INTERLEUKIN RECEPTOR TARGETED MOLECULES FOR TREATMENT OF INFLAMMATORY ARTHRITIS

Abstract: The invention features a method for treating a patient having inflammatory arthritis, the method includes administering to the patient a molecule which is capable of specifically binding to a proteinaceous cell receptor expressed on a lymphocyte of the patient and which contributes to the inflammatory arthritis of the patient, the molecule being capable of decreasing the viability of the lymphocyte.

Description: INTERLEUKIN RECEPTOR TARGETED MOLECULES FOR TREATMENT OF INFLAMMATORY ARTHRITIS Background of the Invention The field of the invention is treatment of inflammatory arthritis. Inflammatory arthritis is a family of arthritic diseases characterized by lymphokine-mediated inflammation of the joints. Inflammatory arthritis is often autoimmune in origin, as is the case with rheumatoid arthritis, psoriatic arthritis, and lupus- associated arthritis. The most common form of inflammatory arthritis is rheumatoid arthritis which occurs in approximately 1 percent of the population. Rheumatoid arthritis is characterized by persistent inflammation of the joints. Inflammation can eventually lead to cartilage destruction and bone erosion. Stiikel et al. (Immunology 64:683, 1988) report that a monoclonal antibody directed against the interleukin-2 receptor inhibits passively transferred adjuvant arthritis in rats (i.e, adjuvant arthritis induced by transfer of spleen cells from rats having adjuvant arthritis to naive rats) , but is not active in suppressing the development of adjuvant arthritis in rats. Case et al. (Proc. Natl . Acad. Sci . USA 86:287, 1989) report that a cytotoxic interleukin-2-Pseudomonas exotoxin fusion protein administered prior to the establishment of overt clinical disease mitigates adjuvant-induced arthritis in rats. Summary of the Invention In general, the invention features a method for treating a patient having inflammatory arthritis, the method includes administering to the patient a molecule which is capable of specifically binding to a proteinaceous cell receptor expressed on a lymphocyte of the patient and which contributes to the inflammatory arthritis of the patient, the molecule being capable of decreasing the viability of the lymphocyte. By "cell receptor" is meant a molecule which is encoded by cellular DNA, binds a ligand, and is expressed so that at least a portion of the molecule is exposed on the cell surface. By "specifically binding" is meant that the molecule does not substantially bind to other cell receptors or cell surface proteins. By "reduces viability" is meant kills or interferes with proliferation. By "ligand" is meant a molecule which is capable of binding to a protein. In various preferred embodiments the inflammatory arthritis is rheumatoid arthritis; the inflammatory arthritis is systemic lupus erythematous-associated arthritis; the inflammatory arthritis is psoriatic arthritis; the proteinaceous cell receptor is the high affinity interleukin-2 receptor; the molecule kills lymphocytes bearing the cell receptor; and the molecule is a hybrid molecule which includes a first and a second portion joined together covalently, the first portion includes a molecule capable of decreasing cell viability and the second portion includes a molecule capable of specifically binding to the cell receptor; the molecule is administered in conjunction with cyclosporin A, the cyclosporin A being administered at a substantially non¬ toxic dosage; and the molecule is administered to the patient until the patient's arthritic condition has substantially improved, following which cyclosporin A is administered to the patient, the cyclosporin A being administered at a substantially non-toxic dosage. In more preferred embodiments, the second portion of the molecule includes all or a binding portion of an antibody specific for the cell receptor; the second portion of the molecule includes all or a binding portion of a ligand for the cell receptor; the ligand is an interleukin; the first portion of the molecule includes a cytotoxin; the cytotoxin is a fragment of a peptide toxin which is enzymatically active but which does not possess generalized eukaryotic receptor binding activity; and the fragment of a peptide toxin comprises fragment A of diphtheria toxin and enough of fragment B of diphtheria toxin to form a pore in a cell membrane. In still more preferred embodiments, the molecule is DAB 486 IL-2; the molecule is DAB 389 IL-4; the molecule is DAB 389 IL-6; the interleukin is interleukin-4; and the interleukin is interleukin-6. In other preferred embodiments, the molecule comprises all or a binding portion of an antibody specific for the cell receptor; and the antibody is a complement activating antibody. In a related aspect, the invention features a method of reducing bone erosion in a patient having inflammatory arthritis, the method includes administering to the patient a molecule which is capable of specifically binding to an interleukin receptor expressed on a lymphocyte of the patient and which contributes to the inflammatory arthritis of the patient, the molecule being capable of decreasing the viability of the lymphocyte. In preferred embodiments, the inflammatory arthritis is rheumatoid arthritis; the molecule is DAB 486 IL-2; and the molecule is DAB 389 IL-2. In a related aspect, the invention features a method for reducing pain in a patient having inflammatory arthritis, the method comprising administering to the patient a molecule which is capable of specifically binding to a proteinaceous cell receptor expressed on a lymphocyte of the patient and which contributes to the inflammatory arthritis of the patient, the molecule being capable of decreasing the viability of the lymphocyte. In a related aspect, the invention features a method of using cyclosporin A to treat a patient with inflammatory arthritis; the method includes administering to the patient a molecule which is capable of specifically binding to a proteinaceous cell receptor expressed on a lymphocyte of the patient and which contributes to the inflammatory arthritis of the patient, the molecule being capable of decreasing the viability of the lymphocyte, the cyclosporin A being administered at a substantially non-toxic dosage. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Detailed Description The figures are first briefly described. Figure 1 is a graphical representation of the effect of treatment with DAB 48g IL-2 on induction of adjuvant arthritis. The arthritic index (assessed as described below) is presented as a function of the number of days after immunization with adjuvant. Animals were treated with TRIS-buffered saline (solid line) or 0.5 mg/kg DAB 48g IL-2 (broken line) on days -1 to 9. Figure 2 is a pair of radiographs of the hind limbs of day 22 adjuvant-induced arthritic rats treated with buffer (panel A) or DAB 48g IL-2 (pane B) on days -1 to 9. Figure 3 is a set of photographs of ankle joint sections taken from adjuvant arthritis induced rats on day 22 post-immunization. Rats were treated with TRIS- buffered saline (panels A and B) or DAB 48g IL-2 (panels C and D) on days -1 to 9. Figure 4 is a graphical representation of the proliferative response of popliteal lymph node lymphocytes isolated from adjuvant arthritis induced rats and stimulated with ConA or M. butyricum. The stimulation index is indicated for cells treated with buffer only (open bars) or DAB 48g IL-2 (striped bars) . Figure 5 is a graphical representation of the effect of DAB 48g IL-2 on the induction of adjuvant arthritis in rats with pre-existing antibodies to diphtheria toxin. The arthritic index (assessed as described below) is presented as a function of the number of days after immunization with adjuvant. Naive animals were treated with TRIS-buffered saline (solid line. A) or 0.5 mg/kg DAB 48g IL-2 (broken line, D) on days -1 to 9. Preimmune animals were similarly treated with TRIS- buffered saline (broken line, B) or 0.5 mg/kg DAB 48g IL-2 (dotted line, C) on days -1 to 9. Figure 6 is a graphical representation of the effect of DAB 48g IL-2 on existing adjuvant arthritis. The arthritic index (assessed as described below) is presented as a function of the number of days after immunization with adjuvant. Animals were treated on days 11 to 21 with TRIS-buffered saline (solid line) or 0.5 mg/kg DAB 48g IL-2. Figure 7 is a graphical representation of the effect of delayed treatment with DAB 48g IL-2 on radiographic features of adjuvant arthritis. Radiographs of hind limbs were taken on day 22 from rats treated with buffer (open bars) or DAB 48g IL-2 (striped bars) on days 11 to 21. Radiographs were graded as follows: 0 - no evidence of disease, 1 = soft tissue swelling, 2 = soft tissue swelling accompanied by mild new bone formation, and 3 = severe soft tissue swelling with extensive new bone formation. The Effect of DAB 40C IL-2 on Rat Adjuvant Arthritis Chronic adjuvant arthritis is an autoimmune disease that can be experimentally induced in genetically susceptible rat strains by immunization with mycobacterial adjuvant. The disease is characterized by subacute polyarthritis involving the distal extremities which is similar clinically and pathologically to human rheumatoid arthritis. Similarities include synovitis, pannus formation, cartilage destruction and bone erosion (Holoshitz et al.. Lancet, 2.∑305, 1986). Involvement of activated T lymphocytes in the pathogenesis of disease has been documented by the observation that adjuvant arthritis, like certain other experimental autoimmune animal models (e.g., experimental allergic encephalomyelitis, streptococcal cell wall induced arthritis, collagen type II induced arthritis and non- obese diabetes) , can be passively transferred to recipient animals by injection of freshly isolated lymphocytes or selected T cell clones from diseased animals (Prud'homme et al. , .Lab. Jnvest., 59:158, 1988). Adjuvant arthritis was induced in female Lewis rats (100 to 125 g; Harlan Sprague-Dawley Inc. , Indianapolis, IN) by injecting a 10 mg/ml suspension of killed, dried Mycobacterium butyricum (Difco, Detroit, MI) in heavy mineral oil (Sigma Chemical Co., St. Louis, MO) . One hundred microliters of the suspension was injected on day 0 intradermally at four to six sites on the lower back while animals were under light methoxyflurane anesthesia. Each rat was evaluated daily for clinical signs of arthritis. Severity of arthritis was quantified by scoring each paw on a scale of 0 to 4 which indicated the severity of peripheral joint swelling and erythema (0=no signs of disease, l=disease evident in a small number of distal joints of a paw, 2=disease evident in all the distal joints of the paw, 3=disease evident in the entire paw and 4=severe disease evident in the entire paw (Trentham et al., J. Exp. Med. ,146:857) . The arthritic index was defined as the sum of the scores of all four paws for each animal with a maximal possible score of 16. Animals were scored by several different observers over the duration of each experiment. Rats immunized with mycobacterial adjuvant typically develop signs of peripheral disease approximately on Day 10 postimmunization. The severity of swelling and erythema of the paws rapidly increases until Day 20 to 25, with individual arthritic indices as high as 10 to 14. Clinical symptoms then gradually decrease to a level which is approximately 50% of the peak b" Day 40. Rats were randomly assigned to experimental groups (10 animals/group) were treated with DAB 48g IL-2 in 0.02M TRIS (pH 8.2) 0.15M NaCl or buffer alone. Treatment occurred during the induction phase of the disease (Day -1 to 9) or after symptoms of arthritis had developed (Days 11 to 21) . Daily subcutaneous administration of 0.5 mg/kg of DAB 48g IL-2 from the day prior to adjuvant administration to Day 9 postimmunization markedly decreased the severity of inflammation associated with disease. Onset of disease was delayed by approximately two days and the degree of peripheral joint swelling and erythema was two to four-fold less severe than in buffer treated animals. In the study depicted in Fig. 1, animals treated with DAB 48g IL-2 had a peak mean arthritic index of 3.0 on Day 22, which later decreased so that animals attained a mean score of approximately 1.6 by Day 40. In contrast, buffer treated control animals had a peak mean arthritic index of 8.5 which decreased to a mean value of only 3.8. A subsequent study demonstrated that treatment with DAB 48g IL-2 on Days 0 to 9 gave comparable results to treatment on Days 1 to 9. Radiographic analysis of the hind limb of buffer treated control animals, terminated on Day 22 postimmunization, showed extensive soft tissue swelling throughout the paw and ankle region (Fig. 2, panel A). This finding was accompanied by narrowing of the joint spaces and moderate new bone formation as shown by irregular areas of greater radiodensity in the metatarsal region. In contrast, radiographs of hind limbs from animals treated with DAB 48g IL-2 during induction (Day -1 to 9) showed only mild soft tissue swelling in the ankle region and no bone destruction or new bone formation (Fig. 2, panel B. For histopathological analysis, hind limbs were preserved in Telly's fixative (glacial acetic acid: formaldehyde: 70% ethanol at 1:2:10). The limbs were then decalcified, paraffin embedded, sectioned along the midline through the metatarsal region and stained with hematoxylin and eosin. Assessment of the sections was based on inflammatory mononuclear cell infiltration, joint space narrowing and periosteal new bone formation. Microscopic examination of sections from buffer treated control animals, terminated on Day 22 postimmunization, revealed extensive signs of disease. Findings included a thickened synovial joint lining, widespread inflammatory infiltrate forming granulomatous lesions and severe proliferative osteomyelitis characterized by bone destruction and new bone formation perpendicular to and impinging on the existing bone (Fig. 3, panel A; Fig. 3, panel B) . Examination of the joints of animals treated with DAB 48g IL-2 during the induction phase (Day -1 to 9) indicated that DAB 48g IL-2 treatment completely inhibited the extensive bone remodeling characteristic of adjuvant arthritis, confirming the radiological results described above. The only evidence of arthritis in animals treated with DAB 48g IL-2 during the induction phase was a mild thickening of the synovial lining (Fig. 3, panel C; Fig. 3, panel D) . The proliferative response of popliteal lymph node cells isolated from animals 10 days postimmunization with mycobacterial adjuvant was assessed. Animals were euthanized, and popliteal lymph nodes were removed under aseptic conditions. The nodes were teased to obtain single cell suspensions. The cells were cultured at 1 x 10 6 /ml in 96 well U-bottomed microtiter plates in RPMI 1640 containing 5% fetal calf serum and 2 x 10" 5 M 2-mercaptoethanol. One hundred miσroliters of Con A (2 μg/ml) , M . butryicum (80 μg/ml) or media alone was then added to quadruplicate wells containing 100 μl of the cell suspension. The cells were incubated for 72 hr, pulsed overnight with 0.625 μCi/well [methyl- 3 H]thymidine (specific activity of 20 Ci/mM) , harvested and assessed for radioactive incorporation. Cells isolated from buffer treated animals responded briskly to stimulation by M. butryicum (Fig. 4) . In contrast, cells derived from DAB 48g IL-2 treated animals had a significantly depressed proliferative response to the specific mycobacterial antigen. This observation is not the result of general depression of the proliferative response since Con A stimulation of cells from DAB 48g IL-2 treated animals was equivalent to that of cells from buffer treated controls (Fig. 4) . The proliferative response of cells from animals not immunized with complete adjuvant but treated with buffer or DAB 48g IL-2 was also evaluated and comparable results obtained. In order to evaluate the potential effect of anti- diphtheria toxin antibodies on DAB 48g IL-2 activity, rats were immunized with diphtheria toxoid prior to immunization with adjuvant and DAB 48g IL-2 treatment. Rats (75 to 100 g) received 100 μg of diphtheria toxoid (Massachusetts State Laboratories, Boston, MA) intramuscularly on five consecutive days. Ten days later, blood samples were obtained from each animal and analyzed for anti-DAB 48g IL-2 antibody levels by an ELISA assay. Briefly, ninety-six-well flexible plates were coated overnight with 1 μg per well of DAB 48g IL-2, blocked with 0.1% gelatin prepared in 0.05% Tween-20 (Sigma) in PBS and washed with 0.05% Tween-PBS. Eight serial five¬ fold dilutions of each serum sample were added to the coated plate (100 μl per well) . The plates were incubated for one hour at room temperature, washed, incubated with an appropriate dilution of alkaline phosphatase-conjugated, affinity purified, goat anti-rat immunoglobulin (Cappel, West Chester, PA) , and developed with alkaline phosphatase substrate (Kirkegaard and Perry, Gaithersburg, MD) . The antibody titer was determined as the greatest dilution of serum which produced an absorbance at 405 nm greater than or equal to 0.1 when analyzed with an automated plate reader. Antibodies that are capable of neutralizing the biological activity of DAB 48g IL-2 for a human IL-2 receptor expressing cell line were measured in an in vitro assay. Two-fold dilutions of each serum sample were prepared in RPMI 1640 media with 15% fetal calf serum and incubated with an equal volume of DAB 48g IL-2 (340 ng/ml) for one hour at 37°C. The preincubated mixture was then added to duplicate V-bottomed microtiter wells containing 10 5 C91/PL cells (13). The cells were incubated overnight, pulsed for two hours with 2.5 μCi/ml[ 1 C]leucine (specific activity of 300 mCi/mM) in leucine free MEM (GIBCO, Grand Island, NY) , harvested an assessed for radioactive incorporation. The end point for this assay is defined as the greatest dilution of serum which protects the cells such that their level of incorporation is greater than or equal to 20 percent of the value for control cells. Results from this assay are reported in units of neutralizing activity. One international neutralizing unit of diphtheria antitoxin is defined as the amount of antibody that will neutralize 2.5 μg of toxin. We have extended this definition to the amount of antibody that will neutralize an equivalent amount on a molar basis (2.7 μg) of DAB 48g IL-2. At the time of immunization with M. butryicum, these animals had moderate levels of antibodies to diphtheria toxin and DAB 48g IL-2 (average ELISA titer of 1:125 and a neutralizing antibody level of at least 0.01 units/ml) . However, the presence of antibodies to diphtheria toxin did not alter the efficacy of treatment with DAB 48g IL-2 during the induction phase of disease (Fig. 5) . The clinical course for both naive and preimmune animals was identical. DAB 48g IL-2 treated animals, both preimmune and naive, had a peak mean arthritic index of 4 while control animals, both preimmune and naive, had a peak mean arthritic index of 13. In order to further investigate the ability of DAB 48g IL-2 to impact on the underlying bone destruction of adjuvant-induced arthritis, the effect of DAB 48g IL-2 treatment during established disease was examined. Unlike the effect observed with DAB 48g IL-2 treatment during the induction phase of disease, administration of the same dose of DAB 48g IL-2 starting after arthritic symptoms had developed on Day 11 and continuing until Day 21, did not alter the measurable clinical signs of disease (Fig. 6) . Histological sections of joints from rats which had received delayed treatment showed widespread mononuclear inflammatory infiltrate consistent with clinical signs of inflammation. However, radiographic analyses demonstrated that bone erosion and new bone formation was observed in a smaller percentage of DAB 48g IL-2 treated animals (30%) than in buffer treated control animals (60%) , despite similar clinical arthritic indices (Fig. 7) . DAB 4CC IL-2 for Treatment of Rheumatoid Arthritis DAB 48g IL-2 has been tested in the treatment of rheumatoid arthritis in a human clinical trial. Thirteen patients with severe rheumatoid arthritis were subjected to a 3 week washout from other drugs used to treat their disease (usually methotrexate or cyclosporin) and treated with DAB 48g IL-2 intravenously at doses of 0.075 mg/kg/day or 0.1 mg/kg/day given over 1 hour for seven consecutive days. After a single course of therapy 4 patients had a substantial response (greater than 50% improvement in joint swelling and pain) , one had a meaningful response (greater than 25% improvement in joint swelling and pain and greater than 25% improvement in at least 2 of 4 other measurable parameters (sedimentation rate, grip strength 50• walk time, and observer assessment) , six patients had a biological effect (greater than a 25% improvement in 3 parameters within 3 weeks) and one patient did not respond. Three of the thirteen patients underwent a second course of therapy. Two of these had a substantial response, and one had a biological response. Molecules Useful in the Method of the Invention In general, there are three ways in which the molecules useful in the invention can act: (1) the molecule can kill a cell because the molecule has a cytotoxic domain; (2) the molecule (an antibody) can cause cell lysis by inducing complement fixation; and (3) the molecule can block binding or uptake of receptor's ligand. In all three cases the molecule must be targeted to receptor bearing cells; this is accomplished by including the receptor's ligand (or a portion or derivative thereof) or an anti-receptor antibody as part of the molecule. Interleukin-2 receptor targeted molecules useful for treatment of inflammatory arthritis provide examples of each of these three approaches. A fusion molecule which includes the IL-2 receptor binding portion of IL-2 and a cytotoxin can be used to kill activated lymphocytes and monocytes/macrophages associated with inflammatory arthritis. Likewise, the second type of molecule described above, a complement fixing antibody, in this instance directed against the IL-2 receptor, can eliminate IL-2 receptor-bearing cells. In this example, the third type of molecule could be a molecule that blocks binding of IL-2 to its receptor. This molecule would prevent infected cells from receiving a proliferation signal from IL-2 and thus could suppress the inflammatory response. Molecules useful for treating patients with inflammatory arthritis can take a number of forms. When IL-2 itself is the targeting agent, the molecule can be a cytotoxic hybrid molecule in which IL-2 is fused to a toxin molecule, preferably a polypeptide toxin. Derivatives of IL-2 which bind to IL-2R, lack IL-2 activity and block binding and/or uptake of bona fide IL- 2 are useful in the method of the invention because they will prevent IL-2-induced proliferation of IL-2R bearing cells. When an anti-IL-2R antibody is the targeting agent, a cytotoxic hybrid molecule can be formed by fusing all or part of the antibody to a cytotoxin. The effectiveness of such an antibody/toxin hybrid, like that of an IL-2/toxin hybrid, depends on the hybrid molecule being taken up by cells to which it binds. Anti-IL-2R antibodies which block binding and/or uptake of IL-2 are also useful in the method of the invention. Lytic anti- IL-2R antibodies are useful in the invention because they can cause complement-mediated lysis of IL-2R-bearing cells. Some of the molecules can be hybrid molecules formed by the fusion of all or part o? two or more molecules. The hybrid molecule can be a hybrid protein encoded by a recombinant DNA molecule, in which case the two domains are joined (directly or through an intermediary domain) by a peptide bond. Alternatively, two domains can be produced separately and joined by a covalent bond in a separate chemical linkage step. In some cases, the cytotoxic domain of a hybrid molecule may itself be derived from two separate molecules. Interleukin-2 as a Targeting Agent IL-2 or any IL-2 receptor binding derivative thereof can be used as a targeting agent for a cytotoxin. The DNA and amino acid sequences of IL-2 are known (Tadatsugu et al.. Nature 302:305, 1983), and its structure has been predicted by x-ray crystallography (Brandhuber et al. , Science 238:1707, 1987). Analysis of genetically engineered variants of IL-2 has provided some information concerning which residues are important for IL-2R binding (Collins et al. , Proc. Natl . Acad. Sci . USA 85:7709, 1988) and bioactivity (Cohen et al. Science 234:349, 1989; Collins et al., supra) . Variants of IL-2 which are useful in the invention include deletion mutants (Genbauffe et al., USSN 388,557, hereby incorporated by reference) which lack one or more amino acid residues in the region between residue 74 and residue 79 (numbering according to Williams et al., Nucl . Acids Res. 16:1045, 1988). These mutants effectively target toxins to IL-2R-bearing cells (Genbauffe et al. , supra) . Generally, IL-2 variants useful for targeting a cytotoxin must efficiently bind IL-2R and be endocytosed. The ability of various derivatives to bind to the IL-2 receptor can be tested with an IL-2R binding assay described below. In designing molecules targeted to cells bearing the IL-2 receptor it must be recognized that the IL-2 receptor, like other receptors, has several forms; and it may be desirable to target cells bearing one form and not another. The human interleukin-2 receptor has a high-, an intermediate-, and a low-affinity form. The high affinity receptor has an apparent K d of -10 M and is composed of two subunits, p55 and p75 (also called p70) . When expressed on the cell surface, both the p75 and p55 subunits are capable of binding IL-2. The p75 subunit corresponds to the intermediate affinity receptor (K d ~ 8.2 x 10~ 10 M) , and p55 subunit corresponds to the low affinity receptor (K d ~ 1-3 x 10~ 8 M) . The p75 subunit is expressed on the surface of resting T cells, natural killer cells monocytes/macrophages, and lymphokine- activated killer (LAK) cell precursors, while the high affinity receptor is expressed on activated T- and B- cells. In the method of the invention it may be desirable to target only cells bearing the high affinity receptor. In these circumstances useful molecules will eliminate or neutralize cells bearing the high affinity IL-2 receptor at a concentration which leaves cells bearing the intermediate or low affinity receptor largely unaffected. When the molecule, like IL-2 itself, has affinity for all three classes of IL-2 receptor, selectivity can be accomplished by administering the molecule at a concentration which does not permit significant binding to cells bearing lower affinity receptors. A hybrid molecule may have altered receptor affinities compared to IL-2. Such hybrid molecules may be more or less selective for cells bearing the high affinity IL-2 receptor. For example, cells bearing the high-affinity receptor are 500-1000 times more sensitive to DAB 48g IL-2, a fusion protein consisting of part of diphtheria toxin and part of IL-2, than are cells bearing the intermediate- affinity receptor (Waters et al., Eur. J. Immunol . 20:785, 1990). A cytotoxin can be attached to an IL-2 derivative in a number of ways. Preferably, an IL-2/toxin hybrid is a hybrid protein produced by the expression of a fused gene. Alternatively, the cytotoxin and the IL-2 derivative can be produced separately and later coupled by means of a non-peptide covalent bond. Linkage methods are described below. Interleukin-4 and Interleukin-6 as a Targeting Agents Interleukin-4 (IL-4) is a cytokine which acts on a variety of cell types. Its receptor is expressed on a number of cell types, including CD4+ T cells and monocytes. IL-4 can act as a T cell growth factor and it is thought to have an influence on IL-2 induced lymphocyte proliferation. High levels of interleukin-6 (IL-6) have been detected in the synovial fluid of patients with active rheumatoid arthritis (Hirano et al. , Eur. J. Immunol . 18:1797, 1988). A cytotoxin directed against IL-4 receptor-bearing cells or IL-6 receptor-bearing cells may enhance the effectiveness of molecules directed against IL-2R-bearing cells. The protein and DNA sequence of IL-4 and IL-6 are known (Lee et al., J * . Biol . Chem. 263:10817, 1988; Hirano et al. , Nature 324:73, 1986). These lymphokines can be used to create hybrid lymphokine/toxin molecules similar to IL-2/toxin hybrid molecules. Monoclonal Antibodies as Targeting Agents Monoclonal antibodies directed against the lymphokine receptor of choice can be used to direct toxins to cells bearing that receptor. These antibodies or antibody fragments can be fused to a cytotoxin either by virtue of the toxin and the antibody being encoded by a fused gene which encodes a hybrid protein molecule, or by means of a non-peptide covalent bond which is used to join separately produced ligand and toxin molecules. Several useful toxins are described below. Antibody/toxin hybrids can be tested for their ability to kill receptor bearing cells using a toxicity assay similar to that which is described below for IL-2R bearing cells. Toxins The toxin molecules useful in the method of the invention are preferably toxins, such as peptide toxins, which are significantly cytotoxic only when present intracellularly. Of course, under these circumstances the molecule must be able to enter a cell bearing the targeted receptor. This ability depends on the nature of the molecule and the nature of the cell receptor. For example, cell receptors which naturally allow uptake of a ligand are likely to provide a means for a molecule which includes a toxin to enter a cell bearing that receptor. Preferably, a peptide toxin is fused to an IL-2R binding domain by producing a recombinant DNA molecule which encodes a hybrid protein molecule. Such an approach ensures consistency of composition. Many peptide toxins have a generalized eukaryotic receptor binding domain; in these instances the toxin must be modified to prevent intoxication of cells not bearing the targeted receptor (e.g. , to prevent intoxication of cells not bearing the IL-2 receptor but having a receptor for the unmodified toxin) . Any such modifications must be made in a manner which preserves the cytotoxic functions of the molecule (see U.S. Department of Health and Human Services, U.S. Serial No. 401,412) . Potentially useful toxins include, but are not limited to: cholera toxin, ricin, O-Shiga-like toxin (SLT-I, SLT-II, SLT II V ) , LT ->xin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, Pseudomonas exotoxin, alorin, saporin, modeccin, and gelanin. Diphtheria Toxin-based Molecules Diphtheria toxin can be used to produce molecules useful in the method of the invention. Diphtheria toxin, whose sequence is known, is described in detail in Murphy U.S. Patent 4,675,382, hereby incorporated by reference. The natural diphtheria toxin molecule secreted by Corynejacteriuiπ diphtheriae consists of several functional domains which can be characterized, starting at the amino terminal end of the molecule, as enzymatically-active Fragment A (amino acids Gly j - Arg 193 ) and Fragment B (amino acids Ser 194 - Ser 535 ) , which includes a translocation domain and a generalized cell binding domain (amino acid residues 475 through 535) . The process by which diphtheria toxin intoxicates sensitive eukaryotic cells involves at least the following steps: (i) the binding domain of diphtheria toxin binds to specific receptors on the surface of a sensitive cell; (ii) while bound to its receptor, the toxin molecule is internalized into an endocytic vesicle; (iii) either prior to internalization, or within the endocytic vesicle, the toxin molecule undergoes a proteolytic cleavage between fragments A and B; (iv) as the pH of the endocytic vesicle decreases to below 6, the toxin crosses the endosomal membrane, facilitating the delivery of Fragment A into the cytosol; (v) the catalytic activity of Fragment A (i.e., the nicotinamide adenine dinucleotide - dependent adenosine diphosphate (ADP) ribosylation of the eukaryotic protein synthesis factor termed "Elongation Factor 2") causes the death of the intoxicated cell. It is apparent that a single molecule of Fragment A introduced into the cytosol is sufficient to inhibit the cell's protein synthesis machinery and kill the cell. The mechanism of cell killing by Pseudomonaε exotoxin A, and possibly by certain other naturally-occurring toxins, is very similar. DAB 48g IL-2, a fusion protein in which the receptor binding domain of diphtheria toxin has been replaced by a portion of human IL-2 (Williams et al., J * . Biol . Chem . 35:20673, 1990; see also Williams et al.. Protein Eng. 1:493, 1987), is an example of a molecule useful in the method of the invention. This molecule selectively kills IL-2R-expressing tumor cells and lymphocytes (Waters et al., Eur. J. Immunol . 20:785, 1990; Kiyokawa et al., Cancer Res. 49:4042, 1989). Because of its ability to kill activated lymphocytes, DAB 48g IL-2 has been used to control graft rejection (Pankewycz et al.. Transplantation 47:318, 1989; Kickman et al., Transplantation 47:327, 1989) and to treat certain autoimmune disorders (Forte et al., 2nd International Symposium on Immunotoxins, 1990) . DAB 48g IL-2 is a chimeric molecule consisting of Met followed by amino acid residues 1 through 485 of the mature diphtheria toxin fused to amino acid residues 2 through 133 of IL-2. Thus, DAB 48g IL-2 includes all of diphtheria toxin fragment A, which encodes the enzymatically active portion of the molecule, and a portion of fragment B. The portion of fragment B present in DAB 48g IL-2 does not include the generalized receptor binding domain but does include the translocation domain which facilitates delivery of the enzymatically active portion into the cytosol. Preparation of DAB 40( .IL-2 DAB 48g IL-2 was produced in E. coli harboring the DAB 48g IL-2 encoding plasmid, pDW24 (Williams et al., J. Biol. Chem. 265:20673, 1990, except amp r is replaced by kan r ) . The protein was purified by immunoaffinity chromatography and high pressure liquid chromatography (Williams et al., supra) . Preparation of DAB 3C0 IL-4 A synthetic gene encoding human interleukin-4 was synthesized (Milligen/Biosearch 7500 DNA synthesizer) . The IL-4 sequence (Yodota et al., Proc Nat ' l Acad Sci . USA, 83:58994, 1986) was modified to incorporate E. coli- preferred codon usage (deBoer et al., in Maximizing Gene Expression, Reznikioff et al., eds., 1986, Butterworths, Boston) , and restriction endonuclease cleavage sites were added to facilitate subsequent cloning steps. IL-4 coding sequence (His 1 to Ser 129 ) was inserted into pDW27 plasmid. pDW27 is derived from pDW24 (Williams et al. , J. Biol. Chem. 265:11885, 1990) by deleting DNA corresponding to amino acids 388 to 485 of native diphtheria toxin. Cytotoxicity of DAB 3CJ IL-4 The ability of DAB 389 IL-4 to reduce viability of various cell types was measured using an inhibition of protein synthesis assay; the results of this assay are presented in Table 3. 1C 50 (M) is the concentration of DAB 38g IL-4 required for a 50% decrease in protein synthesis. The rat, Con A-activated, normal splenic lymphocytes were far less sensitive to DAB 389 IL-4 than any of the other cells or cell lines. Since the rat interleukin-4 receptor does not bind human interleukin- 4, this result demonstrates the specificity of DAB 389 IL- 4. These rat cells are sensitive to a diphtheria toxin/rat interleukin-2 hybrid molecule. Table 3: DAB 389 IL-4 Sensitivity of Normal and Neoplastic Cells and Cell Lines Cell or Cell Classification IC 50 (M) Line T cell origin HUT 102/6TG Human, CTCL, HTLV-I "1" 2.9 X 10 -11 C91/PL Human, HTLV-I + , 6.3 X 10-11 Human, histiocytic Normal PBMC lymphoma 1.6 X 10 ■10 PHA activated T cells Human Non-primate >10 -7 Con A- activated normal splenic Rat T cells Preparation of DAB 30J IL-6 A synthetic gene encoding human interleukin-6 was synthesized (Milligen/Biosearch 7500 DNA synthesizer) . The IL-6 sequence (Revel et al., EPA 8611404.9) was modified to incorporate E. Coli preferred codon usage (deBoer et al., supra) , and restriction endonuclease cleavage sites were added to facilitate subsequent cloning steps. The entire IL-6 coding sequence was inserted into pDW27 plasmid as described above for DAB 389 IL-4. Mixed Toxins The cytotoxic portion of some molecules useful in the invention can be provided by a mixed toxin molecule. A mixed toxin molecule is a molecule derived from two different polypeptide toxins. Generally, as discussed above in connection with diphtheria toxin, polypeptide toxins have, in addition to the domain responsible for generalized eukaryotic cell binding, an enzymatically active domain and a translocation domain. The binding and translocation domains are required for cell recognition and toxin entry respectively. The enzymatically active domain is the domain responsible for cytotoxic activity once the molecule is inside a cell. Naturally-occurring proteins which are known to have a translocation domain include diphtheria toxin, Pseudomonas exotoxin A, and possibly other peptide toxins. The translocation domains of diphtheria toxin and Pseudomonas exotoxin A are well characterized (see, e.g. , Hoch et al. , Proc. Natl . Acad. Sci . USA 82:1692, 1985; Colo batti et al. , J. Biol . Chem. 261:3030, 1986; and Deleers et al., FEBS Lett . 160:82, 1983), and the existence and location of such a domain in other molecules may be determined by methods such as those employed by Hwang et al. Cell 48:129, 1987); and Gray et al. Proc. Natl . Acad. Sci . USA 81:2645, 1984). One useful IL-2/mixed toxin hybrid molecule is formed by fusing the enzymatically active A subunit of E. coli Shiga-like toxin (Calderwood et al. , Proc. Natl . Acad. Sci . USA 84:4364, 1987) to the translocation domain (amino acid residues 202 through 460) of diphtheria toxin, and to IL-2. This three-part hybrid molecule, SLT-A/DTB*/IL-2, is useful in the method of the invention in the same way as DAB 48g IL-2 described above. The IL-2 portion of the three-part hybrid causes the molecule to attach specifically to IL-2R-bearing cells, and the diphtheria toxin translocation portion acts to insert the enzymatically active A subunit of the Shiga- like toxin into the targeted cell. The enzymatically active portion of Shiga-like toxin, like diphtheria toxin, acts on the protein synthesis machinery of the cell to prevent protein synthesis, thus killing the cell. The difference between these two types of hybrid toxins is the nature of their enzymatic activities: the enzymatic portion of DAB 48g IL-2 catalyzes the ADP- ribosylation by nicotinamide adenine dinucleotide of Elongation Factor 2, thereby inactivating this factor which is necessary for protein synthesis, while the enzymatic portion of SLT-A/DTB'/IL-2 is a ribonuclease capable of cleaving ribosomal RNA at a critical site, thereby inactivating the ribosome. SLT-A/DTB'/IL-2 hybrid would therefore be useful as a treatment for the same indications as DAB 48g IL-2, and could be substituted or used in conjunction with it if, for example, a patient's activated T-cells develop a resistance to DAB 48g IL-2. Linkage of Toxins to Binding Ligands The binding ligand and the cytotoxin of useful hybrid molecules can be linked in several ways. If the hybrid molecule is produced by expression of a fused gene, a peptide bond serves as the link between the cytotoxin and the binding ligand. Alternatively, the toxin and the binding ligand can be produced separately and later coupled by means of a non-peptide covalent bond. For example, the covalent linkage may take the form of a disulfide bond. In this case, if the binding ligand is a protein, e.g., IL-2, the DNA encoding IL-2 can be engineered to contain an extra cysteine codon as described in Murphy et al. U.S. Serial No. 313,599, hereby incorporated by reference. The cysteine must be positioned so as to not interfere with the IL-2R binding activity of the molecule. For example, the cysteine codon can be inserted just upstream of the DNA encoding Pro 2 of the mature form of IL-2. The toxin molecule must be derivatized with a sulfhydryl group reactive with the cysteine the modified IL-2. In the case of a peptide toxin this can be accomplished by inserting a cysteine codon into the DNA sequence encoding the toxin. Alternatively, a sulfhydryl group, either by itself or as part of a cysteine residue, can be introduced using solid phase polypeptide techniques. For example, the introduction of sulfhydryl groups into peptides is described by Hiskey (Peptides 3:137, 1981). Derivatization can also be carried out according to the method described for the derivatization of a peptide hormone in Bacha et al. U.S. Patent No. 4,468,382, hereby incorporated by reference. The introduction of sulfhydryl groups into proteins is described in Maasen et al. (Eur. J. Biochem. 134:32, 1983). Once the correct sulfhydryl groups are present, the cytotoxin and IL-2R binding ligand are purified, both sulfur groups are reduced; cytotoxin and ligand are mixed;, (in a ratio of about 1:5 to 1:20) and disulfide bond formation is allowed to proceed to completion (generally 20 to 30 minutes) at room temperature. The mixture is then dialyzed against phosphate buffered saline to remove unreacted ligand and toxin molecules. Sephadex chromatography or the like is then carried out to separate on the basis of size the desired toxin-ligand conjugates from toxin-toxin and ligand-ligand conjugates. Assays for IL-2 Receptor Binding and IL-4 Receptor Binding The IL-2R binding ability of various molecules can be measured using an IL-2R assay for 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). The IL-4R binding activity of various molecules can be measured using the assay described by Park et al. (J * . Exp. Med. 166:176, 1984) or the assay described by Foxwell et al. (Eur. J. Immunol . 19:1637, 1989). Assays for Toxicity Molecules of the invention (both antibodies and hybrid molecules) can be screened for the ability to decrease viability of cells bearing the targeted receptor by means of assays such as those described below. Toxicity towards IL-2R bearing cells can be tested as follows. Cultured HUT 102/6TG (Tsudo et al. , Proc. Natl . Acad . Sci . USA 83:9694, 1986) or YT2C2 (Teshigiwari et al., J. Exp. Med. 165:223, 1987) cells are maintained in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 25 mM HEPES (pH 7.4), 2mM 1-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal calf serum (Hazelton, Lenexa, KS) . Cells are seeded in 96-well V-bottomed plates (Linbro-Flow Laboratories, McLean, VA) at a concentration of 1 x 10 5 per well in complete medium. Putative toxins are added to varying concentrations (10~ 12 M to 10 ""6 M) and the cultures are incubated for 18 hrs. at 37°C in a 5% C0 2 atmosphere. Following incubation, the plates are centrifuged for 5 min. at 170 x g, and the medium removed and replaced with 100 μl leucine-free medium (MEM, Gibco) containing 8 μCi/ml ( 3 H-leucine; New England Nuclear, Boston, MA) . After an additional 90 min. at 37°C, the plates are centrifuged for 5 min. at 170 x g, the medium is removed, and the cells are collected on glass fiber filters using a cell harvester (Skatron, Sterling, VA) . Filters are washed, dried, and counted according to standard methods. Cells cultured with medium alone serve as the control. Toxicity towards cells bearing IL-4R may be tested by an assay similar to that described above for IL-2R bearing cells, but utilizing MLA144 cells (Rabin et al. J * . Immunol . 127:1852,1981) or HUT 102/6TG cells, seeded aatt 11 xx :10 5 cells per well and incubated for 40 hours. Therapy Generally, the molecules of the invention will be administered by intravenous infusion. They may also be administered subcutaneously or injected directly into the inflamed joint. Dosages of molecules useful in the methods of the invention will vary, depending on factors such as whether the substance is a cytotoxin, a lytic antibody, or an cell receptor blocking molecule. In the case of toxic molecules the extent of cell uptake is an important factor; less permeable molecules must be administered at a higher dose. Other Embodiments The molecules described above act to decrease cell viability by directing a cytotoxin (or a lytic antibody) to a targeted cell. Also useful in the method of the invention are molecules which interfere with the targeted cell's ability to utilize a cytokine. Derivatives of IL-2 or other cytokines which block utilization of endogenous cytokine are useful for preventing proliferation of targeted cells. For example, activated cells deprived of IL-2 fail to proliferate and, in the absence of the essential anabolic stimulus provided by IL-2, will eventually die. The ability of a given IL-2 derivative to interfere with IL-2 function can be tested in an IL-2 bioactivity assay such as the one described by Ju et al. (J " . Biol . Chem. 262:5723, 1987). Hybrid molecules in which the toxin has been rendered inactive can be also used to block a cytokine receptor. A non-toxic mutant diphtheria toxin molecule has been described (Uchida et al. J * . Biol . Chem. 248:3838, 1973), and this molecule can be used to produce a non-toxic IL- 2/diphtheria toxin hybrid. See Svrluga et al. U.S. Serial No. 590,113, hereby incorporated by reference, for an example of such a hybrid molecule. Monoclonal antibodies can be used to kill or neutra. 1 cytokine receptor-bearing cells in a number of ways. As described above, anti-cytokine receptor antibodies fused to a toxin molecule can be used to deliver the toxin to receptor-bearing cells. Lytic anti- cytokine receptor antibodies can themselves kill cytokine receptor-bearing cells by activating complement. For example, monoclonal antibodies which activate complement can be used to destroy IL-2R-bearing cells. Complement inducing antibodies are generally.those of the IgGl, lgG2, IgG3, and IgM isotypes. Monoclonal anti-IL-2R antibodies can be screened for those able to activate complement using a complement-dependent cytotoxicity test, as follows. Human T-lymphocytes and EBV transformed B- lymphocytes are labeled with 51 Cr sodium chromate and used as target cells; these cells are incubated with hybridoma culture supematants and with complement, and then the supematants are collected and counted with a gamma counter. Those supematants exhibiting toxicity against activated T-lymphocytes, but not resting T- or B- lymphocytes, are selected (described in detail in by Leonard et al., Proc. Natl . Acad. Sci . USA 80:6957, 1983) . The desired anti-IL-2 receptor antibody is purified from the supematants using conventional methods. The specificity of the antibody can be demonstrated by showing that the activity is blocked by exogenous IL-2. Also useful are antibodies whish block binding and/or uptake of a cytokine. For example, monoclonal antibodies which interfere with the binding and/or uptake of IL-2 are useful in the method of the invention because IL-2R bearing cells deprived of IL-2 fail to proliferate. Blocking monoclonal antibodies (and other blocking molecules) can be tested for their ability to interfere with IL-2 bioactivity using the method of Ju et al., (supra) . Generally, assays useful for blocking molecules will be competitive binding assays which measure the ability of the molecule being to interfere with binding of one or more of the receptor's natural ligands. Monoclonal antibodies useful in the method of the invention can be made by immunizing mice with human IL- 2R + T-lymphocytes, fusing the murine splenocytes with appropriate myeloma cells, and screening the antibodies produced by the resultant hybridoma lines for the requisite IL-2R binding properties by means of an ELISA assay. Antibody production and screening can be performed according to Uchiyama et al. (J. Immunol . 126:1393, 1981). Alternatively, useful antibodies may be isolated from a combinatorial library produced by the method of Huse et al. (Science 246:1275, 1989). The invention can employ not only intact monoclonal or polyclonal antibodies, but also an immunologically-active antibody fragment, for example, a Fab or (Fab) 2 fragment; an antibody heavy chain, an antibody light chain; a genetically engineered single- chain Fv molecule (Ladner et al., U.S. Patent No. 4,946,778) ; or a chimeric antibody, for example, an antibody which contains the binding specificity of a murine antibody, but in which the remaining portions are of human origin. Under some circumstances it may be desirable to administer cyclosporin A at a non-nephrotoxic dosages (e.g., preferably not more than 5 mg/kg/d) in conjunction with the molecules described above. For example, cyclosporin A can be administered subsequent to treatment with one of the above-described molecules. Such subsequent treatment can take place after the arthritic condition has substantially improved as a result of treatment with the above-described molecules. Immunosuppressive compounds with cyclosporin A - like activity may be used in place of cyclosporin A. The administration of cyclosporin A or cyclosporin A - like molecules should be at an effective yet substantially non-toxic dosage. What is claimed is:

Claims: Claims 1. A method for preparing a medicament for treating a patient having inflammatory arthritis, said method comprising admixing with a pharmaceutically acceptable carrier substance a molecule which is capable of specifically binding to a proteinaceous cell receptor expressed on a lymphocyte of said patient and which contributes to the inflammatory arthritis of said patient, said molecule being capable of decreasing the viability of said lymphocyte. 2. The method of claim 1 wherein said inflammatory arthritis is rheumatoid arthritis. 3. The method of claim 1 wherein said inflammatory arthritis is systemic lupus erythematosus- associated arthritis. 4. The method of claim l wherein said inflammatory arthritis is psoriatic arthritis. 5. The method of claim 1 wherein said proteinaceous cell receptor is the high affinity interleukin-2 receptor. 6. The method of claim 1 wherein said molecule kills lymphocytes bearing said cell receptor. 7. The method of claim 1 wherein said molecule is a hybrid molecule comprising a first and a second portion joined together covalently, said first portion comprising a molecule capable of decreasing cell viability and said second portion comprising a molecule capable of specifically binding to said cell receptor. 8. The method of claim 7 wherein said second portion comprises all or a binding portion of an antibody specific for said cell receptor. 9. The method of claim 7 wherein said second portion comprises all or a binding portion of a ligand for said cell receptor. 10. The method of claim 9 wherein said ligand is an interleukin. 11. The method of claim 7 wherein said first portion comprises a cytotoxin. 12. The method of claim 11 wherein said cytotoxin is a fragment of a peptide toxin which is enzymatically active but which does not possess generalized eukaryotic receptor binding activity. 13. The method of claim 12 wherein said fragment of a peptide toxin comprises fragment A of diphtheria toxin and enough of fragment B of diphtheria toxin to form a pore in a cell membrane. 14. The method of claim 13 wherein said molecule is DAB 48g IL-2. 15. The method of claim 13 wherein said molecule is DAB 389 IL-4. 16. The method of claim 13 wherein said molecule is DAB 389 IL-6. 17. The method of claim 10 whdrein said interleukin is interleukin-4. 18. The method of claim 10 wherein said interleukin is interleukin-6. 19. The method of claim 1 wherein said molecule comprises all or a binding portion of an antibody specific for said cell receptor. 20. The method of claim 19 wherein said antibody is a complement activating antibody. 21. A method of preparing a medicament for reducing bone erosion in a patient having inflammatory arthritis, said method comprising admixing with a pharmaceutically acceptable carrier substance a molecule which is capable of specifically binding to an interleukin receptor expressed on a lymphocyte of said patient and which contributes to the inflammatory arthritis of said patient, said molecule being capable of decreasing the viability of said lymphocyte. 22. The method of claim 21 wherein said inflammatory arthritis is rheumatoid arthritis. 23. The method of claim 21 wherein said molecule is DAB 48g IL-2. 24. The method of claim 21 wherein said molecule is DAB 389 IL-2. 25. A medicament for the treatment of inflammatory arthritis, comprising cyclosporin A and a molecule which is capable of specifically binding to an interleukin receptor expressed on a lymphocyte of said patient and which contributes to the inflammatory arthritis of said patient, said molecule being capable of decreasing the viability of said lymphocyte. 26. The method of claim 1 wherein said is molecule is administered to said patient until said patient's arthritic condition has substantially improved, following which cyclosporin A is administered to said patient, said cyclosporin A being administered at a substantially non-toxic dosage. 27. A method of using cyclosporin A to treat a patient having inflammatory arthritis, said method comprising administering to said patient the molecule of claim 1 in combination with cyclosporin A, said cyclosporin A being administered at substantially non¬ toxic dosage. 28. A method for making a medicament for reducing pain in a patient having inflammatory arthritis, said method comprising admixing with a pharmaceutically acceptable carrier substance a molecule which is capable of specifically binding to a proteinaceous cell receptor expressed on a lymphocyte of said patient and which contributes to the inflammatory arthritis of said patient, said molecule being capable of decreasing the viability of said lymphocyte.

Inventors: BACHA PATRICIA A, NICHOLS JEAN C, WOODWORTH THASIA G, BACHA, PATRICIA, A., NICHOLS, JEAN, C., WOODWORTH, THASIA, G.
Applicants: SERAGEN INC, SERAGEN, INC.
Application year: 1992
Main classification: C07K13
IPC codes: A61K35, A61K38, A61K39, A61K47, A61P29, A61P43, C07K14