can choose a maximum of 4 separate doses in the range from 1 to 1000 mg per patient. However, the pharmaceutical composition of the present invention for the treatment of reumatoidea arthritis these dosages are not limited to.

The pharmaceutical composition of the present invention for the treatment of rheumatoid arthritis may be prepared in conventional ways. For example, the composition for injection is prepared by dissolving a purified antagonist of IL-6 in such Ratin 80, gelatin, human serum albumin (HSA) or a similar substance, and the mixture may be pre-liofilizovane and used for recovery solution. Excipient used for lyophilization can be a sugar alcohol such as mannitol or glucose, or sugar.

EXAMPLES

The present invention will now be explained more in detail with the following examples, reference examples and experimental examples, and it should be borne in mind that these examples in no way limit the invention.

REFERENCE EXAMPLE 1

To obtain soluble human IL-6 receptor

Soluble IL-6R receive (Yasukava et al., J. Biochem. 108:673-676, 1990) PCR (polymerase chain reaction) using plasmids pBSF2R, 236, containing cDNA encoding human IL-6 receptor (IL-6R), obtained by the method Yamasaki with TCS. (Science 241:825-828, 1988).

The above-mentioned plasmid pBSF2R.236 digested with the restriction enzyme SphI and receive a fragment of the cDNA of IL-6R, which is then inserted into mpl8 (Amersham With. ). Synthetic oligoprimers ATATTCTCTAGAGAGATTCT designed to introduce a stop codon in the cDNA of IL-6R, used for the introduction of mutations into the cDNA of IL-6R by using PCR method with opolskie amino acids 345 to obtain cDNA, encoding soluble IL-6R (slL-6R).

To Express the cDNA of sIL-6R in CHO cells (Chinese hamster ovary), the above-mentioned cDNA sIL-6R, cut Hindlll-Sall, insert in plasmid pECEdhfr (Clauser et al., Cell, 45:721-635, 1986), which contains cDNA encoding dihydropyrimidinase (dhfr), inserted into the cleavage site of the restriction enzyme Pvul, and get the expression plasmid CHO cells pECEdhfr344.

Using 10 μg of plasmid pECEdhfr 344 for transfection of cell lines dhfr CHO DXB-II (Urland et al.,Proc.Natl.Acad. Ssi.USA 77,4216-4220, 1980) by the method of precipitation of calcium phosphate (Chen et ai., Mol.Cell.Biol. 7:2745-2751, 1987).

Transfection CHO cells cultured for 3 weeks in binucleated selective culture medium MEM containing 1 mm glutamine, 10% cialisbuynow fetal calf serum (FCS), 100 u/ml penicillin and 100 μg/ml streptomycin. Selected cells CHO sceneroot using the method of serial dilutions and get a single monoclonal cell line CHO. The cell clone CHO amplified in methotrexate (MTX) at a concentration of 20 nm - 200 nm and receive cell line CHO E producing human sIL-6R.

Cell shower CHO E cultivate in the environment Dulbecco modified by the method of Claims (IMDM, about the supernatant using ELISA method (enzyme-linked immunosorbent assay) in accordance with the usual procedure.

REFERENCE EXAMPLE 2

Obtaining antibodies to human IL-6

Antibody to human IL-6 receive Matsuda method with TCS. (Fur. J. Immunol. 18:951-956, 1988).

BALB/c mice subjected to immunization 10 µg of recombinant IL-6 (Harada efc al., Immunol. Lett., 17:41,1988) together with complete adjuvant's adjuvant and it continue to exercise once a week until then, until you find the blood serum of antibodies against IL-6.

The immune cells are extracted at the regional lymph nodes and to merge with the line of myeloma P3U1 cells using polyethylene glycol 1500. Hybridoma selected by the method Oi with TCS. (Selective Methods in Cellular lmmunology, W. H. Freeman and Co. , San Francisco, 351, 1980) using cultural GAT environment and determine the line of hybridomas producing antibodies to human IL-6. Hybridoma producing antibodies to human IL-6, analyze binding of IL-6 as described below.

Namely soft polyvinyl 96-well microplate (product Dynatech Laboratories, Inc. , Alexandria, Virginia) sensibiliser during the night of 100 ál of goat antibodies against mouse Ig (immunoglobulin) (10 μl/ml, a product of Cooper Biochemical, Inc>, Malvern, PA) in 0.1 M carbonate-bicarbonate buffer solution (pH 9,6) at 4oC. Then the tablet for 2 h at room SFR to each well add 100 μl of hybridoma culture supernatant and spend incubation overnight at 4oC.

Then the tablets are washed and in each well labeled add1251 recombinant IL-6 to 2000 pulses/min /0.5 ng/ well, and after washing measure the radioactivity of each well counter gamma-quanta (Beckam Gamma 9000, Beckman Instruments, Fullerton, CA). 216 hybridoma clones 32 are positive in the analysis of the binding of IL-6. Among these clones get, finally, a stable clone MN.BSF2. Antibody to IL-6 MN produced this hybridomas has a subtype IgGlk.

Then use the mouse IL-6-dependent hybridoma cell line MN.BSF2 (Matsuda et al, Eur. J. lmmunol. 18:1951-956, 1988) to determine the neutralizing activity of antibodies MN for growth of hybridomas. The MH60 cells. BSF2 metered quantity 1104/200 µl/well, add them to the sample containing the antibody MN, carry out cultivation for 48 h and add 15,1 CI/mmol3H-thymidine (New England Nuclear, Boston, Minnesota), after which the cultivation continued for 6 hours

Cells are placed on a filter paper of glass fiber and process automatic harvester (Labo Mash Science Co., Tokyo, Japan). As a control using rabbit antibody against IL-6. The result reveals that the antibody MN inhibits sings the activity of IL-6.

REFERENCE EXAMPLE 3

Obtaining antibodies to human IL-6 receptor

Antibody against IL-6 MT create method Hirata with TCS. (J. Immunol., 143:2900-2906, 1989), is fixed on sepharose 4B (a product of Pharmacia Fine Chemicals, Piscatway, NJ), CNBr activated, according to the supplied instructions, and fixed complex used for the purification of IL-6R (Yamasaki et al. Science 241:825-828, 1988).

Line human U266 myeloma cells solubilizer 1 mm hydrochloride p - paraaminometribensole (product of Wako Chemicals) containing 1% of digitonin (product of Wako Chemicals), 10 mm triethanolamine (pH 7.8) and 0.15 M NaCI (digitalonly buffer solution), and mixed with the antibody MT, fixed on the granules sepharose 4B. Then the granules are washed 6 times digitanium buffer solution, and get partially purified IL-6 for immunization.

BALB/c mice subjected to immunization 4 times every 10 days partially purified IL-6R obtained from the 3109cells U266, and then get hybridoma conventional methods. Cultural supernatant hybrid from growth-positive wells screened for binding activity of IL-6 in the following way. After tagging 5107cells U26635S-methionine (2.5 MCI) of their solubilizing above digitanium buffer solution. With Atego washing digitanium buffer solution labeled35S-methionine IL-6R wash 0.25 ml digitalanalog buffer solution (pH 3,4) and neutralize 0,025 ml of 1 M Tris (pH 7,4).

Mixing 0.05 ml of hybridoma culture supernatant with 0.01 ml protein-G - sepharose (product of Pharmacia). After washing sepharose incubated with 0,005 ml 35S-labeled IL-6R obtained previously. The substance formed immunoprecipitate analyze SDS-PAGE (polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate), and check the response of hybridoma culture supernatants with IL-6R. In result define clone hybridomas positive PM-1. Antibody RM-1 to IL-6R, produced by hybridomas RM-1 has a subtype IgGLK.

Inhibitory activity of antibodies produced by hybridomas RM-1, against linking IL-6 to human IL-6 explore using lines of human U266 myeloma cells. Human recombinant IL-6 are obtained from E. coli (Harada et al., Immunol. Lett., 17:41, 1988), and labeled1251 reagent Bolton-hunter (New England Nuclear, Boston, Minnesota) (Taga et al., J. Exp.Med. 166:967, 1987).

At room temperature for one hour cultivate 4105of U266 cells in the presence of 100-fold excess of its IL-6 together with 70% (V/o) cultural supernat roujou plastic tube, download 70 μl of the sample, and after centrifugation measure the radioactivity of the cells.

The results showed that antibodies produced by hybridomas RM-1, inhibit the binding of IL-6 to IL-6R.

REFERENCE EXAMPLE 4

Obtaining antibodies to mouse IL-6 receptor

Monoclonal antibodies against mouse IL-6-receptor produced by the method described in the patent application of Japan N 6-134617.

Following the method of Saito with TCS. (J. lmmunol., 147, 168-173, 1993), cell SNO, producing murine soluble IL-6 receptor, is cultivated in the medium FCS containing 10% FCS, and mouse soluble IL-6 receptor purified from culture supernatant using antibodies RS12 mouse soluble IL-6 receptor (see Saito et al., CIT.above) and immovable column effigiem 10 (Biorad).

Mix received 50 µg of mouse soluble IL-6 receptor with complete adjuvant's adjuvant and injected administered intraperitoneally to Wistar rats (Nihon Charles River Co.). After two weeks of repeated immunization with incomplete adjuvant's adjuvant. 45 day rats are killed, and about 2 x 108their splenic cells used for fusion with 1 x 107mouse myeloma cells P3U1 in the usual way using 50% PEG (Berlinger Manheim), then g is neoplanet, sensitized with rabbit antibody against rat IgG(Cappel Co. is injected into the reaction murine soluble IL-6 receptor, and hybridoma producing antibodies against murine soluble IL-6 receptor, is subjected to the screening method is ELISA using rabbit antibodies against mouse IL-6 receptor and sheep anti-rabbit IgG labeled with alkaline phosphatase. Hybridoma clones, which confirmed the production of antibodies, twice subjected to subscribing and get one hybridoma clone. This clone is called MR16-1.

Neutralizing activity produced this hybridoma antibodies against signal transduction of murine IL-6 exploring through the introduction of3H-thymidine using MH60 cells.BSF2 (Matsuda et al., J. Immunol. 18, 951-956, 1988). The MH60 cells.BSF2 added to 96-well microtiter plate in the amount of 1104cells/200 μl/well, and then add murine IL-6 (10 mg/ml) and antibody MR16-1 or RS12 to 12.3-1000 ng/ml, before cultivation at 37oC in 5% CO2for 44 hours, then add3H-thymidine (1 µci/well), and after 4 h measured absorption. In the find that the antibody MR16-1 inhibits the absorption of3H - thymidine by the cells MN. BSF2.

EXPERIMENT 1

Predelovalne cells

Synovial tissue obtained when surgery on the joint of the patient with rheumatoid arthritis. Synovial tissue is cut with scissors into small pieces and then subjected to enzymatic degradation by incubation for one hour at 37oC with 5 mg/ml collagenase type 1 (Sigma Chemical Co. ) and 0.15 mg/ml bovine pancreatic Gnkazy (product of Sigma Chemical Co. in IMDM (environment Dulbecco, modified by the method of Claims), pass through a sieve and get a separate cell. The resulting cells are then cultivated overnight in a flask for culturing using IMDM containing 5% FCS, after which non-stick cells are removed and receive synovial cells. Synovial cells passedout 3-6 times and used for the next experiment.

(2) the Production of IL-6 by synovial cells

Synovial cells obtained as described above, suspended in culture medium IMDM containing 5% FCS (Hyclone Laboratories Inc.), 10 u/ml penicillin G and 100 μg/ml streptomycin, up to the number 3103cells/well, and then cultured in 96-well titration the microplate (Falcon Co. ) where add human interleukin-1B (IL-1B), human - tumor necrosis factor (TNF), celati 0.01 or 0.1, 0.1 or 1.1 or 10 1 or 10 ng/ml, respectively, and after culturing at 37oC for 72 h, harvested culture supernatant.

Add 100 µl of antibody MN (1 µg/ml) against human IL-6 in 96-loony tablet for ELISA (Immunoplate, Nunc Co.) and incubated at 4oC for 24 h and Then each well was washed with SFR containing 0.05% tween-20, and blocked with 4oC SFR containing 1% BSA. Then obtained earlier cultural supernatant bred SFR containing 1% BSA, added to the wells and then incubated at room temperature for 2 hours After washing SFR containing 0.05% tween-20, add 2.5 µg/ml of rabbit polyclonal antibodies against human IL-6, purified on a column with 100 μl of protein a (product of Pharmacia).

After incubation at room temperature for 2 hours, the rabbit polyclonal antibody against IL-6 binding to IL-6 in the culture supernatant is injected into reaction with alkaline phosphatase, associated with the antibody against rabbit IgG (Tago Co. ). And finally add 1 mg/ml substrate of alkaline phosphatase Sigma 104 (a product of Sigma Co.) in accordance with the attached instructions) and measure the absorption at 405-600 nm microtransmitters MPR A4 (product of Tosoh Co.).

The results show that IL-1 significantly promotiom production of IL-6 by synovial cells

EXAMPLE 1

(1) Synovial cells obtained in experiment 1 (3103/well), suspended in culture medium IMDM containing 5% FCS (Hyclone Laboratories, Inc. ), 10 u/ml penicillin G and 100 μg/ml streptomycin, and then added to 96-well titration microplate (N 3072, Falcon product. ) and cultured for 5 days in the presence of different concentrations of the same IL-6 or sIL-6R, or in the presence of both IL-6 and sIL-6R. At 72 h after the start of cultivation in each well add3H-thymidine (Amersham International pIc)to a concentration of 1 µci/well, and after cultivation measure the radioactivity of the cells scintillation counter. The results are shown in Fig.1.

As a result, the absorption of3H-thymidine synovial cells with low one IL-6 or sIL-6R and growth of synovial cells is not observed. On the contrary, in the presence of at least 10 ng/ml IL-6 and 100 ng/ml sIL-6R significant compared with the control group absorption3H-thymidine. Thus, while essentially no detectable effect on ROS the article synovial cells.

(2) Synovial cells (3103/well) were cultured in the presence of an amount of IL-sufficient for production of IL-6 (0.1 ng/ml), 100 ng/ml of sIL-6R and 25 μg/ml of antibodies to IL-6 or 25 μg/ml of antibodies to IL-6R. At 72 h after the start of cultivation in each well add3H-thymidine to a concentration of 1 µci/well, and after cultivation measure the radioactivity of the cells scintillation counter. The results are shown in Fig. 2. Adding antibodies to IL-6 or antibodies to IL-6R completely inhibits the growth of synovial cells, increase slL-6R.

(3) Synovial cells (3103/well) were cultured in the presence of 100 ng/ml IL-6 (Genzyme Co.), 100 ng/ml sIL-6 and 25 μg/ml of antibodies to IL-6 or antibodies to IL-6R, which are the above-mentioned reference examples. At 72 h after the start of cultivation in each well add3H-thymidine to a concentration of 1 µci/well, and after cultivation measure the radioactivity of the cells scintillation counter. The results are shown in Fig. 3. Adding antibodies to IL-6 or antibodies to IL-6R completely inhibits the growth of synovial cells, increase of sIL-6R.

EXAMPLE 2

The inhibitory effect of antibodies to IL-6 receptor on the development of the arthritis research (4 mg/ml), prepared in 0.1 N aqueous solution of acetic acid, and complete adjuvant H37Ra (DIFCO) are mixed in equivalent amounts and receive adjuvant. Injected subcutaneously with 100 μl of adjuvant at the base of the tail 8-9-week-old female mice DBA/IJ (Charles River Japan). After 20 days injected an additional 100 ml under the skin on the back to cause arthritis.

When the first collagen sensitization antibody MR16-1 to mouse IL-6 receptor is administered intravenously at 2 mg per mouse, and then each mouse is injected subcutaneously another 0.5 mg (n=5) per week for 7 weeks. As a control using the antibody against DNP KN-5 (Chugai Seiyaku) of the same isotype (n=5)

The severity of arthritis assessed on a 4-point scale on each limb, just 16 points for individuals. The evaluation standards are the following:

0,5: Erythema was observed on one side of the joint.

1: Erythema was observed on both sides of the joint or redness of the dorsal surfaces, but without swelling.

2: moderate swelling.

3: Severe swelling of the dorsal surfaces of the feet, but does not cover the fingers.

4: Severe swelling of the dorsal surfaces of the feet and toes.

The results are shown in Fig. 4. The development of sravnenie group, which was injected control antibody.

On the other hand, the results of measurement of the titer of antibodies against collagen type 11 in the blood of mice show a significant recovery from the early stages of arthritis in the group which was administered antibody to IL-6 receptor, compared with the group that was administered to the control antibody (Fig. 5).

Mice killed on the 35th day after immunization with collagen, and the rear legs fixed with 20% formalin. Then they are subjected to demineralization in solution edtc (pH 7.6) and dehydrated alcohol. Then they are covered with wax and make the cuts with a thickness of 2 μm. Sections stained with hematoxylin (NOT) and eosin and consider when increasing h (Fig. 6). Now you see that the invasion of granulation tissue in cartilage and bone, i.e., proliferative synovitis, is suppressed in the group which was administered antibody to IL-6 receptor, compared with the group that was administered to the control antibody.

IL-6 is a cytokine that induces differentiation of b-cells in the cells that produce antibodies. IL-6 also promotiom proliferation of synovial cells in the presence of IL-6-receptor. As in the mouse model of collagen arthritis antibody against IL-6-receptor substantially podgruppoi, which was injected control antibody, it is assumed that the inhibition of the production of antibodies, antibody against IL-6 receptor is one of the factors responsible for the inhibitory effect on arthritis. In addition, while suppressing the production of antibodies is not observed on the 49th day after collagen sensitization, the fact that an overwhelming effect on the development of arthritis is manifested even during this time, and that the painting is NOT of tissue surrounding the tarsal bone, shows suppression of invasion of granulation tissue in cartilage and bone in the group which was administered antibody against IL-6 receptor, compared with the control group, suggesting that the inhibitory effect on the growth of synovial tissue contributes to the effect of suppression of arthritis.

Industrial applicability

Synovial cells of patients with rheumatoid arthritis grow in the presence of both IL-6 and sIL-6R. The fact that the synovial fluid of patients with rheumatoid arthritis contains the amount of IL-6 and sIL-6R, sufficient to induce the growth of synovial cells, suggests that signal transduction through IL-6 is involved in the abnormal growth of synovial cells in rheumatoid altercation effective component is the antagonist of IL-6, according to the present invention inhibits the growth of synovial cells in patients with rheumatoid arthritis in the presence of IL-6 and sIL-6R and thus has a therapeutic effect against rheumatoid arthritis. Therefore, the antagonist of IL-6 of the present invention is suitable as a therapeutic agent against rheumatoid arthritis, in which there is abnormal growth of synovial cells. Return to index

1. Pharmaceutical composition for the treatment of chronic rheumatoid arthritis, containing as an effective component an antibody against the receptor of interleukin-6.

2. Pharmaceutical composition for the treatment of chronic rheumatoid arthritis under item 1, characterized in that the antibody against the receptor of interleukin-6 has the ability to suppress abnormal growth of synovial cells, occurring in the case of chronic rheumatoid arthritis.

3. Pharmaceutical composition for the treatment of chronic rheumatoid arthritis under item 1 or 2, characterized in that said interleukin-6 is an interleukin-6 persons.

4. Pharmaceutical composition for the treatment of chronic rheumatoid arthritis PP.1,2 or 3, characterized in that said receptor intergame antagonist of interleukin-6, selected from the group consisting of antibodies, interleukin-6, antibodies to the receptor of interleukin-6, antibodies gp 130, the modified interleukin-6, and receptor antisense interleukin-6, partial peptides of Il-6 and a partial peptide of the receptor of interleukin-6, as an effective component.

6. Inhibitor of growth of synovial cells under item 5, wherein the antagonist of interleukin-6 is an antibody interleukin-6 or antibody of receptor of interleukin-6.

 

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