CA2108886A1

CA2108886A1 - Interleukin receptor targeted molecules for treatment of inflammatory arthritis

Info

Publication number: CA2108886A1
Application number: CA002108886A
Authority: CA
Inventors: Thasia G. Woodworth; Jean C. Nichols; Patricia A. Bacha
Original assignee: Individual
Current assignee: Seragen Inc
Priority date: 1991-05-03
Filing date: 1992-05-04
Publication date: 1992-11-04
Also published as: NO933960L; JPH06510750A; AU665763B2; BR9205967A; NO933960D0; EP0668774A1; AU1992792A; WO1992019259A1; EP0668774A4

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

,- ~W092~192S9 æl~8~ PCr/US92/03~14 INTE:P~LE~I~IN RE:CEP~OR TARGJ3TE~D MOLEC~LE8 FOR TP~:ATll:E:NT C)F INFL~TORY A~ITI~
Background of the_Invention 5The field of the invention is treatment of inflammatory arthritis.
Inflammatory arthritis is a family of arthritic diseases characterized by lymphokine-metliated 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 ~y persistent inflammation of the joints. Inflammati .l can eventually lead to cartilage destruction and bone erosi~n.
StUXel et al. (Im~unology 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 inflamm~tory 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 " :

_ WO92/19~9 PCT/~S92/~3714 the patient and which contributes to the inflammatory arthrîtis of the patient, the molecule ~eing 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 e~ressed so that at least a portion of the molecule is expo-;ed on the cell surface. By "specifically binding" is Dleant 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 bindinq 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 e~bodiments, 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 inc}udes all or a binding portion ~ WO9tJl92S9 2 ~ ~ 8 8 8 ~ PcT/us92/037l~
~,,, 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 frayment 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 DAB486IL-2; the molecule is DAB389IL-4; the molecule is DAB389IL-6; the interleukin is interleukin-4; and the interleukin i5 interleukin-6.
In other preferred embodiments, the molecule comprises all or a binding portion of an antibody speci~ic for the cell recepcor; and the antibody i~ a complement activating antibody.
In a related aspect, the invention features a method of reducing ~one 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 o~ the lymphocyte.
In preferred embodiments, the inflammatory arthritis is rheumatoid arthritis; the ~olecule is DAB~A6IL-2; and the molecule is DAE3389IL-2.
In a related aspect, the invention features a method for reducing pain in a patient having inflammatory arthritis, the method comprising a~ministering 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 W092/l9~9 '~ 1 ~ 8 ~

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.
De~ailed ~escri~tion The figures are first briefly described.
Figure 1 i8 a graphical representation of the effect of treatment with DAB~86IL-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~86IL-2 (broken line) on days -1 to 9.
Figure 2 is a pair of radiographs of the hind limb8 of day 22 adjuvant-induced arthritic rats treated with buffer (panel A) or DAB~6IL-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 ~) or DABA86IL-2 (panels C
and D) on days -1 to 9.
Figure 4 is a graphical representation of the proliferative response of popliteal lymph node ~ WO92/19~ PCT/US92/03714 . .

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~86IL-2 ~striped bars).
Figure 5 is a graphical representation of the effect of DAB~86IL-2 on the induction of adjuvant arthritis in rats with pre-existing antibodies to dipht~eria toxin. The arthritic index (assessed as described below) is presented as a function of the n~ber of days after immunization with adjuvant. Naive animals were treated with TRIS-buffered saline ~solid line, A) or 0.5 mg/kg DAB486IL-2 (broken line, D) on days -1 to 9.
Prei~mune animals ~ere similarly treated with ~RIS-buffered saline (broken line, B) or 0.5 mg/kg DAB486IL-2 (dotted line, C) on days -1 to 9.
Figure 6 is a graphical representation of the effect of DAB~8fiIL-2 on existing adjuvant arthritis. The arthritic index (asse5sed as described ~elow) 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~86IL 2.
Figure 7 i6 a graphical representation of the effect of delayed treatment with DAB4a6IL-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~86IL-2 (striped bars) on days ll to 21. Radiographs were graded as follows: 0 - no evidence of disease, 1 = soft tis6ue swelling, 2 - soft tissue swelling accompanied by mild new bone formation, and 3 = severe soft tissue swelling with extensive new bone formation.
The ~ffect of DAB486I~-2 on Rat Adiuvant Arthritis Chronic adjuvant arthritis is an autoimmune disease that can be experimentally induced in genetically W092/l9~9 PCT/US92/03714 c~ 8 6 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, ~artilage destruction and bone erosion (Holoshitz et al., Lance~, 2:305, 1986j. 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 arthriti~, collagen type II induced arthritis and non-obese diabetes), can be passively transferred to recipient animals by injection o~ freshly isolated lymphocytes or selected T cell clones from diseased animals (~rudlhomme et al., Lab. Invest., S9:158, 1988).
Adjuvant arthritis was induced in female Lewis rats (100 to 125 g; Harlan Sprague-Dawley In~., Indianapolis, IN) by injecting a 10 mg/ml suspension of killed, dried Myco~acterium butyricum (Difco, Detroit, MI) in heavy mineral oil (Sigma Chemical Co., St. Louis, MO). One hundred microliters of the suspen~ion was injected on day 0 intradermally at four to 6iX 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, 1=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 ., 14 6: 857 ) .
The arthritic index was defined as the sum of the scores WO92J19~9 PCT/VS92/0371 of all four pa~s 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 di.sease approximately on Day l0 posti~muni~ation. The severity of swelling and erythema of the paws rapidly increases until Day 20 to 25, with individual arthritic indices as high as l0 to l4. Clinical symptoms then gradually decrease to a level which is approximately 50% of the peak by Day 40. Rats were randonly assigned to experimental groups (l0 animals/group) were treated with DAB~6IL-2 in 0~02M TRIS (pH 8.2) 0.l5M NaCl or buffer alone. Treatment occurred during the induction phase of the disease (Day -l to 9) or after symptoms o~ arthritis had developed (Days l to 21).
Daily subcutaneous administration of 0.5 mg/kg of DAB~86IL-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 leqs severe than in buffer treated a~imals.
In the study depicted in Fig. l, animals treated with DA~3~86IL-2 had a peak mean arthritic index of 3.0 on Day 22, which later decreased so that animals attained a mean score oS approximately l.6 by Day 40. In contrast, bu~fer 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~86IL-2 on Days 0 to 9 gave comparable results to treatment on Days l to 9.
Radiographic analysis of the hind limb of buffer treated control animals, terminated on Day 22 postimmunization, showed extensive soft tissue swelling : w092/l9~9 PCT/US92J0371~
'.. , 2 ~ 6 throughout the paw and ankle region (Fig. 2, panel A).
This ~inding 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~86IL-2 during induction (Day -l to 9) showed only mild soft tissue swelling in the ankle region and no bone de~truction or new bone formation (Fig. 2, panel B.
For histopathological analysis, hind limbs wexe preserved in Telly's fixative tglacial acetic acid:
formaldehyde: 70~ ethanol at 1:2:10). The li~bs were then decalcified, paraffin embedded, sectioned along the midline through the metatarsal region and 6tained 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 le~ions and 6evere 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 ~oints of animals treated with DAB~a6~L-2 during the induction phase (Day -1 to 9) indicated that D~3~86IL-2 treatment complete; inhibited the extensive bone remodeling characteristic of adjuvant arthritis, confirming the radiological results described above. The only evidence of a-thritis in animals treated with DAB~86~L-2 during the ind_:tion phase was a mild thickening of the synovial lining (Fig. 3, panel C; Fig. 3, panel D).

; WO92/19~9 PCT/US~/03~1 _ 9 _ The proliferative response of popliteal lymph node cells isolated from animals 10 days postimmunization with mycobacterial adjuvant was asse~sed.
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 106/ml in 96 well U-bot:tomed microtiter pl~tes in RPMI 1640 containing 5% fetal calf serum and 2 x 10 5M 2-mercaptoethanol. One hundredl microliters of Con A ~2 ~g/ml), M. butryicum (80 ~gl~l) or media alone was then added to quadruplicate wells containing lOo ~l of the cell suspension. The cells were incubated for 72 hr, pulsed overnight with 0.625 ~Ci/well [methyl-3H~thymidine (specific activity of 20 Ci/mM), harvested and assessed for radioactive incorporation. Cells isolated from buf~er treated animals responded briskly to stimulation by M.butry~cum (Fig. 4). In contrast, cells derived from DAB4 6IL-2 treated animals had a 5ignificantly 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~SIL-2 treated animals was equivalent to that of cells fro~ buffer treated controls (Fig. 4). The proliferative response of cells ~rom animals not i~munized with complete adjuvant but treated with bu~fer or DAB~86IL-2 was also evaluated and comparable results obtained.
In order to evaluate the potential e~fect of anti-diphtheria toxin antibodies on DAB~86IL-2 activity, rats were immunized with diphtheria toxoid prior to immunization with adjuvant and DAB~a6IL-2 treatment. Rats (75 to 100 g) received 100 ~g of diphtheria toxoid (Massachusetts State Laboratories, Boston, HA) intramuscularly on five consecutive days. Ten days later, blood samples were obtained from each animal and ; w092/19~9 PCT/US92/~3714 `` 2~a8~

analyzed for anti-DAB686IL-2 antibody levels by an ELISA
assay. Briefly, ninety-six-well flexible plates were coated overnight with 1 ~g per well of DAB~86IL-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 ~l per well). The plates were ,ncubated 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 e~ual to 0.1 when analyzed with an automated plate reader.
Antibodies that are capable of neutralizing the biological activity of DAE3~86IL-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,86IL-2 (340 ng/ml~ for one hour at 37C. The preincubated mixture was then added to duplicate V-bottomed microtiter wells containing 10S C91/PL cells (13). The cells were incubated overnight, pulsed for two hours with 2.5 ~Ci/ml~l~C]leucine (specific activity of 300 mCi/~M) in leucine free MEM (GIBC0, 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 -WO92/19~9 ~0 8 8 ~ ~ PCT/US92~03714 is defined as the amount of antibody that will neutralize 2.5 ~g of toxin. We have extended this definition to the amount of antibody that will neutralize an equivalent amount on a molar basis (2.7 ~g) of DAE~8~IL-2.
At the time of immunization with M. butryicum, these animals had moderate levels of antibodies to diphtheria toxin and DAB~86IL-2 tave agle ELISA titer of l:i25 and a neutralizing antibody level of at least o.o units/ml). However, the presence of antibodies to diphtheria toxin did not alter the efficacy of treatment with DAB486IL-2 during the induction phase of disease (Fig. 5). The clinical course for both naive and preimmune animals was identical. DA~486IL-2 treated animals, both preimmune and naive, had a peak mean arthritic index of 4 while control animais, both preimmune and naive, had a peak mean arthritic index of 13.
In order to ~urther investigate the ability of DAB~06IL-2 to impact on the underlying bone destruction of adjuvant-induced arthritis, the effect of DAB~86IL-2 treatment during established disease was examined.
Unlike the effect obser~ed with DAB~86IL-2 treatment during the induction pha~e of disease, administration of the same dose of DA3~86I~-2 starting after arthritic symptoms had developed on Day ll and continuing until Day 21, did not alter the measurable clinical signs of disease (Fig. 6). Histological sections of ~oints 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 percenta~e of DAB486IL-2 treated animals (30%) than in buffer treated control animals (60%), despite similar clinical arthritic indices (Fig. 7).

', W092/l9~9 PCTtUS~2~03714 - ~: 210~

DAB686IL-2 for Treatment of Rheumatoid Arthritis DAB~86IL-2 has been tested in the treatment of rheumatoid arthritis in a human clinical trial. Thirteen patients with severe rheumatoid arthrit$s were subjected to a 3 weeX washout from other drugs used to treat their disease (usually methotrexate or cyclosporin) and treated with DAB4R6IL_2 intravanously at doses of 0.075 mg/kg/day or 0.1 mg/Xg/day given over l hour for ~even consecutive days. After a single course of therapy 4 patients had a substantial response (greater than 50% improvement in joint swelling and p~in), 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 comp}ement fixation; and (3) the ~olecule can block binding or uptake of receptor's -ligand. In all three cases the molecule must be targeted to receptor ~earing 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.
InterleuXin-2 receptor targeted molecules useful for treatment of inflammatory arthritis provide examples of each of these three approaches. A fusion molecule ~ ~ WO92~19~9 2 ~ ~ 8 ~ ~ ~ PCT/U~92/03714 which includes the IL-2 receptor binding portion of IL-2 and a cytotoxin can be used to kill actlvated lymphocytes and monocytes/macrophages associated with inflammatory arthritis. Likewise, the second t pe of molecule described above, a complement fixing ant:ibody, 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-2b 2 are useful in the method of the invention because theywill prevent IL-2-induced proliferation of IL-2R bearing cells. When an anti-I;-2R antihody is the targeting agent, a cytotoxic hybrid molecule can be formed by fu~ing 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, depend6 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 ~ecause 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 of two or more molecules. The hybrid molecule can be a hybrid protein WO92/19~9 2 1 ~ 8 8 ~ ~ PCT/US92/03714 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 lir~age step. In some cases, the cytotoxic domain of a hybrid molecule may itself be derived from two separate molecules.
InterleuXin-2 as a ~arqeting A~ent 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 in~ormation 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 residuss in the region between residue 74 and residue 79 (numbering according to Williams et al., ~ucl.
25 Acids Re~. 16:1045, 1988). These mutants effectively target toxins to IL-2R-bearing cells (Genbauffe et al., supr~). Generally, IL-2 variants useful for targeting a cytotoxin must ef~iciently 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 w092~l9~9 PCT/US92~03~1~
.. 21~88~6 another. The human interleukin-2 receptor has a high-, an intermediate-, and a low-affinity form. The high affinity receptor has an apparent X~ of ~10 lOM 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 (Kd ~
8.2 x 10 lOM), and p55 subunit corresponds to the low affinity receptor (Kd - 1-3 x 10 ~). The p75 subunit is expressed on the surface of resting T cells, natural killer cells monocytes/macrophages, and lymphokine-activated killer (LAX) 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 moleculec 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 DAB486IL-2, a fusion protein consisting of part of diphtheria toxin and part of IL-2, than are ce}ls bearing the intermediate- affinity receptor (Waters et al., ~ur. J.
Immunol. 20:785, 1990).

W~92/19~9 PCTtUS9~03714 - 16 ~
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 la~er coupled by means of a non-peptide covalent bond. Linkage methods are described below.
Interleu~in-4 and Interleukin-6 as a Targe~lnq Aqents Interleukin-4 ~IL-4) is a cytokine which acts on a variety of cell types. Its receptor is e~pressed 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. Immul~ol . 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 tLee 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 Tar~etinq Aqents 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.

~ WO92/19~9 PCT/US92/03714 2~ 08886 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 peptidP 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 mo}ecule which includes a toxin to enter a cell bearing that receptor.
Pre~erably, 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 o- -omposition.
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, 0-Shiga-like toxin (SLT-I, SLT-II, SLT IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, Pseudomonas exotoxin, alorin, saporin, modeccin, and gelanin.

- wo 92/19~g 2 1 ~ ~ 8 8 ~ PCT/US92/03~14 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 4,67s,382, hereby incorporated by reference.
The natural diphtheria toxin molecule secreted by Corynebacterium diphtheriae consists of sev~ral functional domains which can be characterized, starting at the amino terminal end of the molecule, as enzymatically-active Fragment A (amino acids Glyl ~
Argl93) and Fragment B (a~ino acids Serlg~ - Ser535), 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 bindin~ domain of diphtheria toXin binds to specific receptors on the sur~ace 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 decrea~es 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 (~DP) ribosylation of the eukaryotic protein synthesis factor termed "Elongation Factor 2") causes the death o~
~0 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 Pseudomonas exotoxin A, and possibly by . ' ~' "' ' " ' ' ' ~ :
.

, . WO 92/192S9 2 ~ ~ 8 ~ ~ ~ PCT~US92/03714 ! ~

certain other naturally-occurring toxins, is very similar.
DAB~86IL-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; sse also Williams et al., Protein ~ng.
l:493, 1987), is an example of a molecu:le useful in the method of the invention. This molecul~ selec ively kills IL-2R-expressing tumor cells and lymphocytes (Waters et al ., Eur. J . Immunol . 20:785, l990; Kiyokawa et al., Cancer Res. 49:4042, 1989). Because of its ability to kill activated lymphocytes, DA~3~86IL-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 certa~n autoimmune disorders ~Forte et al., 2nd International Symposi um on Immunotoxins, 1990).
DAB~86IL-2 i6 a chimeric molecule consisting o~
Met followed ~y amino acid residues 1 through 485 of the mature diphtheria toxin fused to amino acid residues 2 through 133 of IL-2. Thus, DAB~86IL-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 DA~3~86IL-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 cyt~ol.
Preparation of DAB~86._~-2 DA~3~86IL-2 was produced in E. coli harboring the DAi3~6IL-2 encoding plasmid, pDW24 (~illiams et al., J.
Biol. Chem. 265:20673, 1990, except ampr is replaced by kanr). The protein was purified by immunoaffinity chromatography and high pressure liquid chromatography (Williams et al., supra).

. . . : .

WO92/19~9 PCT~US92/0371~
- ~ 2 ~

Preparation of DAB389IL-~
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, RezniXioff et al., eds., 1986, Butterworths, Boston), and restriction endonuclease cleavage sites were added to facilitate subsequent cloning steps. IL-4 coding sequence (Hisl to Serl29) 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.
Cytotoxici~y of DAB389IL-4 The ability of DAB389IL-4 to reduce viability of various cell types was measured using an inhibition o~
protein synthesis assay; the results of this assay are presented in Tab}e 3. IC50 (M) is the concentration of DAB389IL-4 required for a 50% decrease in protein synthesis. The rat, Con A-activated, normal splenic lymphocytes were far less sensitive to DAB389IL-4 than any of the other cells or cel} lines. Since the rat interleukin-4 receptor does not bind human interleuXin-4, this result demonstrates the specificity of DAB389IL-4. These rat cel}s are sensitive to a diphtheria toxin/rat interleukin-2 hybrid molecule.

'~ ' ' ' . ;.

.

wos2/ls~s PcT/US92/03714 8 ~ ~ ~

~abl0 3:
DAB~gIL~4 Sensitivity of Normal and Neoplastic Cells and Cel Lines 5 Cell or Cell Classificatlon ICso (M) Line T coll origi~
HUT 102/6TG Human, CTCL, HTLV-I+ 2.9 X 10 C91/PL Human, HTLV-I+, 6.3 X 10-transformed coll origi~
Raji 7.2 X lo 10 Human, Burkitt's ~yolomononuclear lymphoma EBV+
c-ll 2.0 X 10 9 Human, histiocytic Nor~al PB~C l~vmphoma 1.6 X 10 10 PHA activated T cells Human 20 ~o~-primat~ >10-7 Con A-activated normal splenic Rat T cells -PreParation of ~A33891~=~
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 tdeBoer et al., supr~), 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 Mixed Toxins The cytotoxic portion of some molecules useful in the invention can be provided by a mixed toxin moleculeO
A mixed toxin molecl-le is a molecule derived from two W092tl9~9 PCT/US92/03714 ~888~

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 ~inding 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 ~iphtheria toxin and Pseudomonas exotoxin A are well characterized (see, e.g., Hoch et al., Proc . Natl . Acad . Sci . USA 82:1692, 1985; Colombatti et al., J. Biol. Chem. 261:3030, 1986;
and Deleers et al., FE~S Lett. 160:82, 1983), and the existence and location of such a domain in other molecules may be determined by ~ethods 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 enzymatirally active A subunit of E.
col l 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 sam~ way as DAB~86IL-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 : ~in 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, liXe diphtheria - W092/l9~9 PCT/US92/03~1~
: `~ 2 ~

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 en~ymatic portion of DAB186IL-2 catalyzes the ADP-ribosylation by nicotinamide adenine dinu~leotide 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~6IL-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~86IL-2-Linkaqe of Toxins to ~ inq Liqan~
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 liqand 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 Pro2 of the mature form of IL-2. The toxin molecule must W092/19259 2 ~ ~ 8 8 8 ~ PCT/US92/037l~

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 insert:ing 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 tPeptides 3:137, 198}).
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 gro~ps 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 Bindin~ and IL~4 Receptor Bindin~
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 .
VSA 84: 2002, 1987). The IL-4R binding activity of various molecules can be measured usi.~g the assay described by Park et al. (J. Exp. Med. 166:176, 1984) or ,-' ' ', .
.:

~ W092/l9~9 PCT/US9ZtO371~
2 ~ ~

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 descri.bed 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 YT2CZ ~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), 2m~ 1-glutamine, 100 U/ml penicillin, 100 ~g/ml streptomycin, and 10 fetal calf serum (Hazelton, Lenexa, KS). Cells are seeded in 96-well V-bottomed plates (L~nbro-Flow ~aboratories, McLea~, VA) at a concentration of 1 x 105 per well in complete medium. Putative toxins are added to varying concentrations (10 12M to 10 6M) and the cultures are incubated for 18 hrs. at 37C in a 5% C02 atmosphere. Following incubation, the plates are centrifuged for 5 min. at 170 x g, and the medium removed and replaced with 100 ~1 leucine-free medium (MEM, Gibco) containing 8 ~Ci/ml (3H-leucine; New England Nuclear, Boston, MA). After an additional 90 min. at 37C, the plates are centrifuged for 5 min. at 170 x g, the medium i9 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 ~or IL-2R
bearing cells, but utilizing MIA144 c~_ls (Rabin et al.

.

WO92/19~9 PCT/~S9~037t4 J. Immunol . 127:1852~1981) or HUT 102/6TG cells, seeded at 1 x 105 cells per well and incubatecl for 40 hours.
Therapy Generally, the molecules of the invention will be administered by intravenous in~usion. They may also be administered subcutaneously or injectecl directly into the inf lamed joint. Dosages of molecules useful in the methods of the invention t~ill 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; }ess 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 cyto~ines 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 inter~ere 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 prodL_e a non-toxic IL-2/diphtheria toxin hybrid. See Svrluga et al. U.S.

.

:

W0~2/192~9 ~ ~ 8 8 ~ ~ P~T/VS92/0371 Serial No. 590,113, hereby incorporated ~y reference, for an example of such a hybrid molecule.
Monoclonal antihodies-can be used to kill or neutralize 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 ~ill cytokine receptor-bearing cells by activating complement. For tO example, monoclonal antibodies which activate complement can be used to destroy IL-2R-bearing cells. Complement inducing antibodies are generally those of the IgGl, IgG2, IgG3, and IgM isotypes. Monoclonal anti-IL-2R
antibodies can be screened for th~se able to activate complement using a complement-dependent cytotoxicity test, as follows.
Hu~an T-lymphocytes and EBV transformed ~-lymphocytes are labeled with 5lCr sodium chromate and used as target cells; these cells are incubated with hybridoma culture supernatants and with complement, and then the supernatants are collected and counted with a gamma counter. Those supernatants 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 supernatants 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 which b}ock binding and/or uptake of a cytokine. For example, monoclonal antibodies which interfere with the binding and/or uptake of Ih-2 are useful i~ the method of the invention because IL-2R bearing cells deprived of IL-2 fail to proliferate.

~ Wo92/19~ 2 ~ ~ 8 8 ~ ~ P~T/US92J~37~4 Blocking monoclonal antibodies (and other blocking molecules) can be tested for their ability to interfere with IL-2 b~oactivity using the method of Ju et al., (supra) . Generally, assays useful for blocking S molecules will be competitive binding assays which measure the ability of the molecule be:ing to in~erfere 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-lymphocyt~s, fusing the murine splenocytes with appropriate myeloma cells, and screening the antibodies produced by the resultant hybridoma lines for the requisite I~-2R binding properties by means of an ELISA
assay. Antibody production and screening can be performed according to Uchiyama et al. ~J. Im~unol.
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 (~adner 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 tAe molecules described above. For example, cyclosporin A can be administered subsequent to treatment with one of the above-described molecules. Such WO92/19~9 PCT/U592/03714 2 ~ ~8~

subsequent treatment can take place after the arthritic condition has substantially improved as a result of treatment with the above-described molec:ules.
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

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 1 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 DAB486IL-2.

15. The method of claim 13 wherein said molecule is DAB389IL-4.

16. The method of claim 13 wherein said molecule is DAB389IL-6.

17. The method of claim 10 wherein 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 DAB486IL-2.

24. The method of claim 21 wherein said molecule is DAB389IL-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.