JPH06510750A - Molecules targeting interleukin receptors to treat inflammatory arthritis - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Molecular invention targeting interleukin receptors to treat inflammatory arthritis background The field of this invention is the treatment of inflammatory arthritis.

Inflammatory arthritis is a type of arthritis characterized by inflammation of the joints mediated by lymphokines. It is a joint disease. Inflammatory arthritis includes osteoarthritis, psoriasis arthritis, and lupus-related arthritis. The origin is often autoimmune, as in the case of . The most common type of inflammatory arthritis The type is osteoarthritis, which affects about 1% of the population. Osteoarthritis is a joint It is characterized by persistent inflammation. Inflammation can ultimately lead to cartilage destruction and bone erosion. .

5Tukel et al. (Immunology 64:683. 1988) Monoclonal antibodies against tarleukin-2 receptors were passively transferred to rats. Adjuvant arthritis (i.e., tiles taken from rats with adjuvant arthritis) Adjuvant joints induced by transferring visceral cells into naive rats However, the activity of suppressing the development of adjuvant arthritis in rats was It is reported that there is no.

Ca5e et al. (Pric, Natl, Acad, ScL, USA 86:287, (1989) showed that cytotoxic interleukin-2- When Pseudomonas exotoxin fusion protein was administered to rats, adjuvant-induced joint It has been reported to reduce flames.

Summary of the invention In general, the invention features a method of treating a patient with inflammatory arthritis; has the ability to specifically bind to protein-like cell receptors expressed on the patient's lymphocytes. and involves administering to a patient a molecule that contributes to the patient's inflammatory arthritis.

This molecule has the ability to reduce lymphocyte survival. “Cellular receptors are cell receptors. encoded by the DNA of the molecule, binds a ligand, and when expressed, at least one of the molecules also refers to molecules that are partially exposed on the cell surface. What is “specific binding”? , the molecule does not substantially bind to other cell receptors or cell surface proteins. means. “Reducing survival ° means killing or inhibiting proliferation. It means to do something. A “ligand” is a molecule that has the ability to bind to a certain protein. means.

In various preferred embodiments, the inflammatory arthritis is osteoarthritis, and the inflammatory arthritis is This is systemic lupus erythematosus-related arthritis, and inflammatory arthritis is psoriatic arthritis. There is also. The proteinaceous cell receptor is the high affinity interleukin-2 receptor. , a molecule that kills cell receptor-bearing lymphocytes, which are covalently bonded to each other. It is a hybrid molecule that contains a first and a second part combined, the first part being The second part contains a molecule capable of reducing the survival rate, and the second part binds specifically to the cell receptor. Contains molecules that can be combined. This molecule is administered with cyclosporin A and Rosporin A is administered at sufficiently non-toxic doses. The molecule is designed to improve the patient's arthritis symptoms. following which cyclosporine A is administered to the patient until the patient improves, Cyclosporine A is administered at sufficiently non-toxic doses.

In a further preferred embodiment, the second portion of the molecule is an antibody specific for a cellular receptor. Including the whole or combined parts. The second part of the molecule is the ligand for the cell receptor including the whole or combined parts. This ligand is an interleukin. this The first part of the molecule contains the cytotoxin. Cytotoxins are fragments of peptide toxins that are enzymatically active, but not binding to generalized eukaryotic receptors.

and fragments of the cytotoxin are sufficient to form pores in the cell membrane with fragment A of diphtheria toxin. Contains fragment B of diphtheria toxin.

In an even more preferred embodiment, the molecule is DAB4861L-2. The molecule is D AB IL-4, and the molecule may also be DAB3891L-6. Inter entrance Ikin is interleukin-4, and interleukin is interleukin-4. It is Kin-6.

In other preferred embodiments, the molecule is a whole or a specific antibody directed against a cellular receptor. This antibody is an antibody that activates complement.

In a related aspect, the invention reduces bone erosion in patients with inflammatory arthritis. It features a method of This method uses interleukin cells expressed on patient's lymphocytes. It has the ability to specifically bind to Ikin receptors and may contribute to inflammatory arthritis in patients. administration of a molecule to a patient. This molecule reduces lymphocyte survival. have the ability to

In a preferred embodiment, the inflammatory arthritis is osteoarthritis. The molecule is DA 848 61L-2, and the molecule is DAB3891L-2.

In a related aspect, the invention provides a method for reducing pain in patients with inflammatory arthritis. It is characterized by This method targets protein-like cell receptors expressed on the patient's lymphocytes. molecules that can specifically bind and contribute to inflammatory arthritis in patients. This includes administration to individuals. This molecule has the ability to reduce lymphocyte survival.

In a related aspect, the invention provides a system for treating patients with inflammatory arthritis. Features a method using Rosporin A. This method uses It can specifically bind to protein-like cell receptors in patients with inflammatory arthritis. Involves administering the contributing molecule to the patient. This molecule reduces lymphocyte survival. can be reduced. Cyclosporine A is administered at sufficiently non-toxic doses.

Other features and advantages of the invention appear in the following description of preferred embodiments and in the claims. It will be clear from the range.

Detailed description of the invention First, I will give a brief explanation of the diagram.

Figure 1 shows the effect of treatment with DA84861L-2 on the induction of adjuvant arthritis. This is a graphical representation of the results. Arthritis index (measured as described below) Expressed as a function of days post-immunization. Animals were Tris-buffered from days −1 to 9. With saline solution (solid line) or 0.5mg/kg DAB4861 L-2 (dashed line) treated.

Figure 2 shows that the buffer solution (Figure A) or DAB4861L-2 (Figure A) was used on days -1 to 9. A pair of laminae of the 22nd day hindlimb of an adjuvant-induced arthritic rat treated with side B). It is a geograph.

Figure 3 shows samples taken from rats with arthritis induced with adjuvant on day 22 after immunization. This is a set of photographs of ankle joints. Rats received Tris-buffered saline ( Figures A and B) or treated with DAB4861L-2 (Figures C and D).

Figure 4 shows the results of adjuvant arthritis induced ConA or M, butyri. Graph of the proliferative response of popliteal lymph node lymphocytes isolated from rats stimulated with cum. This is a blank display. The irritation index was determined by buffer alone (open bars) or DAB48. IL -2 (hatched bar) treated cells.

FIG. Figure 2 is a graphical representation of the effect of DAB4861L-2 on arthritis induction. arthritic fingers The number (measured as below) is expressed as a function of the number of days after adjuvant immunization. There is. Experiment-naive animals received Tris-buffered saline (solid line, A) from day -1 to day 9. or treated with 0.5 mg/kg DAB4861L-2 (dashed line, D). pre-immunization Animals were similarly treated with Tris-buffered saline (dashed line, B) or with 0.000 ml on days 11 to 9.　 Received treatment with 5 mg/kg DAB IL-2 (dotted line, C).

Figure 6 shows the effects of DAB and 881L-2 on adjuvant arthritis that has already developed. This is a graphical representation of the results. Arthritis index (measured as described below) Expressed as a function of the number of days after. Animals were placed in Tris-buffered saline ( solid line) or treated with 0.5 m g/k gDAB4sa I L 2 Ta.

Figure 7 shows DAB for radiographic features of adjuvant arthritis, 861L- 2 is a graphical representation of the effect of delayed treatment. Hindlimb radiograph number 1 Buffer solution (white outline) or DA848B IL 2 for 1 to 21 days. Photographs were taken on day 22 from rats treated with (hatched bar). The radiograph is Divided into two categories: 〇-no evidence of disease, 1-soft tissue swelling, 2-mild Soft tissue swelling with neoplasia, 3- Severe soft tissue swelling with extensive neoplasia.

Effect of DA84861L-2 on adjuvant arthritis in rats Chronic adjuvant Arthritis is an autoimmune disease, and genetically susceptible rats are treated with bacterial-derived arthritis. can be induced experimentally by immunization with adjuvants.

The disease is characterized by subacute polyarthritis involving the distal extremities, with one case of osteoarthritis in humans. Clinically and pathologically similar. Similarities include periostitis, cunus formation, soft Includes bone fracture and bone erosion (Holoshitz et al., Lnc. etc.

2:305. 1986). Activated T lymphocytes are involved in the pathogenesis of diseases. , adjuvant arthritis, but as in certain other autoimmune disease animal models (e.g. , allergic brain myelitis, streptococcal cell wall-induced arthritis, collagen type II induced arthritis, and non-obese diabetes), freshly isolated lymphocytes from affected animals, or passively by injecting selected T cell clones into recipient animals. This is evidenced by the observation that it can be imported (Prud'homme et al. al., Lab, Invest, 59:158. 1988).

Adjuvant arthritis was performed using female Lewis rats (100-125g; HarI an Sprague-Dawley Inc., Indianapolis , IN) and sterilized and dried Mycobacterium butyricum ( Difco, Detroit, MI) 10 mg/ml heavy mineral oil suspension (Sig Injecting ma Chemical Co, St, Louts, MO) and was induced by. 100 μl of the suspension was added to a light methoxyflurane anesthesia solution. The animals were given sarcastic injections on day 0 in 4 to 6 locations on the upper back. each rat every day The clinical symptoms of arthritis were evaluated. The severity of arthritis was rated on a scale of 0 to 4 for the 6 feet. quantified. This indicates the severity of swelling and erythema in the distal joints (〇 - disease disease). No symptoms, 1 - disease is manifest in a few fixed distal joints, 2 - disease is manifest in all distal joints of the foot , 3- Disease is manifest in all feet, and 4- Severe disease is manifest in all feet (Trent bam et al., J. Exp. Med., 146:857). joint The flame index is defined as the sum of all limb scores for each animal, with a maximum possible score of 16. It was done. Animals were evaluated by several different observers over the duration of each experiment .

Rats immunized with mycobacterial adjuvant were typically found at about 10 days post-immunization. signs of eastern end disease appear. The severity of rhinoceros swelling and erythema increases rapidly from day 20 to day 25. The arthritis index of each individual also becomes high, ranging from 10 to 14. Clinical symptoms then gradually diminish; By the 40th day, the peak level will be approximately 5096. Rats are indiscriminate experimental subjects. (10 animals/group), 0.02 M1-squirrel (pH 8,2)- DA 84861 L2 dissolved in 0°15M NaCl or buffer solution treated only with Treatment is during the induction period of the disease (-1 to 9 days) or during the arthritic symptoms. was carried out after the expression (11 to 21 days).

After immunization with 0.5 mg/kg DAB4861L-2 from the day before adjuvant administration. Daily subcutaneous injections up to day 9 significantly reduced disease-related inflammation. beginning of disease The onset of swelling was delayed by approximately 2 days, and the degree of distal joint swelling and erythema was similar in buffer-treated animals. It wasn't as bad, 2 to 4 times as bad. In the study shown in Figure 1, DAB Animals treated with 486IL-2 showed the highest mean arthritis index of 3.0 on day 22. However, this value subsequently decreased, with animals reaching an average score of approximately 1.6 on day 40. Reached. In contrast, buffer-treated control animals had a highest mean arthritis index of 8.5. , and this value decreased to only 3.8 on average. after that Treatment with DA848BIL-2 for days 0 to 9, depending on the study, It was proven to give results comparable to a 9 day treatment.

Radiographic analysis of the hind limbs of the animals after buffer treatment ended on day 22 post-immunization revealed that the paws and There was extensive soft tissue swelling throughout the ankle region (Figure 2, Panel A). This view In line with the findings, narrowing of the joint spacing and irregularities with greater radioactivity density in the midfoot Moderate neoformation was seen as indicated by regular areas. In contrast, induction During the period (-1st to 9th), DAB48. taken from animals treated with IL-2 A radiograph of the hindlimb showed only mild soft tissue swelling in the ankle area, with no bone loss. No destruction or de novo formations were observed (Figure 2, panel B).

For histopathological analysis, the hind limbs were treated with Te1ly's fixative (glacial acetic acid:formaldehyde). It was stored at a hydride:70% ethanol ratio of 1:2:10). The limb is then decalcified processed, embedded in paraffin, sectioned along the midline through the metatarsal region, and hemographed. It was stained with Tokinrin and Eosin. Sections were evaluated for inflammatory mononuclear cell infiltration, interarticular It was performed based on septal narrowing and periosteal neo-1 formation.

Microscopic examination of sections from buffer-treated control animals terminated 22 days after immunization. Signs of a wide range of illnesses were revealed. Among the findings: thickened synovial joint lining , an extensive inflammatory infiltrate forms granulomatous lesions, and with destruction and perpendicular to the existing bamboo, and abutment. It involved severe proliferative osteomyelitis characterized by penetrating neoplasia (Figure 3, panel A1 Figure 3, panel B). During the induction period (days −1 to 9), the animals were treated with DA8486. Examination of the joints of the animals showed that DA84861L-2 treatment significantly reduced adjuvant arthritis. This suggests that it completely inhibits the wide variety of dishes that are characteristic of this disease, and the radiological I received the test results sometime ago. Animals treated with DAB4861 L-2 during the induction period The only evidence of arthritis in the patient was synovial fluid partial pressure and mild thickening of the intima (Fig. 3, Panel C; Figure 3, Panel D).

Popliteal lymph node cells isolated from animals 10 days after immunization with mycobacterial adjuvant The proliferative response of the cells was evaluated.

Animals were euthanized and popliteal lymph node cells were harvested under sterile conditions. Lymph nodes are torn A single cell suspension was obtained. Cells were grown in 96-well U-bottom microtiter plates. 596 fetal bovine serum and 2X10-5M 2-mercaptoethanol. In RPMI 1640, IX 10G/ml at 41 degrees T culture.

Next, 100 μm of ConA (g1g/ml), M, butyricum ( 80/7g/ml) or culture medium alone into four cells containing 1QQ711 cell suspension. were added to a set of wells.

The cells were injected into the ink for 72 hours at 0.62571 Ci/liter [methyl-3 Pulse overnight with H cotimine (specific radioactivity, 20 Ci/n1M), were collected and radioactivity uptake was measured. Cells isolated from buffer-treated animals were M. butyricum responds actively to stimulation (Figure 4). In contrast to this 2, DA 848 BI L-, 2, cells from animals treated with showed a significantly suppressed proliferative response to target mycobacterial antigens. This discovery It is not the result of a general suppression of the proliferative response. Is it a DAB4861L-2 treated animal? ConA stimulation of these cells was comparable to the response of cells from buffer-treated animals. (Figure 4). Not immunized with complete adjuvant, buffer or DA The proliferative response of cells from animals treated with , 84861 L, -2 was also evaluated. , comparable results were obtained.

To assess the potential effect of anti-diphtheria toxin antibodies on DAB4861L-2 activity. In order to Immunized with phtheria toxoid. Rats (75-100g) received 100μg of diphenyl Terrier Toxoid (MaSsachusetts 5tate Laborat ories, Boston, MA) for 5 consecutive days. 1 After day 0, superficial fluid samples were collected from each animal and anti-DAB4861L-2 antibody levels were determined. Analyzed by ELISA assay. Briefly, a 96-well flexible plate was Coat overnight with lug of DAB4861L-2 per well, 0.005% in PBS. Prepared by dissolving in Tween 20 (S i gma) 1-7 0.1% gelatin and washed with 0.05% Tw6en-PBS. each serum Eight serial 5-fold dilutions of the sample were added to the coated plate (10 per well). 0μm). Plates were incubated for ] hours at room temperature, washed, and washed with alkaline phosphatide. Fatase-conjugated and affinity-purified goat anti-rat immunoglobules Appropriately diluted phosphorus (Cappel, West Chester, PA) and alkaline phosphatase substrate (Kirkeguard and Perry.

Gaitersburg, MD). Antibody titers are automatically plated and read When analyzed with a filter, the absorbance at 405 μm is greater than 0.1 or It was determined that the maximum dilution would be equal to that.

Biological analysis of DAB4861 L-2 on human IL,=2 receptor-expressing cell lines Antibodies capable of neutralizing activity were determined in an in vitro assay. Each serum test The sample was diluted 2 times with RPM+1640 medium containing 15% fetal bovine serum (-1 in the same volume). DAB48 bottom microda microtiter well (13). Cells were incubated overnight. and 2°5zCi/ml [14C] oisine (specific radioactivity 300mC1/m h4/> and leucine-free MEM (GIBCO, Grand l5land, NY) for 2 hours, pulse-labeled, harvested, and measured radioactivity uptake.

The assay endpoint is whether the level of uptake is greater than 20% of the value in control cells. , or the maximum dilution of serum that protects the cells to be equal to . The results of this assay are reported as units of neutralizing activity. diphtheria antitoxin 1 The international neutralization unit is defined as the amount of antibody that neutralizes 2.5 μg of toxin. we Extending this definition, the equivalent of (2,7I1g) DAB4861 L-2 on a molar basis is the amount of antibody that neutralizes.

N1. At the time of immunization with butryicum, these animals were exposed to diphtheria toxin. and had moderate levels of antibodies against DA84861L-2 (]:125 ELISA value and neutralizing antibody level of at least 0.01 unit/ml).

However, the presence of antibodies against diphtheria toxin suggests that DAB did not change the effect of 4861L-2 treatment (Figure 5). Experiment-naive and pre-immune animals The clinical course for both was identical. DAB4861L-2 treated animals were immunized with The highest mean arthritis index was 4 for both pre- and naive animals, whereas control animals The highest mean arthritis index was 13 for both pre-immune and unexperimented subjects.

DA84861L affects bone destruction underlying adjuvant-induced arthritis To further examine the ability of -2 to DAB during disease establishment, 861 The effect of L-2 treatment was investigated. DAB4861L-2 treatment during the disease induction period In contrast to the observed effects, administration of the same dose of DAB 4g61L2 Even if arthritis symptoms begin after the onset of the 11th day and continue until the 21st day, the onset of the disease does not occur. No noticeable changes occurred in bed symptoms (Figure 6). Rats receiving delayed treatment? Histological sections of the joint taken from the patient showed an extensive mononuclear cell inflammatory infiltrate, indicating the onset of inflammation. It was consistent with the floor symptoms. However, radiographic analysis does not allow clinical arthritic index Bamboo corrosion and neotubule formation were significantly lower than in buffer-treated control animals (60%), although they were similar. a lower percentage (30%) in DA 8486I L-2 treated animals than in DA 8486I L-2 treated animals. This proved that what was observed was observed (Figure 7).

DAB for osteoarthritis, 861L-2DA B486I L-2 is deformed It was investigated in human clinical trials for the treatment of arthritis. Thirteen patients with severe osteoarthritis Other drugs used to treat the disease (usually methotrexate or cyclosporin) phosphorus) was washed out for 3 weeks, and 0.075 mg of DA 84861 L2 /kg/B or 0.1 mg/kg/day intravenously over 1 hour. The fire continued for 7F-1. After 1 course of treatment, 4 patients had a significant response. 5006 or more improvement in joint swelling and pain), 1 patient had a significant response (joint swelling and pain). 2500 or more improvement in tension and pain, and at least 2-] or 4 other measurements Possible parameters (settling speed, grasping force, 50 second walking time, and observer rating) 2506 or more improvement in 6 patients showed biological effect (for 3 parameters). showed an improvement of 2,596 or more within 3 weeks), but one patient did not respond. .

Three of the 13 patients received a second course of treatment. Two of these people are equivalent One person gave a physical response.

Generally, there are three means by which molecules useful in this invention may act: (1) Molecules has a cytotoxic domain, so the molecule kills cells; (2) the molecule ( antibodies) can cause cell lysis by inducing complement fixation; and (3) the molecule can block the binding or uptake of the receptor's ligand. Ru.

In all three cases, the molecule is directed to target the receptor-bearing cells. There must be. This is the receptor's ligand (or part of it or its inducer). conductor) or by including an anti-receptor antibody as part of the molecule. Ru.

Helpful in treating inflammatory arthritis and appears to target interleukin-2 receptors The molecules provided provide examples of each of these three solutions. IL-2 of IL-2 A fusion molecule containing a binding moiety to a receptor and certain cytotoxins may be used to treat inflammatory arthritis. used to kill associated activated lymphocytes and monocytes/macrophages. I can do it. Similarly, complement for the second type molecule, in this case the IL-2 receptor. The combination is! L-2 receptor retaining cells can be removed. In this example, the third A type molecule is one that inhibits IL-2 from binding to its receptor.

This molecule prevents diseased cells from receiving proliferation signals from [L-2, thereby causing inflammation. It can suppress the symptomatic reaction.

Molecules useful in treating patients with inflammatory arthritis can take several forms. Ru. If IL-2 itself is the targeting reagent, the molecule child, preferably a hybrid molecule fused to a polypeptide toxin .

It binds to IL-2H, lacks IL-2 activity, and inhibits authentic IL-2 binding and/or IL-2 derivatives that inhibit or block uptake are useful in the methods of the invention. So This is because these derivatives inhibit IL-2-induced proliferation of summer L-2R-retaining cells. It is.

If the anti-IL-2R antibody is a targeting reagent, the entire antibody or a portion thereof can be Forming cytotoxic hybrid molecules by fusing to totoxin Can be done. The effectiveness of such antibody/toxin hybrids is due to the As in the case of thin hybrids, the hybrid molecule is taken up by the cells to which it binds. It depends on what is included. Blocking IL-2 binding and/or uptake Anti-IL-2R antibodies are also useful in the methods of the invention. Cytolytic anti-IL- 2H antibodies can cause complement-mediated lysis of IL-2R-bearing cells. Therefore, it is useful for the present invention.

Some molecules are a fusion of all or part of two or more molecules. It can be made by The hybrid molecule is coded by a recombinant DNA molecule. It can also be a hybrid protein that is coded. In this case, the two domains are linked by domain bonds (directly or through intermediate domains). Instead In other words, the two domains are made separately and covalently bonded by separate chemical linking means. It is also possible to connect more. In some cases, the cytotoxic domain of the hybrid molecule The molecule itself may be derived from two separate molecules.

Interleukin-2I L-2 or IL-2 receptor as a targeting reagent Its derivatives that bind to cells are used as targeting reagents for cytotoxins. can do. The DNA and amino acid sequences of IL-2 are known (Tad atsugu et al, Nature302:305. 1983), Its structure has been predicted by X-ray crystallography (Brandhuber et al. 5science 238:1707゜1987). Genetic manipulation of IL-2 Analysis of the operational variants revealed which residues interact with IL-2R (Collins et al. l,, Proc, Natl, Acad.

Sci, USA 85 Near 709. (1988) and biological activity (Cohene tall, 5science 234:349. 1989; Co11in s, supra).

IL-2 variants useful in the present invention include the region between residues 74 and 79 (numbering is Wi lliams et al, Nucl, Ac1ds Res, 16:104 Deletions lacking one or more amino acid residues (according to 5.1.988) Mutant (Genbauffe et al., USSN 388:557゜ (incorporated by reference into this application). These mutants contain toxins ( toxin) to effectively target IL-2R-bearing cells (Genbauffe et al. et al, supra). Generally useful for targeting cytotoxins II, -2 mutant efficiently binds to L-2H and is endocytosed. There must be. The IL-2 receptor binding ability of various derivatives is determined by the following IL-2 receptor binding ability: Can be tested in a 2H binding assay.

In designing molecules that target IL-2 receptor-bearing cells, IL-2 It should be recognized that, like other receptors, there are several types of receptors. Although it is desirable to target cells that carry one type and not the other, unknown. The human interleukin-2 receptor has high affinity, intermediate affinity, and There is a type with low affinity. High-affinity receptors have an apparent K of ~10”M and 2 It consists of two subunits, p55 and p75 (also called p70).

When expressed on the cell surface, both p75 and p55 subunits bind IL-2. This corresponds to M). The p75 subunit is used in resting T cells and natural killer cells. Killer (L) activated by monocytes/macrophages and lymphocytes AK) is expressed on the surface of cell precursors, but the high affinity receptor is present on activated T cells. and expressed on B cells.

In the method of the present invention, it is desirable to target only high-affinity receptor-bearing cells. It might be true. Under these circumstances, useful molecules have intermediate or low affinity. high-affinity receptors at concentrations that have little effect on cells that harbor sexual receptors. Remove or neutralize retained cells. If the molecule is IL-2, like IL-2 itself, 2 has affinity for all three types of receptors, selectivity is By administering the molecule at a concentration that does not significantly bind to the cells that hold it. Therefore, it can be achieved. The hybrid molecule has less receptor capacity than IL-2. It may change the affinity. Such hybrid molecules are more or less , may be selective for high-affinity IL-2 receptor-bearing cells. for example , high-affinity receptor-bearing cells are more sensitive to DAB486 than intermediate-affinity receptor-bearing cells. 1L-2, that is, a fusion protein consisting of part of diphtheria toxin and part of IL-2. 500-1000 times more sensitive to white matter (Waters et al. Eur.

J, Immunol, 20 Near 85. 1990).

Cytotoxins can bind to IL-2 derivatives in several ways.

Desirably, the IL-2/toxin hybrid is produced by expression of an artificial fusion gene. It is a hybrid protein created by Instead, cytotoxin and IL-2 derivatives can be made separately and later linked by a non-peptide covalent bond. I can do it. The connection method is described below.

Interleukin-4 and Interleukin-6 Interleukin as Targeting Reagents IL4 (IL4) is one of the cytokines that acts on various cell types. the The receptor is expressed on several cell types including CD4 T cells and monocytes. IL -4 can act as a growth factor for T cells and L7, leading to IL-2 induction. It is thought to have an effect on lymphocyte proliferation. High levels of interleukin-6 ( IL-6) has been detected in the synovial fluid of patients with active osteoarthritis (Hirano et al, Eur, J, Immunol, 18:1797゜19 88).

Cytotope for IL-4 receptor-holding cells or IL-6 receptor-holding cells. Xins can enhance the q-childhood of molecules for IL-2R-bearing cells. IL-4 ,as well as! The protein and DNA sequences of L-6 are known (Lee et al. .

J. Biol. Chem. 263:10817. 1988; Hira no et al,, Nature 324 Near 3. 1986) o These Lymphokines are molecules similar to the 11-2/toxin hybrid molecules. It can be used to create unique lymphokine/toxin molecules.

Monoclonal antibodies as targeting reagents Monoclonal antibodies directed against specifically selected lymphokine receptors are It can be used to direct toxins to the cells. These antibodies or The fragment is an artificial fusion gene that encodes a toxin and antibody/X hybrid protein molecule. coded by the child, or generated separately or in Gantt and Tortoise. Cytotoxicity is achieved through non-peptide covalent bonds used to link toxic molecules. Can be fused with Shin. Some useful toxins are listed below.

Antibody/toxin hybrids are similar to the test for IL-2R-retaining cells described below. Similar toxicity tests can be used to test the ability to kill receptor-bearing cells.

toxin Toxin molecules useful in the methods of the invention are desirable only when present intracellularly. Toxins such as peptide toxins are toxic to humans. Of course, such provisions Under the conditions, the molecule must be able to enter the cell that harbors the targeted receptor. No. This ability depends on the nature of the molecules and cellular receptors. For example, the ligand The cellular receptors that naturally allow for uptake require that molecules containing toxins retain their receptors. It holds promise to provide a means of entering cells that harbor cells. Preferably, hybrid proteins By producing recombinant DNA molecules that encode white matter molecules, Syn is fused with a domain that binds to IL-2R. Such means Ensure consistency of composition.

Many togycins have binding domains for generalized eukaryotic receptors. this In such cases, the toxin prevents poisoning of cells that do not carry the targeted receptor. must be modified to retain IL-2 receptors (e.g., to retain IL-2 receptors). but not to prevent intoxication of cells that retain receptors for unmodified toquinone. ).

Such modifications are in any case done in a manner that preserves the cytotoxic function of the molecule. must be carried out (U, S, Department of Healt h and Human 5 services, U, S, 5erial No.

401.412). Potentially cited toxins include, but are not limited to: However, they include: cholera toxin, ricin, 0-hemlock-like toxin ( SLT-1,5LT-11,5LT-order v), LT human sin, C3) xin, cycin Gutkin, pertussis toxin, tetanus toxin, Shutomonas exotoxin, allo phosphorus, savorin, modexin, and geranin.

Diphtheria toxin-based molecules Diphtheria toxin can be used to produce molecules useful in the methods of the invention.

Diphtheria toxin, the sequence of which is known, is disclosed in Murphy's U.S. Patent No. 4゜675.382, which is incorporated by reference into this application. . Corynebacterium Schiff area (Corynebacterium di The natural diphtheria toxin secreted by some Consisting of functional domains, they are enzymatically determined starting from the amino terminus of the molecule. Active fragment A (amino acid Gly-Arg193), and fragment B(amic) acid Ser-3et), and the latter is a translocation domain. , and a general cell-binding domain (amino acid residues 475 to 535) .

The process by which diphtheria toxin poisons susceptible eukaryotic cells involves at least the following steps: (1) The binding domain of diphtheria toxin binds specifically to susceptible cells. Binds to a receptor: (ii) While bound to its receptor, the toxin is endocytosed. Endocytic (endocytic) taken up inside the vesicle; (order I) after internalization Before or within the endocytotic vesicle, the toxin molecule is located between fragments A and B. , undergo proteolytic cleavage: (iv) Endocyto-cis vesicles have a pH of 6 or higher. Going down, the toxin crosses the endosomal membrane into the cytosol of Fragment A. facilitates the release of (V) Catalytic activity of fragment A (i.e. “elongation factor 2′ A eukaryotic protein synthesis factor called nicotinamide adenine dinucleotide Existing adenone diphosphate (ADP) ribonlation results in the death of poisoned cells. vinegar.

Only one molecule of fragment A introduced into the cytosol is the protein synthesis mechanism of the cell. is clearly sufficient to inhibit cell death. Outside Pseudomonas Killing of cells by toxin A1 and possibly certain other toxins found in nature The mechanics are very similar.

DAB IL-2 combines the receptor binding domain of diphtheria toxin with human IL-28. B (Williams et al., J.B. iol, Chem, 35:20673. 1990; Williamse tal, Protein Eng, 1:493. See also 1987 ) are examples of molecules useful in the methods of the invention. This molecule is selective Kills L-2R expressing cancer cells and lymphocytes (Waters et al., Eu r, J, Immunol, 20 Near 85. 1990; Kiyokawa a et al, Cancer Res, 49:4042. 1989) .

Due to its ability to kill activated lymphocytes, DAB IL-2 inhibits graft rejection. B Absolute ill (Pankewycz et at, Transp+antat i.

n 47:3] 3. 1989; Kickman et al, Tran plantation 47:327. 1989), and certain types of autoimmunity Treatment of diseases (Forte et al., 2nd International Symposium on Immunotoxins, 1990) I got A.

DAB, 861L-2 is a chimeric molecule, with Met followed by amino acid residues of IL-2. Amino acid residues 1 to 485 of the mature diphtheria toxin fused to groups 2 to 133 It consists of DA84861L-2 is thus the enzymatically active portion of the molecule. Contains the entire fragment A and part of fragment B of diphtheria toxin encoding I'm reading. The part of fragment B present in DAB4861L-2 has been generalized. does not contain a receptor-binding domain, but allows the release of enzymatically active moieties into the cytosol. Contains a translocation domain that facilitates translocation.

DanΔdan, 86 Preparation of IL-2 DAB IL-2 is a plasmid encoding DAB4861 L-2, pDW2 produced in E. coli harboring 4 (Williams et al.

J, Biol, Chem, 265: 20673.　1990. example (As an exception, amp has been replaced with kan). Protein has immune affinity Purified by chromatography and high pressure liquid chromatography (Wi lliams et al, supra).

Preparation of 891L-4 A synthetic gene encoding human interleukin 4 has been synthesized (Millig en/Biosearch 7500 DNA Synthesizer). IL-4 distribution Column (Yodota et al, Proc, Natl Acad Sc t, USA, 83; 58994. (1986) has been modified to be suitable for E. coli. Incorporating codon usage, deBoer et al., in Maximizi ng Gene Expression, Reznikoff et al, .

eds, 1986. Butterworths, Boston), limited A endonuclease cleavage site was introduced to facilitate subsequent cloning steps.

The IL-4D-ding sequence (His' to 5er129) was inserted into pDW27 plasmid. I inserted it into the card. pDW27 is pDW24 (Wtlliams et al.

J. Biol. Chem. 265:11885. 1990) since natural Diphtheria toxin was derived by removing amino acids 388 to 485.

DanΔdan. 89 IL 4 cytotoxicity The ability of DAB IL-4 to reduce the viability of various cell types is due to inhibition of protein synthesis. It was measured using a test. The test results are shown in Table 3. IC5o(M) is protein t , the concentration of AB3891L-4 required for a 50% reduction in D formation. rat C0 Normal lymphocytes that have undergone nA activation are different from any other cell or cell line. It was also much less sensitive to DA83891 L-4. rat interface -This result is because the leukin-4 receptor does not bind human interleukin-4. This proves the specificity of DAB IL-4. These cavities and sotho cells are diphtheria Sensitive to toxin/rat interleukin two-hybrid molecules.

Table 3 DAB3891L-4 sensitive cells or tumor cells and cell lines Class IC5o (M) cell line T cell origin HLIT 102/6TG human, cTCL, HTLV-12,9x 10” C91/PL human, HTLV-1+ 6. 3xlO-11 transformed B cell origin Ra　j　i　7.2X10” Pulp mononuclear cell human, Burkitt U937’) Mumba tumor EBV+ 2.0XIO-9 normal PBMC human, histiocytes PHA activation lymphoma 1.6xlO”T cells non-primate ConA activation human >10' -Miss 1 town l----Two legs h---1---Miss 11---Once Δ 1, 89 Preparation of IL-6 A synthetic gene encoding human interleukin 6 has been synthesized (Millig en/Biosearch 7500 DNA Synthesizer), IL-6 version Column (Revel et al., EPA 8611404.9) is qualified Incorporating E. coli preferred codon usage (deBoer et al., supra), restricting enzymatic A donuclease cleavage site was introduced to facilitate subsequent cloning steps. . The entire sequence encoding IL-6 was compared to the above DAB3891 L-4. and inserted into pDW27 plasmid.

mixed toxins The cytotoxic portion of some molecules useful in the present invention can be combined with mixed toxin molecules. can be provided. Mixed toxin molecules are two different polypeptide toxins It is a molecule derived from. generally as described above in connection with diphtheria toxin. In sea urchins, polypeptide toxins contain domains responsible for general eukaryotic cell binding. In addition to the protein, it has an enzymatically active domain and a transport domain. Binding and transport The domains are required for cell recognition and toxin import, respectively. The enzymatically active domain is , the domain responsible for cytotoxic activity once the molecule enters the cell. Import Naturally occurring proteins known to have transport domains include diphtheria toxin. Peptidomitoxin, Pseudomonas exotoxin A, and possibly other peptide toxins. Ji The transport domains of Phtheria toxin and Pseudomonas exotoxin A are well characterized. (e.g. Hoch et al, Proc, Natl, Acad .

Sci, USA 82:1692. 1985; Colombatti e tal, J. Biol. Chem. 261:3030.　1986; and Deleers et al, FEBS Lett, 160:82゜1 983), the presence and localization of such domains in other molecules has shown that Hw a n g et al. (Cell 48:129. 1987) and Gray et al. (Pr .

c, Natl, Acad, Sci, USA 81:2645. 1984 ) can probably be determined by the method adopted by

One useful IL-bipartite mixed toxin hybrid molecule is E. coli (E. coli). li) Shiga-like toxin (Calderwood et al., Proc.

Natl, Acad, Sci, USA 84:4364. 1987) The enzymatically active A subunit is linked to the diphtheria toxin transport domain (amino acid residues 202 to 460) and by fusion to IL-2.

This three-part hybrid molecule, 5LT-A/DTB-/IL-2, is It is useful in the method of the invention in a similar manner as DA84861L-2. Part 3? A hybrid made up of! The L-2 moiety specifically attaches the molecule to IL-2R-bearing cells. The diphtheria toxin transport portion is the enzymatically active A subunit of shiga toxin. It works by inserting the target into the target cell. The enzymatically active part of Nga-like toxin is , like diphtheria toxin, acts on the protein synthesis mechanism of cells and inhibits protein synthesis. , thereby killing the cells. Differences between these two types of hybrid toxins lies in the nature of their enzymatic activity. D A B 4861 Enzyme part of L2 ADP-ribolysis of elongation factor 2 with nicotinamide adenine dinucleotide oxidation, thereby inactivating this factor required for protein synthesis. on the other hand , the enzyme part of 5LT-A/DTB''/IL-2 converts liposomal RNA into important sites. It is a ribonuclease that can cleave liposomes with Make inert.

Therefore, the 5LT-A/DTB71L-2 hybrid is DA848G L-2. It is useful as a treatment for similar symptoms and if, for example, the patient's activation If T cells develop resistance to DAB4861L-2, they can be replaced with DAB4861L-2 or can also be used with it.

Linkage of Tokin/N to binding ligand Although not useful hybrid molecules, binding ligands and cytotoxins are bound in several ways. Can be connected. If the hybrid molecule is caused by the expression of the artificial fusion gene, If a peptide bond is generated between the cytotoxin and the bound ligand, then Acts as a connector. Alternatively, the toxin and binding ligand can be made separately and then It is also possible to be linked to by a non-peptide covalent bond.

For example, a covalent a-bond may take the form of a disulfide bond. In this case, if If the bound ligand is a protein, such as IL-2, Murphy et al. tal, ) U.S. Application No. 313.599, incorporated herein by reference. The DNA encoding IL-2 was manipulated to remove an extra cysteine codon. can be made to include. Cysteine controls the IL-2 binding activity of this molecule. Must be located so as not to interfere. For example, the cysteine codon is mature Can be inserted immediately upstream of the DNA encoding type I L-2 PtO2 .

The toxin molecule has a sulfhydryl group that reacts with the modified IL-2 cysteine. must be derivatized by In the case of peptidetoxins, this This is achieved by inserting a cysteine codon into the DNA sequence encoding the toxin. can be achieved. Alternatively, the sulfhydryl group may be can be introduced as part of the protein residues using solid phase polypeptide synthesis.

For example, the introduction of sulfhydryl groups into peptides can be done using Hiskey (P eptides 3:137. (1981). Derivatization also described Bacha et al. on the derivatization of peptide hormones. l,) described in U.S. Pat. No. 4,468,382, incorporated herein by reference. It can be carried out according to the method. The introduction of sulfhydryl groups into proteins is Maasen et al. (Eur, J. Biochem, 13 4:32, 1983). On the one hand, the correct sulfhydryl group is present. Then, the cytotoxin and the IL-2 moiety-binding ligand are purified, and both sulfur groups is reduced, and the cytotoxin and ligand are mixed (at a ratio of about 1:5 to 1=20). ), and disulfide bond formation is allowed to proceed to completion at room temperature (generally at 20 30 minutes). The mixture was then dialyzed against phosphate buffered saline to remove unreacted ligand. and remove toxin molecules. Next, Sephadex chromatography, Alternatively, a similar method can be used to identify the desired toxin based on molecular size. Separating the ligand complex from toxin-toxin and ligand-ligand complexes Ru.

Assay of IL-2 receptor binding and IL-4 receptor binding IL-2 moiety binding of various molecules ability has high affinity (Ju et al., J. Biol. Chem., 262 +5723. 1987), or intermediate affinity receptors (Rob et al. , Proc, Natl, Acad, Sci, USA 84: 2002 ．． 1987) using the IL-2R assay. various The IL-4 binding activity of the molecule was determined by Park et al. (J. Ex. p. Med, 166:176, 1984). Foxwell et al. (Eur, J, I mmunol, 13:1637. Using the assay method described by (1989) can be measured.

Toxicity assay The molecules of the invention (both antibodies and hybrid molecules) can be used to target receptor-bearing cells. The ability to reduce survival rate can be screened using the following assay method. Ru.

Toxicity to IL-2R-retaining cells can be assayed as follows. HUT 10 2/6TG (Tsudo et al, Proc, Natl, Acad.

Sci, USA 83:9694. 1986) or YT2C2 (Tes Higiwari et al, J, Exp, Med, 165:223 (1987) cultured cells in 25mM HEPES (pH 7,4), 2mM 1-glutamine, 100 U/ml penicillin, 100 μg/ml strepto mycin, and 10% fetal bovine serum (Hazelton, Lenexa.

RPM11640 medium (Gibco, Grand l5lan) supplemented with d, NY). Cells were grown in a 96-well ■bottom plate (Linbr).

-Flow Laboratories, McLean, VA); Inoculate at a concentration of 1 x 105 per cell in complete medium. Various estimated toxins (from 10”’M to 10−6M) and cultured in a 5% CO2 atmosphere. Incubate for 18 hours at 37°C. After incubation, the plate is 17 Centrifuge at 0xg for 5 minutes to remove the medium, and add 8 μCi/ml (3H-leucine; Ne Leuci-free, including w England Nuclear, Boston, MA) Replace with medium (MEM, Gibco) 1.00111. 9 more at 37℃ After 0 min, the plate was centrifuged at 170xg for 5 min, the medium was removed, and the cells were transferred to a cell harvester. Glass fiber using Star (Skatron Sterling, VA) – Collect on filter. Filters were washed, dried and counted according to standard methods do.

Cells cultured in medium alone serve as a control.

Toxicity to IL-4R-retaining cells was determined by the above method for IL-2R-retaining cells. Although tested by a similar assay, MLA144 cells (Rabin et al. , J. Immunol, 127:1852. 1981) or HU T102/6TC; using cells, inoculating 1 x 105 cells per well, 4 Incubate for 0 hours.

treatment Generally, molecules of the invention are administered by intravenous infusion. These molecules can also be injected subcutaneously. It may be administered by injection or directly into the inflamed joint. Molecules useful in the methods of the invention The dose of It may vary depending on various factors such as children. For toxic molecules, The degree of uptake by the cells is an important factor, with molecules with lower permeability having higher must be administered in doses.

Other Q-sama The molecules target cytotoxins (or cytolytic antibodies) to target cells. act to reduce cell survival rate. Cytokine availability of target cells Also useful in the methods of the invention are molecules that interfere with .

Induction of IL-2 or other cytokines that block the utilization of endogenous cytokines Conductors are useful for interfering with target cell proliferation. for example,! L-2 was taken away Activated cells are no longer able to proliferate and meet the needs provided by IL-2. In the absence of assimilative stimulation, it ultimately dies. L of specific IL-2 derivatives -2 function-interfering ability was determined by Ju et al. (J, Biol, Che. m.

262:5723. IL-2 biology as described by (1987) It can be tested using standard assay methods. Hybrid with inactivated toxin Molecules can also be used for cytokine receptor blockade. detoxified mutant The molecule of diphtheria toxin has been described (Uchida et al., J.

Biol, Chem, 248:3838. (1973), this molecule is non-toxic1 1,-2/diphtheria toxin hybrid. child As an example of a hybrid molecule such as Sverga et al. See U.S. Application No. 590:113.

Monoclonal antibodies can kill or kill cytokine receptor-bearing cells in several ways. can be used to neutralize. fused to a toxin molecule, as described above. Anti-tokine receptor antibodies can be used to transport toxins to receptor-bearing cells. can. Soluble anti-cytokine receptor antibodies activate complement on their own and activate cytotoxicity. Can kill tocaine receptor-bearing cells. For example, complement activation clone anti- The body can be used to destroy IL-2R-bearing cells. Complement-induced antibodies are one Generally IgG1, 1gG2, 1gG3. and 1 gM isotype antibody. Single The loan anti-IL-2R antibody was tested using a complement-dependent cytotoxicity assay as follows: Those with activation potential can be screened to find them.

Human T lymphocytes and EBV mutant B lymphocytes were treated with 51c, sodium chromate. Label and use as target cells. These cells were incubated with hybridoma culture supernatant and Incubate with complement, then collect the supernatant and measure in a gamma counter. do. It shows toxicity when lit in an activated 1926 bulb, but T or B phosphorus in a static 1F state Select the supernatant that does not show up in the cells (Leonard et al., Pro c.

Natl, Acad, Sci, USA 80:6957. Detailed in 1983 ). The desired anti-IL-2 receptor antibody is purified from the supernatant by a standard method. It will be done. The specificity of the antibody indicates that its activity is inhibited by exogenous IL-2. It can be proven by

Antibodies that inhibit cytokine binding and/or uptake are also useful.

For example, monoclonal antibodies that interfere with IL-2 binding and/or uptake are Quoted in Method of Invention. That is, IL-2R-retaining cells deprived of IL-2 This is because they cannot proliferate. During blocking, clonal antibodies (and other blocking molecules) The biological activity of IL-2 can be tested using the jj method of et al. (supra). can.

In general, useful assays for blocking molecules are those in which the molecule has one or more receptor sites. This would be a competitive binding assay that measures the ability to interfere with the binding of a natural ligand.

Monoclonal antibodies useful in the method of the present invention can be used to transform mice into human IL-2R+T-phosphorylated antibodies. The spleen cells of two mice were immunized with vesicles and fused with appropriate myeloma cells, resulting in Antibodies produced by the ibritoma system were detected using the standard ELISAI method. can be created by screening for the required FjlL-2R binding properties. . Antibody generation and screening was performed by Uchida et al. J.

Immunol, 126:1393. (1981) . Alternatively, useful antibodies may be described by Huse et al. ce 246/1275.1989) combination library generated by the method of It can also be isolated from

The present invention is directed not only to intact monoclonal or polyclonal antibodies, but also to immunological Biologically active antibody fragments, such as Fab or (F a b ) 2 flag ment, antibody heavyweights, antibody light chains, genetically engineered single chain Fv molecules (Ladne r etal, U.S. Patent No. 4,946,778), or chimeric antibodies, For example, a mouse antibody that contains the binding specificity, but other parts are of human origin. Antibodies are also used. Under certain circumstances, cyclosporin A, along with the above molecules, can be used as a non-nephrotoxic agent. (e.g., preferably no more than 5 mg/kg/day). is preferable. For example, cyclosporine A can be used following treatment with one of said molecules. can be administered. This subsequent treatment is expected to result in significant arthritic symptoms as a result of treatment with the molecule. It can be done after being improved. Immunosuppression with cyclosporine ALff activity agents may be used in place of cyclosporin A. cyclosporine A or cyclosporine Rosporin A-like molecules must be administered at effective yet sufficiently non-toxic doses .

The claims are as follows: r::LIre! Day FIG, 3A FIG, 3C Fi；Sore4 Town Uπ5 Day Fi；vre　6 Day Fj, ;ure　7 Skua Continuation of front page (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IT, LU, MC, NL, SE), 0A (BF , BJ, CF, CG, CI, CM, GA, GN, ML, MR, SN, TD, TG. ), AT, AU, BB, BG, BR, CA, CH, C3, DE.

DK, ES, FI, GB, HU, JP, KP, KR, LK, LU, MG, MN, MW, NL, No, PL, RO, RU, SD, SE

Claims (28)

[Claims] 1. A method for preparing a medicament for treating a patient with inflammatory arthritis, comprising: specifically binds protein-like cell receptors expressed on the patient's lymphocytes. and the molecule responsible for the patient's inflammatory arthritis is pharmaceutically acceptable. be mixed with a carrier material to ensure that the molecule reduces the survival rate of the lymphocytes. How to characterize it. 2. 2. The method of claim 1, wherein the inflammatory arthritis is osteoarthritis. A method of characterizing something. 3. 2. The method of claim 1, wherein the inflammatory arthritis is caused by systemic erythema. A method characterized by matosus-related arthritis. 4. 2. The method of claim 1, wherein the inflammatory arthritis is psoriatic arthritis. A method of characterizing something. 5. 2. The method of claim 1, wherein the proteinaceous cell receptor is a high affinity A method characterized in that it is a sexual interleukin 2 receptor. 6. 2. The method of claim 1, wherein the molecule is A method characterized by killing lymphocytes. 7. 2. The method of claim 1, wherein the molecules are covalently linked to each other. A hybrid molecule consisting of a first and second part linked together, the first part being comprising a molecule capable of reducing cell viability, the second portion comprising a molecule capable of reducing cell viability; A method characterized in that the method comprises a molecule capable of specifically binding to a cell receptor. . 8. 8. The method of claim 7, wherein the second portion A method characterized in that it consists of the whole or binding part of an antibody specific for. 9. 8. The method of claim 7, wherein the second portion A method characterized in that the method comprises the entire or binding portion of the ligand. 10. 10. The method of claim 9, wherein the ligand is an interleukin. A method characterized in that: 11. 8. The method of claim 7, wherein the first portion is cytotoxic. A method characterized by comprising the steps of: 12. 12. The method of claim 11, wherein the cytotoxin is a peptide. A fragment of dotoxin that has enzymatic activity but no general eukaryotic receptor binding activity. A method characterized by the absence of gender. 13. 13. The method of claim 12, wherein said fragment of a peptide toxin contains diphtheria toxin fragment A and enough diphtheria toxin to create a pore in the cell membrane. A method characterized in that it consists of fragment B. 14. 14. The method of claim 13, wherein the molecule is DAB486IL. -2. 15. 14. The method of claim 13, wherein the molecule is DAB389IL. -4. 16. 14. The method of claim 13, wherein the molecule is DAB389IL. -6. 17. 11. The method according to claim 10, wherein the interleukin is A method characterized in that Tarleukin 4 is used. 18. 11. The method according to claim 10, wherein the interleukin is A method characterized in that Tarleukin-6 is used. 19. 2. The method of claim 1, wherein the molecule is directed against the cellular receptor. A method characterized in that the method comprises the whole or a binding portion of a specific antibody. 20. 20. The method of claim 19, wherein the antibody is a complement activating antibody. A method of characterizing something. 21. In a method of preparing a drug that reduces bone erosion in patients with inflammatory arthritis The method includes interleukin receptors expressed in lymphocytes of the patient. a molecule that can specifically bind to and is responsible for the inflammatory arthritis in the patient. into a pharmaceutically acceptable carrier material, and the molecule increases the survival rate of the lymphocytes. A method characterized by lowering. 22. 22. The method of claim 21, wherein the inflammatory arthritis is a degenerative joint. A method characterized by being a flame. 23. 22. The method of claim 21, wherein the molecule is DAB486IL. -2. 24. 22. The method of claim 21, wherein the molecule is DAB389IL. -2. 25. A drug for the treatment of inflammatory arthritis, comprising cyclosporin A and the patient's can specifically bind to interleukin receptors expressed on lymphocytes, The molecule is the cause of inflammatory arthritis in the patient, and the molecule is responsible for the inflammatory arthritis of the patient. A drug characterized by its ability to reduce survival rate. 26. 2. The method of claim 1, wherein the molecule is associated with joint inflammation in the patient. Cyclosporin A is administered to the patient until the patient's condition is sufficiently improved. and wherein said cyclosporin A is administered at a sufficiently non-toxic dose. how to. 27. A method of using cyclosporine A for the treatment of patients with inflammatory arthritis, , the method comprises administering to the patient the molecule described in claim 1 together with cyclosporin A. cyclosporine A is administered at a dose sufficient to ensure that the cyclosporin A is non-toxic. How to characterize that. 28. A method of preparing a drug that reduces pain in patients with inflammatory arthritis. Therefore, the method involves detecting proteinaceous cell receptors expressed on lymphocytes of the patient. A molecule that can specifically bind to a molecule that causes inflammatory arthritis in the patient. The molecule is incorporated into a pharmaceutically acceptable carrier material, and the molecule increases the survival rate of the lymphocytes. A method characterized by being able to reduce