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  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
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  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
  • A61K47/6817—Toxins
  • A61K47/6829—Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
• • A—HUMAN NECESSITIES
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  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/5406—IL-4
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  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/5412—IL-6
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/55—IL-2
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
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  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
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  • C07K2319/00—Fusion polypeptide
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  • C07K2319/00—Fusion polypeptide
  • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
  • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
  • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

• 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.
• 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.
• 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.
• specifically binding is meant that the molecule does not substantially bind to other cell receptors or cell surface proteins.
• reduceds viability is meant kills or interferes with proliferation.
• ligand is meant a molecule which is capable of binding to a protein.
• 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.
• 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.
• 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.
• the molecule comprises all or a binding portion of an antibody specific for the cell receptor; and the antibody is a complement activating antibody.
• 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.
• the inflammatory arthritis is rheumatoid arthritis; the molecule is DAB 486 IL-2; and the molecule is DAB 389 IL-2.
• 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.
• 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.
• 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.
• 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.
• DAB 40C IL-2 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).
• 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.
• 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) .
• 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.
• the cells were incubated for 72 hr, pulsed overnight with 0.625 ⁇ Ci/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) .
• 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.
• Rats were immunized with diphtheria toxoid prior to immunization with adjuvant and DAB 48g IL-2 treatment. Rats (75 to 100 g) received 100 ⁇ g 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.
• diphtheria toxoid Massachusetts State Laboratories, Boston, MA
• 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 ⁇ Ci/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 ⁇ g of toxin.
• DAB 48g IL-2 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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)
• 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.
• LAK lymphokine- activated killer
• 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.
• an IL-2/toxin hybrid is a hybrid protein produced by the expression of a fused gene.
• 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 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 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.
• cytotoxin 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.
• toxins such as peptide toxins
• 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.
• 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.
• 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.
• 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.
• cholera toxin ricin
• O-Shiga-like toxin SLT-I, SLT-II, SLT II V
• LT ->xin C3 toxin
• Shiga toxin pertussis toxin
• tetanus toxin tetanus toxin
• Pseudomonas exotoxin alorin
• saporin modeccin
• gelanin gelanin.
• 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 Corynejacteriui ⁇ 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 -
• 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) .
• 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 eukary
• 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.,
• 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.
• 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.
• 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
• DAB 389 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 Table 3:
• 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
• 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.
• 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 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
• 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.
• 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.
• the covalent linkage may take the form of a disulfide bond.
• 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.
• 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.
• a sulfhydryl group either by itself or as part of a cysteine residue, can be introduced using solid phase polypeptide techniques.
• 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.
• 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).
• Molecules of the invention 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 ⁇ g/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 ⁇ l leucine-free medium (MEM, Gibco) containing 8 ⁇ Ci/ml ( 3 H-leucine; New England Nuclear, Boston, MA) . After an additional 90 min.
• MEM leucine-free medium
• 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.
• 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.
• the molecules described above act to decrease cell viability by directing a cytotoxin (or a lytic antibody) to a targeted cell.
• molecules which interfere with the targeted cell's ability to utilize a cytokine are useful in the method of the invention 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 .
• 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.
• antibodies whish block binding and/or uptake of a cytokine 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) .
• 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).
• 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.
• a non-nephrotoxic dosages e.g., preferably not more than 5 mg/kg/d
• 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.

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• 1992
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