NZ614207B2 - Treatment of osteoarthritis and pain - Google Patents

Abstract

Disclosed is the use of a binding protein for the manufacture of a medicament for treating pain and/or osteoarthritis wherein the binding protein binds both IL-1alpha and IL-1beta.

Description

TREATMENT OF OSTEOARTHRITIS AND PAIN Cross-Reference to Related Application This application claims the benefit of priority to United States Provisional Application No. 61/440,853, filed February 8, 2011, the entire contents of which are orated herein by reference.

Fieid 0f the Invention The t invention relates to the treatment of osteoarthritis and pain, and more specifically to the use of proteins that bind lL-loc andlor IL-lB to treat osteoarthritis and pain.

The articular age, or “hyaline cartilage”, of healthy vertebrates (including humans and other mammals) is a semi-transparent, opalescent connective tissue characterized by a columnar growth pattern of chondrocytes in an extracellular matrix (ECM) composed predominantly of proteoglycans, type II collagen, and water. Articular cartilage provides an effective weight-bearing cushion to prevent contact between opposing bones in a joint and thus is critical to the normal function of the joint. Articular cartilage is not only tible to damage by joint trauma, but also to a gradual process of erosion. lly, such an n may be simply an asymptomatic "partial thickness defect" in which an area of reduced hyaline cartilage does not penetrate completely to the subchondral bone. Such partial thickness defects are usually not painful and typically are only detected during arthroscopic examination. However, if the erosive process is not treated, the base of 2O a partial thickness defect may continue to wear away and the diameter of the defect may increase such that the defect eventually progresses to a "full thickness defect" that penetrates the underlying bone. Such full thickness defects may become sufficiently large that surfaces of opposing bones of the joint make t and begin to erode one another, leading to inflammation, pain, and other degenerative changes, i.e., the c symptoms of osteoarthritis (OA). Osteoarthritis is thus a degenerative, progressive, and crippling disease that results in joint deformity, instability, impairment, and pain. ally, joint replacement surgery may be the only practical recourse for restoring, at least in part, some level of mobility to an individual.

The lL—l superfamily is sed of ors of inflammatory processes with a wide range of ical and physiological effects, including fever, prostaglandin sis (in, e.g., fibroblasts, muscle cells and endothelial cells), T-lymphocyte activation, and eukin-2 production. The original members of the IL-1 superfamily are IL-la, IL-1 [3, and the IL-1 Receptor Antagonist (IL-1Ra, IL—lRA, IL—lra, lL-lRa). lL-la and IL-B are flammatory cytokines involved in immune defense against infection. The IL-lRoc competes for receptor binding with IL- lot and lL-lB, ng their role in immune activation. Other members of the IL-1 superfamily include lL-18 (see Dinarello (1994) FASEB J. 8(15):1314—1325; Huising et al. (2004) Dev. Comp.

Immunol. 28(5):395—413) and six additional genes with ural homology to IL-la, IL-lB, or IL- lRA, named ILlFS, IL1F6, IL1F7, IL1F8, ILlF9, and ILlFlO. In accordance, IL-la, IL-lB, and IL- lRA have been renamed IL-lFl, IL—1F2, and IL—1F3, respectively (see Sims et al. (2001) Trends Immunol. :536—537; Dunn et al. (2001) Trends Immunol. 22(10):533—536). A further putative member of the IL-1 family has been described called IL-33 or IL-lFl 1, although this name is not officially accepted in the HGNC gene family nomenclature database.

Both lL—lo. and IL—lB are produced by macrophages, monocytes, and dendritic cells. They form an important part of the inflammatory response of the body against infection. These cytokines increase the expression of adhesion factors on endothelial cells to enable transmigration of leukocytes to sites of infection and re-set the hypothalamus thermoregulatory center, leading to an increased body ature which ses itself as fever. IL-1 is therefore called an endogenous pyrogen. The increased body temperature helps the body’s immune system to fight infection. IL-1 is also important in the regulation of hematopoiesis. IL-lB production in peripheral tissue has also been associated with hyperalgesia (increased sensitivity to pain) associated with fever (Morgan et al. (2004) Brain Res. 1022(1-2):96—100). IL—l upregulates expression of cyclooxygenase-2 (COX-2) associated with pain. For the most part, lL-loc and IL—ll3 bind to the same cellular receptor. This receptor is ed of two related, but non-identical, subunits that transmit intracellular signals via a pathway that is shared in large part with certain other receptors. These include the Toll family of innate immune receptors and the IL—18 receptor. IL—ld and IL-lB also possess similar biological 2O properties, including induction of fever, slow wave sleep, and neutrophilia, T- and B-lymphocyte tion, fibroblast proliferation, cytotoxicity for certain cells, induction of collagenases, synthesis of hepatic acute phase proteins, and increased tion of colony stimulating s and collagen. cDNAs encoding IL-10. and lL—IB have been isolated and expressed; these cDNAs represent two different gene products, termed IL—loc (Lomedico et al. (1984) Nature 312:45 8) and lL-1[3 (Auron et al. (1984) Proc. Natl. Acad. Sci. USA 81:7909). Eight interleukin 1 family genes form a cytokine gene cluster on chromosome 2. IL—l[3 is the predominant form ed by human tes both at the mRNA and protein levels. The two forms of human IL-1 share only 26% amino acid homology. Despite their ct polypeptide sequences, the two forms of IL-1 have structural rities (Auron et al. (1985) J. Mol. Cell Immunol. 2: 169), in that the amino acid homology is confined to discrete regions of the IL-1 molecule. lL-la and IL-lB are produced as precursor peptides. In other words they are made as a long protein that is then processed to release a shorter, active molecule, which is called the mature protein. IL-lu is ed as a proprotein that is proteolytically processed by n and released in a mechanism that is still not well studied. Mature IL-lB, for e, is released from -lB following cleavage by a certain member of the caspase family of proteins, called caspase-1 or the interleukin-1 converting enzyme (ICE). The 3-dimensional ure of the mature forms of each member of the human IL-1 superfamily is composed of 12-14 B-strands producing a barrel-shaped protein.

IL-lo. was originally termed "catabolin" because of its effect in increasing cartilage resorption, but also as "monocyte cell factor" (MCF) because of its stimulatory effect on collagenase and prostaglandin in synovial cells, and as "leukocyte endogenous factor" (LEM) having a stimulatory effect on acute phase reactions. IL—la has a broad spectrum of biological activities, since lL-la is synthesized by many different cells, such as monocytes, macrophages, fibroblasts, endothelial cells and lymphocytes, and many cells possess specific receptors for IL-la. IL-la ates thymocyte proliferation by inducing [L-2 release, B-cell maturation and proliferation, and fibroblast growth factor activity. IL-lot proteins were identified as endogenous pyrogens and are reported to stimulate the release of prostaglandin and collagenase from synovial cells. Thus, IL-lu also es a central on as the trigger for various disorders and symptoms of disorders.

These disorders are often predominantly serious disorders for which there is little or no treatment. It has been suggested that the polymorphism of these genes is associated with rheumatoid arthritis and Alzheimer's disease. IL-1 in l has been implicated in many human es, including arthritis, pulmonary fibrosis, diseases of the central nervous system, diabetes mellitus, and certain cardiovascular diseases. The undesirable s of IL—la are described in, for example, Oppenheim et al. (1986) Immunol. Today 7:45-56, Durum et al. (1985) Ann. Rev. Immunol. 3:263-287 and 2O Symons et al. (1989) Lymphokine Res. 8:365-372.

The initiation, maintenance, and progression of 0A is ed by a x cascade of mechanical and mical pathways in which IL—l plays a pivotal role . IL-loc and IL-lB are produced not only by monocytes, macrophages, and neutrophils, but by cells in joint tissues, such as chondrocytes, synovial lasts, and osteoclasts (see, e.g., Dinarello et al. (2009) Ann. Rev. l. 27: 519-550). In vitro, lL—l can stimulate chondrocytes and synoviocytes to produce proteinases involved in cartilage destruction leading to OA (see, e.g., Dayer et al. (1977) Science 195: 181-183; Dayer et al. (1984) Biochem. Pharmacol. 33: 2893-2899; McGuire-Goldring et al. (1984) Arthritis Rheum. 27: 654-662), as well as inhibit synthesis of proteoglycan and collagen type II, the main components of the extracellular matrix (ECM) of normal hyaline cartilage (see, e.g., 3O Goldring et a1. (1987) J. Biol. Chem. 262: 16724-16729; Goldring et al. (1988) J. Clin. Investig. 82: 2026-2037). Preclinical and clinical s have provided further evidence of IL-1 in the pathogenesis of 0A. For e, intra-articular (ia) injection of IL-1 into animal knees resulted in leukocyte infiltration and cartilage loss (Pettiphar et al. (1986) Proc. Natl. Acad. Sci. USA 83: 8749- 8753). In st, ia injection of IL—1 nist resulted in significant reduction in the progression of experimental OA (see, e.g., Pelletier et al. (1997) Arthritis Rheum. 40: 1012-1019; Caron et al.

[Annotation] ggg (1996) Arthritis Rheum. 39: 544); des et al. (1999) Am. J. Pathol. 154: 11590-11690); Zhang et al. (2006) Biochem. Biophys. Res. Commun. 341: 202-208). In addition, IL-1 knockout (KO) mice were found to be resistant to surgically d cartilage damage when compared to their ype counterparts (Glasson et al. (2009) Osteoarthritis Cartilage, 18: 572-580).

Both IL-1α and IL-1β are sed in synovial membranes, cartilage, and synovial fluid of human OA patients (see, e.g., Farahat et al. (1993) Ann. Rheum. Dis. 52: 870-875).

The IL-1 antagonist, Anakinra, which is an IL-1 or antagonist, and AMG-108, which is an IL-1 receptor monoclonal dy, have demonstrated some efficacy in OA trials with respect to symptoms and chondroprotection ("Results from a Randomized Controlled Trial of AMG 108 (a fully human monoclonal antibody to IL-1R type I) in Patients With rthritis of the Knee" Cohen et al., ACR2007). Both of these proposed therapies await additional studies to demonstrate clear and robust clinical efficacy.

A need remains for new and ive methods and compositions for treating individuals afflicted with osteoarthritis.

Summary of the Invention The invention provides methods for ng osteoarthritis (OA) and for treating pain. Such methods comprise administering to an individual (human or other mammal) one or more binding proteins that bind IL-1α and IL-1β.

In an aspect of the ion, there is provided use of a binding n that binds both IL-1α and IL-1β for the manufacture of a medicament for treating pain in an individual.

In an aspect of the invention, there is provided use of a binding protein that binds both IL-1α and IL-1β for the manufacture of a medicament of treating osteoarthritis in an individual.

In an aspect of the invention, a method for treating osteoarthritis in an individual (human or other mammal) comprises the step of administering to the individual a binding protein that binds IL-1α in combination with (e.g., in a mixture, by successive administration, or by concurrent administration with) a binding protein that binds IL-1β or administering to the individual a binding protein that binds both IL-1α and IL-1β. (10761989_1):GGG In an ment, a method for treating osteoarthritis in an individual comprises administering to the individual a g protein that binds IL-1α, wherein the binding protein is an antibody to IL-1α, for example, a monoclonal antibody to IL-1α. In another embodiment, a method for treating osteoarthritis in an dual comprises stering to the individual a binding protein that binds IL-1β, wherein the binding protein is an antibody that binds IL-1β, for example, a monoclonal antibody that binds IL-1β.

In another aspect of the invention, a method of treating osteoarthritis in an individual (human or other mammal) comprises the step of administering to the individual a g protein that binds both IL-1α and IL-Ιβ. Preferably, the binding protein is a dual variable domain immunoglobulin binding protein (also ed herein as "DVD-Ig™" or "DVD-Ig" binding protein or molecule) that comprises at least one binding site that binds IL-1α and at least one binding site (10084870_1):GGG that binds . More preferably, the DVD—Ig binding protein comprises two binding sites that bind IL-loc and two binding sites that bind IL-1 [3 In another embodiment, a method according to the invention for treating osteoarthritis in an individual comprises administering to the individual a pharmaceutical composition comprising a binding protein that binds IL—loc, a binding protein that binds IL-1 [3, a combination of a binding protein that bind IL-lOL and a binding protein that binds IL—l[3, or a binding protein that binds both IL-la and lL-1[3; and a ceutically acceptable carrier.

In an embodiment of the ion, a method for treating osteoarthritis comprises administering to an individual a crystallized binding protein that binds IL-loc and a crystallized binding protein that binds IL- 1 [3, or a crystallized binding protein that binds both IL-loc and .

Such crystallized binding proteins useful in the invention include, but are not limited to, a crystallized antibody IL-loc, a crystallized dy to IL-l[3, and a crystallized DVD-Ig binding protein that binds both IL—loc and IL—l[3.

Compositions useful in methods of the invention for ng osteoarthritis in an individual include a composition for the e of a llized binding protein that binds IL-l (x, a crystallized g protein that binds IL—l[3, a combination of a crystallized binding protein that binds IL-l 0c and a llized binding protein that binds IL-1 [3, or a crystallized binding protein that binds both ll-loc and IL-1 [3.

In an embodiment, a composition useful in a method for treating osteoarthritis according to the invention comprises: (a) a formulation, wherein said formulation comprises a llized binding protein that binds IL-loc, a crystallized binding protein that binds IL—1[3, a combination of both a crystallized g protein that binds IL-lOt, and a crystallized binding protein that binds IL-l[3, or a crystallized binding n that binds both Il-los and IL—l[3; and, optionally, an ingredient; and (b) at least one polymeric carrier.

In an ment, a composition described above comprises a combination of both a crystallized binding protein that binds IL—loc and a crystallized binding protein that binds IL-1[3. In another embodiment, the crystallized binding protein that binds IL—loc is a crystallized dy, for example, a crystallized monoclonal antibody that binds IL-loc and the crystallized binding protein that binds lL-1[3 is a llized antibody, for example, a monoclonal antibody that binds IL-1[3.

In an ment, a composition described above comprises a crystallized binding protein that binds both ll-loc and lL—1[3. In another embodiment, the crystallized binding protein is a crystallized dual variable domain (DVD-lg) binding protein the binds both IL-loc and IL-l[3-Ig.

In r embodiment, a method for treating osteoarthritis in an dual comprises administering to the individual a composition comprising a crystallized binding protein that binds IL-loc, a crystallized binding protein that binds , a combination of both a crystallized g protein that binds IL-lot and a crystallized binding protein that binds IL-lB, or a crystallized binding protein that binds both Il-l (X, and lL—l [3; wherein said at least one ric carrier is a polymer selected from one or more of the group consisting of poly ic acid), poly (cyanoacrylates), poly (amino acids), poly (anhydrides), poly (depsipeptide), poly s), poly (lactic acid), poly (lactic- co-glycolic acid) or PLGA, poly (b-hydroxybutryate), poly (caprolactone), poly (dioxanone); poly (ethylene glycol), poly ((hydroxypropyl) methacrylamide, poly [(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleic anhydride- alkyl vinyl ether copolymers, pluronic polyols, albumin, alginate, cellulose and cellulose derivatives, en, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfated polysaccharides, blends and copolymers f.

In an embodiment, when the optional ingredient is present in a ition described above, the ingredient is selected from the group consisting of albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-[3-cyclodextrin, methoxypolyethylene glycol and polyethylene glycol.

In an embodiment, a method for treating osteoarthritis described above further comprises administering to the individual a second agent, wherein the second agent provides an additional ble property to the method or composition used in the method. Such a second agent can be one 2O or more molecules in the group consisting of side, epidermal growth factor, corticosteroids, cyclosporin, sulfasalazine, aminosalicylates, aptopurine, azathioprine, idazole, lipoxygenase inhibitors, mesalamine, olsalazine, balsalazide, antioxidants, thromboxane inhibitors, IL-1 receptor antagonists, anti-IL—lB monoclonal antibodies, L-6 monoclonal antibodies, growth s, elastase inhibitors, pyridinyl-imidazole nds, antibodies of TNF, LT, IL-2, IL- 6, IL-7, IL-8, IL-12, IL-13, IL—15, lL-l6, IL—18, IL—23, EMAP-II, GM-CSF, FGF, and PDGF, antibodies of CD2, CD3, CD4, CD8, CD-19, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their s, methotrexate, cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide, an NSAID, ibuprofen, corticosteroids, prednisolone, phosphodiesterase inhibitors, adensosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, IRAK, NIK, IKK, p38, MAP kinase inhibitors, IL-1[3 converting enzyme inhibitors, TNFOL converting enzyme inhibitors, T-cell signalling inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine receptors, soluble p55 TNF receptor, soluble p75 TNF receptor, le-lRI, sIL-lRII, sIL—6R, anti-inflammatory cytokines, IL-4, IL-10, IL- 11, IL-13, and TGFB.

In another aspect of the invention, a method for treating osteoarthritis described herein comprises administering to an individual one or more binding proteins described herein or a ition described herein by at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, epatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, pinal, intrasynovial, intrathoracic, terine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal.

In an aspect of the invention, a method for treating pain in an individual (human or other mammal) comprises the step of administering to the individual a g protein that binds IL-loc in combination with (e.g., in a mixture, by successive administration, or by rent administration with) a binding protein that binds lL-1|3 or administering to the individual a binding protein that binds both IL-loc and IL-lB.

Methods and compositions according to the invention can be used to treat pain in an individual having any form of pain, ing pain from cancer, neuropathic pain, muscle pain, joint pain, bone fracture pain, wound pain, pain from surgery, headache, migraine, as well as pain conditions such as nia (allodynic pain), hyperalgesia, and a combination of allodynia and hyperalgesia.

In an embodiment, a method for treating pain in an individual comprises administering to the individual a binding protein that binds IL—loc, wherein the binding n is an antibody to IL-loc, for example, a monoclonal dy to IL—loc. In another embodiment, a method for treating pain in an individual comprises administering to the dual a binding protein that binds IL-1[3, wherein the binding protein is an antibody that binds , for example, a monoclonal antibody that binds IL- 1 B.

In another aspect of the invention, a method of treating pain in an individual comprises the step of administering to the dual a binding protein that binds both IL-loc and IL-lB.

Preferably, the binding protein is a dual variable domain imrnunoglobulin binding protein (also referred herein as "DVD-IgTM" or “DVD-1g" binding protein or molecule) that comprises at least one binding site that binds IL-lOL and at least one binding site that binds IL-1 [3 Preferably, the DVD-Ig g protein comprises two binding sites that bind IL-loc and two binding sites that bind IL-IB.

In another embodiment, a method according to the invention for treating pain in an individual ses administering to the individual a ceutical composition comprising a binding protein that binds , a binding protein that binds IL-1I3, a combination of a binding protein that bind IL-loc and a g protein that binds IL-lB, or a binding protein that binds both IL—loc and IL-lB; and a pharmaceutically acceptable carrier.

In an embodiment of the invention, a method for treating pain comprises administering to an individual a crystallized binding protein that binds IL—loc and a crystallized binding protein that binds IL-lB, or a crystallized binding protein that binds both IL-loc and IL-lB. Such crystallized g proteins useful in the invention e, but are not limited to, a crystallized antibody IL-loc, a crystallized antibody to lL-lB, and a crystallized DVD—Ig binding n that binds both IL-loc and IL- 1 B Compositions useful in methods of the invention for treating pain in an individual include a composition for the release of a llized binding protein that binds IL-1 on, a llized g protein that binds IL—lB, a combination of a crystallized binding protein that binds IL-loc and a crystallized binding protein that binds IL—lB, or a crystallized binding protein that binds both ll-loc and IL- 1 B.

In an embodiment, a composition useful in a method for treating pain according to the invention comprises: (a) a formulation, wherein said formulation comprises a crystallized binding protein that binds IL-loc, a crystallized binding protein that binds IL—lB, a combination of both a llized binding protein that binds IL-loc and a llized binding protein that binds IL-lB, or a llized binding protein that binds both 11-100 and IL—1 B; and, optionally, an ingredient; and (b) at least one ric carrier.

In an embodiment, a composition described above ses a combination of both a crystallized binding protein that binds IL—loc and a crystallized binding protein that binds IL-lB. In another embodiment, the crystallized binding protein that binds IL-loc is a crystallized antibody, for example, a crystallized monoclonal antibody, that binds IL—loc and the crystallized g protein that binds IL-lB is a crystallized antibody, for example, a monoclonal antibody that binds IL-lB.

In an embodiment, a composition described above comprises a crystallized binding protein that binds both ll-loc and lL-lB. In another embodiment, the crystallized binding protein is a crystallized dual variable domain (DVD-lg) binding protein the binds both IL-loc and IL-lB-Ig.

In another ment, a method for treating pain in an individual comprises administering to the individual a composition comprising a crystallized binding protein that binds IL-loc, a crystallized binding protein that binds IL-lB, a combination of both a crystallized binding protein that binds lL-loc and a crystallized binding protein that binds IL-l B, or a crystallized binding protein that binds both ll-loc and IL—lB; n said at least one polymeric r is a polymer selected from one or more of the group consisting of poly (acrylic acid), poly (cyanoacrylates), poly (amino acids), poly (anhydrides), poly (depsipeptide), poly (esters), poly (lactic acid), poly (lactic-co- glycolic acid) or PLGA, poly (b-hydroxybutryate), poly (caprolactone), poly none); poly (ethylene glycol), poly ((hydroxypropyl) methacrylamide, poly [(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleic anhydride- alkyl Vinyl ether copolymers, pluronic polyols, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfated polysaccharides, blends and copolymers thereof.

In an embodiment, when the optional ingredient is present in a composition described above, the ingredient is selected from the group consisting of albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-l?)-cyclodextrin, methoxypolyethylene glycol and polyethylene glycol.

In an embodiment, a method for ng pain described above r ses administering to the individual a second agent, wherein the second agent es an additional desirable property to the method or composition used in the method. Such a second agent can be one or more les in the group consisting of budenoside, epidermal growth factor, corticosteroids, cyclosporin, sulfasalazine, aminosalicylates, 6-mercaptopurine, azathioprine, metronidazole, lipoxygenase inhibitors, mesalamine, olsalazine, balsalazide, antioxidants, thromboxane inhibitors, IL-1 receptor antagonists, anti-IL—ll3 monoclonal antibodies, anti-IL-6 monoclonal antibodies, growth s, se inhibitors, pyridinyl-imidazole compounds, antibodies of TNF, LT, IL-2, IL- 6, IL-7, IL-8, IL-l2, IL-l3, IL-15, lL-16, IL—18, IL—23, EMAP-H, GM-CSF, FGF, and PDGF, 2O antibodies of CD2, CD3, CD4, CD8, CD-19, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their s, methotrexate, cyclosporin, FK506, rapamycin, mycophenolate mofetil, mide, an NSAlD, ibuprofen, corticosteroids, prednisolone, phosphodiesterase inhibitors, adensosine agonists, antithrombotic agents, complement inhibitors, rgic agents, IRAK, NIK, IKK, p38, MAP kinase inhibitors, IL-lB converting enzyme inhibitors, TNFoc converting enzyme inhibitors, T-cell signalling inhibitors, metalloproteinase tors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine receptors, e p55 TNF or, soluble p75 TNF receptor, , sIL-lRII, sIL-6R, anti-inflammatory cytokines, IL-4, IL-10, IL- 11,IL-13, and TGFB.

In another aspect of the invention, a method for treating pain as described herein comprises administering to an individual one or more binding proteins described herein or a composition described herein by at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, articular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, elial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, yocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal.

In another embodiment, the invention provides a method of treating pain in an individual ing from a disease or disorder associated with lL-l expression. Such IL-1 expression in the individual can result in increased IL-1 levels in the plasma and/or local tissue of the individual.

In an embodiment, the methods and compositions described herein are used to treat pain in an individual suffering from a disease or disorder selected from the group comprising osteoarthritis, rheumatoid arthritis, juvenile chronic arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflammatory bowel disease, insulin dependent diabetes mellitus, thyroiditis, asthma, ic diseases, psoriasis, dermatitis, derma, graft versus host disease, organ transplant rejection, acute or c immune disease associated with organ transplantation, sarcoidosis, sclerosis, disseminated intravascular coagulation, Kawasaki's disease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schoenlein purpurea, microscopic vasculitis of the kidneys, chronic active tis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, infectious diseases, tic diseases, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignancies, heart failure, myocardial tion, Addison‘s disease, sporadic polyglandular ency type I, andular deficiency type II (Schmidt’s syndrome), adult (acute) respiratory distress syndrome, alopecia, ia areata, seronegative arthropathy, arthropathy, 's disease, psoriatic pathy, ulcerative c arthropathy, pathic synovitis, Chlamydia-associated arthropathy, Yersinia-associated arthropathy, Salmonella-associated arthropathy, spondyloarthropathy, atheromatous disease/arteriosclerosis, atopic allergy, autoimmune bullous disease, pemphigus is, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune haemolytic anaemia, Coombs positive haemolytic anaemia, acquired pernicious anaemia, juvenile pernicious a, c encephalitisiRoyal Free disease, chronic mucocutaneous candidiasis, giant cell tis, primary sclerosing hepatitis, cryptogenic mune hepatitis, acquired immunodeficiency syndrome, acquired immunodeficiency related diseases, hepatitis B, hepatitis C, common varied immunodeficiency (common variable mmaglobulinaemia), dilated cardiomyopathy, female infertility, ovarian failure, premature n failure, fibrotic lung disease, cryptogenic fibrosing alveolitis, post-inflammatory interstitial lung disease, interstitial pneumonitis, connective tissue e associated interstitial lung disease, mixed connective tissue disease associated lung disease, systemic sclerosis associated interstitial lung disease, rheumatoid arthritis associated titial lung disease, systemic lupus erythematosus associated lung disease, dermatomyositis/polymyositis associated lung disease, Sjogren's disease ated lung disease, ankylosing spondylitis associated lung e, vasculitic diffuse lung disease, haemosiderosis ated lung disease, drug-induced interstitial lung disease, fibrosis, radiation fibrosis, bronchiolitis obliterans, chronic philic pneumonia, lymphocytic infiltrative lung disease, postinfectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, type-l autoimmune hepatitis (classical autoimmune or lupoid hepatitis), type-2 mune hepatitis (anti-LKM antibody hepatitis), autoimmune mediated hypoglycemia, type B insulin resistance with acanthosis nigricans, rathyroidism, acute immune disease associated with organ transplantation, chronic immune disease associated with organ transplantation, osteoarthrosis, primary sclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathic leucopaenia, autoimmune neutropaenia, renal disease NOS, glomerulonephritides, microscopic itis of the kidneys, Lyme disease, discoid lupus erythematosus, male infertility idiopathic or NOS, sperm munity, multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease, Goodpasture's syndrome, pulmonary manifestation of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sis, Sjorgren's syndrome, Takayasu's disease/arteritis, autoimmune ocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroid disease, hyperthyroidism, us autoimmune hypothyroidism (Hashimoto's disease), atrophic mune hypothyroidism, primary myxoedema, phacogenic uveitis, primary vasculitis, vitiligo, acute liver disease, c liver diseases, lic cirrhosis, alcohol-induced liver injury, cholestasis, idiosyncratic liver disease, drug-induced hepatitis, non-alcoholic steatohepatitis, allergy, group B streptococci (GBS) infection, mental disorders (e.g., depression and schizophrenia), Th2 2O Type and Th1 Type mediated diseases, acute and chronic pain rent forms of pain), cancers such as lung, breast, stomach, bladder, colon, pancreas, ovarian, prostate and rectal cancer and hematopoietic malignancies (leukemia and lymphoma), abetalipoproteinemia, acrocyanosis, acute and chronic parasitic or infectious processes, acute leukemia, acute lymphoblastic leukemia (ALL), acute d leukemia (AML), acute or chronic bacterial infection, acute pancreatitis, acute renal failure, adenocarcinomas, atrial ectopic beats, AIDS dementia complex, alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis, allograft rejection, alpha-l- antitrypsin deficiency, amyotrophic lateral sclerosis, anemia, angina pectoris, anterior horn cell degeneration, anti-CD3 therapy, antiphospholipid me, anti—receptor ensitivity reactions, aortic and peripheral aneurysms, aortic tion, arterial hypertension, arteriosclerosis, 3O arteriovenous fistula, ataxia, atrial fibrillation (sustained or paroxysmal), atrial flutter, atrioventricular block, B cell lymphoma, bone graft rejection, bone marrow transplant (BMT) ion, bundle branch block, Burkitt's lymphoma, burns, cardiac hmias, cardiac stun syndrome, cardiac tumors, cardiomyopathy, cardiopulmonary bypass inflammation response, cartilage lant rejection, cerebellar cortical degenerations, cerebellar disorders, chaotic or multifocal atrial tachycardia, chemotherapy associated disorders, chronic myelocytic ia (CML), chronic alcoholism, c atory pathologies, chronic lymphocytic leukemia (CLL), chronic obstructive pulmonary disease (COPD), chronic salicylate intoxication, colorectal carcinoma, congestive heart failure, conjunctivitis, contact dermatitis, cor pulmonale, coronary artery e, Creutzfeldt-Jakob disease, culture negative sepsis, cystic fibrosis, ne therapy associated ers, dementia pugilistica, demyelinating diseases, dengue hemorrhagic fever, dermatitis, dermatologic conditions, diabetes, diabetic arteriosclerotic disease, diffuse Lewy body disease, dilated congestive cardiomyopathy, ers of the basal ganglia, Down's syndrome in middle age, drug- d movement disorders induced by drugs which block CNS dopamine receptors, drug sensitivity, eczema, encephalomyelitis, rditis, inopathy, epiglottitis, Epstein-Barr virus infection, erythromelalgia, extrapyramidal and cerebellar disorders, familial hemophagocytic lymphohistiocytosis, fetal thymus implant rejection, Friedreich's , functional peripheral arterial disorders, fungal , gas ne, gastric ulcer, glomerular nephritis, graft rejection of any organ or tissue, gram negative sepsis, gram positive sepsis, granulomas due to intracellular organisms, hairy cell leukemia, Hallervorden-Spatz disease, oto's thyroiditis, hay fever, heart transplant rejection, hemochromatosis, hemodialysis, hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura, hemorrhage, hepatitis A, His bundle arrhythmias, HIV infection/HIV neuropathy, Hodgkin's disease, hyperkinetic movement disorders, hypersensitivity reactions, hypersensitivity nitis, hypertension, hypokinetic movement disorders, hypothalamic-pituitary-adrenal axis evaluation, idiopathic Addison's disease, idiopathic pulmonary fibrosis, antibody mediated xicity, asthenia, infantile spinal muscular atrophy, inflammation of the aorta, influenza A, ionizing radiation exposure, iridocyclitis/uveitis/optic neuritis, ia- reperfusion injury, ischemic stroke, juvenile rheumatoid tis, juvenile spinal muscular atrophy, Kaposi's sarcoma, kidney transplant rejection, legionella, leishmaniasis, leprosy, lesions of the corticospinal system, lipedema, liver lant rejection, lymphedema, malaria, malignant lymphoma, malignant histiocytosis, malignant melanoma, meningitis, meningococcemia, metabolic migraine he, idiopathic migraine headache, mitochondrial ystem disorder, mixed connective tissue disease, monoclonal gammopathy, multiple myeloma, multiple systems degenerations (Menzel, Dejerine-Thomas, Shy-Drager, and Machado-Joseph), myasthenia graVis, mycobacterium avium intracellulare, mycobacterium tuberculosis, ysplastic syndrome, myocardial infarction, myocardial ischemic ers, nasopharyngeal carcinoma, al chronic lung disease, nephritis, sis, neurodegenerative diseases, neurogenic muscular atrophies, neutropenic fever, non- Hodgkin's lymphoma, occlusion of the abdominal aorta and its branches, occlusive arterial disorders, OKT3® therapy, orchitis/ epididymitis, orchids/vasectomy reversal procedures, organomegaly, osteoporosis, pancreas transplant rejection, pancreatic oma, paraneoplastic syndrome/hypercalcemia of malignancy, parathyroid transplant rejection, pelvic inflammatory e, perennial rhinitis, pericardial disease, peripheral atherosclerotic disease, eral vascular disorders, nitis, pernicious anemia, pneumocystis i pneumonia, pneumonia, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes syndrome), post perfusion me, post pump syndrome, post-MI cardiotomy syndrome, preeclampsia, progressive supranucleo palsy, primary pulmonary hypertension, radiation therapy, d‘s phenomenon, Raynaud's e, Refsum's disease, regular narrow QRS ardia, renovascular ension, reperfusion injury, restrictive cardiomyopathy, sarcomas, senile chorea, senile dementia of Lewy body type, gative arthropathies, shock, sickle cell anemia, skin allograft rejection, skin changes syndrome, small bowel transplant rejection, solid tumors, specific arrhythmias, spinal ataxia, spinocerebellar degenerations, streptococcal is, ural lesions of the cerebellum, te sclerosing panencephalitis, syncope, is of the cardiovascular system, systemic anaphylaxis, ic inflammatory response syndrome, systemic onset juvenile toid arthritis, T-cell or FAB ALL, telangiectasia, thromboangiitis obliterans, thrombocytopenia, toxicity, transplants, trauma/hemorrhage, type III hypersensitivity reactions, type IV hypersensitivity, unstable , uremia, urosepsis, urticaria, valvular heart diseases, varicose veins, vasculitis, venous diseases, venous thrombosis, ventricular fibrillation, viral and fungal infections, viral encephalitisiaseptic itis, viral-associated hemophagocytic syndrome, Wernicke- Korsakoff me, Wilson's disease, xenograft rejection of any organ or tissue, acute coronary syndromes, acute idiopathic polyneuritis, acute inflammatory demyelinating polyradiculoneuropathy, acute ischemia, adult Still’s disease, alopecia , anaphylaxis, anti-phospholipid antibody syndrome, ic anemia, arteriosclerosis, atopic eczema, atopic dermatitis, autoimmune dermatitis, autoimmune disorder associated with ococcus infection, autoimmune enteropathy, autoimmune hearing loss, autoimmune lymphoproliferative syndrome (ALPS), autoimmune myocarditis, autoimmune premature ovarian failure, blepharitis, bronchiectasis, s pemphigoid, cardiovascular disease, catastrophic antiphospholipid syndrome, celiac disease, cervical spondylosis, chronic ischemia, cicatricial pemphigoid, clinically isolated syndrome (CIS) with risk for multiple sclerosis, childhood onset psychiatric disorder, chronic obstructive pulmonary disease (COPD), dacryocystitis, dermatomyositis, diabetic pathy, disk herniation, disk se, drug induced immune hemolytic anemia, endocarditis, endometriosis, endophthalmitis, episcleritis, erythema multiforme, erythema multiforme major, gestational goid, Guillain-Barré syndrome (GBS), hay fever, Hughes syndrome, thic Parkinson’s disease, idiopathic interstitial pneumonia, IgE-mediated allergy, immune hemolytic anemia, inclusion body myositis, infectious ocular inflammatory disease, inflammatory inating disease, inflammatory heart disease, inflammatory kidney disease, IPF/UIP, iritis, keratitis, keratojunctivitis sicca, Kussmaul disease or Kussmaul-Meier disease, Landry's paralysis, Langerhan's cell histiocytosis, livedo reticularis, macular ration, microscopic polyangiitis, Morbus Bechterev, motor neuron disorders, mucous membrane pemphigoid, multiple organ failure, myasthenia gravis, myelodysplastic syndrome, myocarditis, nerve root disorders, neuropathy, non-A non-B hepatitis, optic neuritis, osteolysis, ovarian cancer, pauciarticular JRA, peripheral artery occlusive disease (PAOD), peripheral vascular disease (PVD), peripheral artery disease (PAD), phlebitis, polyarteritis nodosa (or periarteritis nodosa), polychondritis, polymyalgia rheumatica, poliosis, polyarticular JRA, polyendocrine deficiency me, polymyositis, polymyalgia rheumatica (PMR), post-pump syndrome, primary Parkinsonism, prostate and rectal cancer and hematopoietic malignancies (leukemia and ma), prostatitis, pure red cell aplasia, primary l insufficiency, recurrent yelitis optica, restenosis, rheumatic heart disease, SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis), secondary dosis, shock lung, scleritis, ca, secondary adrenal insufficiency, silicone associated connective tissue disease, Sneddon- Wilkinson dermatosis, spondylitis ankylosans, Stevens-Johnson syndrome (SJS), systemic inflammatory response syndrome, temporal arteritis, toxoplasmic retinitis, toxic epidermal ysis, transverse myelitis, TRAPS (tumor-necrosis factor receptor type 1 (TNFR)-associated periodic me), type 1 allergic reaction, type II diabetes, urticaria, usual interstitial pneumonia (UIP), itis, vernal conjunctivitis, viral retinitis, Vogt-Koyanagi-Harada syndrome (VKH syndrome), wet macular degeneration, and wound healing.

In an embodiment, the methods and compositions bed herein are used to treat pain in an individual suffering The method for treating pain in an individual according to claim 16, n the individual is suffering from a disease selected from the group consisting of a primary , a metastatic cancer, breast , colon cancer, rectal cancer, lung cancer, oropharynx cancer, hypopharynx cancer, esophagus cancer, stomach , pancreatic cancer, liver cancer, gallbladder cancer, bile duct , small intestine cancer, colon cancer, urinary tract cancer, kidney , bladder cancer, urothelium cancer, female genital tract cancer, cervical cancer, uterine cancer, ovarian cancer, choriocarcinoma, gestational trophoblastic disease, male genital tract cancer, prostate cancer, seminal vesicle cancer, ular cancer, germ cell tumor, endocrine gland cancer, thyroid cancer, adrenal cancer, pituitary gland cancer, skin cancer, hemangioma, melanoma, a, bone cancer, soft tissue cancer, Kaposi’s sarcoma, tumor of the brain, nerve cancer, eye cancer, cancer of the meninges, astrocytoma, glioma, glioblastoma, retinoblastoma, neuroma, lastoma, Schwannoma, meningioma, a solid tumor arising from hematopoietic malignancy, leukemia, Hodgkin's lymphoma, and non -Hodgkin's lymphoma. 3O Brie Descri tion 0 the Drawin s Figure 1A is a schematic representation of Dual Variable Domain Immunoglobulin (DVD- Ig) constructs and shows the strategy for generating a DVD-lg molecule from two parent antibodies.

Figure 1B shows schematic representations of c constructs for DVDl-Ig, DVD2—Ig, and two chimeric, ecific, monoclonal dies 2D13.E3 lL—loc) and 13F5.G5 (anti- IL—1[3). "VHB" and "VL[3" indicate, respectively, a heavy chain variable domain and a light chain le domain of an antigen g site of the 13F5.G5 antibody that binds IL-[3. "VHoc" and "VLoc" indicate, respectively, a heavy chain le domain and a light chain variable domain of the 3D12.E3 antibody that binds IL-lot. "L" indicates a leader ce. In m of genetic construct for DVD2-Ig, horizontal markings between "VHB" and "VHoc" and between "VLB" and "VLoc" indicate presence of a linker sequence.

Figure 2 shows bar graphs of histology scoring of articular cartilage of mice in a joint instability model (JIM) of osteoarthritis. Each set of bar graphs provides scoring for articular cartilage in lmees of mice in four treatment groups: treatment with phosphate buffered saline (PBS) vehicle only ("PBS"), treatment with an anti-IL-la monoclonal antibody ("anti-IL-la mAb"), treatment with an anti-IL-lB monoclonal antibody ("anti-IL-lB mAb"), and treatment with a combination of anti-IL—lot and anti-IL-lB monoclonal antibodies ("anti-IL-lu mAb + anti-IL-IB mAb"). Individual bar graphs within each set show a total histology score and scores for three separate zones, wherein each zone is one-third of the area of the medial tibia cartilage (zone 1: inside zone, zone 2: middle zone, and zone 3: outside zone). From left to right: bar graph of total score for medial tibia zones 1, 2, and 3; bar graph of score for medial tibia zone 1; bar graph of score for medial tibia zone 2; and bar graph of score for medial tibia zone 3. See e 3.3.1.

Figure 3 shows bar graphs of ogy scoring of articular cartilage of mice in a joint instability model (JIM) of osteoarthritis. Each set of bar graphs provides scoring for articular cartilage in knees of mice in four ent groups: treatment with PBS vehicle only cle"), 2O treatment with a combination of anti-IL—la monoclonal antibody and anti-IL-l B monoclonal antibody ("anti-IL-loc mAb (6 mg/kg) + anti—IL-lB mAb (6 mg/kg)"), treatment with mIL-loc/[3 DVD-Ig binding protein at 6 mg/kg ("IL—1(x/[3 DVD-Ig (6 mg/kg)"), and treatment with mIL-loc/[3 DVD-Ig binding protein at 12 mg/kg ("IL—la/B DVD-Ig (12 mg/kg)"). Individual bar graphs within each set show a total histology score and scores for three separate zones, wherein each zone is one- third of the area of the medial tibia cartilage (zone 1: inside zone, zone 2: middle zone, and zone 3: outside zone). From left to right: bar graph of total score for medial tibia zones 1, 2, and 3; bar graph of score for medial tibia zone 1; bar graph of score for medial tibia zone 2; and bar graph of score for medial tibia zone 3. See Example 3.3.2.

Figure 4 shows bar graphs of ogy scoring of articular cartilage of mice in a joint instability model (JIM) of osteoarthritis. Each set of bar graphs provides scoring for articular cartilage in lmees of mice in four treatment groups: treatment with PBS vehicle only cle"), treatment with a combination of anti-IL-la monoclonal antibody (6 mg/kg) and anti-IL-IB monoclonal dy (6 mg/kg) ("anti-IL—loc mAb (6 mgi’kg) + anti-IL-ll3 mAb (6 mg/kg)"), ent with an L—lB monoclonal antibody (12 mg/kg) ("anti-IL-lB mAb (12 mg/kg)"), treatment with an anti-IL—lB monoclonal antibody (6 mg/kg) ("anti-IL-lB mAb (6 mg/kg)"), and treatment with an anti-IL-lB monoclonal antibody (3 mg/kg) ("anti-IL-lB mAb (3 mg/kg)").

Individual bar graphs within each set show a total histology score and scores for three separate zones, wherein each zone is one-third of the area of the medial tibia cartilage (zone 1: inside zone, zone 2: middle zone, and zone 3: outside zone). From left to right: bar graph of total score for medial tibia zones 1, 2, and 3; bar graph of score for medial tibia zone 1; bar graph of score for medial tibia zone 2; and bar graph of score for media] tibia zone 3. See Example 3.3.3.

Figure 5 shows the level of zymosan—induced IL—6 production ) in animals in a joint instability model of osteoarthritis when treated with PBS vehicle only ("Vehicle"), with a combination of anti-IL-la onal antibody (6 mgfkg) and anti-IL-lB monoclonal antibody (6 mg/kg) ("anti-IL-IOL mAb (6 mg/kg) + anti-IL-ll3 mAb (6 mg/kg)"), With mIL-loc/B DVD-lg binding protein at 6 mg/kg ("lL—loc/B DVD (6 mg/kg)"), and treatment with mIL-loc/B DVD-lg binding protein at 12 mg/kg ("IL-10MB DVD (12 "). See Example 3.3.4.

Figure 6 shows total sum score for cartilage degeneration in animals in a destabilization of medial meniscus (DMM) model of osteoarthritis treated the PBS vehicle ("Vehicle"), with a combination of anti-lL-ld monoclonal antibody (6 mg/kg) and L-lB monoclonal antibody (6 mg/kg) ("anti-lL-loc mAb (6 mg/kg) + anti-IL—ll3 mAb (6 mg/kg)"), and with mlL-loc/B DVD-lg binding protein at 6 mg/kg oc/[3 DVD (6 mg/kg)"). See Example 3.3.5.

Figure 7 shows the results of an 8 week study of various treatments on cartilage degeneration in animals in a ilization of medial meniscus (DMM) model of osteoarthritis. 2O Each bar graph indicates the mean sum score for cartilage degeneration in animals treated with PBS vehicle only ("Vehicle"), with anti-IL—la onal antibody (6 mg/kg) ("anti-IL-loc mAb 6 mg/kg"), with anti-IL-lB monoclonal antibody (6 mg/kg) ("anti-lL-lB 6 mg/kg"), with a combination of anti-IL-lo. monoclonal antibody (6 mg/kg) and anti-IL-lB monoclonal antibody (6 mg/kg) ("anti-IL-loc mAb (6 mg/kg) + anti-IL—ll3 mAb (6 mgi’kg)"), With mIL-loc/[3 DVD-lg g protein at 1.5 mg/kg ("IL-10MB DVD 1.5 mg/kg"), with mIL-loc/B DVD-lg binding n at 3 mg/kg ("IL-10MB DVD 3 mg/kg"), or with mIL-loc/B DVD-lg binding protein at 6 mg/kg oc/B DVD 6 mg/kg"). See Example 3.3.6.

Figure 8 shows the s of a 4 week study of ng animals in a destabilization of medial meniscus (DMM) model of osteoarthritis with vehicle alone ("Vehicle"), doxycycline (30 3O mg/kg) ("Doxycycline (30 mg/kg)“), or a combination of an anti-IL-la monoclonal antibody (6 mg/kg) and an anti-IL-IB monoclonal antibody (6 mg/kg) ("anti-IL-l on mAb (6 mg/kg) + anti-lL-IB mAb (6 mg/kg)"). See e 33?.

Figure 9 shows bar graphs of paw withdrawal old (grams, "g") in paws of mice with DMM surgery on Days 7, 14, 21, 28, and 35. Paw withdrawal threshold of DMM paw ("DMM") was significantly decreased as compared to paws from lateral (non-surgical) paw ("Contra") and sham surgery ("Sham"). DMM paws were allodynic as early as day 7 and exhibited this pain behavior through day 35. See Example 4.

Figure 10 shows bar graphs of paw withdrawal threshold (grams, "g") in DMM mice following treatment vehicle alone ("PBS Control"), with IgG isotype control (positive control for established disease and pain "IgG Control"), or with a combination of anti-IL-lu and anti-IL-lB monoclonal antibodies ("anti-IL—los mAb (6 mg/kg) + anti-IL—lB mAb (6 mg/kg)") after 5 weeks (day 35) compared to paws from contralateral (non—surgical) paw ("Contralateral") and sham surgery ("Sham"). The data indicate that neutralization of both IL-loc and IL-IB significantly prevented the development of allodynia. Similar efficacy was observed in animals treated after one week (not shown). See Example 4.

Figure 11 shows bar graphs of paw withdrawal threshold , "g") in DMM mice previously treated with IgG isotype control (positive control for established disease and pain) for 35 days and then dosed with a combination of anti-lL—lo or L-lB monoclonal antibodies and tested for paw withdrawal threshold 24 hours later at day 36 ("anti-lL-loc mAb (6 mg/kg) + anti-IL- IB (6 mg/kg) (24 hours post dosing)"). For comparison, paw withdrawal threshold values at day 35 are provided for contralateral (non-surgical) paw ("Contralateral"), sham y paw ("Sham"), vehicle only treatment ("PBS Control"), IgG isotype ent ("IgG Control"), and al paw d with a combination of L-la and anti-IL—lB monoclonal antibodies -IL-loc mAb (6 mg/kg) + anti-lL-lB mAb (6 mg/kg)") after 5 weeks (day 35). The data indicate that the 2O neutralization of both IL-loc and IL—lB significantly reversed allodynia in mice with established pain. See Example 4.

Figure 12 shows bar graphs of paw withdrawal threshold (grams, "g") in DMM mice treated with PBS vehicle only ("Vehicle"), with anti-IL—la monoclonal antibody (6 mg/kg) ("anti-IL-loc mAb (6 mg/kg)"), with anti-IL-lB onal antibody (6 mg/kg) ("anti-IL-lB mAb (6 mg/kg)"), or with a combination of anti-lL—lo. monoclonal antibody (6 mgi’kg) and anti-IL-lB monoclonal antibody (6 mg/kg) ("anti-lL-loc mAb (6 mg/kg) + anti-IL—lB mAb (6 mg/kg)") stered intraperitoneally (ip) twice per week for four weeks. Animals were tested for allodynia on day 28.

"Contralateral" bar graph provides paw withdrawal threshold of a representative rgical a lateral) limb. The teral (surgical) limb of animals in groups treated with vehicle was allodynic 3O (painful) compared to the contra lateral (non-surgical) limb or compared to animals that had undergone sham y ("Sham"). See Example 5.

Figure 13 shows bar graphs of paw withdrawal threshold (grams, "g") in DMM mice treated with PBS vehicle only ("Vehicle") or with a combination of anti-lL-lu monoclonal antibody and anti-lL-lB monoclonal antibody, both administered eritoneally (ip) every four days for four weeks at 1 mg/kg ("anti-IL—loc mAb (1 mg/kg) + anti-IL—ll3 mAb (1 mg/kg)"), at 3 mg/kg ("anti-IL- 10c mAb (3 mg/kg) + anti-lL-lB mAb (3 mg/kg)"), or at 6 mg/kg ("anti-lL-loc mAb (6 mg/kg) + anti- IL-1[3 mAb (6 mg/kg)"). Animals were tested on day 28. The ipsilateral (surgical) limb of animals in groups treated with PBS vehicle only was allodynic (painful) compared to the contra lateral (non- al) limb ("Contralateral") or ed to animals that had undergone sham surgery ("Sham").

The results show that treatment with a ation of anti-IL-l (1 monoclonal antibody and anti-IL- 1B monoclonal antibody prevents allodynia development in DMM mice in a dose related manner.

See Example 6.

Figure 14 shows bar graphs of paw withdrawal threshold (grams, "g") when DMM mice with ished osteoarthritis and mechanical allodynia were treated at day 27 with a combination of anti-lL-la monoclonal antibody and anti-IL-lB monoclonal antibody, both stered intraperitoneally (ip) at 1 mg/kg (“anti-IL-lrx mAb (1 mg/kg) + anti-IL-IB mAb (1 mg/kg)"), at 3 mg/kg ("anti-lL—loc mAb (3 mg/kg) + anti-lL-ll3 mAb (3 mgi’kg)"), or at 6 mg/kg ("anti-IL-loc mAb (6 mg/kg) + anti-lL-lB mAb (6 mg!kg)") at 24 hours prior to testing for allodynia on day 28. The ipsilateral (surgical) limb of animals in groups treated with vehicle ("Vehicle") was allodynic (painful) compared to the contra lateral (non-surgical) limb ("Contralateral") or compared to animals that had undergone sham surgery ("Sham"). The s show that treatment with a combination of anti-lL-la monoclonal antibody and anti-lL—l B monoclonal antibody reverses established nia in DMM mice with established e in a dose d manner. See Example 7.

Figure 15 shows bar graphs of paw withdrawal latency (seconds, "s") in animals in a mouse 2O carrageenan-induced inflammatory pain (hyperalgesia) model. At 30 hours after intraplantar carrageenan injection, mice were treated with anti-IL-la monoclonal antibody alone (900 ug) - IL-loc mAb"), anti-lL-lB monoclonal dy alone (900 ug) ("anti-IL-lB mAb"), a combination of anti-lL-la monoclonal antibody (900 ug) and anti-IL—lB monoclonal antibody (900 ug) ("anti-IL-loc mAb + anti-lL-lB mAb"), PBS vehicle ("PBS"), or IgG isotype control ("IgG"). Another group was administered a dose (30 mg/kg) of diclofenac (non-steroidal anti-inflammatory analgesic control) at 47 and 95 hours after intraplantar administration of carrageenan. Thermal lgesia testing using radiant heat stimulus was performed 48 hours (Figure 15A) and 96 hours (Figure 15B) after intraplantar administration of carrageenan. Filled bars show paw withdrawal latency for l paw (no eenan). Open bars show paw withdrawal latency for paw administered carrageenan. See e 8.

Figure 16 shows bar graphs of paw withdrawal latency (seconds, "s") in s in a mouse carrageenan-induced inflammatory pain (hyperalgesia) model in mice. At 30 hours after intraplantar carrageenan injection, mice were treated with e ("PBS"), IgG isotype control ("IgG"), or one of three doses of a combination of anti-lL-la monoclonal antibody and anti-IL-IB monoclonal antibody, wherein each monoclonal antibody was administered at 100 ug ("anti-IL-loc mAb (100 ug) + anti-IL-lB mAb (100 ug) "), at 300 ug ("anti—IL-loc mAb (300 Mg) + anti-lL-lB mAb (300 ug)"), or at 900 ug ("anti-IL-loc mAb (900 ug) + L—ll3 mAb (900 ug)"). Another group was administered a dose (30 mg/kg) of diclofenac teroidal anti-inflammatory analgesic positive control) at 47 and 95 hours after intraplantar administration of carrageenan. Thermal hyperalgesia testing using radiant heat stimulus was performed at 48 hours (Figure 16A) and 96 hours (Figure 16B) after intraplantar administration of carrageenan. Filled bars show paw withdrawal latency for control paw (no carrageenan). Open bars show paw withdrawal y for paw administered eenan. See Example 9.

Figure 17 shows results of testing anti-IL-loc and anti-IL-ll3 monoclonal antibody combination therapy in a CFA ("Complete Freund's Adjuvant) inflammatory pain model in mice. At hours after intraplantar CFA injection, mice were treated with a combination of L-la monoclonal antibody (900 ug) and anti-IL-IB monoclonal antibody (900 ug), with PBS vehicle ("PBS"), or with IgG isotype control ("IgG"). Another group was administered a dose (30 mg/kg) of diclofenac (non-steroidal, anti-inflammatory analgesic positive control) at 47 hours after intraplantar administration of CFA. Mechanical allodynia testing of animals using a von Frey monofilament was performed at 48 hours after CFA administration. Figure 17A shows bar graphs of paw withdrawal threshold (grams, "g"). Filled bars show paw withdrawal threshold for contra lateral (no CFA) l paws. Open bars show paw withdrawal threshold for paw administered CFA in animals of treatment groups. Figure 17B shows bar graphs for magnitude of efficacy (% MPE) for s of 2O ent groups. See Example 10.

Figure 18 shows bar graphs of paw withdrawal threshold (grams, "g") in animals of an L5/L6 spinal nerve ligation (SNL) mouse model of neuropathic pain. Animals were treated at day 6 after SNL surgery with a combination of anti-lL-la monoclonal antibody (900 ug) and anti-lL-lB onal antibody (900 ug) ("anti—IL—loc mAb + anti-IL—lB mAb"), PBS vehicle ("PBS"), or IgG isotype control ("IgG"). ical allodynia testing of animals using a von Frey monofilament was performed at 24 hours (Figure 18A) and 72 hours (Figure 18B) after SNL surgery. Another group was treated with gabapentin (100 mg/kg, "Gabapentin") at 1 hour prior to testing as a positive control. See Example 11.

Figure 19 shows bar graphs for magnitude of efficacy (% MPE) for animal treatment groups at 24 and 72 hours as bed for Figure 18. "Vehicle" indicates SNL animals treated with PBS vehicle d. "IgG" indicates SNL animals treated with IgG isotype control. "IL-locB" indicates animals treated with ation anti-IL-la monoclonal antibody (900 ug) and L-ll3 onal antibody. See Example 11. ed Description of the Invention The invention is based on the discovery that blocking the function of interleukin-1 (IL-1) can be an effective means to treat osteoarthritis (0A) in an individual (human or other ).

According to the invention, blocking IL—l function for treating OA may be achieved by administering to an individual one or more proteins that bind IL-loc and IL-1 [3. Such a "dual- specific" therapy can be achieved by stering to an OA patient a binding n (e.g., an antibody) that binds lL-loc and a binding protein (e.g., an antibody) that binds IL-lB or by administering a multivalent and multispecific binding protein that binds both IL-loc and IL-1[3. Such a multivalent and multispecific binding protein useful in the ion includes a dual le domain immunoglobulin binding protein (also referred herein as "DVD-IgTM" or "DVD-lg" binding protein or molecule). See, e.g., PCT Publication No. and Wu et a1. (2007) Nature Biotech. 25(11): 1290-1297. Whether a particular combination of IL-loc and IL-ll3 binding proteins or a specific DVD-lg molecule that binds both lL-loc and IL—lB will be useful for the treatment of OA can be assessed using an animal model of OA, such as the joint instability model (JIM) of 0A or the destabilization of medial meniscus (DMM) model of 0A (Glasson et al. (2007) Osteoarthrit.

Cart. 1061-9).

Unless otherwise defined herein, scientific and cal terms used in connection with the present invention shall have the meanings that are ly understood by those of ry skill in the art. The g and scope of the terms should be clear, however, in the event of any latent 2O ambiguity, definitions ed herein take precedent over any dictionary or extrinsic ion.

Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of the term "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including", as well as other forms, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" encompass both ts and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.

Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and nucleic acid hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present invention are lly performed according to conventional s well lmown in the art and as described in various general and more specific references that are cited and sed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and ly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, ation, and delivery, and treatment of patients.

That the present invention may be more readily understood, select terms are defined below.

The term "polypeptide" means any polymeric chain of amino acids. The terms "peptide" and "protein" are used interchangeably with the term polypeptide and also refer to a polymeric chain of amino acids. The term eptide“ encompasses native or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. A polypeptide may be monomeric or polymeric.

The term "isolated protein“ or "isolated ptide" means a protein or polypeptide that by virtue of its origin or source of derivation is not associated with naturally associated components that accompany it in its native state, is substantially free of other proteins from the same species, is expressed by a cell from a different s, or does not occur in nature. Thus, a ptide that is chemically synthesized or sized in a cellular system different from the cell from which it naturally originates will be "isolated" from its naturally associated ents. A protein may also be ed substantially free of naturally ated components by isolation, using protein purification techniques well known in the art.

The term "recovering" means the process of rendering a chemical species such as a polypeptide substantially free of naturally associated components by isolation, e.g., using protein purification ques well known in the art.

The term "human IL-loc" (also abbreviated herein as "hIL-loc" or "IL-10c"), includes a pleiotropic cytokine involved in various immune responses, inflammatory processes, and hematopoiesis. For example, IL—loz includes the human cytokine produced by activated macrophages; it ates thymocyte proliferation by inducing IL-2 release, B-cell maturation and proliferation, and fibroblast growth factor activity. The term "human IL-loc" is intended to include recombinant human IL-10L("rh IL—los") that can be prepared by standard recombinant expression methods.

The term IL—1[3" (also abbreviated herein as "hIL—l [3", or "IL—lb") includes a pleiotropic cytokine involved in various immune responses, inflammatory processes, and hematopoiesis. The term human “IL-15" includes recombinant human IL-ll3 ("rh IL-ll3") that can be prepared by rd inant sion methods.

The amino acid sequences of human IL-lu and IL-lB are shown in Table l.

Table 1: ces of Human IL-la and IL-lfi Protein Sequence Sequence Identifier 123456789012345678901234567890 Human pro IL—la SEQ ID N021 MAKVPDMFEDLKNCYSENEEDSSSIDHnSL NQKSFYHVSYGPLHEGCMDQSVSLSISETS KTSKLTFKESMVVVATNGKVLKKRRLSLSQ SITDDDLEAIANDSEEEIIKPRSAPFSFLS NVKYNFMRIIKYEFILNDALNQSIIRANDQ YLTAAALHNLDEAVKFDMGAYKSSKDDAKI TVILRISKTQLYVTAQDEDQPVLLKEMPEI PKTITGSETNLLFFWETHGTKNYFTSVAHP NLFIATKQDYWVCLAGGPPSITDFQILENQ Human mature Residues 113- SAPFSFLSNVKYNFMRIIKYEFILNDALNQ IL—la 271 of SEQ ID SIIRANDQYLTAAALHNLDEAVKFDMGAYK no;1 SSKDDAKITVILRISKTQLYVTAQDEDQPV LLKEMPEIPKTITGSETNLLFFWETHGTKN YFTSVAHPNLFIATKQDYWVCLAGGPPSIT DFQILENQA Human mature SEQ ID NO:2 APVRSLNCTLRDSQQKSLVMSGPYELKALH IL—lB LQGQDMEQQVVFSMSFVQGEESNDKIPVAL GLKEKNLYLSCVLKDDKPTLQLESVDPKNY PKKKMLKRthNKIbINNKthbSAQtPNW YISTSQAENMPVFLGGTKGGQDITDFTMQF The term "biological ty" refers to all inherent ical properties of the cytokine.

Biological properties of IL-lu and lL-IB include, but are not limited to, binding to an IL-1 receptor.

"Biological activity" refers to all inherent biological ties of IL-loc. ical properties of IL-loc include, but are not limited to, binding to the IL-loc or; stimulating thymocyte proliferation by inducing IL—2 release, B-cell maturation and proliferation, and fibroblast growth factor activity.

The terms "specific binding" or fically binding", in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, mean that the interaction is ent upon the presence of a particular structure (e.g., an antigenic inant or epitope) on the chemical species, for example, an antibody recognizes and binds to a c protein structure rather than to proteins generally. If an antibody is specific for e "A", the presence of a molecule containing epitope A (or free, unlabeled A), in a on containing labeled "A" and the antibody, will reduce the amount of labeled A bound to the antibody.

The term "antibody", broadly refers to any immunoglobulin (lg) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivative thereof, that retains the essential e binding features of an lg molecule. Such mutant, variant, or derivative antibody formats are known in the art, nonlimiting embodiments of which are discussed below.

In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is sed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more ved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRI, CDRl, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgAl and IgA2) or subclass.

The term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the y to specifically bind to an antigen (e.g., hlL-loc). The n-binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments may also have bispecific, dual specific, or multi-specific formats, specifically binding to two or more ent ns. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) an Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment sing two Fab fragments linked by a disulfide bridge at the hinge region; 2O (iii) an Fd fragment consisting of the VH and CH1 s; (iv) an FV fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al. (1989) Nature 341 :544-546, PCT Publication No. WO 90/05144), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two s of the Fv fragment, VL and VH, are coded for by separate genes, they can be , using recombinant methods, by a synthetic linker that s them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain FV (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85 :5 879-5 883). Such single chain antibodies (scFvs) are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Other forms of single chain dies, such as diabodies are also encompassed. Diabodies are bivalent, ific antibodies in which VH and VL s are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and ng two antigen binding sites (see, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al. (1994) Structure 2:1121-1123).

Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (Springer-Verlag, New York, 2001) (ISBN 341354-5».

The term "antibody uct" refers to a polypeptide comprising one or more the antigen binding portions of the invention linked to a linker polypeptide or an immunoglobulin constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Such linker polypeptides are well known in the art (see, e.g., Holliger et a1. (1993) Proc. Natl. Acad. Sci. USA 4-6448; Poljak et a1. (1994) Structure 2:1121-1123). An immunoglobulin constant domain refers to a heavy or light chain nt . Human IgG heavy chain (gamma) and light chain (kappa and ) constant domain amino acid sequences are lmown in the art and represented in Table 2.

Table 2: Sequences of Human IgG Heavy and Light Chain Constant Domains Sequence Sequence Identifier 123456789012345678901234567890 Ig gamma—l SEQ ID N013 ASTKGPSVFFLAPSSKS-SGG-AALGCLVK constant region DYFPEPVTVSW.SGALTSGVJTFPAVAQSS GLYSLSSVVTVPSSSLGTQTYICNV iKPS 2HN<OWN<m wW U] 0UWHE I]0w 'U0www' ‘J mGG PSVFLFPPKPKJLLMISRLPL CVVVDVS HEDPEVKFNWYVJGVLVJNA< <PRddQY STYRVVSVLTVAAQDWL GKE <C<VSN<A LPAPIEKTISKAKGQPRdPQVY -PPSR** MTKNQVSLTCAVKGFYPSDIAV W451 GQP ENNYKTTPPVADSDGSFFLYSKJTVD<SRW QQGNVFSCSV iEALiNiY"QKS-S-SPG< Ig gamma—l SEQ ID NO:4 ASTKGPSVFPAAPSS<S SGG AA-GC-V< constant region VTVSW GV4"FPAV4QSS mutant GLYSLSSVVTVPSSSAG Q YIC V {<PS NTKVDKKVEPKSCDKTi"CPPCPAPEAAGG PSVFLFPPKPKDTLMISRLPLV CVVVDVS HEDPEVKFNWYVDGVLVHNA<1<PR 1 *QY STYRVVSVLTVVHQDWVNGKTY<C<VS <A LPAPIEKTISKAKGQ?RFPQVY -PPSR** MTKNQVSLTCLVKGFY?SDIAV WdSVGQP1 ENNYKTTPPVLDSDGSTFLYSKLTVD{SRW QQGNVFSCSVWHEALHVHYTQKSLSLS?G< Ig Kappa constant SEQ ID N025 TVAAPSVFIF3?SDEQLKSGTASVVCVVVW_ region FYPREAKVQWKVDNALQSGNSQESVTE 35 KDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSTQRGECL Ig Lambda SEQ ID N026 QPKAAPSVTLT?PSSEELQANKATLVCLISL constant region DFYPGAVTVAWKADSSPVKAGVETTTPSKQ ASSYLSLTPEQWKSHRSYSCQVTH EGSTVEKTVAPTECS Still further, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody, or antigen binding portion thereof, with one or more other ns or peptides. Examples of such immunoadhesion les include use of the avidin core region to make a tetrameric scFV molecule (Kipriyanov, S. et al. (1995) Human Antibod. Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, S. et al. (1994) Mol. Immunol. 31: 1047-105 8). Antigen binding portions of antibodies, such as Fab and 2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, tively, of whole antibodies.

Moreover, antibodies, n binding portions thereof, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds hIL-loc is substantially free of antibodies that specifically_bind antigens other than hIL-loc). An isolated antibody that specifically binds hIL-loc may, however, have cross-reactivity to other antigens, such as IL-loc molecules from other species. Moreover, an ed antibody may be substantially free of other cellular material and/or chemicals.

The term "human antibody" includes dies having variable and nt s derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., ons introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody", does not include antibodies in which CDR sequences derived from the germline of r mammalian species, such as a mouse, have been grafted onto human framework sequences. 2O The term "recombinant human antibody" includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a inant expression vector transfected into a host cell (described further in Section II C, , antibodies isolated from a recombinant, combinatorial human dy library (Hoogenboom, H. (1997) Trends Biotechnol. 15: 62-70; Azzazy and ith (2002) Clin. Biochem. 35: 425-445; Gavilondo and Larrick (2000) BioTechniques 29: 128-145; Hoogenboom and Chames (2000) l. Today 21: 371-378 ), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20: 6287-6295; Kellermann and Green (2002) Curr. Opin. Biotechnol. 13: 593—597; Little et al. (2000) Immunol.

Today 21:364-370) or antibodies prepared, expressed, created or isolated by any other means that involves ng of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin ces. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro nesis (or, when an animal transgenic for human lg sequences is used, in viva somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody ne repertoire in vivo.

The term "chimeric antibody" refers to antibodies that comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions.

The term "CDR-grafted antibody" refers to antibodies that comprise heavy and light chain variable region ces from one species but in which the sequences of one or more of the CDR regions of VH and/or VL regions are replaced with CDR sequences of another species, such as antibodies that have human heavy and light chain variable regions in which one or more of the human CDRs (e.g., CDR3) has been replaced with murine CDR sequences, for example, as obtained from a murine monoclonal antibody to human IL-loc.

As used herein, the term "CDR" refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDRl, CDR2, and CDR3, for each of the variable regions. The term "CDR set" as used herein refers to a group of three CDRs that occur in a single variable region (i.e., VH or VL) of an antigen binding site. The exact boundaries of these CDRs have been defined differently according to different systems. The system bed by Kabat (Kabat et al. (1987, 1991) ces of Proteins of Immunological Interest (National utes of Health, 2O Bethesda, Maryland) not only provides an guous residue numbering system applicable to any variable region of an antibody, but also provides e e boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917 and Chothia et al. (1989) Nature 342: 877-883) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as L1, L2, and L3 or H1, H2, and H3, where the "L" and the "H" designates the light chain and the heavy chains regions, respectively. These regions may be ed to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries ng CDRs pping with the Kabat CDRs have been described by Padlan et al. (1995) FASEB J. 9: 133-139 and MacCallum 3O (1996) J. Mol. Biol. 262(5): 732-?45). Still other CDR ry definitions may not strictly follow one of the above systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact n binding. The methods used herein may utilize CDRs defined according to any of these systems, gh certain embodiments use Kabat or Chothia defined CDRs.

The terms "Kabat numbering", "Kabat definition" and "Kabat labeling" are used interchangeably herein. These terms refer to a system of numbering amino acid residues which are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable s of an antibody, or an n binding portion thereof (Kabat et al. (1971) Ann. NY Acad. Sci. 190: 382-391 and Kabat, E. et a1. (1991) Sequences of Proteins of Immunological Interest Fifth Edition, US. Department of Health and Human es, NIH Publication No. 91- 3242). For the heavy chain variable region, the hypervariable region ranges from amino acid positions 31 to 35 for CDRl, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3. For the light chain variable region, the hypervariable region ranges from amino acid ons 24 to 34 for CDRl, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.

The growth and analysis of extensive public databases of amino acid sequences of variable heavy and light regions over the past twenty years have led to the understanding of the typical boundaries between framework regions (FR) and CDR sequences within variable region sequences and enabled persons skilled in this art to tely determine the CDRs according to Kabat numbering, Chothia numbering, or other systems. See, e.g., Martin, "Protein Sequence and Structure Analysis of Antibody Variable Domains," In Kontermann and Dubel, eds., Antibody ering (Springer-Verlag, Berlin, 2001), r 31, pages 432—433. A useful method of determining the amino acid sequences of Kabat CDRs within the amino acid sequences of variable heavy (VH) and 2O variable light (VL) regions is provided below: To identify a CDR-L1 amino acid sequence: Starts approximately 24 amino acid residues from the amino terminus of the VL region; Residue before the CDR—Ll sequence is always cysteine (C); Residue after the CDR-L1 sequence is always a phan (W) residue, typically Trp-Tyr-Gln (W-Y-Q), but also Trp-Leu-Gln (W-L-Q), Trp-Phe- Gln (W-F-Q), and Trp-Tyr-Leu (W-Y-L); Length is typically 10 to 17 amino acid residues.

To identify a CDR-L2 amino acid sequence: 3O Starts always 16 residues after the end of ; Residues before the CDR-L2 sequence are lly Ile-Tyr (I-Y), but also Val-Tyr (V-Y), Ile-Lys (I-K), and fle-Phe (I-F); Length is always 7 amino acid es.

To identify a CDR-L3 amino acid sequence: Starts always 33 amino acids after the end of CDR-L2; Residue before the CDR-L3 amino acid sequence is always a cysteine (C); Residues after the CDR—L3 sequence are always y-X-Gly (E-G-X-G) (SEQ ID NO:7), where X is any amino acid; Length is typically 7’ to 11 amino acid residues.

To identify a CDR-Hl amino acid sequence: Starts approximately 31 amino acid residues from amino us of VH region and always 9 residues after a cysteine (C); Residues before the CDR-Hl sequence are always Cys—X—X-X-X-X-X-X-X (SEQ ID NO: 151), where X is any amino acid; Residue after CDR-Hl sequence is always a Trp (W), typically Trp-Val (W- V), but also Trp-He (W-I), and Trp-Ala (W-A); Length is typically 5 to 7 amino acid residues.

To identify a CDR-H2 amino acid sequence: Starts always 15 amino acid es after the end of CDR-Hl; Residues before CDR-H2 sequence are typically Leu-Glu-Trp-Ile-Gly (L-E- W-I—G) (SEQ ID NO:8), but other variations also; es after CDR-H2 sequence are LysiArg-Leu/Ile/VaUPhe/Thr/Ala- Thr/Ser/Ile/Ala (K/R-L/I/V/F/T/A—T/S/IiA); Length is typically 16 to 19 amino acid residues.

To identify a CDR-H3 amino acid sequence: 2O Starts always 33 amino acid residues after the end of CDR-H2 and always 3 after a ne (C)' Residues before the CDR—H3 sequence are always Cys-X-X (C-X-X), where X is any amino acid, typically Cys-Ala-Arg ); Residues after the CDR—H3 sequence are always Trp-Gly-X-Gly (W-G-X- G) (SEQ ID NO:9), where X is any amino acid; Length is typically 3 to 25 amino acid residues.

As used , the terms “acceptor" and "acceptor antibody" refer to the antibody or nucleic acid sequence providing or ng at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequences of one or more of the framework regions. In some embodiments, the term "acceptor“ refers to the antibody amino acid or nucleic acid sequence providing or encoding the constant region(s). In yet another embodiment, the term "acceptor" refers to the antibody amino acid or nucleic acid sequence providing or encoding one or more of the framework regions and the constant region(s). In a specific embodiment, the term "acceptor" refers to a human antibody amino acid or nucleic acid sequence that provides or encodes at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequences of one or more of the framework s. In accordance with this embodiment, an acceptor may contain at least 1, at least 2, at least 3, least 4, at least 5, or at least 10 amino acid residues that does (do) not occur at one or more specific ons of a human dy. An acceptor framework region and/or acceptor constant region(s) may be, e.g., derived or obtained from a germline antibody gene, a mature dy gene, a functional antibody (e.g., antibodies well-known in the art, antibodies in development, or antibodies commercially available).

As used herein, the term “canonical" residue refers to a residue in a CDR or framework that defines a particular canonical CDR structure as defined by Chothia et a1. (1987) J. Mol. Biol. 196:901-917 and Chothia et a1. (1992) J. Mol. Biol. 9-817). According to Chothia et a1., critical portions of the CDRs of many antibodies have nearly identical peptide backbone confirmations despite great diversity at the level of amino acid sequence. Each canonical structure specifies ily a set of peptide backbone torsion angles for a contiguous segment of amino acid residues g a loop.

As used herein, the terms "donor" and "donor antibody" refer to an antibody ing one or more CDRs. In one embodiment, the donor antibody is an antibody from a s different from the antibody from which the framework regions are obtained or derived. In the context of a humanized dy, the term "donor antibody" refers to a non-human antibody providing one or more CDRs.

As used herein, the term "framework" or "framework sequence" refers to the remaining sequences of a le region minus the CDRs. Because the exact definition of a CDR sequence 2O can be determined by different systems, the meaning of a ork sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, -L2, and -L3 of light chain and CDR-H1, -H2, and -H3 of heavy chain) also divide the framework regions on the light chain and the heavy chain into four sub-regions (FRl, FR2, FR3 and FR4) on each chain, in which CDRl is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4.

Without specifying the particular sub—regions as FRl, FR2, FR3 or FR4, a framework region, as referred by , represents the combined FR's within the le region of a single, naturally occurring immunoglobulin chain. As used herein, a FR represents one of the four sub- regions, and FRs represents two or more of the four sub— regions constituting a framework region.

Human heavy chain and light chain acceptor sequences are known in the art. In one embodiment of the invention the human heavy chain and light chain acceptor sequences are selected from the sequences described in Table 3 and Table 4.

Table 3: Heavy Chain Acceptor Sequences SEQ Protein region Sequence I 123456789012345678901234567890 I VH2—70/JH6 FRl EVTLRESGPALVKPTQTLTLTCTFSGFSLS I VH2—70/JH6 FRZ WIRQPPGKALEWLA SEQ n region ce 123456789012345678901234567890 12 VH2—70/JH6 FR3 RLTISKDTSKNQVVLTMTNWDPVDTATYYC 13 VH2—70/JH6 FR4 WGQGTTVTVSS 14 VH2—26/JH6 FRl EVTLKESGPVLVKPTETLTLTCTVSGFSLS VH2—26/JH6 FR2 WIRQPPGKALEWLA 16 VH2—26/JH6 FR3 DTSKSQVVLTMTNWDPVDTATYYC 17 VH2—26/JH6 FR4 WGQGTTVTVSS 18 VH3—72/JH6 FRl EVQLVESGGGLVQPGGSLRLSCAASGFTFS 19 VHa—vz/Jna FR2 VH3—72/JH6 FR3 RFTISRDDSKNSLYLQMNSLKTEDTAVYYC 21 VHa—vz/Jna FR4 22 VH3—21/JH6 FRl EVQLVESGGGLVKPGGSLRLSCAASGFTFS 23 VH3—21/JH6 FR2 WVRQAPGKGLEWVS 24 Vh3—21/JH6 FR3 RFTISRDNAKNSLYLQMNSARAEDTAVYYC Vh3—21/JH6 FR4 WGQGTTVTVSS 26 Vhl—69/JH6 FRl EVQLVQSGAEVKKPGSSVKVSCKASGGTFS 27 Vhl—69/JH6 FR2 WVRQAPGQGLEWMG 28 Vhl—69/JH6 FR3 DKSTSTAYMELSSLRSEDTAVYYC 29 Vhl—69/JH6 FR4 WGQGTTVTVSS so vu—ls/Jms m 31 Vhl—l8/JH6 FR2 WVRQAPGQGLEWMG 32 Vhl—l8/JH6 FR3 RVTMTTDTSTSTAYMELRSLRSJDTAVYYC 33 Vhl—l8/JH6 FR4 WGQGTTVTVSS 34 1/JH6 FRl QVQLVQSGSELKKPGASVKVSC<ASGYTFT VH7—4.1/JH6 FR2 WVRQAPGQGLEWMG 36 1/JH6 FR3 RFVFSLDTSVSTAYLQISSLKAEDTAVYYC 37 VH7—4.l/JH6 FR4 WGQGTTVTVSS Table 4: Light Chain Acceptor Sequences SEQ Protein region Sequence 123456789012345678901234567890 38 B3/JK4 FRl DIVMTQSPDSLAVSLGERATINC 39 B3/JK4 FR2 WYQQKPGQPPKLLIY 40 B3/JK4 FR3 GVPDRFSGSGSGTDFTLTISSLQAEDVAVY 41 B3/JK4 FR4 VEIKR 42 L2/JK4 FRl EIVMTQSPATLSVSPGERATLSC 43 L2/JK4 FR2 WYQQKPGQAPRLLIY 44 L2/JK4 FR3 GIPARFSGSGSGTEFTLTISSLQSEDFAVY 45 L2/JK4 FR4 FGGGTKVEIKR 46 L15/JK4 FRl DIQMTQSPSSLSASVGDRVTITC 47 L15/JK4 FR2 WYQQKPEKAPKSLIY 48 L15/JK4 FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATY 49 L15/JK4 FR4 FGGGTKVEIKR 50 L5/JK4 FRl DIQMTQSPSSVSASVGDRVTITC 51 L5/JK4 FR2 WYQQKPGKAPKLLIY SEQ Protein region Sequence 123456789012345678901234567890 52 L5/JK4 FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATY 53 L5/JK4 FR4 FGGGTKVEIKR 54 l8/JK2 FRl DIQMTQSPSSLSASVGDRVTITC 55 l—33/Ol8/JK2 FR2 WYQQKPGKAPKLLIY 56 1—33/018/JK2 GVPSRFSGSGSGTDFTFTISSLQPEDIATY FR3 YC 57 1—33/018/JK2 FGQGTKLEIKR 58 1—33/018/JK4 FGGGTKVEIKR As used herein, the term “germline dy gene" or "gene fragment" refers to an globulin sequence encoded by non- lymphoid cells that have not undergone the maturation process that leads to genetic rearrangement and mutation for expression of a particular globulin (see, e.g., Shapiro et al. (2002) Crit. Rev. Immunol. 22(3): 183-200; Marchalonis et al. (2001) Adv. Exp. Med. Biol. 484:13-30). One of the advantages provided by s embodiments of the present invention stems from the recognition that germline antibody genes are more likely than mature antibody genes to conserve essential amino acid sequence structures teristic of duals in the species, hence less likely to be recognized as from a foreign source when used therapeutically in that species.

As used herein, the term "key" residues refer to certain residues within the variable region that have more impact on the binding specificity and/or affinity of an dy, in particular a humanized antibody. A key residue includes, but is not limited to, one or more of the following: a residue that is adjacent to a CDR, a potential glycosylation site (can be either N— or O-glycosylation site), a rare residue, a residue capable of interacting with the n, a residue capable of interacting with a CDR, a canonical residue, a contact residue between heavy chain variable region and light chain variable region, a residue within the Vernier zone, and a residue in the region that overlaps between the Chothia definition of a variable heavy chain CDRl and the Kabat tion of the first heavy chain framework. 2O The term "humanized antibody" refers to antibodies that comprise heavy and light chain variable region sequences from a man species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more "human-like", i.e., more similar to human germline variable sequences. One type of zed antibody is a CDR-grafted antibody, in which mmhmmmCDmewmwamhMmmwdmwhmmnVHmmVquwmmMommmeme corresponding non-human framework (FR) sequences. For example, a "humanized antibody" is an antibody or a variant, derivative, , or nt thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a mentary determining region (CDR) having substantially the amino acid sequence of a non-human dy. As used herein, the term "substantially" in the context of a CDR refers to a CDR having an amino acid sequence at least 80%, at least 85 %, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human dy CDR. A zed antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab’, F(ab‘)2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non—human immunoglobulin (i.e., donor antibody) and all or ntially all of the framework regions are those of a human immunoglobulin consensus sequence. In an ment, a humanized antibody also comprises at least a n of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, a humanized antibody contains both the light chain as well as at least the variable domain of a heavy chain. The dy also may include the CH1, hinge, CH2, CH3, and CH4 s of the heavy chain. In some embodiments, a humanized antibody only contains a humanized light chain. In some embodiments, a humanized antibody only contains a humanized heavy chain. In specific embodiments, a humanized antibody only contains a zed variable domain of a light chain andior humanized heavy chain.

The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including without limitation IgGl, IgG2, IgG3, and IgG4.

The humanized antibody may comprise sequences from more than one class or isotype, and 2O ular constant domains may be selected to optimize desired effector functions using techniques well-known in the art.

The framework and CDR regions of a zed dy need not correspond precisely to the parental sequences, e.g., the donor antibody CDR or the consensus framework may be mutagenized by substitution, insertion and/or deletion of at least one amino acid residue so that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. In one embodiment, such mutations, however, will not be extensive.

Usually, at least 80%, at least 85%, at least 90%, and at least 95% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences. As used herein, the term "consensus framewor " refers to the framework region in the consensus immunoglobulin sequence.

As used herein, the term "consensus immunoglobulin sequence" refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (see, e.g., Winnaker (1987) From Genes to Clones gsgesellschaft, Weinheim, Germany». A "consensus immunoglobulin sequence" can thus comprise a nsus variable domain" and/or a "consensus constant domain". A "consensus variable domain" can in turn comprise one or more "consensus framework s" andior one or more "consensus CDRs". In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus ce.

As used herein, "Vernier" zone refers to a subset of framework residues that may adjust CDR structure and fine-tune the fit to antigen as described by Foote and Winter (1992) J. Mol. Biol. 224:487-499. Vernier zone residues form a layer underlying the CDRs and may impact on the ure of CDRs and the affinity of the antibody.

The term "multivalent binding protein" is used in this specification to denote a g protein comprising two or more antigen binding sites. The multivalent binding protein is engineered to have the three or more antigen binding sites, and is generally not a naturally occurring antibody.

The term "multispecific binding n" refers to a binding n capable of binding two or more related or unrelated targets. Dual variable domain (DVD) binding proteins are binding proteins that comprise two or more antigen binding sites and are alent or multivalent binding proteins. Such DVD binding proteins may be monospecific, i.e., e of binding one antigen or pecific, i.e., capable of binding two or more antigens. DVD binding proteins sing two heavy chain DVD polypeptides and two light chain DVD polypeptides are referred to as a DVD-IgTM molecule.

Each half of a DVD-IgTM molecule comprises a heavy chain DVD polypeptide, and a light chain DVD polypeptide, and two antigen binding sites. Each binding site comprises a heavy chain variable domain and a light chain variable domain with a total of 6 CDRs involved in antigen binding per antigen binding site. DVD binding ns and methods of making DVD binding 2O proteins are disclosed in U.S. Patent No. 7,612,181.

One aspect of the invention ns to a DVD binding protein comprising binding proteins capable of binding human IL-lot. In another aspect, the DVD binding protein is capable of binding IL-loc and a second . In one embodiment, the DVD binding protein is capable of binding IL-loc and IL-lB.

The term "neutralizing" refers to neutralization of biological activity of a cytokine when a binding protein specifically binds the cytokine. In an embodiment, a neutralizing binding n is a neutralizing antibody, whose binding to hlL-l on results in inhibition of a biological ty of hIL- 10L. In an embodiment, the neutralizing binding protein binds hIL—l 0c and reduces a biologically activity of hIL-lOL by at least about 20%, at least about 40%, at least about 60%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%. Inhibition of a biological activity of L by a neutralizing binding protein can be assessed by ing one or more indicators of hIL—loc biological activity well known in the art.

The term "epitope" includes any polypeptide determinant e of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. An e thus ts of the amino acid residues of a region of an antigen (or fragment thereof) known to bind to the complementary site on the specific binding r. An nic fragment can contain more than one epitope. In certain embodiments, an antibody is said to specifically bind an n when it recognizes its target antigen in a complex mixture of proteins and/or macromolecules. Antibodies are said to "bind to the same epitope" if the antibodies cross- compete (one ts the binding or modulating effect of the other). In addition structural definitions of epitopes apping, similar, identical) are informative, but functional definitions are often more relevant as they encompass structural (binding) and functional (modulation, competition) parameters.

The term "surface plasmon nce", refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein trations within a biosensor matrix, for example using the BIACORETM system (Biacore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, New Jersey). For further descriptions, see J6nsson, U. et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U. et al. (1991) BioTechniques 11: 620-627; Johnsson, B. et al. (1995) J. Mol. it. 8: 125-131; and Johnsson, B. et al. (1991) Anal. Biochem. 198: 268-277.

The term "Kon" refers to the on rate constant for association of a binding protein (e.g., an antibody) to the antigen to form the, e.g., antibody/antigen complex as is known in the art. The "Kon" also is known by the terms "association rate constant," or "ka," as used interchangeably herein. This value indicating the g rate of an antibody to its target antigen or the rate of complex formation between an antibody and antigen also is shown by the equation: Antibody ("Ab") + n ("Ag")—>Ab-Ag.

The term "Koff" refers to the off rate nt for dissociation of a binding protein (e.g., an antibody) from the, e.g., antibody/antigen complex as is known in the art. The "Koff" also is known by the terms "dissociation rate constant" or "kd" as used interchangeably herein. This value indicates the dissociation rate of an antibody from its target antigen or separation of Ab-Ag complex over time into free antibody and antigen as shown by the equation below: Ab + Ag<—Ab-Ag.

The terms "equilibrium dissociation constant" or "KD," as used interchangeably herein, refer to the value obtained in a titration measurement at equilibrium, or by dividing the dissociation rate constant (koff) by the association rate constant (kon). The association rate nt, the dissociation rate constant, and the equilibrium dissociation constant are used to represent the binding affinity of an antibody to an antigen. s for determining association and dissociation rate constants are well known in the art. Using fluorescence—based techniques offers high sensitivity and the ability to examine samples in physiological buffers at equilibrium. Other experimental ches and instruments such as a BIACORETM (biomolecular interaction analysis) assay can be used (e.g., instrument available from Biacore International AB, a GE Healthcare company, Uppsala, Sweden).

Additionally, a KinExA® tic on Assay) assay, available from Sapidyne Instruments (Boise, Idaho) can also be used.

The term "labeled binding protein" as used herein, refers to a protein with a label incorporated that provides for the identification of the binding protein. In one aspect, the label is a able marker, e.g., incorporation of a radiolabeled amino acid or ment to a polypeptide of biotinyl es that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 358, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and 153Sm); fluorescent labels (e.g., FITC, rhodamine, and nide phosphors), enzymatic labels (e.g., adish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl ; ermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, and epitope tags); and magnetic agents, such as gadolinium chelates.

The term "antibody ate" refers to a binding protein, such as an antibody, chemically linked to a second chemical moiety, such as a therapeutic or cytotoxic agent. The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological als. In one aspect the therapeutic or cytotoxic agents include, but are not limited to, pertussis toxin, taxol, cytochalasin B, gramicidin D, um bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, drotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.

The terms "crystal" and "crystallized" refer to an antibody, or antigen binding portion thereof, that exists in the form of a crystal. Crystals are one form of the solid state of matter, which is distinct from other forms such as the amorphous solid state or the liquid crystalline state. ls are composed of regular, repeating, dimensional arrays of atoms, ions, molecules (e.g., proteins such as antibodies), or molecular assemblies (e.g., antigen/antibody complexes). These three- dimensional arrays are arranged according to specific mathematical relationships that are well- tood in the field. The fundamental unit, or building block, that is repeated in a l is called the tric unit. Repetition of the asymmetric unit in an arrangement that conforms to a given, well-defined crystallographic symmetry provides the "unit cell" of the crystal. Repetition of the unit cell by r translations in all three dimensions provides the l. See Giegé and Ducruix (1999) Chapter 1, In Crystallization of c Acids and Proteins, a cal Approach, 2nd ed., (Ducruix and Giegé, eds.) (Oxford University Press, New York, 1999) pp. l-l6.

The term "polynucleotide" means a polymeric form of two or more nucleotides, either cleotides or deoxynucleotides, or a modified form of either type of nucleotide. The term includes single and double ed forms of DNA or RNA, but in an embodiment is - stranded DNA.

The term "isolated polynucleotide" means a cleotide (e.g., of genomic, cDNA, or synthetic origin, or a combination thereof) that is not associated with all or a portion of a polynucleotide with which it is associated in nature, with which it is operably linked to in nature, or with which it occurs in nature as part of a larger sequence.

The term "vector" refers to a nucleic acid le capable of orting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be d. Another type of vector is a Viral vector, wherein additional DNA segments may be ligated into the Viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other s (e. g., isomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked.

Such vectors are ed to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, id" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression s, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The term "operably linked“ refers to a positioning of components such that they function in their intended manner. A control sequence "operably linked" to a coding ce is ligated in such 3O a way that sion of the coding sequence is achieved under conditions compatible with the control sequences. "Operably linked“ sequences include expression control sequences that are contiguous with a nucleic acid of interest, expression control sequences that act in trans, i.e., are located on a different nucleic acid molecule than a nucleic acid of interest but nevertheless exert control over the nucleic acid of interest, and expression control sequences that are located on the same nucleic acid molecule as, but at a distance from, a nucleic acid of interest. The term "expression control sequence" as used herein refers to polynucleotide ces that are necessary to effect the sion and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription tion, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein ion. The nature of such control sequences differs depending upon the host organism; in yotes, such control ces generally include er, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination ce. The term "control sequences" is intended to include components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

"Transformation" refers to any process by which ous DNA enters a host cell. ormation may occur under natural or artificial conditions using various methods well known in the art for the ion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell, for example. The method is selected based on the host cell being ormed and may include, but is not limited to, viral infection, electroporation, lipofection, and particle bombardment. Such "transformed" cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. They also include cells which ently express the inserted DNA or RNA for limited periods of time.

The term "recombinant host cell" (or simply "host , as used herein, is intended to refer to a cell into which exogenous DNA has been introduced. Such terms are intended to refer not only to the particular subject cell, but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still ed within the scope of the term "host cell" as used herein. In one aspect, host cells include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life. Eukaryotic cells e t, fungal, plant and animal cells. In another aspect, host cells include, but are not limited to, the prokaryotic cell line Escherichia coli; mammalian cell lines CHO, HEK 293, and COS; the insect cell line Sf9; and the fungal cell 3O Saccharomyces cerevisiae.

Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and ormation (e.g., electroporation and lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing ques and procedures may be lly performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed.

(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).

The term "transgenic organism" refers to an organism having cells that contain a transgene, wherein the transgene introduced into the organism (or an ancestor of the organism) ses a polypeptide not naturally expressed in the organism. A "transgene" is a DNA construct that is stably and operably integrated into the genome of a cell from which a transgenic organism develops, directing the expression of an d gene product in one or more cell types or tissues of the transgenic organism.

The terms "regulate" and “modulate" are used interchangeably and refer to a change or an alteration in the activity of a molecule of interest (e.g., the biological activity of hIL-lu). Modulation may be an increase or a decrease in the magnitude of a certain activity or function of the molecule of st. Exemplary ties and functions of a molecule e, but are not limited to, binding characteristics, enzymatic activity, cell or activation, and signal transduction.

Correspondingly, the term "modulator" is a compound capable of changing or altering an activity or function of a molecule of st (e.g., the ical ty of c). For example, a modulator may cause an increase or decrease in the magnitude of a certain activity or function of a molecule compared to the magnitude of the activity or function observed in the absence of the modulator. In certain embodiments, a modulator is an inhibitor, which decreases the magnitude of at least one ty or function of a molecule. Exemplary inhibitors include, but are not limited to, proteins, peptides, antibodies, peptibodies, carbohydrates or small organic molecules. Peptibodies are described, e.g., in PCT Publication No. WO 01/83525.

The term "agonist" refers to a modulator that, when contacted with a molecule of interest, causes an increase in the magnitude of a certain activity or function of the le compared to the magnitude of the activity or function observed in the absence of the agonist. Particular ts of interest may include, but are not limited to, a polypeptide, a c acid, a carbohydrate, or any other molecule that binds to IL—loc andior IL—l[3.

The term "antagonist" or “inhibitor" refers to a modulator that, when contacted with a molecule of interest, causes a decrease in the magnitude of a certain ty or function of the molecule compared to the ude of the activity or function observed in the absence of the antagonist. nists include those that block or modulate the biological or immunological activity of IL-lOL and/or IL-ll3. Antagonists and inhibitors of IL-loc may include, but are not limited to, a polypeptide, a nucleic acid, a carbohydrate, or any other molecule that binds to lL-loc. nists and inhibitors of IL—lB may e, but are not limited to, a polypeptide, a nucleic acid, a carbohydrate, or any other molecule that binds to IL-1 [3.

The term "effective amount" refers to the amount of a therapy that is sufficient to reduce or ameliorate the severity and/or duration of a er or one or more symptoms thereof, t the advancement of a disorder, cause regression of a disorder, prevent the ence, development, onset or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or improve the lactic or therapeutic effect(s) of another y (e.g., prophylactic or therapeutic agent).

The term "sample" is used in its broadest sense. A gical sample" includes, but is not limited to, any quantity of a substance from a living thing or ly living thing. Such living things e, but are not limited to, humans, mice, rats, monkeys, dogs, rabbits and other animals.

Such substances include, but are not limited to, blood, serum, urine, al fluid, cells, organs, tissues, bone marrow, lymph nodes, and spleen.

An "affinity matured" antibody is one with one or more alterations in one or more CDRs or frameworks thereof which result in an improvement in the affinity of the antibody for antigen compared to a parent antibody that does not possess such alteration(s). red affinity matured dies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are ed by procedures known in the art. Marks et al. (1992) BioTechnology 10: 779- 783 describes y maturation by VH and VL domain shuffling. Random mutagenesis of CDR and/or framework residues is described by Barbas et al. (1994) Proc Nat. Acad. Sci. USA 91: 3809- 3813; Schier et al. (1995) Gene 169: 147-155; Yelton et al. (1995) J. Immunol. 155: 1994-2004; 2O n et al. (1995) J. Immunol. 154(7): 3310-3319; and Hawkins et al. (1992) J. Mol. Biol., 226: 889-896.

Pain that persists in an individual after an initial cause for pain has disappeared is no longer a symptom but a recognized disease in its own right. The term "allodynia" as used herein refers to a condition in which an individual experiences a painful se to a normally innocuous stimulus, typically of a mechanical nature, such as brushing of the skin. Allodynic pain does not involve nociceptors and therefore may also be referred to as "non-nociceptive" pain. The term "hyperalgesia" as used herein refers to a condition in which an individual has an increased sensitivity to pain that results from a noxious stimulus, especially a stimulus that activates nociceptors, such as a painful mechanical, thermal, or chemical stimulation. A stimulus that activates nociceptors causes 3O pain. Hyperalgesia is a condition in which an dual has an increased pain response to a normally noxious stimulus. An dual may suffer from allodynia, hyperalgesia, or a combination of allodynia and hyperalgesia. 1. Generation of DVD-IgTM Binding Proteins that Bind IL-loc and IL-ll3 The design and production of dual variable domain immunoglobulin (DVD-IgTM) binding proteins that are capable of binding one or more target antigens (or epitopes) have been described (see, e.g., PCT Publication No. ). A DVD—lg binding protein useful in the methods and compositions described herein for treating osteoarthritis binds IL-loc and IL-ll3. In an embodiment, a TM binding protein comprises at least two polypeptide chains, wherein the first ptide chain comprises l)n-VD2-C-(X2)n, wherein VDl is a first heavy chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant , X1 is a linker with the proviso that it is not CH1, and X2 is an Fc region; and wherein said second polypeptide chain comprises VDl-(Xl)n-VD2—C-(X2)n, n VDl is a first light chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant , X1 is a linker with the proviso that it is not CH1, and X2 does not comprise an Fc region; and n is 0 or 1. A DVD-lgTM g protein consisting of the first and second polypeptide chains has two antigen binding sites.

In another embodiment, a DVD-lgTM binding protein comprises four polypeptide chains wherein each of the first two polypeptide chains comprises VDl -(Xl)n-VD2-C-(X2)n, respectively wherein VDl is a first heavy chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant domain, X1 is a linker with the proviso that it is not CH1, and X2 is an Fc ; and each of the second two polypeptide chains comprises VDl-(X1)n-VD2-C-(X2)n respectively, wherein VDl is a first light chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant domain, X1 is a linker with the proviso that it is not CH1, and X2 does not comprise an Fc region; and n is 0 or 1. Such a DVD-IgTM g protein has four antigen binding sites. 2O By way of example, a DVD-Ig binding protein useful in the methods and compositions described herein for treating osteoarthritis in an dual may be engineered to have a g site for IL-1[3 formed by the ation of the VDl variable domains of the two (first and second polypeptides) and a binding site for IL—loc formed by the association of the VD2 variable domains of the two (first and second polypeptides). In an ative arrangement, a DVD-Ig binding protein useful in the methods and compositions described herein for treating osteoarthritis in an individual may have a binding site for IL—loc formed by the association of the VDl variable domains of the two (first and second polypeptides) and a binding site for IL—l[3 formed by the association of the VD2 variable domains of the two (first and second polypeptides).

A. Generation of Parent Monoclonal Antibodies The variable domains of the DVD binding protein can be ed from parent antibodies, including polyclonal and onal antibodies capable of binding antigens of interest. These antibodies may be naturally occurring or may be generated by recombinant technology.

Monoclonal antibodies can be prepared using a wide y of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A tory Manual, 2nd ed., (Cold Spring Harbor Laboratory Press, 1988); Hammerling, et al., Monoclonal Antibodies and T-Cell Hybridomas (Elsevier, N.Y., 1981), pages 563-581 (said references orated by reference in their entireties). The term "monoclonal antibody" (abbreviated "mAb") as used herein is not limited to antibodies produced h hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any otic, prokaryotic, or phage clone, and not the method by which it is produced. Hybridomas are selected, cloned and further screened for desirable characteristics, including robust oma growth, high antibody production and desirable antibody characteristics using standard methods. Hybridomas may be cultured and expanded in vivo in syngeneic animals, in animals that lack an immune system, e. g., nude mice, or in cell culture in vitro. Methods of selecting, cloning, and expanding hybridomas are well known to those of ordinary skill in the art. In a red embodiment, hybridomas are mouse hybridomas. In another ment, hybridomas are produced in a non-human, non-mouse species such as rats, sheep, pigs, goats, cattle, or horses. In r embodiment, the omas are human hybridomas in which a human non-secretory a is fused with a human cell expressing an antibody capable of binding a specific desired antigen, such as IL-10t or IL-1B.

Recombinant monoclonal antibodies are also generated from , isolated lymphocytes using a procedure referred to in the art as the selected lymphocyte antibody method (SLAM), as described in U.S. Patent No. 5,627,052, PCT Publication WO 92/02551 and Babcook et al. (1996) 2O Proc. Natl. Acad. Sci. USA 93:7843-7848. In this method, single cells secreting antibodies of interest, e.g., lymphocytes derived from an immunized animal, are identified, and, heavy- and light- chain le region cDNAs are rescued from the cells by reverse transcriptase-PCR and these variable s can then be expressed, in the t of appropriate immunoglobulin constant regions (6. g., human constant regions), in mammalian host cells, such as COS or CHO cells. The host cells transfected with the amplified immunoglobulin sequences, derived from in viva selected lymphocytes, can then undergo further analysis and selection in vitro, for e by panning the transfected cells to isolate cells expressing antibodies to the antigen of interest. The amplified immunoglobulin sequences further can be manipulated in vitro, such as by in vitro ty maturation methods such as those described in PCT Publication No. WO 97/29131 and PCT Publication No. WO 00/56772.

Monoclonal antibodies are also produced by immunizing a non-human animal comprising some or all of a human immunoglobulin locus with an antigen of interest. In an embodiment, the non-human animal is a XENOMOUSE transgenic mouse, which is an engineered mouse strain that comprises large fragments of the human immunoglobulin loci and is ent in mouse antibody production. See, e.g., Green et al. (1994) Nature Genetics 7:13-21 and US Patent Nos. 5,916,771; 598; 5,985,615; 5,998,209; 6,075,181; 6,091,001; 6,114,598; and 6,130,364. See also PCT Publication Nos. W0 91/10741; W0 94/02602; W0 96/34096; W0 96/33735; W0 98/16654; W0 98/24893; W0 33; W0 99/45031; W0 99/53049; W0 00/09560; and W0 00/037504. The XENOMOUSE transgenic mouse produces an adult-like human oire of fully human antibodies, and generates antigen-specific human monoclonal antibodies. The XENOMOUSE transgenic mouse contains approximately 80% of the human antibody repertoire through uction of se sized, germline configuration YAC fragments of the human heavy chain loci and x light chain loci. See, Mendez et al. (1997) Nature Genet. 15: 146-156 and Green and Jakobovits (1998) J. Exp. Med. 188: 483-495.

In vitro s also can be used to make the parent antibodies, wherein an antibody library is screened to identify an dy having the desired binding specificity. Methods for such screening of recombinant antibody libraries are well known in the art and include methods described in, for example, US Patent No. 5,223,409; PCT Publication Nos. W0 92/18619; W0 91/17271; W0 92/20791; W0 92/15679; W0 93/01288; WO 92/01047; W0 92/09690; W0 97/29131; and Fuchs et al. (1991) Bio/Technology 9: 1369-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3: 81-85; Huse et al. (1989) Science 246: 1275-1281; McCafferty et al. (1990) Nature 348: 4; Griffiths et al. (1993) EMBO J. 12: 725-734; Hawkins et al. (1992) J. Mol. Biol. 226: 889-896; Clackson et al. (1991) Nature 352: 624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89: 3576-3580; Garrard et al. (1991) Bio/Technology 9: 377; Hoogenboom et al. (1991) Nucl. Acid Res. 19: 2O 4133-4137; and Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88: 7978-7982, and US Patent Publication No. 2003/0186374.

Parent antibodies useful in the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional dy domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.

In a particular, such phage can be utilized to display antigen-binding domains expressed from a oire or atorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of st can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from 3O phage with Fab, Fv or disulfide ized FV antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et a1. (1995) J. l. s 182:41-50; Ames et al. (1995) J. Immunol. Methods 184: 177-186; Kettleborough et a1. (1994) Eur. J. Immunol. 24: 952-958; Persic et al. (1997) Gene 187: 9-18; Burton et al. (1994) Adv. l. 57: 0; PCT Application Nos. PCT/GB91i01134; PCT Publication Nos. W0 90/02809; W0 91/10737; W0 92/01047; WO 92/18619; W0 93/11236; W0 95/15982; and W0 01; and US Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,821,047; ,571,698; 5,427,908; 5,516,637; 5,780, 225; 5,658,727; 5,733,743 and 108.

As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole dies including human antibodies or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab', and F(ab’)2 fragments can also be employed using methods lmown in the art such as those disclosed in PCT Publication No. WO 92/22324; Mullinax et a1. (1992) BioTechniques 12(6): 864-869; and Sawai et a1. (1995) AJRI 34: 26-34; and Better et a1. (1988) Science, 240: 1041-1043. Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in US Patent Nos. 4,946,778 and 5,258,498; Huston et a1. (1991) Methods Enzymol. 203: 46-88; Shu et al. (1993) Proc. Natl. Acad. Sci. USA 90: 7995-7999; and Skerra et al. (1988) Science 240: 1038-1041.

Alternative to screening of recombinant antibody libraries by phage display, other methodologies known in the art for screening large combinatorial libraries can be applied to the identification of parent antibodies. One type of alternative expression system is one in which the recombinant antibody library is sed as RNA—protein fusions, as described in PCT Publication No. WO 98/31700 by Szostak and Roberts, and in Roberts, R.W. and Szostak, J.W. (1997) Proc.

Natl. Acad. Sci. USA, 94: 12297-12302. In this system, a covalent fusion is created between an 2O mRNA and the e or protein that it encodes by in vitro translation of synthetic mRNAs that carry puromycin, a peptidyl or otic, at their 3‘ end. Thus, a specific mRNA can be enriched from a complex mixture of mRNAs (e.g., a combinatorial library) based on the properties of the encoded peptide or protein, e.g., dy, or portion thereof, such as binding of the antibody, or portion thereof, to the dual icity antigen. Nucleic acid sequences encoding antibodies, or portions thereof, recovered from screening of such libraries can be expressed by recombinant means as bed above (e.g., in mammalian host cells) and, moreover, can be subjected to further affinity maturation by either additional rounds of screening of mRNA-peptide fusions in which mutations have been introduced into the originally selected sequence(s), or by other s for affinity maturation in vitro of recombinant antibodies, as described above. 3O In another approach the parent antibodies can also be generated using yeast display methods lmown in the art. In yeast display methods, genetic s are used to tether antibody s to the yeast cell wall and display them on the surface of yeast. In particular, such yeast can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody y (e. g., human or ). Examples of yeast display methods that can be used to make the parent antibodies e those sed in US Patent No. 6,699,658.

The antibodies described above can be further modified to te CDR-grafted and humanized parent antibodies. CDR-grafted parent antibodies comprise heavy and light chain variable region sequences from a human antibody wherein one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of murine antibodies capable of binding antigen of st. A framework sequence from any human antibody may serve as the te for CDR- grafting. However, straight chain ement onto such a framework often leads to some loss of binding affinity to the antigen. The more homologous a human antibody is to the original murine antibody, the less likely the possibility that combining the murine CDRs with the human framework will introduce distortions in the CDRs that could reduce affinity. ore, it is preferable that the human variable framework that is chosen to replace the murine variable framework apart from the CDRs have at least a 65% sequence identity with the murine antibody variable region framework. It is more preferable that the human and murine variable regions apart from the CDRs have at least 70% sequence identify. It is even more preferable that the human and murine variable regions apart from the CDRs have at least 75% sequence identity. It is most preferable that the human and murine variable s apart from the CDRs have at least 80% sequence identity. Methods for producing such antibodies are known in the art (see, EP 0; PCT Publication WO 91/09967; US Patent Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan (1991) Mol. Immunol. 28(4/5): 489-498; Studnicka et al. (1994) Protein Engineering 7(6): 805-814; Roguska et al. (1994) Proc. Natl. Acad. Sci. USA 91: 969-973), and chain shuffling (US. 2O Patent No. 5,565,352).

Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non- human s and framework regions from a human immunoglobulin molecule. Known human lg sequences are disclosed, e.g., www.ncbi.nlm.nih.gov/entrez- /query.fcgi; www.atcc.org/phage/hdbhtml; iquest.coml; www.abcam.coml; www.antibodyresource.com/onlinecomp.html; www.public.iastate.edul.about.pedroiresearch_tools.html; www.mgen.uni- heidelberg.de/SD/IT/IThtml; www.whfreeman.com/immunology/CH— 05/kuby05.htm; brary.thinkquest.org/ l 2429/Immune/Antibodyhtml; www.hhmi.org/grants/lectures/ l996/vlab/; www.path.cam.ac.uk/.about.mrc7/m- geshtml; www.antibodyresource.com/; mcb.harvard.edu/BioLinks/Innnuno- logy.html.www.innnunologylink.com/; pathbox.wustl.edu/.about.hcenter/index.- html; www.biotech.ufl.edu/.about.hcl/; www.pebio.com/paf3409 13.13409 l3.html- ; l.usda.gov/awic/pubs/antibodyi; www.m.ehime-u.acjp/.about.yasuhito- /Elisa.html; www.biodesign.com/table.asp; www.icnet.uk/axp/facsidavies/lin- ks.html; www.biotech.ufl.edu/.about.fccl/protocol.html; www.isac—net.org/sites_geo.html; aximtl.imt.uni- marburg.de/.about.rek/AEP- Start.html; baserv.uci.kun.nl/.about.jraats/linksl.html; www.recab.uni- hd.de/immuno.bme.nwu.edu/; www.mrc—cpe.cam.ac.uk/imt-doc/pu- blic/INTROhtml; www.ibt.unam.mX/vir/V_mice.html; imgt.cnusc.fr:8104l; www.biochem.ucl.ac.uk/.about.martin;’abs/index.html; antibody.bath.ac.uk/; abgen.cvm.tamu.edu/lab/wwwabgenhtml; izh.ch/.about.honegger/AHOsem— inar/Slide01.html; www.cryst.bbk.ac.uk;’.about.ubcg07s/; www.nimr.mrc.ac.uk/CC/ccaewgiccaewghtm; www.path.cam.ac.uk/.about.mrc7/h- umanisation/TAHHPhtml; t.unam.mx/vir/structure/stat_aim.html; www.biosci.missouri.edu/smithgp/indexhtml; www.cryst.bioc.cam.ac.uk/.abo- ut.fmolina/Web- pages/Pept/spottechhtml; www.jerini.deffr roducts.htm; wwwpatents.ibm.com/ibm.html.Kabat et al., Sequences of Proteins of Immunological Interest, US. Dept. Health (1983), each entirely orated herein by nce. Such imported sequences can be used to reduce genicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic, as known in the art.

Framework residues in the human framework regions may be tuted with the corresponding residue from the CDR donor dy to alter, preferably improve, antigen binding.

These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at ular 2O positions. See, e.g., US Patent No. 089; Riechmann et a1. (1988) Nature 332: 323. Three- dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional mational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In l, the CDR es are directly and most substantially ed in influencing antigen binding. Antibodies can be humanized using a variety of ques lmown in the art, such as but not d to those described in Jones et al. (1986) Nature 321 :522; Verhoeyen et a1. (1988) Science 239: 1534; Sims et al. (1993) J. Immunol. 151: 2296; Chothia and Lesk (1987) J. Mol. Biol. 196:901; Carter et al. (1992) Proc. Natl. Acad. Sci. USA 89:4285; Presta et a1. (1993) J. Immunol. 15122623; Padlan (1991) Mol. Immunol. 28(4/5):489-498; Studnicka et a1. (1994) Protein Engineering 7(6):805-814; Roguska, et al. (1994) Proc. Natl. Acad. Sci. USA 91:969- 973; PCT ation Nos. WO 91I09967; W0 43; W0 90/14424; W0 90/14430; WO 99/06834 (PCT/US98/16280); WO 97/20032 (PCTiUS96/18978); WO 92/11272 (PCT/US91/09630); WO 92/03461 (PCT/US91/05939); W0 94/18219 (PCT/US94/01234); WO 92/01047 (PCT/GB91/01134); and WO 93/06213 (PCT/GB92/01755); EP 0 592 106; EP 0 519 596; EP 0 239 400; US Patent Nos. 5,565,332; 5,723,323; 862; 5,824,514; 483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 370; 5,693,762; ,530,101; 5,585,089; 539; and 4,816,567.

Parent monoclonal antibodies may be selected from various monoclonal antibodies capable of binding lL—loc or lL-lf).

Parent onal antibodies may also be selected from various therapeutic antibodies ed for use, in clinical trials, or in development for clinical use.

B. Construction of DVD-lg les A dual variable domain immunoglobulin (DVD-lg) molecule is designed such that two different light chain variable s (VL) from the two different parent monoclonal antibodies are linked in tandem directly or Via a short linker by recombinant DNA techniques, followed by the light chain constant domain. Similarly, the heavy chain comprises two different heavy chain variable domains (VH) linked in tandem, followed by the constant domain CH1 and Fc . See, PCT Publication No. WO 24715.

The variable domains can be obtained using recombinant DNA techniques from a parent antibody generated by any one of the methods described above. In a red embodiment the variable domain is a murine heavy or light chain variable domain. More preferably the variable 2O domain is a CDR grafted or a humanized variable heavy or light chain domain. Most preferably the variable domain is a human heavy or light chain variable domain.

In one embodiment the first and second variable s are linked directly to each other using recombinant DNA techniques. In another embodiment the variable domains are linked via a linker sequence. Preferably two variable domains are linked. Three or more variable domains may also be linked directly or via a linker sequence. The variable domains may bind the same antigen or may bind ent antigens. DVD-lg molecules of the invention may include one immunoglobulin variable domain and one non- immunoglobulin variable domain such as ligand binding domain of a or, active domain of an enzyme. DVD—lg molecules may also comprise 2 or more non-Ig domains. 3O The linker sequence may be a single amino acid or a polypeptide sequence. Examples of linker sequences that can be used in designing and producing DVD-Ig g proteins useful in the methods and compositions described herein include, but are not limited to, AKTTPKLEEGEFSEAR (SEQ ID NO:59); AKTTPKLEEGEFSEARV (SEQ ID NO:60); AKTTPKLGG (SEQ ID NO:61); SAKTTPKLGG (SEQ ID NO:62); SAKTTP (SEQ ID NO:63); RADAAP (SEQ ID NO:64); RADAAPTVS (SEQ ID NO:65); AGGPGS (SEQ ID NO:66); RADAAAA(G4S)4 (SEQ 1D NO:67); SAKTTPKLEEGEFSEARV (SEQ ID NO:68); ADAAP (SEQ ID NO:69); ADAAPTVSIFPP (SEQ ID NO:70); TVAAP (SEQ ID NO:71); TVAAPSVEIPPP (SEQ ID NO:72); QPKAAP (SEQ ID NO:73); QPKAAPSVTLFPP (SEQ ID NO:74); AKTTPP (SEQ ID NO:75); AKTTPPSVTPLAP (SEQ ID NO:76); AKTTAP (SEQ ID NO:77); AKTTAPSVYPLAP (SEQ ID NO:78); ASTKGP (SEQ ID NO:79); ASTKGPSVFPLAP (SEQ ID NO:80); GGSGGGGSG (SEQ ID NO:81); GGGGSGGGGSGGGGS (SEQ ID NO:82); GENKVEYAPALMALS (SEQ ID NO:83); GPAKELTPLKEAKVS (SEQ ID NO:84); GHEAAAVMQVQYPAS (SEQ ID NO:85); TVAAPSVFIFPPTVAAPSVFIFPP (SEQ 1D NO:86); and ASTKGPSVFPLAPASTKGPSVFPLAP (SEQ ID NO:87). The choice of linker sequences is based on crystal structure analysis of several Fab les. There is a natural flexible linkage n the variable domain and the CH1/CL constant domain in Fab or antibody molecular structure. This natural linkage comprises approximately 10-12 amino acid residues, contributed by 4-6 residues from C-terminus of V domain and 4-6 residues from the N-terminus of CL/CHl . In an embodiment, a DVD-Ig molecule useful in the invention is generated using N-terminal 5-6 amino acid residues or 11-12 amino acid residues of CL or CH1 as linker in light and heavy chains of a DVD-Ig molecule, respectively. The N—terminal residues of CL or CH1 domains, particularly the first 5-6 amino acid residues, adopt a loop conformation without strong secondary structures, and therefore can act as e linkers between the two variable domains. The N-terminal residues of CL or CH1 domains are natural extensions of the variable domains, as they are part of the immunoglobulin sequences, and ore minimize to a large extent any immunogenicity potentially g from the linkers and ons.

Other linker ces may include any sequence of any length of CL/CHl domain but not all residues of CL/CHl domain; for e the first 5-12 amino acid residues of the CL/CHl domains; the light chain linkers can be from CK or C)»; and the heavy chain linkers can be derived from CH1 of any isotypes, including C71, CYZ, C73, C74, Cocl, COLZ, C5, C8, and Cit. Linker sequences may also be d from other proteins such as Ig-like proteins, (e.g., TCR, FcR, KIR); G/S based sequences (e.g., G4S (SEQ ID NO:88) repeats); hinge region-derived ces; and other natural sequences from other proteins.

In an embodiment, a constant domain is linked to the two linked variable domains using recombinant DNA techniques. Preferably, a sequence sing linked heavy chain variable domains is linked to a heavy chain constant domain and a sequence comprising linked light chain variable domains is linked to a light chain constant domain. Preferably, the constant domains are human heavy chain constant domain and human light chain constant domain, respectively, when the DVD-Ig binding protein is to be used in a human. Preferably, a DVD-lg heavy chain is further linked to an Fc region. The Fc region may be a native ce Fc , or a t Fc region.

Most preferably, the Fc region is a human Fc region. In a preferred embodiment the Fc region includes Fc region from IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

In an embodiment, two heavy chain DVD-Ig polypeptides and two light chain DVD-Ig polypeptides are combined to form a DVD-Ig le. Detailed descriptions of examples of specific DVD-Ig molecules capable of binding specific targets and methods of making the same have been described. See, e.g., PCT ation No. WO 24715.

C. Production of DVD-lg Binding Proteins DVD-1g proteins that bind IL-loc and [L-lB and are useful in the methods and compositions described herein for treating osteoarthritis may be produced by any of a number of techniques known in the art. See, for example, PCT Publication No. 2007/024715. For example, expression from host cells, n expression vector(s) ng the DVD-lg heavy and DVD-lg light chains is (are) transfected into a host cell by standard techniques. The various forms of the term "transfection" are ed to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a yotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, extran transfection and the like. Although it is possible to express DVD-Ig binding proteins in either prokaryotic or eukaryotic host cells, expression of DVD-Ig proteins in eukaryotic cells is preferable, and most preferable in mammalian host cells, because such eukaryotic cells (and in particular mammalian cells) are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active DVD-Ig protein. 2O Preferred mammalian host cells for expressing the recombinant antibodies of the invention include e Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, used with a DHFR selectable marker, e.g., as described in RI. Kaufman and PA. Sharp (1982) Mol. Biol. 159: 601-621), NSO myeloma cells, COS cells, and SP2 cells. When recombinant expression vectors encoding DVD-Ig proteins are introduced into mammalian host cells, the DVD-Ig proteins are produced by culturing the host cells for a period of time sufficient to allow for expression of the DVD-Ig proteins in the host cells or, more ably, secretion of the DVD-lg proteins into the culture medium in which the host cells are grown. DVD-1g proteins can be recovered from the culture medium using standard protein purification methods.

In a red system for recombinant expression of DVD-lg ns useful in the invention, a inant expression vector encoding both the DVD-lg heavy chain and the DVD-lg light chain is uced into dhfr- CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the DVD-lg heavy and light chain genes are each operatively linked to CMV enhancer/AdMLP promoter regulatory elements to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification.

The selected ormant host cells are cultured to allow for expression of the DVD-lg heavy and light chains and intact DVD-lg protein is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the DVD-lg protein from the culture medium. Still further the invention provides a method of synthesizing a DVD-lg n of the invention by culturing a host cell of the invention in a suitable culture medium until a DVD-1g protein of the invention is synthesized. The method can further comprise isolating the DVD-lg n from the culture medium.

An important e of a DVD-lg binding protein is that it can be produced and purified in a similar way as a conventional antibody. The production of DVD-lg binding protein results in a homogeneous, single major product with desired dual-specific activity, without any sequence modification of the constant region or chemical modifications of any kind. Other previously described methods to generate "bi-specific", -specific", and "multi-specific multivalent" full length binding proteins do not lead to a single primary product but instead lead to the intracellular or secreted production of a mixture of led inactive, mono-specific, multi-specific, multivalent, full length binding proteins, and multivalent full length binding proteins with combination of ent binding sites. As an example, based on the design described by Miller and Presta (PCT Publication No. WO2001/077342, there are 16 possible ations of heavy and light chains. 2O Consequently only 6.25% of protein is likely to be in the desired active form. Separation of fully active forms of the protein from inactive and partially active forms of the n using standard chromatography techniques, typically used in large scale manufacturing, is yet to be trated. singly, the design of the DVD—lg binding proteins, which are "dual-specific multivalent full-length binding proteins", led to a dual variable domain light chain and a dual variable domain heavy chain that assemble primarily to the d "dual-specific multivalent full length binding proteins", i.e., a functional DVD-lg binding protein. See, PCT ation No. WO 2007/024715.

II. lline and Derivatized Binding Proteins The invention includes methods and compositions for ng osteoarthritis that a binding 3O protein that is a l. In an embodiment, the crystallized binding protein has a greater half-life in vivo than the soluble counterpart of the binding protein. In another embodiment, the binding protein retains biological activity after crystallization.

Crystallized binding proteins useful in the invention may be produced according to s known in the art and as disclosed in PCT Publication No. WO 02072636, incorporated herein by reference.

Another embodiment of the invention employs a ylated binding protein wherein a binding protein or antigen-binding portion thereof comprises one or more carbohydrate residues.

Nascent in viva protein production may undergo further sing, known as post-translational modification. In particular, sugar (glycosyl) es may be added enzymatically, a process known as glycosylation. The resulting proteins bearing covalently linked oligosaccharide side chains are known as glycosylated proteins or glycoproteins. Antibodies are glycoproteins with one or more carbohydrate residues in the Fc domain, as well as the variable domain. Carbohydrate residues in the Fc domain have important effect on the effector function of the Fc domain, with minimal effect on antigen binding or half-life of the antibody (Jefferis (2005) Biotechnol. Prog, 21: 11—16). In contrast, glycosylation of the variable domain may have an effect on the antigen g activity of the antibody. ylation in the variable domain may have a negative effect on antibody binding affinity, likely due to steric hindrance (Co et al. (1993) Mol. Immunol. 30: 1361-1367), or result in increased ty for the antigen (Wallick et al. (1998) Exp. Med. 99-1109; Wright et al. (1991) EMBO J. 10:27 17-2723).

It is also le to generate glycosylation site mutants in which an O- or N—linked glycosylation site of s g protein has been mutated. One skilled in the art can te such s using standard technologies. ylation site mutants that retain the ical activity but have increased or sed binding activity may also be used in the s and compositions described herein for treating rthritis. 2O The glycosylation of a binding protein or antigen-binding portion thereof useful in the methods and compositions of the invention may be modified. For example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid tutions can be made that result in elimination of one or more variable region glycosylation sites to thereby eliminate glycosylation at that site.

Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in PCT Publication No. WO 20031016466 and US Patent Nos. 5,714,350 and 6,350,861. 3O Additionally or alternatively, a modified binding protein useful in the invention can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNAc structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.

Such carbohydrate modifications can be lished by, for example, expressing the binding protein in a host cell with altered ylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered ylation.

See, for example, Shields et al. (2002) J. Biol. Chem. 277: 26733-26740; Umana et al. (1999) Nat.

Biotech. 17: 176-181, as well as, European Patent No. EP 1,176,195; and PCT Publication Nos. WO 03/035835 and WO 99/5434280.

Protein glycosylation depends on the amino acid sequence of the protein of st, as well as the host cell in which the protein is expressed. Different organisms may produce different glycosylation enzymes (e.g, glycosyltransferases and glycosidases), and have different substrates (nucleotide sugars) available. Due to such factors, protein glycosylation pattern, and ition of glycosyl residues, may differ depending on the host system in which the ular protein is expressed. Glycosyl residues of a g protein useful in the invention may include, but are not limited to, glucose, galactose, mannose, fucose, ylglucosamine and sialic acid. Preferably the glycosylated g protein comprises glycosyl residues such that the glycosylation pattern is human.

It is known to those skilled in the art that differing protein ylation may result in differing protein characteristics. For instance, the efficacy of a therapeutic protein produced in a microorganism host, such as yeast, and glycosylated utilizing the yeast endogenous pathway may be d compared to that of the same protein expressed in a mammalian cell, such as a CHO cell line. Such glycoproteins may also be immunogenic in humans and show reduced half-life in viva after administration. Specific receptors in humans and other animals may recognize specific 2O glycosyl residues and e the rapid clearance of the protein from the bloodstream. Other adverse effects may include changes in protein folding, solubility, susceptibility to proteases, trafficking, transport, compartmentalization, ion, recognition by other proteins or factors, antigenicity, or allergenicity. Accordingly, a practitioner may prefer a therapeutic protein with a specific ition and n of glycosylation, for example glycosylation composition and pattern identical, or at least similar, to that produced in human cells or in the s-specific cells of the intended subject animal.

Expressing ylated proteins different from that of a host cell may be achieved by genetically modifying the host cell to express logous glycosylation enzymes. Using techniques lmown in the art a practitioner may generate dies or antigen-binding portions 3O thereof exhibiting human protein glycosylation. For example, yeast strains have been genetically modified to express turally ing glycosylation s such that glycosylated proteins (glycoproteins) produced in these yeast strains exhibit protein glycosylation identical to that of animal cells, especially human cells (US Patent Publication Nos. 2004/0018590 and 2002/0137134 and PCT Publication No. WC 2005!1005 84).

Further, it will be appreciated by one skilled in the art that a protein of interest may be expressed using a library of host cells genetically engineered to express various glycosylation s, such that member host cells of the library produce the protein of interest with variant ylation patterns. A practitioner may then select and isolate the n of interest with particular novel glycosylation patterns. Preferably, the protein having a particularly selected novel glycosylation pattern exhibits improved or altered biological properties.

IV. Pharmaceutical Compositions The methods of the invention employ pharmaceutical compositions for treating rthritis or pain in an individual that comprise one or more binding proteins that bind IL-loc and/or IL-1 [3.

Such compositions can also se a pharmaceutically acceptable carrier, a diluent, and/or excipient, or any other compound(s) that es a desirable therapeutic, pharmaceutical, or pharmacological benefit to the ition other than the activity of the one or more binding proteins to bind IL—loc and/or IL—lfi. In an embodiment, a composition useful in treating osteoarthritis or pain in an individual according to the invention comprises a binding protein that binds lL-loc, for example an L—loc antibody, and a binding protein that binds lL-lB, such as an anti-lL-lB antibody, or a binding protein that binds both IL-loc and/or IL-lB, such as an IL-loc/B DVD-lg binding protein.

Binding proteins useful in the methods of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, a ceutical composition comprises one or more binding proteins that bind IL-loc and/or IL-lB and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" broadly includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable rs e one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include an isotonic agent, for example, sugar, polyalcohol (e.g., mannitol or sorbitol), or sodium chloride in a composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying , preservatives or s, which enhance the shelf life or effectiveness of the antibody or dy portion.

Various delivery systems are known and can be used to administer one or more binding proteins useful in the invention or a combination of one or more binding proteins and a prophylactic agent or other therapeutic agent useful for preventing, ng, treating, or ameliorating osteoarthritis or pain in an individual, e.g., ulation in liposomes, microparticles, microcapsules, inant cells capable of expressing the binding protein or fragment, and receptor-mediated endocytosis (see, e.g., Wu and Wu (1987) J. Biol. Chem, 262: 4429-4432), construction of a nucleic acid as part of a iral or other vector, etc. Methods of administering to an individual a binding protein that binds lL-loc and/or IL-lB include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural administration, intratumoral administration, and mucosal administration (e. g., intranasal and oral routes). In addition, pulmonary administration can be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., U.S. Patent Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064; 913; and 5,290,540; and PCT ation Nos. W0 44, WO 9782572, WO 97/44013, WO 98/31346, and WO 99/66903.

In one embodiment, a binding protein useful in the invention is administered using Alkermes AIR® pulmonary drug delivery technology (Alkermes, Inc., dge, Mass.). In a specific embodiment, a binding protein useful in the invention is administered uscularly, intravenously, umorally, orally, intranasally, pulmonary, or subcutaneously. A binding n may be administered by any ient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and inal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.

In a specific embodiment, it may be desirable to administer a binding protein locally to the area in need of treatment, such as ly into the joint area. This may be ed by, for example, and not by way of limitation, local infusion, by injection, or by means of an implant, wherein the t is a porous or non-porous material, including membranes and matrices, such as tic membranes, polymers, fibrous matrices (e.g., Tissuel®), or collagen matrices. In an embodiment, an 2O effective amount of one or more binding proteins is administered locally to the affected joint to treat, manage, ameliorate, and/or prevent r progression of cartilage degeneration as otherwise occurs in osteoarthritis, including pain. In r embodiment, an effective amount of one or more binding proteins is administered locally to an affected joint in combination with an effective amount of one or more other therapies (e. g., one or more prophylactic or therapeutic agents) other than the binding protein(s) to prevent, treat, manage, andfor ameliorate osteoarthritis or one or more symptoms thereof, including pain.

In another embodiment, IL—loc and lL-l[3 binding proteins can be delivered in a controlled release or sustained release system. In one ment, a pump may be used to achieve controlled or sustained release (see Langer (1990) Science 249: 1527-1533; Sefton (1987) CRC Crit. Rev. 3O Biomed. Eng, 14: 201-240; Buchwald et al. (1980) Surgery, 88: 507-516; Saudek et a1. (1989) N.

Engl. J. Med., 321: 574-579). In r embodiment, polymeric materials can be used to e controlled or sustained release of binding proteins useful in the invention (see, e.g., Medical Applications of Controlled Release, (Langer and Wise, eds.) (CRC Press, Inc., Boca Raton, 1984); Controlled Drug Bioavailability, Drug Product Design and Performance, (Smolen and Ball, eds.) (Wiley, New York, 1984); Langer and Peppas (1983) J. Macromol. Sci. Rev. Macromol. Chem.

Phys., C23: 61-126; see also Levy et al. (1985) Science 228:190-192; During et al. (1989) Ann. ., 25: 351-356; Howard et al. (1989) J. Neurosurg. -112); U.S. Patent Nos. 377; ,916,597; 5,912,015; 5,989,463; and 5,128,326; and PCT Publication Nos. WO 99/15154 and WO 99/20253. Examples of polymers that be used in ned release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, - vinyl pyrrolidone), poly(vinyl l), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In an embodiment, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In yet another embodiment, a lled or sustained release system can be placed in proximity of the prophylactic or eutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, J .M., Chapter 6, In Medical Applications of lled Release, Vol. II, Applications and Evaluation, (Langer and Wise, CRC Press, Inc., Boca Raton, 1984), pp. 115-138).

Controlled e systems are discussed in the review by Langer, Science, 249: 1527-1533 (1990). Any technique known to one of skill in the art can be used to e sustained release formulations comprising one or more therapeutic agents of the invention. See, e.g., U.S. Patent No. 4,526,938; PCT ation Nos. WO 91/05548 and WO 96/20698; Ning et al. (1996) Radiother.

Oncol., 39: 179-189; Song et al. (1996) PDA J. Pharm. Sci. Technol., 50: 372-377; Cleek et al. 2O (1997) d. Int'l. Symp. Control. Rel. Bioact. Mater. 24: 853-854; and Lam et al. (1997) Proceed. Int'l. Symp. Control Rel. . Mater., 24: 759-760.

In a specific embodiment, where a composition useful in the invention is a nucleic acid encoding a protein that binds IL-lot and/or IL—l[3, the c acid can be administered in vivo to promote expression of its encoded prophylactic or therapeutic agent, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see, e.g., U.S. Patent No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, DuPont), or coating with lipids or cell- surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide, which is lmown to enter the nucleus (see, e.g., Joliot et al. (1991) Proc. Natl. Acad. Sci. USA 3O 88:1864-1868). atively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression by homologous recombination.

A pharmaceutical composition useful in the invention for treating osteoarthritis or pain is formulated to be compatible with its intended route of administration. Examples of routes of stration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. In a specific embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to human beings. Typically, compositions for enous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocamne to ease pain at the site of the ion.

If the compositions useful in the invention for treating osteoarthritis or pain are to be administered topically, the compositions can be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well- known to one of skill in the art. See, e.g., Remington’s Pharmaceutical Sciences and uction to Pharmaceutical Dosage Forms, 19th ed., (Mack Publishing Co., Easton, Pennsylvania, 1995). For non-sprayable l dosage forms, s to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic ity preferably greater than water are typically employed. Other suitable formulations include, without limitation, suspensions, s, liniments, salves, and the like. In an embodiment, such ations are sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, such as, for example, osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, for example, in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized le (e.g., a s propellant, such as FREON®) or in a squeeze bottle.

Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well known in the art.

If a method of the invention for treating rthritis or pain ses intranasal administration of a composition, the composition can be formulated in an aerosol form, spray, mist, or in the form of drops. In particular, a binding protein for use according to the present invention can be conveniently red in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and dges (composed of, e.g., n) for use in an r or insufflator may be formulated containing a powder mix of the nd and a suitable powder base such as lactose or starch.

If a method of the invention for treating osteoarthritis or pain comprises oral administration, a composition can be formulated orally in the form of tablets, capsules, cachets, gelcaps, solutions, suspensions, and the like. Tablets or capsules can be prepared by conventional means with ceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); s (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch ate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid ations for oral administration may take the form of, but not limited to, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by tional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or enated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily , ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p- hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow e, controlled release, or sustained release of a binding protein(s) useful in the invention for treating osteoarthritis or pain.

A method for treating osteoarthritis or a method of treating pain according to the invention may comprise ary administration, e.g., by use of an inhaler or nebulizer, of a composition formulated with an aerosolizing agent. See, e.g., US Patent Nos. 6,019, 968; 5,985,320; 5,985,309; 272; 5,874,064; 5,855,913; and 5,290,540; and PCT Publication Nos. WO 92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903. In a specific embodiment, a 2O binding protein that binds lL-loc and/or IL—lB or combination therapy for treating osteoarthritis is administered using Alkermes AIR® pulmonary drug delivery logy (Alkermes, Inc., Cambridge, Massachusetts).

A method of the invention may comprise administration of a composition sing a g protein that binds IL-lot andt’or IL—1[3 that is formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multi-dose ners) with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, a g protein may be in powder form for constitution with a suitable e (e.g., e pyrogen-free water) before use.

Methods of the invention may additionally comprise of administration of compositions formulated as depot preparations. Such long acting formulations may be administered by tation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compositions may be formulated with suitable polymeric or hydrophobic materials (e. g., as an on in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt). s of the invention encompass administration of compositions formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

Generally, the ients of compositions are supplied either separately or mixed together in unit dosage form, for e, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or te indicating the quantity of active agent.

Where the mode of administration is infusion, composition can be dispensed with an infusion bottle containing sterile ceutical grade water or saline. Where the mode of administration is by injection, an e of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The invention also provides one or more binding proteins that bind IL-1oc and/or IL-1[3 or a pharmaceutical composition comprising such binding proteins packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of the agent. In an embodiment, one or more binding proteins that bind IL—loc and/or IL—lB or a pharmaceutical composition comprising such binding protein(s) is ed as a dry sterilized lyophilized powder or water free concentrate in 2O a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject to treat tis or pain. In an embodiment, one or more g proteins that bind IL-lot and/or IL—1[3 or a pharmaceutical ition comprising such binding protein(s) is supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least about 5 mg, at least about 10 mg, at least about 15 mg, at least about 25 mg, at least about 35 mg, at least about 45 mg, at least about 50 mg, at least about 75 mg, or at least about 100 mg. The lyophilized binding protein(s) or ceutical composition comprising such binding protein(s) should be stored at between about 2°C and about 8°C in its original container and binding protein(s) or pharmaceutical composition comprising such binding protein(s) should be administered within 1 week, within 5 days, within 72 hours, within 48 hours, within 24 hours, within 3O 12 hours, within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being tituted.

In an alternative embodiment, one or more binding proteins that bind IL-loc and/or IL-ll3 or a pharmaceutical composition comprising such binding protein(s) is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the agent. In an embodiment, the liquid form of the administered composition is ed in a hermetically sealed container at least about 0.25 mg/ml, at least about 0.5 mg/ml, at least about 1 mg/ml, at least about 2.5 mg/ml, at least about 5 mg/ml, at least about 8 mg/ml, at least about 10 mg/ml, at least about 15 mg/kg, at least about 25 mg/ml, at least about 50 mg/ml, at least about 75 mg/ml or at least about 100 mg/ml. The liquid form should be stored at between about 2°C and about 8°C in its original ner.

Binding proteins useful in the methods and itions described herein can be incorporated into a pharmaceutical composition suitable for parenteral administration. In one aspect, binding proteins will be prepared as an injectable solution ning about 0.1 mg/ml to about 250 mg/ml antibody. The injectable solution can be composed of either a liquid or lyophilized dosage form in a flint or amber vial, ampoule, or led syringe. The buffer can be L-histidine (about 1 mM to about 50 mM), lly about 5 mM to about 10 mM, at about pH 5.0 to about 7.0 (optimally about pH 6.0). Other suitable buffers include but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. Sodium chloride can be used to modify the ty of the solution at a concentration of about 0 to about 300 mM (optimally about 150 mM for a liquid dosage form). Cryoprotectants can be included for a lyophilized dosage form, principally about 0% to about 10% e (optimally about 0.5% to about 1.0%). Other suitable cryoprotectants include trehalose and lactose. Bulking agents can be included for a lyophilized dosage form, pally about 1% to about 10% mannitol (optimally about 2% to about 4%).

Stabilizers can be used in both liquid and lyophilized dosage forms, pally about 1 mM to about 50 mM L-methionine (optimally about 5 mM to about 10 mM). Other suitable bulking agents 2O include glycine, arginine, can be included as about 0% to about 0.05% polysorbate-80 (optimally about 0.005% to about 0.01%). Additional surfactants include but are not d to polysorbate 20 and BRIJ surfactants.

Compositions useful for treating osteoarthritis or pain according to the invention may be in a variety of forms. These e, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and itories. The particular form depends on the intended mode of administration and therapeutic application. Typical compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with antibodies. The mode of administration is parenteral (e.g., intravenous, subcutaneous, 3O intraperitoneal, intramuscular). In an embodiment, the dy is administered by intravenous infusion or injection. In r embodiment, the antibody is administered by intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, me, or other d structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., antibody, or antigen binding portion thereof) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, ed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile, lyophilized powders for the preparation of sterile injectable solutions, exemplary methods of preparation are vacuum drying and spray-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile—filtered solution thereof. The proper y of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. ged absorption of injectable compositions can be brought about by including, in the composition, an agent that delays absorption, for example, monostearate salts and gelatin.

The binding proteins useful for ng osteoarthritis or pain according to the invention can be administered by a y of methods known in the art, although an ary route/mode of administration is subcutaneous injection, intravenous ion, or infusion. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary ing upon the desired results. In certain embodiments, a binding protein(s) may be prepared with a carrier that will protect the binding protein(s) against rapid release, such as a controlled release formulation, including implants, transdermal s, and microencapsulated delivery systems. Biodegradable, patible polymers can be used, such as ethylene vinyl e, polyanhydrides, polyglycolic acid, en, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., (Marcel Dekker, Inc., New York, 1978).

In certain embodiments, a binding protein(s) useful in the invention may be orally administered, for example, with an inert t or an lable edible carrier. The binding protein(s) (and other ingredients, if d) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the t's diet. For oral therapeutic stration, the compounds may be incorporated with excipients and used in the form of ingestible s, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. 3O To ster a compound of the invention by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.

Supplementary active compounds can also be incorporated into the compositions. In certain embodiments, a binding protein (e.g., an antibody), or antigen binding portion thereof, useful in the invention is coformulated with andfor coadministered with one or more additional therapeutic agents that are useful for treating osteoarthritis or pain. For example, a g protein that binds hIL-loc and/or hIL-1[3, or antigen binding portion(s) thereof, may be coformulated and/or coadministered with one or more additional antibodies that bind other targets (e.g., antibodies that bind other cytokines or that bind cell surface molecules). Furthermore, one or more binding proteins may be used in combination with one or more other therapeutic agents. Such combination ies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.

In certain embodiments, a protein(s) that binds IL— let and/or IL—1[3, or binding portion f, is linked to a half-life extending vehicle lmown in the art. Such vehicles include, but are not d to, the Fc domain, polyethylene , and dextran. Such vehicles are described, e.g., in US. Patent No. 6,660,843.

In a specific embodiment, nucleic acid molecules comprising nucleotide sequences ng one or more polypeptides of a binding protein are administered to treat, prevent, manage, or ameliorate osteoarthritis by way of gene therapy. Gene therapy refers to y performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded binding polypeptide(s) for a g n(s) that binds IL-loc and/or IL-1[3 and mediates a prophylactic or therapeutic effect with respect to osteoarthritis or pain.

Any of the methods for gene therapy available in the art can be used according to the present invention. For general reviews of the methods of gene y, see Goldspiel et al. (1993) Clin. 2O Pharm. 12:488-505; Wu and Wu (1991) rapy 3: 87-95; Tolstoshev (1993) Ann. Rev. col. Toxicol. 32: 573-596; Mulligan (1993) Science 260: 926-932; Morgan and Anderson (1993) Ann. Rev. Biochem. 62:191-21?; and on, C. (1993) Trends Biotechnol. 11(5): 155.

Methods commonly known in the art of recombinant DNA technology which can be used are bed in Ausubel et al. (eds.), Current Protocols in Molecular Biology (John Wiley & Sons, New York, 1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, ton Press, New York, 1990). Detailed descriptions of various methods of gene therapy are disclosed in US patent application publication No. 20030042664.

A binding protein(s) that binds lL—loc and/or IL—lB can be used alone or in combination with one or more additional agents useful in the treatment of osteoarthritis or pain. For example, an additional agent can be a therapeutic agent art-recognized as being useful to treat one or more symptoms of osteoarthritis or to affect a target other than IL-l that is associated with pain. An additional agent also can be an agent that s a beneficial attribute to the therapeutic ition e.g., an agent that affects the viscosity of the composition to be administered to an individual.

It should r be understood that the combinations that are to be included within this invention are those combinations useful for their intended purpose. The agents set forth below are illustrative for purposes and not ed to be limiting. The ations, which are part of this invention, can be a binding protein(s) that binds lL-loc andi'or IL-lB and at least one additional agent that provides a desirable ty. A combination can also include more than one additional agent, e.g., two or three additional agents, if the combination is such that the formed composition can perform its ed function. Preferred combinations to treat osteoarthritis or pain include nonsteroidal anti-inflammatory drug(s) also referred to as "NSAIDS", which include drugs like ibuprofen. Other preferred combinations are nflammatory agents, including corticosteroids, such as solone; the well lmown side-effects of steroid use can be reduced or even eliminated by tapering the steroid dose required when treating patients in combination with a binding protein(s) that binds lL-loc and/or 1L-1[3. Non-limiting es of therapeutic agents for use in treating an individual suffering from osteoarthritis or pain may include, but are not limited to, one or more the following: budenoside, epidermal growth factor, corticosteroids, cyclosporin, sulfasalazine, aminosalicylates, aptopurine, azathioprine, metronidazole, lipoxygenase inhibitors, mesalamine, olsalazine, balsalazide, antioxidants, thromboxane inhibitors, IL-1 receptor antagonists, anti-lL-lB monoclonal antibodies, anti-IL—6 monoclonal antibodies, growth factors, elastase inhibitors, pyridinyl-imidazole compounds, antibodies of TNF, LT, IL-2, IL-6, IL-7, IL-8, lL-l2, IL- 13, lL-15, IL-16, lL-18, lL-23, EMAP-II, GM-CSF, FGF, and PDGF, antibodies of CD2, CD3, CD4, 2O CD8, CD-19, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands, methotrexate, cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide, an NSAlD, ibuprofen, corticosteroids, prednisolone, phosphodiesterase inhibitors, adensosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, IRAK, NIK, IKK, p38, MAP kinase inhibitors, IL- l[3 converting enzyme tors, TNFos converting enzyme inhibitors, T-cell ling inhibitors, oproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, ensin converting enzyme inhibitors, soluble cytokine receptors, soluble p55 TNF receptor, soluble p75 TNF receptor, le-lRI, le-lRH, le-6R, anti-inflammatory cytokines, IL-4, lL-10, IL-1 1, lL-l3, and TGFB.

The pharmaceutical compositions of the invention may include a "therapeutically effective amount" or a "prophylactically effective " of one or more binding protein(s) that binds IL-loc and/or IL-IB. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the binding protein may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the g protein to elicit a desired se in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the g protein(s), or portion(s) thereof, are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic , such as preventing further degeneration or loss of articular cartilage in an affected joint in osteoarthritis or to prevent onset or intensification of pain. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the lactically effective amount will be less than the therapeutically ive amount.

In an embodiment, a method for treating an individual for pain wherein the individual is suffering from a disease or disorder associated with IL-1 accumulation. Such IL-l accumulation in an individual can be the result of reduced IL-1 expression or reduced metabolism of IL-1.

Accumulation of lL-l can occur in the blood (including plasma, serum) or a local tissue of the individual.

In an embodiment, the invention provides a method for treating an individual for pain n the individual is suffering from a disease or disorder associated with IL-1 lation.

In an embodiment, compositions and s described herein can be used to treat pain in an individual suffering a disease or disorder selected from the group comprising osteoarthritis, rheumatoid arthritis, juvenile chronic arthritis, septic tis, Lyme tis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, Crohn's e, ulcerative colitis, inflammatory bowel disease, n dependent diabetes us, thyroiditis, asthma, allergic diseases, psoriasis, dermatitis, scleroderma, graft versus host disease, organ transplant rejection, acute or chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki's disease, s disease, nephrotic syndrome, chronic e syndrome, Wegener's granulomatosis, Henoch-Schoenlein purpurea, microscopic vasculitis of the kidneys, c active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, infectious diseases, parasitic diseases, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer‘s disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignancies, heart failure, myocardial infarction, Addison‘s disease, sporadic polyglandular deficiency type I, polyglandular deficiency type II (Schmidt's me), adult (acute) respiratory distress syndrome, alopecia, alopecia , seronegative arthropathy, arthropathy, Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy, enteropathic synovitis, Chlamydia-associated arthropathy, Yersinia-associated arthropathy, Salmonella-associated arthropathy, spondyloarthropathy, atheromatous diseasefarteriosclerosis, atopic allergy, mune bullous disease, pemphigus is, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune haemolytic anaemia, Coombs positive haemolytic anaemia, acquired pernicious anaemia, juvenile pernicious anaemia, c encephalitisiRoyal Free disease, chronic taneous candidiasis, giant cell tis, primary sclerosing hepatitis, cryptogenic mune hepatitis, acquired immunodeficiency syndrome, acquired immunodeficiency related diseases, hepatitis B, hepatitis C, common varied immunodeficiency (common variable hypogammaglobulinaemia), dilated cardiomyopathy, female infertility, ovarian failure, premature ovarian failure, fibrotic lung e, cryptogenic fibrosing alveolitis, post-inflammatory interstitial lung disease, interstitial pneumonitis, connective tissue disease associated interstitial lung disease, mixed connective tissue disease associated lung disease, systemic sclerosis associated interstitial lung disease, rheumatoid arthritis associated interstitial lung disease, systemic lupus erythematosus associated lung disease, dermatomyositis/polymyositis associated lung disease, n's disease associated lung disease, ankylosing spondylitis associated lung disease, vasculitic diffuse lung disease, haemosiderosis associated lung disease, drug-induced interstitial lung disease, fibrosis, radiation s, bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease, fectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, type-l autoimmune hepatitis ical autoimmune or lupoid hepatitis), type-2 autoimmune tis (anti-LKM antibody hepatitis), mune mediated ycemia, type B insulin resistance with acanthosis nigricans, hypoparathyroidism, acute immune disease ated with organ lantation, chronic immune disease associated with organ transplantation, rthrosis, primary sing gitis, psoriasis type 1, psoriasis type 2, thic leucopaenia, autoimmune neutropaenia, renal disease NOS, glomerulonephritides, microscopic vasculitis of the kidneys, Lyme disease, discoid lupus erythematosus, male infertility idiopathic or NOS, sperm autoimmunity, multiple sis (all 2O subtypes), hetic ophthalmia, pulmonary hypertension ary to connective tissue e, Goodpasture's syndrome, pulmonary manifestation of polyarteritis nodosa, acute tic fever, rheumatoid spondylitis, Still's disease, ic sclerosis, Sjorgren's syndrome, Takayasu's disease/arteritis, mune thrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroid disease, hyroidism, goitrous autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxoedema, phacogenic uveitis, primary vasculitis, vitiligo, acute liver disease, chronic liver diseases, alcoholic cirrhosis, alcohol-induced liver injury, cholestasis, idiosyncratic liver disease, drug-induced hepatitis, non-alcoholic steatohepatitis, allergy, group B streptococci (GBS) infection, mental disorders (e.g., depression and schizophrenia), Th2 Type and Th1 Type mediated diseases, acute and chronic pain (different forms of pain), cancers such 3O as lung, breast, stomach, bladder, colon, as, ovarian, prostate and rectal cancer and hematopoietic malignancies (leukemia and lymphoma), abetalipoproteinemia, acrocyanosis, acute and chronic parasitic or infectious processes, acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute or chronic ial infection, acute pancreatitis, acute renal failure, adenocarcinomas, atrial ectopic beats, AIDS dementia complex, alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis, allograft rejection, alpha-l- antitrypsin deficiency, amyotrophic lateral sclerosis, anemia, angina pectoris, anterior horn cell degeneration, anti-CD3 therapy, ospholipid syndrome, anti-receptor hypersensitivity reactions, aortic and peripheral aneurysms, aortic dissection, arterial hypertension, arteriosclerosis, arteriovenous fistula, ataxia, atrial fibrillation (sustained or paroxysmal), atrial flutter, atrioventricular block, B cell lymphoma, bone graft rejection, bone marrow transplant (BMT) rejection, bundle branch block, Burkitt‘s lymphoma, burns, cardiac arrhythmias, cardiac stun syndrome, cardiac tumors, cardiomyopathy, cardiopulmonary bypass inflammation response, cartilage transplant ion, cerebellar cortical degenerations, llar disorders, chaotic or multifocal atrial tachycardia, chemotherapy associated ers, chronic myelocytic leukemia (CML), chronic alcoholism, chronic inflammatory pathologies, chronic lymphocytic leukemia (CLL), chronic obstructive pulmonary disease (COPD), chronic salicylate intoxication, colorectal carcinoma, congestive heart failure, conjunctivitis, contact dermatitis, cor pulmonale, coronary artery disease, Creutzfeldt-Jakob disease, culture negative sepsis, cystic fibrosis, ne therapy associated ers, dementia pugilistica, inating diseases, dengue hemorrhagic fever, dermatitis, dermatologic conditions, diabetes, ic arteriosclerotic disease, diffuse Lewy body disease, dilated congestive cardiomyopathy, disorders of the basal ganglia, Down's syndrome in middle age, drug- d movement ers induced by drugs which block CNS dopamine receptors, drug sensitivity, eczema, encephalomyelitis, endocarditis, endocrinopathy, epiglottitis, Epstein-Barr virus infection, erythromelalgia, extrapyramidal and cerebellar disorders, al hemophagocytic lymphohistiocytosis, fetal thymus implant rejection, Friedreich's ataxia, functional peripheral arterial disorders, fungal sepsis, gas ne, gastric ulcer, glomerular nephritis, graft rejection of any organ or tissue, gram negative sepsis, gram positive sepsis, omas due to intracellular organisms, hairy cell leukemia, Hallervorden-Spatz disease, oto's thyroiditis, hay fever, heart transplant rejection, hemochromatosis, hemodialysis, hemolytic uremic me/thrombolytic thrombocytopenic purpura, hemorrhage, hepatitis A, His bundle arrhythmias, HIV infection/HIV neuropathy, Hodgkin‘s e, hyperkinetic movement disorders, hypersensitivity reactions, hypersensitivity pneumonitis, ension, hypokinetic nt disorders, hypothalamic-pituitary-adrenal axis evaluation, idiopathic Addison's e, idiopathic pulmonary fibrosis, antibody mediated cytotoxicity, ia, infantile spinal muscular atrophy, inflammation of the aorta, influenza A, ng radiation exposure, iridocyclitis/uveitis/optic 3O neuritis, ischemia- reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma, kidney transplant rejection, legionella, leishmaniasis, leprosy, lesions of the corticospinal , lipedema, liver transplant ion, lymphedema, malaria, malignant lymphoma, malignant histiocytosis, ant melanoma, itis, meningococcemia, metabolic migraine headache, idiopathic migraine headache, mitochondrial multisystem disorder, mixed connective tissue disease, monoclonal gammopathy, multiple myeloma, multiple systems degenerations (Menzel, Dejerine-Thomas, Shy-Drager, and Machado-Joseph), myasthenia gravis, mycobacterium avium intracellulare, cterium tuberculosis, myelodysplastic syndrome, myocardial tion, myocardial ischemic disorders, nasopharyngeal oma, neonatal chronic lung disease, nephritis, nephrosis, neurodegenerative es, enic muscular atrophies, neutropenic fever, non- Hodgkin's lymphoma, occlusion of the abdominal aorta and its branches, occlusive arterial disorders, OKT3® therapy, orchitis/ epididymitis, orchitis/vasectomy reversal procedures, organomegaly, osteoporosis, pancreas transplant rejection, pancreatic carcinoma, paraneoplastic syndrome/hypercalcemia of ancy, parathyroid transplant rejection, pelvic inflammatory disease, perennial rhinitis, pericardial disease, eral atherosclerotic disease, peripheral vascular disorders, peritonitis, pernicious anemia, pneumocystis carinii pneumonia, pneumonia, POEMS me (polyneuropathy, organomegaly, inopathy, monoclonal gammopathy, and skin changes me), post perfusion syndrome, post pump syndrome, post-MI tomy syndrome, preeclampsia, progressive supranucleo palsy, primary pulmonary hypertension, radiation therapy, Raynaud's phenomenon, Raynaud's disease, Refsum's disease, regular narrow QRS tachycardia, renovascular ension, reperfusion injury, restrictive cardiomyopathy, sarcomas, senile chorea, senile dementia of Lewy body type, seronegative arthropathies, shock, sickle cell , skin allograft ion, skin changes syndrome, small bowel transplant rejection, solid tumors, specific arrhythmias, spinal , spinocerebellar degenerations, streptococcal myositis, structural lesions of the cerebellum, subacute sclerosing panencephalitis, syncope, syphilis of the cardiovascular system, systemic anaphylaxis, systemic inflammatory response syndrome, systemic onset juvenile rheumatoid arthritis, T-cell or FAB ALL, telangiectasia, thromboangiitis obliterans, thrombocytopenia, toxicity, transplants, trauma/hemorrhage, type III hypersensitivity reactions, type IV hypersensitivity, unstable angina, uremia, urosepsis, urticaria, valvular heart diseases, varicose veins, vasculitis, venous diseases, venous thrombosis, ventricular fibrillation, viral and fungal infections, viral encephalitis/aseptic meningitis, associated hemophagocytic syndrome, Wernicke- Korsakoff syndrome, 's e, xenograft rejection of any organ or tissue, acute coronary syndromes, acute idiopathic uritis, acute inflammatory demyelinating polyradiculoneuropathy, acute ischemia, adult Still’s disease, alopecia areata, anaphylaxis, anti-phospholipid antibody syndrome, aplastic anemia, arteriosclerosis, atopic , atopic dermatitis, autoimmune dermatitis, autoimmune disorder ated with streptococcus 3O infection, autoimmune enteropathy, autoimmune hearing loss, autoimmune lymphoproliferative me (ALPS), autoimmune myocarditis, mune premature ovarian failure, blepharitis, bronchiectasis, bullous goid, cardiovascular disease, rophic antiphospholipid syndrome, celiac disease, cervical spondylosis, chronic ischemia, cicatricial pemphigoid, clinically isolated syndrome (CIS) with risk for multiple sclerosis, childhood onset psychiatric disorder, chronic obstructive pulmonary disease (COPD), dacryocystitis, dermatomyositis, diabetic retinopathy, disk herniation, disk prolapse, drug induced immune tic anemia, endocarditis, endometriosis, endophthalmitis, episcleritis, erythema multiforme, erythema multiforme major, gestational pemphigoid, Guillain-Barré syndrome (GBS), hay fever, Hughes syndrome, idiopathic Parkinson’s disease, idiopathic interstitial pneumonia, IgE-mediated allergy, immune hemolytic anemia, inclusion body myositis, infectious ocular inflammatory disease, inflammatory demyelinating disease, inflammatory heart disease, inflammatory kidney disease, lPF/UIP, iritis, keratitis, keratojunctivitis sicca, Kussmaul disease or Kussmaul-Meier disease, Landry's paralysis, Langerhan‘s cell histiocytosis, livedo reticularis, macular degeneration, microscopic polyangiitis, Morbus rev, motor neuron disorders, mucous membrane pemphigoid, multiple organ failure, enia gravis, myelodysplastic syndrome, myocarditis, nerve root disorders, neuropathy, non-A non-B hepatitis, optic neuritis, osteolysis, ovarian cancer, pauciarticular JRA, peripheral artery ive disease (PAOD), peripheral vascular disease (PVD), peripheral artery disease (PAD), phlebitis, polyarteritis nodosa (or periarteritis nodosa), polychondritis, polymyalgia rheumatica, poliosis, polyarticular JRA, polyendocrine deficiency syndrome, ositis, polymyalgia rheumatica (PMR), post-pump syndrome, primary Parkinsonism, te and rectal cancer and hematopoietic malignancies (leukemia and ma), prostatitis, pure red cell aplasia, y adrenal insufficiency, recurrent neuromyelitis optica, restenosis, rheumatic heart disease, SAPHO itis, acne, pustulosis, hyperostosis, and osteitis), secondary amyloidosis, shock lung, tis, sciatica, secondary adrenal insufficiency, silicone associated connective tissue disease, Sneddon- Wilkinson dermatosis, spondylitis ankylosans, Stevens—Johnson syndrome (SJS), systemic inflammatory se me, temporal arteritis, asmic tis, toxic epidermal necrolysis, transverse myelitis, TRAPS (tumor-necrosis factor receptor type 1 (TNFR)-associated periodic me), type 1 allergic reaction, type II es, ria, usual titial pneumonia (UIP), vasculitis, vernal conjunctivitis, viral retinitis, Vogt—Koyanagi-Harada syndrome (VKH syndrome), wet macular degeneration, and wound g. itions and methods described herein can be used to treat pain in an individual suffering from a disease selected from the group consisting of primary and metastatic cancers, including carcinomas of breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, gallbladder and bile ducts, small intestine, urinary tract (including kidney, bladder and urothelium), female genital tract (including cervix, uterus, and s as well as choriocarcinoma and gestational trophoblastic disease), male genital tract ding prostate, seminal vesicles, testes and germ cell tumors), endocrine glands (including the thyroid, adrenal, and pituitary ), and skin, as well as hemangiomas, melanomas, sarcomas (including those arising from bone and soft tissues as well as Kaposi’s sarcoma), tumors of the brain, nerves, eyes, and meninges (including astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas, and meningiomas), solid tumors arising from hematopoietic malignancies such as leukemias, and lymphomas (both Hodgkin's and non-Hodgkin's lymphomas).

Dosage ns may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For e, a single bolus may be stered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the cies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.

Dosage unit form as used herein refers to physically te units suited as unitary s for the mammalian subjects to be treated; each unit ning a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of a binding protein(s) that binds IL-loc and/or lL-ll3 and the particular therapeutic or prophylactic effect to be achieved, and (b) the tions inherent in the art of compounding such protein(s) for the treatment of individuals.

An exemplary, miting range for a therapeutically or prophylactically ive amount of a binding protein useful in the treatment of osteoarthritis or pain is 0.1-20 mg/kg, more ably 1-10 mg/kg. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods and compositions of the invention described herein are obvious and may be made using suitable equivalents without departing from the scope of the invention or the embodiments disclosed . Having now described the present invention in detail, the same will be more y understood by reference to the following examples, which are included for purposes of illustration only and are not ed to be limiting of the invention.

Examples e 1. Generation of Dual Variable Domain Immunoglobulin (DVD-lg) Protein A dual variable domain immunoglobulin (DVD-lg) molecule is ed such that two different light chain variable domains (VL) from two different parent mAbs are linked in tandem directly or via a short linker by inant DNA techniques, followed by a light chain constant domain. Similarly, the heavy chain comprises two different heavy chain variable domains (VH) linked in tandem directly or via a short linker, followed by a constant domain CH1 and Fc region.

See Figure 1A. The design and production of DVD-lg binding proteins from parental monoclonal antibodies, including examples of a DVD-lg binding protein that bind to IL-loc and IL-lB as ed from a parental anti-IL-l (x monoclonal antibody and a parental anti-IL-1[3 monoclonal antibody, has been described. See, PCT Publication No. A2 and Wu et al., Nature Biotechnol, 25(11): 1290-1297 (200?), incorporated herein by nce. Descriptions of selected monoclonal antibodies to IL-lot and IL-l[3 and their use as parental monoclonal antibodies for the production of DVD-Ig molecules that bind both IL—loc and IL—l[3 are provided herein. The DVD-Ig molecules were terized for possible therapeutic activity using known animal models for toid arthritis and osteoarthritis.

Example 1.1. Generation of Murine Monoclonal Antibodies to IL-la and IL-1|3 Monoclonal antibodies (mAbs) to lL-loc and IL-ll3 were generated as follows using standard hybridoma technology.

Example 1.1.A. Immunization Of Mice Purified recombinant human IL-loc and murine IL-lB (R&D Systems) were used as immunogens as well as g antigens in titer assays and screening ELISA. Immunizing dosages ranged from 5.0 to 20.0 ug/mouse/injection for all antigens for both primary and boost immunizations. ImmunEasy nt was purchased from Qiagen (Waltham, MA) and used at nt/antigen ratio of 20 ml ImmunEasy adjuvant per 10.0 ug antigen. Each group of animals to be immunized contained five IL-1(x[3 KO mice obtained from Dr. Yoichiro Iwakura (University of Tokyo, -ku, Tokyo, Japan). The mice were immunized according to dosing schedule 2O described below. MRC-S cells were purchased from ATCC sas, VA) and used for IL-1 bioassay. Human IL-8 ELISA kits and control mouse anti-hIL-loc and anti-hIL-1[3 antibodies (MAB200 and MAB201) were purchased from R&D Systems (Minneapolis, MN).

Briefly, adjuvant-antigen mixture was ed by first gently mixing the nt in a vial using a vortex. The desired amount of adjuvant was removed from the vial and put into an aved 1.5 mL microcentrifuge tube. The antigen was prepared in PBS or saline with concentration ranging from 0.5-1.0 mgiml. The calculated amount of antigen was then added to the microcentrifuge tube with the adjuvant and the on was mixed by gently pipetting up and down times. The adjuvant-antigen mixture was incubated at room temperature for 15 minutes and then mixed again by gently pipetting up and down 5 times. The adjuvant-antigen solution was drawn into the proper syringe for animal injection. A total of 5-20 ug of antigen was injected in a volume of 50- 100u1. Each animal was immunized, and then boosted 2 to 3 times depending on the titer. Animals with good titers were given a final intravenous boost before fusion and generation of hybridomas.

Example 1.1.B. Screening Hybridomas Hybridomas, generated as described above, were screened and antibody titer determined using ELISA. Protein antigens were directly coated on ELISA plates for detecting the specific antibodies using standard ELISA procedures. Briefly, ELISA plates were coated with 100 til of either rhIL-loc or rhIL-lB (1.0 ug/ml in PBS) overnight at 4°C. Plates were washed 3 times with 250 til PBS/0.5%Tween20 and blocked with 200 u] blocking buffer (2% BSA in PBS with 0.5 20).

Diluted sera or hybridoma supernatant (100 pl) was added to each well, and incubated at room temperature for 2 hours. Plates were then washed three times with PBS/0.5%Tween20. HRP-goat anti-murine IgG was used for detection, and binding ODs were observed at 450 nm. Hybridoma clones producing dies that showed high specific binding activity in the ELISA were subcloned and ed, and affinity (Biacore) and potency (MRC-S bioassay) of the antibodies were characterized as s. e 1.1.C. Characterization of Murine Monoclonal Antibodies to IL-lot And IL-1|3 The following assays were used to terize the antibodies produced by the hybridomas described in e 1.1.B.

Example 1.1.C.1. Surface Plasmon Resonance Real-time binding interactions between antibody (mouse anti-recombinant mIL-l dy) captured on a biosensor matrix Via goat anti-mouse IgG and rmIL-l were measured by surface plasmon resonance (SPR) using the BIAcore system (Biacore AB, Uppsala, Sweden) according to manufacturer's instructions and standard procedures. Briefly, rmIL-l was diluted in HBS running buffer (Biacore AB) and 50 pl ts were injected through the immobilized protein matrices at a flow rate of 5 ml/minutes. The concentrations of rhIL—l employed were 62.5, 125, 187.5, 250, 375, 2O 500, 750, 1000, 1500, and 2000 nM. To determine the dissociation constant (off-rate), association constant (on-rate), BIAcore kinetic evaluation software (version 3.1) was used.

Example 1.1.C.2. L-l Bioassay The MRC-S cell line is a human lung fibroblast cell line that produces lL-8 in response to human IL-1oc and IL-1[3 in a dose-dependent manner (see, Dinarello, Muegge, and Durum (2000) Curr. Protocols l. 6:1). MRC-S cells were cultured in 10% FBS te MEM and grown at 37°C in a 5% C02 incubator. To determine lizing potencies of the monoclonal antibodies (mAbs) against recombinant human IL—loc or ll.—1[3, different concentrations (0-10 ug/ml) of mAb (50 ill) was added to a 96-well plate and pre-incubated with 50 u] of rhIL—loc or rhIL-lB (10-50 pg/ml) for 1 hour at 37°C. The supematants were harvested, diluted, and IL-8 concentrations ed by ELISA using a standard lL-8 ELISA kit (R&D Systems). dy potency was determined by its ability to inhibit IL-8 production by MRC-S cells.

Based on Biacore and MRC-S bioassay, a number of murine anti-hIL-lu and anti-hIL-lB antibodies with high affinity and potency were identified, as shown in Table 1 below: Table 1. Generation And terization Of Murine Anti-Hil-lot/B Mabs mAb Clone# city KD (M) IC50 (M) 3D12.E3 hIL—loc 1.11 x 10‘9 6.70 x10'10 C8 hIL—loc 5.78 x 10‘10 8.90 x 10'11 6H3.1A4.3E11 hIL—loc 3.54 x10‘10 2.40 x10"10 13F5.G5 hIL—IB 2.91 x 10‘10 6.00 x 10’10 1B12.4H4 hIL—IB 2.13 x10‘10 5.30 x10’10 6B12.4F6 hIL-lfi 5.54 x 10‘10 3.20 x 10'10 Example 1.1.D. Cloning and Sequencing of the Murine Monoclonal Antibodies (mAbs) to IL-loc and IL-1[3 Cloning and sequencing of the variable heavy (VH) and light (VL) genes of all anti-IL-lodB mAbs described in Table 1 ) and additional antibodies were carried out after isolation and purification of the total RNA from the each oma cell line using Trizol reagent (Invitrogen) according to the manufacturer's instructions. ication of both VH and VL genes was carried out using the IgGVH and ngVL oligonucleotides from the Mouse Ig-Primer Set (Novagen, Madison, WI) with One-tube RT-PCR kit (Qiagen) as suggested by the cturer. DNA fragments resulting from productive amplifications were cloned into pCR-TOPO vector (Invitrogen) according to the manufacturer's instructions. Multiple VH and VL clones were then sequenced by the dideoxy chain termination method using an ABI 3000 sequencer (Applied Biosystems, Foster City, CA). The sequences of all mAb VL and VH genes are shown below in Table 2.

TABLE 2. Murine Monoclonal Antibodies Capable 0f Binding Human IL-la 0r IL-1[3 Protein Sequence Identification Number Sequence 12345678901234567890 QIQLVQSGPfiLKKPGfiiVKI SCKASGYTFQNYGMNWVKQA PGKDLKRMAWINTYTGESTY ADDFKGRFATSLETSASTAY VH 3D12.E3 SEQ ID N0:89 LQINNLKNEDTATYFCARGI YYYGSSYAMDYWGQGTSVTV TTSSLSASLGDRVT ISCRASQDISNCLNWYQQKP DGTVKLLIYYTSRLHSGVPS RFSGSGSGTDYSLTISNLEQ VL 3D12.E3 SEQ ID NO:90 EDIATYFCQQGKTLPYAFGG GTKLEINR Protein Sequence Identification Number Sequence 12345678901234567890 EVQLQQSGAELVKPGASVKL LNIKDTYMHWLKQ? PEQGLEWIGQIDPANGNAKY DPRFLGKATITADTSSNTAY VH 18F4.2C8 SEQ ID N0:91 TSEDTAVYYCARGD GNFHTDYWGQGTTLTVSS DIVMTQSQRTMSTSVGDRVS VTCKASQNVGTNIAWYQQKP GQSPRALIYSASYRYSGVPD SEQ ID N0:92 RFTGSGSGTDFTLTISNVQS VL 18F4.2C8 VDLAEYFCQQYTRYPLTFGG GTKLEIKR QVQLQQPGAELVRPGASVKL SCKASGY-E1-YWMNWVKQR PEQGLEW-GR-DPYDSETLY SEQ ID N0:93 SQKtKD_AIL_VDKSSSTAY VH 6H3.1A4.3E11 TSEDSAVYYCARYG FDYWGQG_1L-VSS QIVLTQSPALMSASPGE<VT MTCSASSSVVYMYWYQQ<PR SSPKPWIYLTSN_4ASGVPAR FSGSGSGISYSTIIISS *.A*.

SEQ ID N0:94 VL 6H3.1A4.3E11 DAATYYCQQWNSVPYTFGGG QVQIQQSGAELVRPGSSVKI SC<ASGYAFSSYWMNWVKQR PGQGLEWIGQIYPGDGD"NY NG<FKGKAT_4TADKSSS"SY VH 13F5.G5 SEQ ID N0:95 MQ'ISG ITSEDSA YFCVRFP TGVDYYAMDYWGQGTSV"VS NIVLTQSPASLAVSLGQRAT ISCRASESVDSYGNSYMIWY QQKPGQPPKLLIYLASNLES GVPARFSGSGSRTDFTLTID VL 13F5.G5 SEQ ID N0:96 PVEADDAATYYCQQNNEDPF TFGSGTKLEIKR QVHLKESGPGLVAPSQSLSI TCTVSGFSLTDYGVSWIRQP WLGLIWGGGDTYYN VH 1B12.4H4 SEQ ID N0:97 SPLKSRLSIRKDNSKSQVFL KMNSLQTDDTAVYYCAKQRT LWGYDLYGMDYWGQGTSVTV Protein Sequence Identification Number Sequence 12345678901234567890 ETTVTQSPASLSMAIGEKVT IRCITSTDIDVDMWWYQQKP 4LISQGNTLRPGVPS RFSSSGSGTDFVE‘I IzawMLs VL 1312 4H4‘ SEQ ID N0-98' EDVADYYCLQSDNVPVTFGA GTKLELKR EVQLQQSGPELVKTGTSVKI SCKASGYSFTGYYMHWVRQS HGKSLEWIGYISCYNGFTSY KATFTVDTSSSTAY VH 6B12.4F6 SEQ ID No.99, IQFSRLTSEDSAVYYCARSD YYGTNDYWGQGTTLTVSS QIVLTQSPAIMSASPGEKVT ITCSASSSVSYMHWFQQKPG ASPKLWIYSTSNLASGVPAR FSGSGSGTSYSLTVSRMEAA VL 6B12.4F6 SEQ ID N0:100 CQQRSTYPYTFGGG TKLEIKR Example 1.2. Generation and Characterization of Murine-Human Chimeric Antibodies All mAbs described above were ted to chimeric antibodies (with human constant region) and expressed, purified, and characterized to confirm activity. The antibodies were also used for ls for subsequent DVD-lg binding n analysis. To convert 3D12.E3 into chimeric form, 3D12.E3-VL was PCR amplified using primers P1 and P2; meanwhile human CK gene (in pBOS vector generated in-house, Abbott Bioresearch Center, Worcester, MA) was amplified using primers P3 and P4. Both PCR reactions were performed according to standard PCR techniques and procedures. The two PCR products were rified, and used together as overlapping template for the subsequent overlapping PCR reaction using primers P1 and P4 using rd PCR conditions.

The final PCR product, the chimeric light chain 3D12.E3-VL-hCK, was subcloned into pEF6 TOPO mammalian expression vector (Invitrogen) by TOPO cloning ing to the manufacturer's instructions. Table 3 shows the sequences of each of the PCR s used in this procedure.

Table 3. PCR Primers P3: 5 CGT ACG GTG GCT GCA CCA TCT GTC 3' SEQ ID NO: 103 Pm 5TCAACACTCTCCCCTGTTGAAGC3' SEQHDNOJO4 To convert 3D12.E3 heavy chain into chimeric form, 3D12.E3-VH was PCR amplified using primers P5 and P6; meanwhile human C71 gene (in pBOS vector generated in-house at ABC) was amplified using primers P7 and P8. Both PCR reactions were performed according to standard PCR techniques and procedures. The two PCR products were rified, and used together as overlapping template for the subsequent overlapping PCR reaction using primers P5 and P8 using standard PCR conditions. The final PCR t, the chimeric light chain 3-VH-hCyl, was ned into pcDNA3.l TOPO mammalian expression vector (Invitrogen) according to the manufacturer’s ctions. Table 4 shows the sequences of each of the PCR primers used in this procedure.

Table 4. PCR Primers SEQ ID NO: 105 P6: 5' GGG CCC TTG GTC GAC GCT GAG GAG ACG GTG ACT GAG G 3' SEQ 1]) NO: 106 P7: 5' GCG TCG ACC AAG GGC CCA TCG GTC TTC C 3' SEQ 1]) NO: 107 P8: 5' TC ATT TAC CCG GAG ACA GGG AGA GGC 3' SEQ 1D NO:108 Similarly, chimeric l3F5.G5-VH-Cyl was generated using primers P21/P22 (for VH) and P7/P8 (for hCyl) and cloned into pcDNA3.1 TOPO vector, and chimeric 13F5.G5-VL-CK was generated using primers P23/P24 (for VL) and P3/P4 (for hCK) and cloned into pEF6 TOPO vector.

Table 5 shows the sequences of each of the PCR primers used in this procedure.

Table 5. PCR s P21: 5' ATA GAA TGG AGC TGG GTT TTC CTC 3' SEQ ID NO: 109 P22: 5' GGG CCC TTG GTC GAC GC TGA GGA GAC GGT GAC TGA 3' SEQ ID NO: 110 P23: 5' ATG GTC CTC ATG TCC TTG CTG TTC 3' SEQ ID NO:111 P24: 5' GC AGC CAC CGT ACG CCG TTT TAT TTC CAG CTT TG 3' SEQ ID NO: 112 To express chimeric antibodies, 13F5.G5-VL—CK and 13F5.G5-VH-Cyl were co-expressed in COS cells using Lipofectamin (Invitrogen) for 72 hours, and the medium collected and lgG purified by Protein A chromatography. Similarly, l3F5.G5-VL-CK and l3F5.G5-VH-Cyl were co- 2O expressed in COS using Lipofectamin (Invitrogen) for 72 hours, and the medium ted and IgG d by Protein A chromatography. Both purified chimeric Abs were characterized by Biacore and MRC-S bioassay to confirm activity. The results showed that these chimeric Abs displayed similar affinity and potency to that of the al murine mAbs.

Example 1.3. Construction, sion, and Purification of IL-loz/B Dual Variable Domain Immunoglobulin (DVD-lg) Molecule The construct used to generate DVD-1g binding protein capable of binding hIL- 10c and hIL- l[3 is illustrated in Figure 1B. , parent mAbs including two high affinity murine antibodies, anti-hlL-loc (clone 3D12.E3) and anti-hIL—ll3 (clone 13F5.G5), were obtained by immunizing Balb/c 3O mice with recombinant lL-loc protein (rhlL-loc) and recombinant IL-IB protein (rhIL—l B), respectively. The VL/VH genes of these two hybridoma clones were isolated by RT-PCR using the mouse lg Primer Kit (Novagen, Madison, WI). The VL/VH genes were first ted into chimeric antibodies (with human constant regions) to confirm activity and potency. To generate DVDl-lg binding protein, the VH and VL of 13F5.G5 was directly fused to the N-terminus of the VH and VL of 3D12.E3, respectively (as shown in Figure 1B). The DVD2-lg binding protein was constructed similarly, except that it had a linker between the two variable s in both the light chain (the linker sequence is ADAAP) and the heavy chain (the linker sequence is AKTTPP). These sequences were selected from the N-termini of murine Ck and CH1 sequences. These linker sequences, ed from the N-termini of murine Ck and CH1, are natural extension of the variable domains and exhibit a flexible conformation without significant secondary structures based on the analysis of several Fab crystal structures. The detailed procedures of the PCR cloning are described below. e 1.3.A. Molecular Cloning of hIl-lor/B DVDl-Ig Binding Protein 13F5.G5-VH was PCR amplified using primers P21 and P25. 3D12.E3-VH-hCyl was amplified using primers P14 and P8. Both PCR reactions were performed according to standard PCR techniques and procedures. The two PCR products were gel-purified, and used together as overlapping template for the uent pping PCR reaction using primers P21 and P8 using standard PCR conditions. The final PCR product, the DVDl-Ig heavy chain x/[3DVD1-VH- hCyl, was subcloned into pcDNA3.1 TOPO mammalian expression vector (Invitrogen) according to the manufacturer's instructions. Table 6 shows the sequences of the PCR primers used in this 2O procedure.

Table 6. PCR Primers P14: 5’ CAG ATC CAG TTG GTG CAG TCT GG3’ SEQ ID NO; 113 P25: 5’ CAC CAA CTG GAT CTG TGA GGA GAC GGT GAC TGA GG 3’ SEQ ID NO; 114 To generate hIL-loL/BDVDl—Ig light chain, 13F5.G5-VL was PCR amplified using primers P23 and P26; ile 3D12.E3-VL—hCK was amplified using primers P16 and P4. Both PCR reactions were med according to standard PCR techniques and procedures. The two PCR products were rified, and used together as overlapping template for the subsequent pping PCR reaction using primers P23 and P4 using standard PCR ions. The final PCR product, the z/[3 DVD 1 -Ig light chain hIL- 1 oc/BDVDl-VL-hCK, was subcloned into pEF6 TOPO mammalian expression vector rogen) according to the manufacturer’s instructions.

Table 7 shows the sequences of each of the PCR primers used in this procedure.

Table 7. PCR Primers P16: 5' AAT ATC CAG ATG ACA CAG ACT ACA TCC 3' SEQ ID NO: 115 P26: 5' GTGT CAT CTG GAT ATT CCG TTT TAT TTC CAG CTT TG 3' SEQ ID NO: 116 Example 1.3.B. Molecular cloning of x/B g 13F5.G5-VH was PCR amplified using primers P21 and P17. 3D12.E3-VH-hCy1 was amplified using primers P18 and P8. Both PCR reactions were performed according to standard PCR techniques and procedures. The two PCR products were gel-purified, and used er as pping te for the subsequent overlapping PCR reaction using primers P21 and P8 using standard PCR ions. The final PCR product, the DVD2—Ig heavy chain hIL-l (x/BDVD2-VH- hCyl, was subcloned into pcDNA3.1 TOPO mammalian expression vector (Invitrogen) according to the manufacturer’s instructions. Table 8 shows the sequences of each of the PCR s used in this ure.

Table 8. PCR Primers P17: 5' TGG GGG TGT CGT TTT GGC TGA GG 3' SEQlDNO:117 P18: 5' GCC AAA ACG ACA CCC CCA CAG ATC CAG TTG GTG CAG 3' SEQ ID NO:118 | To generate hIL-1a/[3DVD2-Ig light chain, 13F5.G5-VL was PCR amplified using primers P23 and P19. 3D12.E3-VL—hCK was amplified using primers P20 and P4. Both PCR reactions were performed according to standard PCR techniques and procedures. The two PCR ts were gel- d, and used together as overlapping template for the subsequent pping PCR reaction using primers P23 and P4 using standard PCR conditions. The final PCR product, the hIL- 1(x/[3DVD2-Ig light chain hIL-loc/BDVDZ-VL—hCK, was subcloned into pEF6 TOPO mammalian expression vector (Invitrogen) according to the manufacturer’s instructions. Table 9 shows the sequences of each of the PCR primers used in this procedure.

Table 9. PCR Primers P19: 5’ TGG TGC AGC ATC AGC CCG TTT TAT TTC 3’ SEQ ID NO:119 P20: 5’ GCT GAT GCT GCA CCA AAT ATC CAG ATG ACA CAG 3’ SEQ ID NO: 120 The final sequences of hlL-loni'fiDVDl-Ig and hIL—loc/[3DVD2-Ig are described in TablelO.

Table 10. Amino Acid Sequence of hIl-la/BDVDl-Ig and hIl-la/[SDVDZ-Ig Binding Protein Protein Sequence Protein region Sequence Identifier l 2 3 4 5 6 7 8 9 0 l 2 3 4 5 6 7 8 9 0 QVQLQQSGAELVRPGSSVKI SCKASGYAFSSYWMNWVKQR PGQGLEWIGQIYPGDGDTNY NGKFKGKATLTADKS S STSY DVD-1g HEAVY SEQ ID N03121 MQLSGLTSEDSAMYFCVRFP VARIABLE TGNDYYA_-DYWGQGTSVTVS SQIQLVQSGPELKKPGETVK hIL'l‘x/BDVDI'Ig I SCKASGYTFRNYGMNWVKQ APGKDLKRMAWINTYTGEST YADDFKGRFAF S LET SAS TA Sequence Protein region Sequenceldenfifier 12345678901234567890 YLQINNLKNEDTATYFCARG IYYYGSSYAWDYWGQGTSVT VH 13F5.G5 SEQ ID NO:95 QVQLQQSGAELVRPGSSVKI SCKASGYAFSSYWMNWVKQR PGQGLEWIGQIYPGDGDTNY NGKTKGKATLTADKSSSTSY TSEDSAMYFCVRFP TGNDYYAMDYWGQGTSVTVS Linker None 3D12.E3 VH SEQ ID NO:89 QIQLVQSGPELKKPGETVKI SCKASGYTFRNYGMNWVKQA PGKDLKRMAWINTYTGESTY ADDFKGRFAFSLETSASTAY LQINNLKNEDTATYFCARGI YYYGSSYAMDYWGQGTSVTV SEQ ID NO:122 ASTKGPSVFPAAPSSKS"SG GTAALGCAVKDYtPLPV vs WNSGALTSGV{"FPAV4QSS GLYSLSSVVTVPSSSAG"QT YICNVNH<PSV"<VD<< KSCDKTHTCPPCPAP? PSVFLFPPKP<DTL 18' P VJVS{«JP«V<tww YVDGVLViNA<i<PRn.« w S"YRVVSVLTV.{QDW.WG < EY<C<VSWKA.PAPI?<TIS <A<GQPRnPQVYiLPPSRnn "(WQVS.TC.V<GFYPSDI AVthSNGQPnNVY<iiPPV LDSDGSFFLYSKATVDKSRW QQGVVFSCSV H?A.HN{Y" QKSLSLSPGK VIVLTQSPAS.AVS.GQRA" ISCRASESVDSYGNSYMiWY DVD-Ig LIGHT QQK?GQPPKLVIYLASN.?S GVPARFSGSGSRTDTTLTID SEQ ID N0:123 VARIABLE PVEADDAATYYCQQVNED?F TFGSGTKLEIKRNIQMTQTT hIL-lot/BDVDl-Ig SSLSASLGDRVTISCRASQD ISNCLNWYQQKPDGTVKLLI HSGVPSRFSGSGSG ISNLEQEDIATYFC QQGKTLPYAFGGGTKLEINR 13F5.G5 VL SEQ ID NO:96 SPASLAVSLGQRAT ISCRASESVDSYGNSYMHWY QQKPGQPPKLLIYLASNLES GVPARFSGSGSRTDFTLTID PVEADDAATYYCQQNNEDPF TFGSGTKLEIKR Linker None Sequence Protein region Sequenceldenfifier 12345678901234567890 3D12.E3 VL SEQ ID N0:90 TTSSLSASLGDRVT QDISNC_LVWYQQ<P DGTVKLLIYYTSRLHSGVPS RFSGSGSGTDYSLTISN.?Q EDIATYTCQQGKTLPYATGG GTKLEIVR CL SEQ ID NO:124 TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC QVQLQQSGAELVRPGSSVKI SCKASGYAFSSYWMNWVKQR PGQGLEWIGQIYPGDGDTNY NGKFKGKATLTADKSSSTSY DVD-1g HEAVY SEQ ID NO:125 ”QLSGLTSEDSAMYFCVRFP VARIABLE TGNDYYAHDYWGQG-SV-VS SAKTTPPQIQLVQSGPflIK< hIL-1a/[3DVD2-Ig PGETVKISCKASGYTFRWYG ”NWVKQAPGKDL<RMAWINT YiGES-YADDtKGRbAbSLL TSASTAYIQINNI<NflDlA IYYYGSSYA DYWG QGTSVTVSS 13F5.G5 VH SEQ ID N0:95 QVQLQQSGAELVRPGSSVKI SCKASGYAFSSYWMNWVKQR PGQGLEWIGQIYPGDGDTNY NGKFKGKATLTADKSSSTSY MQTSGITSPDSA YFCVRFP TGNDYYAMDYWGQGTSVTVS Linker SEQ ID N0:75 AKTTPP 3D12.E3 VH SEQ ID N0:89 QIQLVQSGPflLK<PGfliVKI SCKASGYTFRNYGMNWVKQA PGKDLKRMAWINLYLGLS Y ADDTKGRFATSLETSASTAY LQIVNLKNEDTATYFCARGI YYYGSSYAMDYWGQGTSVTV CH SEQ ID NO:122 ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDT'MISRTP EVTCVVVDVSjEDPEVKFNW YVDGVEVflNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS REPQVYLLPPSRfifl SLTCLVKGFYPSDI AVEWESNGQPENNY<TTPPV Sequence Protein region celdenfifier 1234567890123 4567890 LDSDGSFFLYSKLTVDKSRW QQGVVFSCSVMHLAIHNiY" LSPGK SPAS' IIAVS'GQ3A" ISC3ASESVDSYGNSYMHWY QQK?GQPPKLLIYLASNLES GVPARFSGSGSRTDTTLTID SEQ ID NO:126 DVD-lg LIGHT PVEADDAATYYCQQVNEDPF KLEIKRADAAPVIQ VARIABLE HIL- MTQTTSSLSASLGDRVTISC lot/BDVDZ-Ig RASQDISNCLNWYQQKPDGT VKLLIYYTSRLHSGVPSRFS GSGSGTDYSLTISNLEQEDI ATYFCQQGKTLPYAFGGGTK LEINR 13F5.G5 VL SEQ ID NO:96 NIVLTQSPASLAVSLGQRAT ESVDSYGNSYUjWY QQKPGQPPKLLIYLASNLLS GVPARFSGSGSRTDFTLTID PVEADDAATYYCQQNNLDPF TFGSGTKLEI<3 Linker SEQ ID NO:69 ADAAP 3D12.E3 VL SEQ ID NO:90 NIQMTQTTSSLSASLGD3VT ISCRASQDISWCLVWYQQ<P DGTV<LLIYYTS3LHSGVPS 3FSGSGSGTDYS .TISV I3Q LDIATYFCQQGKT.LPYAFGG GTKLLIV3 CL SEQ ID N0:124 "VAAPSVFIFPPS DTQ I<SG "ASVVCI INNFYP3LA<VQW KVDNALQSGNSQ fiSVi*IQDS KDSTYS ISSTTIT IS<ADYLK HKVYACLVTHQG .SSPVT<S FNRGEC Example 1.3.C. Expression and Purification of hIL-lodBDVDl-Ig g Protein The heavy and light chain of each construct was subcloned into pcDNA3.l TOPO and pEF6 TOPO vectors (Invitrogen Inc), respectively, and sequenced to ensure accuracy. The plasmids encoding the heavy and light chains of each construct were transiently expressed using Lipofectamine 2000 and 293fectin reagents, respectively in COS cells as well as human embryonic kidney 293 cells (American Type Culture Collection, Manassas, VA). The cell culture media were ted 72 hour-post transient transfection and antibodies purified using protein A chromatography (Pierce, Rockford, IL) according to manufacturer's instructions. The dies were analyzed by SDS-PAGE and quantitated by A280 and BCA (Pierce, Rockford, IL). Table 11 shows that the expression levels of hIL—lw’BDVDl-Ig and hlL-loc/BDVDZ-Ig are comparable to that of the chimeric antibodies, ting that the DVD-lg binding protein can be expressed efficiently in mammalian cells.

Table 11. Expression and Molecular Weight Analysis of hIL-lu/B DVD-lg Binding Protein Expression Level (ngiml) Molecular Mass (Dalton) yle 293 Light Chain Heavy Chain Full length 0 0 3D12.E3-Ch 2788 3886 23,696 49,914 147,220 13F5.G5-Ch 3260 3562 24,084 49,518 147,204 DVDl-I_ 2988 3300 (35,790) (64,371) (200,521) 36,222 64,976 202,354 DVD2-Ig 2433 (36,220) (64,973) (202,573) The molecular mass of the light chain, heavy chain, and full length of DVDl-lg binding protein and DVD2-Ig binding n determined experimentally by mass spectrometry are shown in parenthesis.

Example 1.4. Mass Spectrometry and SEC Analysis of hIL-la/BDVD-Ig Binding Protein For measuring lar weight (MW) of light and heavy chains of DVD-lg binding protein, 10 ltL of DVD-lg molecule (0.8 ug/uL) was reduced by 1.0 M DTT solution (5 liL). A , 8”, 4000A, and 1 x 150 mm protein column (Michrom ource, Auburn, MA) was used to te heavy and light chains of DVD-lg molecule. Agilent HP1100 Capillary HPLC (Agilent Technologies Inc., Pala Alto, CA) was used with the mass spectrometer QSTAR (Applied Biosystems, Foster City, CA). The valco valve was set at 10 minutes to switch the flow from waste to MS for desalting sample. Buffer A was 0.02% TFA, 0.08% FA, 0.1% ACN and 99.8% HPLC- H20. Buffer B contained 0.02% TFA, 0.08% PA, 0.1% HPLC-H20, and 99.8% ACN. The HPLC flow rate was 50 liL/minute and the sample injection volume was 8.0 mL. The temperature of the column oven was set at 60°C, and separation gradient was: 5%B for 5 minutes; 5%B to 65%B for 35 minutes; 65%B to 95%B for r 5 minutes, and 95%B to 5%B for 5 minutes. TOFMS scan was from 800 to 2500 amu, and cycles were 3600. To determine the MW of full length DVD-1g binding protein, 21 Protein MicroTrap cartridge (Michrom BioResource, Auburn, MA) was used for ing the sample. The HPLC gradient was: 5%B for 5 minutes; 5%B to 95%B in 1 minute; and from 95%B to 5%B in another 4 minutes. The QSTAR TOFMS scan was from 2000 to 3500 amu, and cycles were 899. All MS raw data were ed using the Analyst QS software ed Biosystems). For SEC analysis of the DVD-lg binding protein, purified DVD-lg binding protein and ic Abs, in PBS, were applied on a Superose 6 10i300 G2, 300 x 10 mm column (Amersham Bioscience, Piscataway, NJ). An HPLC instrument, Model 10A (Shimadzu, Columbia, MD) was used for SEC. All ns were determined using UV detection at 280 nm and 214 nm. The elution was isocratic at a flow rate of 0.5 mL/minute. For stability study, samples in the tration range of 0.2-0.4 mg/ml in PBS ent 3 freeze-thaw cycles between -80°C and 25°C, or were ted at 4°C, 25°C, or 40°C, for 4 weeks and 8 weeks, followed by SEC analysis.

DVD-lg binding protein and chimeric antibodies were purified by protein A chromatography. The purification yield (3-5 mg/L) was consistent with hIgG quantification of the expression medium for each protein. The composition and purity of the d DVD-Ig binding proteins and chimeric antibodies were analyzed by SDS-PAGE in both reduced and non-reduced conditions. In non-reduced condition, each of the four proteins migrated as a single band. The DVD-lg proteins showed greater molecular weight than the chimeric dies, as expected. In non-reducing condition, each of the four proteins yielded two bands, one heavy chain and one light chain. Again, the heavy and light chains of the DVD-lg binding proteins were larger in size than that of the ic antibodies. The SDS-PAGE showed that each DVD-lg binding protein is expressed as a single species, and the heavy and light chains are efficiently paired to form an IgG-like molecule. The sizes of the heavy and light chains as well as the full-length protein of two DVD-lg molecules are consistent with their calculated molecular mass based on amino acid sequences (see Table 11).

In order to determine the precise molecular weight of the DVD-lg binding proteins, mass spectrometry was employed. As shown in Table 1, the experimentally determined molecular mass of 2O each DVD-lg binding protein, including the light chain, heavy chain, and the full-length protein, is in good agreement with the ted value. To further study the physical properties of DVD-lg g n in solution, size exclusion tography (SEC) was used to analyze each protein.

Both ic Abs and DVD2—Ig binding protein exhibited a single peak, demonstrating physical homogeneity as monomeric proteins. The 3D12.E3 chimeric Ab showed a r physical size then 13E5.G5 ic Ab, indicating that 3D12.E3 chimeric Ab adopted a more compact, globular shape. DVDl-Ig binding protein revealed a major peak as well as a shoulder peak on the right, suggesting that a portion of DVDl-Ig binding protein is possibly in an aggregated form in current buffer condition.

Example 1.5: Analysis of In Vitro Stability of hIL-lw’B DVD-lg Binding Proteins The physical stability of DVD-lg was tested as follows. Purified antibodies in the concentration range of 0.2-0.4 mg/ml in PBS underwent 3 freeze-thaw cycles between -80°C and °C, or were incubated at 4°C, 25°C, or 40°C, for 4 weeks and 8 weeks, followed by analysis using size exclusion chromatography (SEC) analysis (see Table 12).

Table 12. In Vitro Stability Analysis of hIl-ltx/B DVD-lg by SEC 3D12.E3-Ch 13F5.G5-Ch DVDl-Ig DVD2-Ig Ab Frgm Agg Ab Frgm Agg Ab Frgm Agg 3xFreeze —ThaW . 98.28 0.00 13.0 87.0 0.0 46.50 53.50 0.00 0.0 . l 0‘0 0.0 . . . 100.0 0.0 . .0 .0 _-fl__ _-fl__ _-fl__ 40°C @ 8 Wks 4.74 81.47 13.79 34.6 65.4 0.0 20.55 67.16 12.29 0.0 100.0 0.0 The degree of ation and fragmentation are shown in percentage, whereas the tage of Ab represents intact molecule.

Agg: aggregates; Ab: intact antibody; Frgm: fragments.

Both chimeric antibodies showed minor degrees of aggregation and fragmentation, normal for a regular IgG molecule. DVDl-Ig binding protein showed some aggregation on SEC after purification. In the ity analysis, DVDl-Ig binding protein also showed aggregations in PBS under different conditions; however the percentage of aggregated form of DVDl-Ig binding protein did not increase during prolonged storage or at higher temperatures. The percentage of the fragmented form of DVDl-Ig binding protein was in the normal range, similar to that of the chimeric 3D12.E3 Ab. In contrast, DVDZ-Ig binding protein showed exceptional stability. Neither aggregation nor fragmentation was detected for DVDZ-Ig binding protein in all conditions tested, and 100% of DVDZ-Ig binding protein maintained as intact monomeric molecule.

Example 1.6. Determination of n g Affinity of hIL-lot/B DVD-lg Binding Proteins The cs of DVD-Ig les binding to rhILl—oc and rhILl—B was determined by surface plasmon resonance-based measurements with a Biacore 3000 instrument re AB, a, Sweden) using HBS-EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20) at 25°C. All als were obtained from Biacore AB (Uppsala, Sweden) or otherwise from a different source as described herein. Approximately, 5000 RU of goat anti- human IgG Fey fragment specific polyclonal antibody e Biotechnology Inc, Rockford, IL) diluted in 10 mM sodium acetate (pH 4.5) was directly immobilized across a CM5 research grade biosensor chip using a standard amine coupling kit according to manufacturer’s instructions and procedures at 25 mg/ml. Unreacted es on the biosensor surface were blocked with ethanolamine. Modified carboxymethyl dextran surface in flowcell 2 and 4 was used as a reaction surface. Unmodified carboxymethyl dextran without goat anti-human IgG in flow cell 1 and 3 was used as the reference surface. For kinetic analysis, rate ons derived from the 1:1 Langmuir binding model were fitted simultaneously to association and dissociation phases of all ten injections (using global fit analysis) using the Bioevaluation 4.0.1 software. d DVD-lg samples were diluted in HEPES-buffered saline for capture across goat anti—human IgG Fc specific reaction surfaces and injected over reaction es at a flow rate of 5 ml/minute. The association and dissociation rate constants, kon (M-ls-l) and koff (s-l) were determined under a uous flow rate of 25 ml/minute. Rate constants were derived by making kinetic binding measurements at ten different antigen concentrations ranging from 1.25 to 1000 nM. The equilibrium dissociation constant (M) of the reaction between DVD-Ig le and rhIthx/B was then calculated from the kinetic rate constants by the following formula: KD = on. Aliquots of rhILloc/B samples were also aneously injected over a blank reference and reaction CM surface to record and subtract any nonspecific binding background to eliminate the majority of the refractive index change and injection noise. Surfaces were regenerated with two subsequent 25 ml injections of 10 mM Glycine (pH 1.5) at a flow rate of 5 ml/minute. The anti-Fc antibody immobilized surfaces were completely rated and retained their full capture capacity over twelve cycles. The apparent stoichiometry of the captured DVD-lg- rhIL1(X,/ [3 complex was calculated under saturating binding conditions 2O (steady-state equilibrium) using the following formula: /B se (RU) DVD-lg (MW) Stoichiometry = ———— x ——___ DVD response (RU) rhILl (x/[3 (MW) The Biacore is indicated the chimeric antibodies possessed similar binding kinetics and affinities to IL-1 as the al hybridoma monoclonal antibodies, indicating that the correct VL/VH sequences had been isolated (Table 13). The overall binding parameters of the two DVD-lg binding proteins to hIL—IOL were similar, with the affinities of the DVD—1g binding proteins being only 2-3 fold less than that of the chimeric 3D12.E3 antibody. The binding affinity of DVD2-Ig binding protein to hIL—ll?) was slightly less than the chimeric antibody 13F5.G5, but 3-fold higher than that of DVDl-Ig binding n. The affinity of the two DVD-lg binding proteins to hIL-l as compared to the affinity of chimeric antibodies to hIL-l was similar as ted by the tion of the stoichiometry to IL-1. Both chimeric antibodies, being bivalent ecific, bound to IL-loc and lL-1[3 on Biacore with a stoichiometry of 1.6 and 1.7, respectively. This is common for an IgG due to inter-molecular interference when antibodies are immobilized y on the Biacore sense chip resulting in stoichiometry being in the range from 1.5 to 2.0. The stoichiometries of both DVD- Ig binding proteins for c and hL—IB were similar to that of the two chimeric antibodies, indicating that both DVD-lg binding proteins possessed bivalent binding capability to each antigen.

Table 13. Functional Characterization of IL-1 Binding Proteins IL-l Binding ken koff Kd Stoichio- Potency Protein Antigen (M-l 8—1) (8—1) (M) metry ICso (M) 3D13.E3 hIL-101 6.43 x 10+5 7.13 x 104 1.11 x 10‘9 2.0 6.70 x 10'10 3D12.E3-Ch hIL-loc 4.12 x 10+5 5.52 x 10“ 1.34 x 10'9 1.6 7.00 x 10'10 DVDl-Ig hIL-loc 3.70 x 10+4 1.05 x 10“ 2.83 x 10'9 1.8 2.30 x 10‘9 DVD2-I c 7.35 x 10+4 2.52 x 10“ 3.42 x 10'9 2.0 2.90 x 10‘9 13F5.G5 hIL-1[3 2.13 x 10+6 6.21 x 10“ 2.91 x 10'10 1.8 6.00 x 10-10 13F5.G5-Ch hIL—1[3 1.41 x 10*6 6.54 x 104 4.62 x 10-10 1.7 5.30 x 10'10 g hIL-1[3 6.09 x 10+5 1.59 x 10-3 2.60 x 10-9 1.5 3.10 x 10'9 DVD2-Ig hIL-1[3 1.19 x 10+6 9.50 x 104 7.98 x 10'10 1.8 1.60 x 10'9 Affinity and stoichiometry were measured by Biacore; Potency (ICSO) was determined by MRC-5 bioassay; Ch = chimeric In addition, alent pecific antigen binding of DVD-lg binding protein was also analyzed by Biacore (Table 14). DVD-lg g protein was first captured via a goat anti-human Fc antibody on the Biacore sensor chip, and the first antigen was injected and a binding signal observed. As the DVD-lg binding protein was saturated by the first antigen, the second antigen was then injected and the second signal observed. This was done either by first injecting IL—1[3 then IL—loc or by first injecting lL—loc followed by IL—1[3 for DVD2-Ig binding protein. In either sequence, a dual-binding ty was detected. r results were obtained for DVDl-lg binding protein. Thus, each DVD-lg binding protein was able to bind both antigens simultaneously as a dual-specific tetravalent molecule. As shown in Table 14, the stoichiometry of both DVD-lg binding protein to the first antigen, either c or hlL—IB, were larger than 1.5, similar to that of monospecific bivalent binding. Upon the injection of the second antigen, while DVD-lg binding protein was already occupied by the first antigen, the stoichiometry of both DVD—Igs binding protein to the second antigen (i.e., hIL-IOL or hIL—1[3) was between 1.0 and 1.3. Thus, DVD—1g binding n was able to bind two lL—lOL and two IL-B molecules. DVD-1g binding protein was first captured via a goat anti-human Fc antibody on the e sensor chip, and the first n was injected and a binding signal observed, followed by the injection of the second antigen.

Table 14. Stoichiometry Analysis of hIL-lot/B DVD-lg Binding Protein in Tetravalent Dual-S ecific Bindin_ to IL-10t/ 5 Response Unit Stoichiometry Captured Binding Protein 1st antigen 2nd antigen hIL-10t:DVD-Ig hIL-l S :DVD-Ig DVDl-Ig: 932 hIL-10L: 190 hIL-1[3: 75 DVDl-Ig: 1092 hIL-IB: 141 hIL—loc: 107 DVD2-Ig: 1324 hIL-loc: 209 3: 137 DVD2-Ig: 1184 : hIL—loc: 131 Example 1.7. ination of Functional Homogeneity of DVD-lg Molecules Because DVD2-Ig binding protein was purified by n A chromatography instead of target-specific affinity chromatography, any potential misfolded and/or mismatched VL/VH domains, if present, can be ed by binding s against the two different antigens. Such binding analysis was conduced by size exclusion liquid chromatography (SEC). DVD2-Ig binding protein, alone or after a 120-minute incubation period at 37°C with IL-loc, IL-IB, or both IL-10t and IL-ll3, in equal molar ratio, were applied to the . Each of the antigens was also run alone as controls. The SEC results indicated that DVD2-Ig binding protein was able to bind IL-10t and IL-1[3 in solution, and such binding ed in a shift to the SEC signal indicating an se in the dynamic size of DVD2-Ig binding protein when it was in complex with either antigen. The shift of the DVD2-Ig binding protein signal was 100%, not partial, suggesting all DVD2-Ig molecules were able to bind the n. In the ce of both IL-loc and , there was a further and complete shift of the DVD2-Ig g protein signal, indicating all DVD2-Ig molecules were able to bind both antigens in a uniform fashion. This experiment demonstrated that DVD-lg binding protein was expressed as a functionally homogeneous protein. This has significant implications as it demonstrates that DVD-lg binding protein can be produced as a homogeneous single, functional s, which differs from all previously described cific, multi-specific, and multi-valent immunoglobulin-like and immunoglobulin-derived molecules. 2O Example 1.8. Determination of Biological Activity of DVD-lg Binding Proteins The biological activity of DVD—lg was measured using MRC—S bioassay. The MRC-5 cell line is a human lung fibroblast cell line that produces IL—8 in response to human IL—loc and IL—ll?) in a dose-dependent manner. MRC-5 cells were obtained from ATCC and cultured in 10% FBS complete MEM at 37°C in a 5% C02 incubator. To determine neutralizing activity of the DVD-lg against human IL—loc or 1L—1[3, 50 n1 of dy (1 x 10'7 to 1 x 10‘12 M) in MEM/10%FBS was added to a 96 well plate and pre-incubated with 50 ul of hIL—loc or hIL—ll3 (200 pg/ml) for 1 hour at 37°C, 5% C02. MRC-5 cells at a concentration of 1 x 105fml were then added (100 pl) to all wells, and the plates were incubated overnight at 37°C in a 5% C02 incubator. The supernatants were harvested and human IL-8 production measured by standard ELISA (R&D Systems, Minneapolis, MN). Neutralizing activity of the DVD—lg binding protein was determined by its ability to inhibit IL-8 tion.

As shown in Table 13, both DVD-Ig binding proteins were able to neutralize hlL-loc and hIL-lB. Consistent with the g affinity to hIL—loc, the neutralizing activities of g binding protein and DVD2-Ig binding protein against hIL-loc were also similar, i.e., 3-fold less than that of the ic antibodies (see, Table 13). Consistent with its g affinity for hIL—lB, the neutralizing activity of g binding protein to hIL—IB is slightly less than that of the chimeric Ab 13F5.G5, but 3-fold higher than that of DVDl-Ig binding protein. Overall there was no significant decrease in the biological activities of DVD-lg molecules compared to the original monoclonal antibodies.

To determine if DVD-lg binding protein was able to inhibit IL-8 production in the presence of both IL-loc and IL-l[3, equal s of hIL—loc and hIL-lB were added in the same culture system of MRC-5 assay. Both the DVDl-Ig binding protein and the DVD2-Ig binding protein were able to inhibit IL-8 synthesis by MRC-S cells in the presence of both IL-loc and IL—lB, with activities similar to that of mono-assays where only one cytokine was present (Table 13). In this assay where both IL-loc and IL-l[3 were present, the nhibition activity of DVD2-Ig binding protein (1.2 nM) was higher than that of DVDl-Ig binding protein (2.2 nM).

Example 2. Analysis of Linker Size and Variable Domain Orientation in DVD-lg Binding 2O Proteins onal DVD-Ig molecules with different parent mAb pairs, as shown in Table 15, were constructed. For each pair of mAbs, four different DVD-Ig ucts were generated: 2 with a short linker and 2 with a long linker, each in two different domain orientations: a-b-C (alpha-beta-constant domain) and b-a-C (beta-alpha-constant domain). The linker ces were derived from the N- terminal sequence of human Ck or CH] domain, as follows: Short linker: light chain: TVAAP (SEQ ID ; heavy chain: ASTKGP (SEQ ID NO:79); and Long linker: light chain: TVAAPSVEIFPP (SEQ ID NO:72); heavy chain: ASTKGPSVFPLAP (SEQ ID .

All heavy and light chain ucts were ned into the pBOS expression vector, and expressed in COS cells or freestyle 293 cells.

To construct new DVD-lg clones, the variable domains of the two mAbs, both light chain and heavy chain, were first jointed in tandem using overlapping PCR as described for hIL- 1(x/[3DVD1-Ig and hIL—loc/BDVD2-Ig. The jointed pieces were then subcloned in pBOS vector using homologous recombination. Briefly, vectors were linearized by restriction digestion (2 ug of pBOS-th vector were digested with FspAI and Bsin in 0+ buffer, and 2 ug of pBOS-hCy z,non a vector was digested with FspAI and SaII in 0+ ). The digested samples were run on a 1% agarose gel and the backbone fragment purified in 50 ul water. For homologous recombination and transformation, DHSoc competent cells were thaw on ice, and mixed with 20-50 ng jointed PCR product and 20-50 ng of linearized vector (in every 50 Ml DHSOL cells). The e was mixed gently and incubated on ice for 45 minutes, followed by heat shock at 42°C for 1 minute. Then 100 til SOC medium were added and incubated at 37°C for 1 hour. The transformation culture was inoculated on LB/agar plates containing ampicillin and incubated at 37°C for 18-20 hours. The bacterial clones were isolated, from which DNA was purified and ted to sequencing analysis.

The final sequence-verified clones were co-transfected (matching HV and LC of the same antibody pair) in COS or 293 cells for antibody expression and purification, as usly described.

Characteristics of the purified DVD-lg proteins are summarized in Table 16. The left section of the Table 16 shows the specificity, binding affinity, and neutralization potency of the 2 pairs of mAbs used for the construction of the new hIL-loc/B DVD-lg molecules. Antibodies 18F4.2C8 and 1B12.4H4 (see, Example 1.1.D) were used to construct hlL-la/B DVD3a-Ig, hlL- 10c/[3 Ig, hIL-loc/[3 DVD3b-Ig, and hIL-loc/B DVD4b-lg. hlL-loc/BDVD3a-lg and hlL- 10c/[3DVD4a-Ig were in a-b-C orientation, with a short and long linker, respectively. hlL- DVD3b-Ig and X,/[3DVD4b-Ig were in b-a-C orientation, with a short and long , respectively. Antibodies 6H3.1A4 and 6B12.4F6 were used to construct hlL-1(x/[3DVD5a-Ig, hlL- 1(x/[3DVD6a-lg, hlL-loc/B Ig, and hIL—loc/B DVD6b-Ig. hIL-loc/B Ig and hlL- 1(x/[3DVD6a-lg were in a-b-C orientation, with a short and long linker, respectively. hlL-loc/B 2O DVDSb-Ig and hIL-loc/BDVD6b-lg were in b-a—C orientation, with a short and long linker, respectively. The molecular cloning of these additional hIL-loc/[3 DVD-lg binding proteins were performed using the procedure previously described for hIL-loc/B DVDl-lg (see, Example 1.3), using overlapping PCR procedures. The amino acid sequences of these onal hlL-loc/[3 DVD-lg g proteins are disclosed in Table 15.

Table 15. Amino Acid Sequence of Heavy Chain and Light Chain of Six DVD-Ig Proteins e of Binding IL-10. and IL-1[3 Protein region Sequenceldenfifier 12345678901234567890 EVQLQQSGAELVKPGASVKJ SCTASGLNIKDTYMHWLKQR PEQGLEWIGRIDPANGNAKY KATITADTSSNTAY DVD-Ig HEAVY SEQ ID N0:127 LQLSSLTSEDTAVYYCARGD VARIABLE GNFHFDYWGQGTTLTVSSQS hIL-la/B DVD3a-Ig TKGPQVHLKESGPGLVAPSQ SLSITCTVSGFSLTDYGVSW KGLEWLGLIWGGGD LKSRLSIRKDNSKS QVFLKMNSLQTDDTAVYYCA KQRTLWGYDLYGMDYWGQGT SVTVSS 18F4.2C8 VH SEQ ID NO:91 EVQLQQSGAELVKPGASVKL SCTASGLNIKDTYMHWLKQR PEQGLEWIGRIDPANGNAKY DPRFLGKATITADTSSNTAY LQASSATSED"AVYYCARGD GNFHFDYWGQGTTLTVSS LINKER SEQ ID NO:79 ASTKGP 1B12.4H4 VH SEQ ID NO:97 QViL<ESGPGLVAPSQSDSI iC VSGhSL DYGVSWIQQP PG<G.?WLGIIWGGGDTYYN SPAKSRLSIQKDNSKSQVFL KMVSAQTDD"AVYYCAKQRT LWGYDAYGMDYWGQGTSVTV SEQ ID N0:122 ASTKGPSVFPJAPSS<S"SG GTAAIGCIV<DYEPLPV VS WNSGALTSGV{"FPAV4QSS GLYSLSSVVTVPSSSDG"QT YICVVNH<PSW"KVD<<VEP KSCDKTHTCPPCPAP?.IGG PSVTLFP?KPKDT4WISQTP EVTCVVVDVSHED?EV<FVW YVDGVEVHVAKTK??EEQYN STYRVVSVVTVLHQDWVVGK EYKCKVSWKALPA?IEKTIS KAKGQPRE?QVYTL?PSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT SPGK DIVQTQSQRFMSTSVGDRVS VTCSASQNVGTNIAWYQQKP GQSPRALIYSASYRYSGVPD RtiGSGSG-Db-AiISNVQS SEQ ID N0:128 DVD-Ig LIGHT VDLAEYFCQQYTRYPLTFGG GiKAEIKR-VAAPEiiV QS VARIABLE HIL- PASASMAIGEKVTIRCI"ST Sequence Protein region Sequenceldenfifier 12345678901234567890 lot/[3 DVD3a-Ig DIDVDMVWYQQKPG?PP<.L ISQGNTLRPGVPSRFSSSGS GTDFVFIIENMLSEDVADYY LPLTFGAG"KLE.K 18F4.2C8 VL SEQ ID N0:92 DIVMTQSQRFMSTSVGDRVS VTCKASQNVGTNIAWYQQKP GQSPRALIYSASYRYSGV?D SGTDFTLTISNVQS VDLAEYFCQQYTRYPLTFGG LINKER SEQ ID NO:71 TVAAP 1B12.4H4 VL SEQ ID NO:98 ETTVTQSPASLSMAIGEKVT TDIDVDMNWYQQKP GEPPKLLISQGNTLRPGVPS RFSSSGSGTDFVFIIENMLS EDVADYYCLQSDNLPLTFGA GTKLELKR SEQ ID N0:124 TVAAPSVFIFPPSDEQJKSG TASVVCALNNFYPREA<VQW KVDNALQSGNSQflSVifiQDS KDSTYSASSTITIS<ADYEK HKVYACEVTHQG.SSPVT<S FNRGEC QViL<ESGPGLVAPSQSDSI TCTVSGFSJ"DYGVSWIRQP PG<GIEWIG.IWGGGDTYYN SPJKSRLSIRKDNSKSQVFL DVD-lg HEAVY SEQ ID N0:129 KMVSJQTDD"AVYYCAKQR" VARIABLE YG DYWGQGTSVTV hIL-lot/B DVD3b- SSAST<GPEVQLQQSGA7.VJ. 1g KPGASVKJSCTASGLNI<D" YMHW1<QRPflQGIflWIGRID PANGVAKYDPRFJGKATI"A DTSSVTAYVQussuiSflD A VYYCARGDGVTHTDYWGQGT TLTVSS 1B12.4H4 VH SEQ ID N0:97 QVHLKESG?GLVA?SQSLSI TCTVSGFSLTDYGVSWIQQP PGKGLEWLGLIWGGGDTYYN SPLKSRLSIRKDNSKSQVFL KMNSLQTDDTAVYYCAKQRT LWGYDLYGMDYWGQGTSVTV LINKER SEQ ID NO:79 ASTKGP 18F4.2C8 VH SEQ ID NO:91 EVQLQQSGAELVKPGASVKL SCTASGLNISDTYMHWLKQR WIGRIDPANGNAKY DPRELGKAII-ADISSN-AY LQLSSLTSEDT VYYCARGD GNFHFDYWGQGTTLTVSS CH SEQ ID NO:122 ASTKGPSVFPLAPSSKS"SG GTAALGCLVKDYEPLPV VS Sequence Protein region Sequenceldenfifier 12345678901234567890 WNSGALTSGVHTFPAVJQSS GLYSLSSVVTVPSSSJGTQT YICVVNHKPSNTKVD<<VE KSCDKTHTCPPCPAP7.LGGJ.

PPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFVW YVDGVEVHNAKTKPREEQYN STYQVVSVLTVWHQDWVNGK EYKCKVSNKAL?APIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSD: AVEWESNGQPENNYKTTPPV DSDGSFFLYSKLTVDKSRW QGNVFSCSVMjEALHNHYT XSASLSPGK TTVTQSPASLSMAIGEKVT RCITSTDIDVDMNWYQQKP PPKLLISQGNTARPGVPS SSSGSGTDFVFIIENMJS SEQ ID NO:130 DVD-1g LIGHT VADYYCLQSDNuPITFGA ,_] KEELKRTVAAPDIVMTQS VARIABLE HIL- IOOWSUg—IDHFJIOIOF'U'TJRFMSTSVGDRVSVTCKASQ 1a/[3 Ig NVGTNIAWYQQ<PGQSPRAL IYSASYRYSGVPDRFTGSGS N06) 10 10 ._<Db JIISNVQSVDLAEYF RYPLitGGGiKuflIK H4 VL SEQ ID N0:98 DL‘J/UGDHD 1V QSPASLSMAIGE<VT RCI"STDIDVDMVWYQQKP EPP<.LISQGNT4RPG FSSSGSGTDFVFIIEV LS CIQSDNIPITFGA TK.?.KR LINKER SEQ ID N0:71 TVAAP 18F4.2C8 VL SEQ ID N0:92 DIVMTQSQRF RVS VTCKASQNVGTNIAWYQQKP GQS?RALIYSASYRYSGVPD RFTGSGSGTDTTLTISNVQS VDLAEYTCQQYTQYPLTFGG GTKLEIKR SEQ ID NO:124 TVAAPSVFIF?PSDEQLKSG TASVVCLLNNTY?REAKVQW KVDVALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC SGAELVKPGASVKL SCTASGLNIKDTYMHWAKQR PEQGLEWIGRIDPANGNAKY DPRFLGKATITADTSSNTAY DVD-Ig HEAVY SEQ ID NO:131 LQLSSLTSEDTAVYYCARGD VARIABLE hIL- GNFHFDYWGQGTTLTVSSEE 1(x/[3 DVD4a-Ig TKGPSVFPLAPQVHLKESGP GLVAPSQSLSITCTVSGFSA Sequence Protein region Sequence Identifier 12345678901234567890 TDYGVSWIRQPPGKG.LWLG LIWGGGDTYYVSPLKSQLSI RKDVSKSQVFLKMNSIQTDD TAVYYCAKQRTLWGYDLYGM C8 VH SEQ ID NO:91 EVQLQQSGAELVKPGASVKL SCTASGLNIKDTYMHWLKQR PEQGLEWIGRIDPANGVAKY DPRFLGKATITADTSSNTAY LQLSSLTSEDTAVYYCARGD GNFHFDYWGQGTTLTVSS LINKER SEQ ID NO:80 ASTKGPSVFPLAP 1B12.4H4 VH SEQ ID NO:97 QVH'KESGPGLVAPSQSLSI TCTVSGFSLTDYGVSWIRQP WLGLIWGGGDTYYN SPLSSRLSIRKDNSKSQVFL KMNSLQTDDTAVYYCAKQRT LWGYJLYGMDYWGQGTSVTV CH SEQ ID N0:122 ASTK PSVFPLAPSS<S"SG CLVKDYEPLPV VS WNSGALTSGV{"FPAVLQSS GLYSLSSVVTVPSSSL YICNVVH<PSV"<VD<<VL PSVF_LFPPKP<DTL 18' P LViCVVVDVSiLDPLV<bVW YVDGV.LV{NA<1<PRL.« w STYRVVSVLTvuiQDWIVG < LYKC<VSVKAIPAPIE<TIS {AKGQPRLPQVYLTIPPSRLL WTKVQVSITCIV<GFYPSDI AVEWLSNGQP*.NVYK11PPV .LDSDGSFFLYSKLTVD<S?W SCSV HEALHVHYT QKSLSLSPGK DIVWTQSQRFWSTSVGDRVS VTCKASQNVGTNIAWYQQKP GQS?RALIYSASYRYSGVPD SEQ ID N0:132 RFTGSGSGTDTTLTISVVQS DVD—1g LIGHT VDLAEYTCQQYTRYPLTFGG GTKLEIKRTVAAPSVFIFPP VARIABLE HILETTVTQSPASLSMAIGLKVT 1u/[3 DVD4a-Ig IRCITSTDIDVDMNWYQQKP GEPPKLLISQGNTLRPGVPS RFSSSGSGTDFVFIIENMLS EDVADYYCLQSDNLPLTFGA GTKLELKR 18F4.2C8 VL SEQ ID NO:92 DIVMTQSQRFMSTSVGDRVS QNVGTNIAWYQQKP GQSPRALIYSASYRYSGVPD SGTDFTLTISNVQS VDLAEYFCQQYTRYPLTFGG GTKLEIKR Sequence Protein region Sequence Identifier l 2 3 4 5 6 7 8 9 0 l 2 3 4 5 6 7 8 9 0 LINKER SEQ ID N0:72 TVAAPSVFIFPP 1312.4H4 VL SEQ ID N0:98 ETTVTQSPAS .SMAIGEKVT TDIDVDMEWYQQKP GEPPKLLISQGNTLRPGVPS RFSSSGSGTDEVEIIEEMLS EDVADYYCLQSDNLPLTFGA GTKLELKR SEQ ID N0:124 TVAAPSVFIF?PSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC QVHLSESGPGDVAPSQSLSI TCTVSGFSDTDYGVSWIRQP WLGLIWGGGDTYYN DVD-1g HEAVY SEQ ID N0:133 EDPEESRTEEERKfiiggvfi“Q ‘ Q VARIABLE hIL- LWGYDLYGUDYWGQGTSVTV “VBIDVD4ng GPSVE9.APEVQ.Q QSGAELVKPGASVKSSCTAS GLNIKDTY HW.<QRP?QG.

EWIGRIDPANGEAKYJPRES GKATI"ADTSSETAY.Q.SS LISLD AVYYCARGDGVFHF TTD"VSS 1B12.4H4 VH SEQ ID N0:97 QVH u<T-SGPGT-VAPSQS.JS I TCTVSGFSD"DYGVSWIRQP PGKG.?WLG.IWGGGDTYYN SPL<SRLSIRKDNSKSQVFL KMNSSQTD3"AVYYCAKQRT LWGYDDYG DYWGQGTSVTV LINKER SEQ ID N0:80 ASTKGPSVFPDAP 18F4.2C8 VH SEQ ID N0:91 F-VQTIQQSGAT. .V <PGASVKS SCTASGLNIKDTYMHWLKQR PEQGLEWIGRIDPANGNAKY DPRFLGKATITADTSSNTAY LQLSSLTSEDTAVYYCARGD GNFHTDYWGQGTTLTVSSL CH SEQ ID N0:122 ASTKGPSVE‘?LA?SSKSTSG GTAAVGCVVKDYEPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKD LMISRTP EVTCVVVDVSjEJPEVKFNW YVDGVEVjNAKTKPREEQYN SVLTVLjQDWLNGK EYKCKVSNKADPAPIEKTIS RLPQVYIDPPSRnfi MTKNQVSDTCDVKGFYPSDI AVEWESNGQPENNY<TTPPV Sequence Protein region Sequenceldenfifier 12345678901234567890 LDSDGSFFLYSKLTVD<SRW QQGVVTSCSVMHEALHWHY" QKS'S'ISPGK ETTVTQSPASLSWAIG.LKV IRCITSTDIDVDWVWYQQKP GEP?KLLISQGN LRPGVPS RFSSSGSGTDFVTIIEVMLS SEQ ID N0:134 DVD—1g LIGHT EDVADYYCLQSD LPLTFGA GTK'E'<RTVAA?SVFIFPP VARIABLE HIL- DIVMTQSQRFMSTSVGDRVS 1a/[3 DVD4b-Ig VTCKASQNVGTNIAWYQQKP GQSPRALIYSASYRYSGVPD RFTGSGSGTDFTLTISNVQS VDLAEYFCQQYTRYPLTFGG GTKLEISR 1B12.4H4 VL SEQ ID NO:98 b-1V-QSPASLSMAIGEKVT IRCITSTDIDVDMNWYQQKP GEPPKLLISQGNTLRPGVPS SGTDFVFIIENMLS EDVADYYCLQSDNLPLTFGA GTKLELKR LINKER SEQ ID N0:72 TVAAPSVFIFPP 18F4.2C8 VL SEQ ID NO:92 DLVMTQSQRFMSTSVGDRVS VTCKASQNVGTNIAWYQQKP GQSPQALIYSASYRYSGVPD RFTGSGSGTDF"_LTISVVQS VDIALYECQQY RYPLitGG CL SEQ ID N0:124 "VAAPSVFIFPPSDLQIKSG "ASVVCIINVFYPREA<VQW KVDNALQSGVSQLSVILQDS ISSTITIS<ADYL< LVTIQGISSPVT<S QVQLQQ?GA7.VRPGASV<L SCKASGYTFTTYWMNWVKQ? PEQGLEWIGQIDPYDSL .Y SEQ ID N0:135 SQK?KDTAILTVDKSSS"AY DVD-lg HEAVY MQLSSLTSEDSAVYYCARYG VARIABLE hIL- FDYWGQGTTLTVSSASTKGP 1a/B DVDSa-Ig EVQLQQSGPELV<TGTSVKI SCKASGYSFTGYYMHWVRQS HGKSLEWIGYISCYNGFTSY NPKFKGKATFTVDTSSSTAY IQFSRLTSEDSAVYYCARSD YYGTNDYWGQGTTLTVSS 4.3E11 SEQ ID NO:93 QVQLQQPGAELVRPGASVKL VH SCKASGY-ti-YWMNWVKQR PEQGLEWLGRLDPYDSETLY LAILLVDKSSSTAY MQLSSLTSEDSAVYYCARYG G-1L-VSS LINKER SEQ ID N0:79 ASTKGP 6B12.4F6 VH SEQ ID NO:99 EVQLQQSGPELVKTGTSVKI Protein region Sequenceldenfifier 12345678901234567890 SCKASGYSFTGYYMHWVRQS HGKSLEWIGYISCYNGF"SY NPKFKGKATFTVDTSSS"AY IQFS LTSEDSAVYYCAQSD YYGTVDYWGQGTTLTVSS CH SEQ ID N0:122 ASTKG?SVFPLA?SSKSMSG GTAALGCLVKDYTPEPVHVS WNSGALTSGVHTTPAVLQSS GLYSLSSVVTV?SSSLGMQT HKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVjNAXTXPREEQYN STYRVVSVLTVLjQDWLNGK SNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI MTCSASSSVVY YWYQQ<PR SSP<PWIYLTSVLASGVPAR FSGSGSG SYSuiISS *A* SEQ ID N0:136 DVD-Ig LIGHT DAATYYCQQWNSNPYTFGGG R VAAPQIVLTQSP VARIABLE HIL- AIMSASPGE<VTITCSASSS 1a/[3 DVDSa-Ig VSY PGASP<LWIY STSWLASGVPARFSGSGSG" SYS. VSR DADDAAIYYCQ QRSTYPYTFGGGTKLEI<R 6H3.1A4.3E11 SEQ ID N0:94 QIVL"QSPA.MSASPGL<V VL MTCSASSSVVY YWYQQ<PR SSPKPWIYLTSWLASGVPA? FSGSGSGTSYSLLISS *A* DAATYYCQQWNSNPYTFGGG TKLEMKR LINKER SEQ ID N0:71 TVAAP 6B12.4F6 VL SEQ ID NO:100 QIVLTQSPAIMSASPGEKVT ITCSASSSVSYMHWFQQKPG ASPKLWIYSTSNLASGVPAR FSGSGSGTSYSLTVSRMEAE DAATYYCQQRSTYPYTFGGG TKLEIKR SEQ ID NO:124 TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC EVQLQQSGPELVKTGTSVKI SCKASGYSFTGYYMHWVRQS Sequence Protein region Sequenceldenfifier 78901234567890 HGKSLEWIGYISCYNGF"SY DVD-lg HEAVY SEQ ID N0:137 NPKFKGKATFTVDTSSS"AY VARIABLE hIL- TSEDSAVYYCARSD let/B DVDSb-Ig YYGTNDYWGQGTTLTVSSEE TKGPQVQLQQPGAELVRPGA SVKLSCKASGYTFTTYWMNW VKQRPEQGLEWIGRIDPYDS ETLYSQKFKDTAILTVDKSS STAYMQLSSLTSEDSAVYYC ARYGFDYWGQGTTLTVSS 6B12.4F6 VH SEQ ID NO:99 EVQLQQSGPELVKTGTSVKI SCKASGYSFTGYYMHWVRQS HGKSLEWIGYISCYNGFTSY NPKFKGKATFTVDTSSSTAY IQFSRLTSEDSAVYYCARSD YYGTNDYWGQGTTLTVSS LINKER SEQ ID N0:79 ASTKGP 6H3.1A4.3E11 SEQ ID N0:93 QVQLQQPGAELVRPGASVKD VH SCKASGY-E1-YWMNWVKQR PEQGLEWIGRIDPYDSL LY SQKEKD_AI4 VDKSSS"AY MQLSSL SLDSAVYYCARYG FDYWGQG 14 V88 SEQ ID N0:122 ASTKGPSVFPJAPSS<S"SG GTAAIGC.V<DYEPLPV VS WNSGALTSGV{"FPAV4QSS GLYSJSSVVTVPSSS YICNVNH<PSV"KVD<<VE <8CD<THTCPPCPAP?.IGG PSVFAFPPKP<DTL ISRTP LVICVVVDVSIfiDPflV<bWW YVDGVLVINA<1KPRfifiQYV STYQVVSVLTVIHQDWIWG< SVKAIPAPIE<TIS RflPQVYlLPPSRflfl MTKVQVSVTCIVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFTLYSKLTVDKSRW QQGVVFSCSVMHEALHNHYT QKSLSLS?GK QIVLTQS?AIMSASPGEKVT ITCSASSSVSYMHWFQQKPG ASPKLWIYSTSNLASGVPAR FSGSGSGTSYSLTVSRMEAE DVD—1g LIGHT SEQ ID N0:138 DAATYYCQQRSTYPYTFGGG TKLEIKRTVAAPQIVLTQSP VARIABLE HIL- ALMSASPGEKVTMTCSASSS 1(x/[3 DVDSb-Ig VNYMYWYQQKPRSSPKPWIY LTSNLASGVPARFSGSGSGT SYSLTISSMEAEDAATYYCQ QWNSNPYTFGGGTKLEMKR 6B12.4F6 VL SEQ ID N0:100 QIVLTQSPAIMSASPGEKV" ITCSASSSVSYMHWFQQKPG ASPKLWIYSTSNLASGVPAR Sequence Protein region Sequenceldenfifier 12345678901234567890 FSGSGSGISYSTIVSRM*A* DAATYYCQQRSTYPYTFGGG LINKER SEQ ID N0:71 TVAAP 6H3.1A4.3E11 SEQ ID N0:94 QIVLTQSPALMSASPGEKVT VL MTCSASSSVNYMYWYQQKPR SSPKPWIYLTSNLASGVPAR FSGSGSGTSYSLTISSMEAE CQQWNSNPYTFGGG TKLEMKR CL SEQ ID NO:124 TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC QVQLQQPGAELVRPGASVKL SCKASGY-EI-YWMNWVKQR PEQGLEW-GR-DPYDS.L LY SQKtKD-AIL_VDKSSS"AY DVD-lg HEAVY SEQ ID NO:139 MQLSSLTSEDSAVYYCARYG VARIABLE hIL- FDYWGQG i. VSSAS {GP 10t/[3 DVD6a-Ig SVFPIAPTVQTQQSGPTIVK TGTSVKISC<ASGYSF"GYY MHWVRQSiG<SIEWIGYISC YWP<F<GKATFTVD SSS AYIQESRIISLDSAV YYCARSDYYG"VDYWGQGTT LTVSS 6H3.1A4.3E11 SEQ ID N0:93 PGAELVRPGASVKL VH YTFT"YWMNWVKQ3 PLQGLLWIGRIDPYDS.L LY SQKbKD AIL VDKSSS"AY MQLSS: SLDSAVYYCARYG FDYWGQGTTL"VSS LINKER SEQ ID N0:80 ASTKG3SVT?LAP 6B12.4F6 VH SEQ ID N0:99 EVQLQQSG3ELVKTGTSVKI SCKASGYSTTGYYMHWVRQS HGKSLEWIGYISCYNGFTSY NPKFKGKATFTVDTSSSTAY TSEDSAVYYCARSD YYGTNDYWGQGTTLTVSS CH SEQ ID NO:122 ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPK3KDTLUISRT? EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLVGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYIIPPSRLL Sequence Protein region Sequence Identifier 12345678901234567890 MTKEQVSLTCEVKGEYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGEVESCSVWHEALHNHYT QKSLSLSPGK SPALWSASPGEKVT MTCSASSSVNYMYWYQQKPR SSPKPWIYLTSNLASGVPAR FSGSGSGTSYSLTISSMEAE DVD-1g LIGHT SEQ ID NO:140 DAATYYCQQWNSNPYTFGGG TKLEMKRTVAAPSVFIFPPQ VARIABLE HIL- IVLTQSPAIMSASPGEKVTI 1a/[3 DVD 6a-Ig TCSASSSVSYMHWFQQKPGA SPKLWIYSTSNLASGVPARF TSYSLTVSRMEAED AATYYCQQRSTYPYTFGGGT 6H3.1A4.3E11 SEQ ID NO:94 QIVLTQSPALMSASPGEKV" VL MTCSASSSVNYMYWYQQ<PR SSPKPWIYLTSNLASGVPAR FSGSGSGiSYSTiISSM*A* DAATYYCQQWNSNPYTFGGG TKLEMKR LINKER SEQ ID N0:72 TVAAPSVFIFPP 6B12.4F6 VL SEQ ID NO:100 QIVATQSPAIMSASPGE<VT ITCSASSSVSYMHWFQQ<PG ASP<LWIYSTSNLASGVPAQ FSGSGSGLSYSTILVS? fiA*.

DAATYYCQQRSTYPYTFGGG TKLEIKR CL SEQ ID NO:124 R"VAAPSVFIFPPSD?Q.KS C4LNNFYPREA<VQ WKVDNADQSGNSQESVifiQD SKDSTYSISSTLTIS<ADY? KHKVYACEVTHQGISSPVT< EVQLQQSGPELVKTGTSVKI SCKASGYSFTGYYMHWVRQS HGKSLEWIGYISCYNGFTSY KATFTVDTSSSTAY DVD-lg HEAVY SEQ ID NO:141 TSEDSAVYYCARSD VARIABLE hIL- YYGTNDYWGQGTTLTVSS§§ lot/[3 DVD6b-Ig TKG?SVFPLAPQVQLQQPGA ELVRPGASVKLSCKASGYTF TTYWMNWVKQRPEQGLEWIG RIDPYDSETLYSQKFKDTAI LTVDKSSSTAYMQLSSLTSE DSAVYYCARYGFDYWGQGTT LTVSS 6B12.4F6 VH SEQ ID NO:99 EVQLQQSGPELVKTGTSVKI SCKASGYSFTGYYMHWVRQS HGKSLEWIGYISCYNGFTSY NPKtKGKAibiVD_SSS AY IQFSRLTSEDSAVYYCARSD Sequence Protein region Sequence Identifier l 2 3 4 5 6 7 8 9 0 l 2 3 4 5 6 7 8 9 0 YYGTNDYWGQGTTLTVSS LINKER SEQ ID N0:80 ASTKGP SVF?_4AP 6H3.1A4.3E11 SEQ ID N0:93 QVQLQQPGAELVRPGASVKJ VH SCKASGYTFTTYWMNWVKQ? PEQGLEWIGRIDPYDSETLY TAILTVDKSSSTAY MQLSSLTSEDSAVYYCARYG FDYWGQGTTLTVSS SEQ ID N0:122 ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP HTCPPCPAPELLGG PSVFLFPPKPKDTAMISRT? EVTCVVVDVSjEDPEVKFNW YVDGVEV_NAKT PREEQYN SVATVAjQDWLNGK EYKCKVSNKAAPAPIEKTIS R PQVYIIPPSRflfl 4V<GFYPSDI TPPV LDSDGSFFuYS<ITVD<SRW {EALHW{Y" QKSJSLSPGK QIVJTQSPAI SASPGL<V ITCSASSSVSY iWFQQ<PG ASP<LWIYSTSV4ASGVPAR FSGSGSG SYSILVS? flAfl SEQ ID N0:142 DVD-1g LIGHT DAA"YYCQQRSTYPYTFGGG iKuflIKR VAAPSVFIFPPQ VARIABLE HIL- IVJ"QSPALMSASPGE<VTJ. 1a/[3 DVD6b-Ig TCSASSSVNYMYWYQQ<PRS SPK?WIYLTSN4ASGVPARF SGSGSGTSYSLLISS flAflD AATYYCQQWNSWPYTFGGG" 6B12.4F6 VL SEQ ID N0:100 QIVLTQSPAIMSASPGLKV ITCSASSSVSYWHWFQQKPG ASPKLWIYSTSVLASGVFAR FSGSGSGTSYSLTVSRWEAE CQQRSTYPYTTGGG TKLEIKR LINKER SEQ ID NO:72 TVAAPSVFIFPP 6H3.1A4.3E11 SEQ ID NO:94 QIVLTQSPALMSASPGEKVT VL MTCSASSSVNYMYWYQQKPR SSPKPWIYLTSNLASGVPAR FSGSGSGTSYSLTISSMEAE DAATYYCQQWNSNPYTFGGG TKLEMKR CL SEQ ID N0:124 TVAAPSVFIFPPSDEQAKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTISKADYEK Sequence Protein region Sequence Identifier l 2 3 4 5 6 7 8 9 0 l 2 3 4 5 6 7 8 9 0 EVTHQGLS SPVTKS FNRGEC Characteristics of the new DVD-lg constructs are summarized in Table 16. Affinity (Kd) and biological activity (ICSO) were determined by Biacore and MRC-S bioassay, respectively. SDS- PAGE analysis of all new DVD-lg proteins showed normal migration patterns in both reduced and non-reduced ions, similar to a regular antibody and DVD1/2-Ig.

Table 16. Characterization of New DVD-lg les Derived from New mAb Pairs Affinity (Kd) . Kd IC50 Spec1f. DVD Orient.. Llnker. IL- 1a IL-IB (M) (M) 8.37 6.37 DVD3a a—b—C short X1010 X103 .95 3.30 7.01 9.30 18F4.2C8 rhlL—lot DVD4a a—b—C X1040 X1040 long X1040 X1040 2.61 6.00 1.24 1.90 1B12.4H4 I‘l’llL-lB DVD3b b—a—C short X1040 X1040 X103 X1040 .60 1.28 DVD4b b—a—C long X1040 X1040 .08 1.25 DVD5a a—b—C short ><1 0.10 X10'8 3.54 2.40 1.06 2.09 6H3.1A4 rhlL—lot 10 10 DVD6a a—b—C long X10. X10. X103 X109 .54 4.00 1.32 6.71 6B12.4F6 I‘l’llL-lB 10 10 DVDS]? b—a—C short X10. X10. XlO_8 X1040 8.20 6.97 DVD6b b—a—C long X1040 X1040 mAb = monoclonal antibody; NA = no neutralization activity ed.

The functional terization of the new DVD-lg molecules revealed that with either orientation, DVD-lg molecules with the long linker med better than the ones with the short linker in terms of binding and neutralizing of both antigens. With respect to DVD-1g molecules with the long linkers, those with the b-a—C orientation showed good binding to and lization of both antigens, while the DVD-1g molecules with an a-b-C orientation showed good binding to and neutralization of lL-lOL and reduced binding to and neutralization of IL-ll3 (e. g., DVD4b vs.

DVD4a). The DVD-lg molecule, DVD4b, bound and neutralized both IL-loc and IL-ll3 with sub-nM and fully retained the binding and neutralizing characteristics of the parent mAbs.

Example 3. Generation of mIL-lu/B DVD-lg Molecules To study key issues concerning pharmacokinetics, in Vivo efficacy, tissue penetration, and immunogenicity of DVD-lg molecules, mouse-anti-mouse IL-1(x/[3 DVD-lg molecules were constructed as described below.

Example 3.1. Construction of mIL-lw’B DVD-lg Molecules Mouse anti-mouse IL—1(x/[3 DVD-lg molecules were constructed using two mouse anti- mouse IL-lodB onal antibodies (9H10 and lOGl 1) generated from l3 double KO mice.

Mouse anti-mouse lL-loc, and mouse anti-mouse IL-lB, monoclonal antibodies (mAbs) were generated by immunizing IL-loc/[3 double KO mice with mouse IL-loc, or mouse IL-ll3, respectively.

One mouse anti-mouse IL-10L (Clone 9H10) and one mouse anti-mouse IL-ll3 mAb (clone 10G11), were selected and used to generate mIL-lou’B DVD-lg les. Various linker sizes and different domain ations were tested. The final functional mIL-loc/B DVD-lg molecule was constructed in an orientation of V(anti-m1L-1[3)-linker-V(anti-mIL-loo-murme constant region (CYZa and CK).

The cloning, expression, and purification procedures were similar to that of the hIL-loc/B DVD-lg g protein. The cloning of mIL-IOLIB DVD-lg binding protein was carried out using similar overlapping PCR and homologous recombination as described for hIL-loc/B DVD3-Ig. The sequences of c/B DVD-lg binding proteins are shown below in Table 17.

Table 17. Amino Acid Sequence of mIL-lu/B DVD-lg Binding Protein Sequence Protein region Sequence Identifier l 2 3 4 5 6 7 8 9 0 l 2 3 4 5 6 7 8 9 0 ?VQLQQSGPTLVKPGTSV SCKTSGYTFTSYV'HWV<Q< ?GQGLEWIGYIIPYNDNT<Y mIL-la/[3 DVD-lg SEQ ID NO:143 VE?FK§KAT_JTSD<SSST"AY TSEDSAVYYCARRW HEAVY FFDYWGQGTTLTVS VARIABLE SAKTTAPSVYP'A?QVI.<? SG?GI'QPSQT'S'TCSTSG FSLSTYGTAVNWIRQPSGKG LEWLAQIGSDDQK'YNPT'K SRITLSEDTSNSQVFLKITS ATYYCANGVMEYWG LGTSVTVSS 10G11 VH SEQ ID NO:144 EVQLQQSGPELVKPGTSVKM SCKTSGYTFTSYVMHWVKQK PGQGLEWIGYIIPYNDNTKY NEKFKGKATLTSDKSSSTAY MELSSLTSEDSAVYYCARRN FFDYWGQGTTLTVS LINKER SEQ ID NO:78 AKTTAPSVYPLAP 9H10 VH SEQ ID NO:145 QVILKESGPGILQPSQTLSJ TCSFSGFSLSTYGTAVNWI? Sequence Protein region Sequence Identifier l 2 3 4 5 6 7 8 9 0 l 2 3 4 5 6 7 8 9 0 QPSGKGV?WLAQIGSDDRKL SRIT.S?DTSNSQV FLKITSVDTEDSATYYCANG VMEYWGLGTSVTVSS CH SEQ ID N0:146 AKTTAPSVYPLA?VCGDTTG SSVTLGCLVKGY?PEPVTLT WNSGSLSSGViTTPAVLQSD SVTVTSSTWPSQSI TCNVAHPASSTKVDKKIEPR GPTIKPCPPCKCPAPNLLGG PSVFIFPPKIKDVLMISLSP IVTCVVVDVSEDDPDVQISW FVNNVEVHTAQTQTHREDYN STLRVVSALPIQHQDWMSGK EFKCKVNNKDLPAPIERTIS KPKGSVRAPQVYVAPPPEEE MTKKQVTLTCMVTDFMPEDI NGKTEANYKNTEPV LDSDGSYFMYSKLRVEKKNW VERNSYSCSVVjEGLHwHH" SPASLSASVGLLV ITCRGSGILHNY_4VWYQQKQ GKSPQ. IVYSAKILADGVPS SEKQHDDKMI47 RFSGSGSGTQYSIKINsuQP mIL-la/[3 DVD-lg EDFGSYYCQHFWS"PFTFGS G KuflI<RADAAP"VSIFPP LIGHT VARIABLE SIVMTQTPKFALVSAGDRVT I"C<ASQSVN{DVAWYQQMP GQSP<L4IYFASNRYTGVPD RFTGSGYGTDFTFTIS"VQA EDLAVYFCQQDYSSPYHFGG GTK.:I<R 10G11 VL SEQ ID N0:148 DIQMTQSPAS .SASVGWVT ITCQGSGILHVY_4VWYQQKQ GKS?QL_4VYSAKILADGVPS RFSGSGSGTQYS_4KINSLQP EDFGSYYCQHTWSTPFTFGSL GTKLEIKR LINKER SEQ ID N0:70 ADAAPTVS IF?P 9H10 VL SEQ ID N0:149 S IVMTQTPKFJLVSAGDRVT ITCKASQSVNHDVAWYQQMP GQSPKLLIYFASNRYTGVPD YGTDFTFTISTVQA EDLAVYFCQQDYSSPYTFGG GTKLEIKR CL SEQ ID N0:150 ADAAPTVSIFPPSSEQLTSG GASVVCFLNNFYPKDINVKW KIDGSERQNGVLNSWTDQDS KDSLYSMSS14-L-KDbeR HNSYiCbAiHK-S-SPIVKS FNRNEC Murine mIL-loc/B DVD-1g binding proteins retained affinity/in vitro potency against both murine IL-loc (mIL-loc) and murine IL—IB (m]L-1[3). Table 18 shows the characterization of mAbs 9H10 (anti-mlL-loc), 10G11 (anti-mIL—IB), and mIL-lodl?) DVD-lg binding proteins.

Table 18. Characterization of mDVD4-Ig Antigen KD (M) ICSO (M) miL—ia/sDVD—Ig mIL-loc 7.66 x 10‘10 2.00 x 10'9 —_-694x10“ 800x10” Example 3.2. In Vivo Activity of mIL-lw‘BDVD-Ig Binding Proteins in Rheumatoid Arthritis Model The therapeutic effects of anti-IL—loc, anti-IL—lB, combined anti-IL-loc/anti-IL-IB, and murine IL-loc/B DVD4-Ig, were evaluated in a collagen-induced arthritis mouse model well known in the art. , male DBA-l mice were immunized with bovine type II collagen in CFA at the base of the tail. The mice were boosted with Zymosan intraperitoneally (i.p) at day 21. After disease onset at day 24-27, mice were selected and divided into te groups of 10 mice each.

The mean arthritis score of the control group, and anti—cytokine groups was comparable at the start of treatment. To neutralize IL—l, mice were injected every other day with 1-3 mg/kg of anti-IL-loc mAb, anti-IL-lB mAb, combination of anti-IL—loc and anti-IL-lB mAbs, or murine IL-loc/B DVD4-Ig intraperitoneally. Mice were carefully examined three times a week for the visual appearance of arthritis in peripheral joints, and scores for disease activity determined.

Blockade of IL-1 in the therapeutic mode effectively reduced the ty of arthritis, with anti-IL-lB showing greater efficacy (24% ion in mean arthritis score compared to control 2O group) than anti-IL-loc (10% reduction). An additive effect was observed between anti-IL-loc and anti-IL-IB, with a 40% reduction in mean arthritis score in mice d with both anti-IL-loc and anti-IL-IB mAbs. Surprisingly, at the same dose level, the treatment of mDVD-Ig g protein exhibited 47% reduction in mean arthritis score, demonstrating the in vivo therapeutic efficacy of g binding protein. Similar efficacy was also observed in the measurements of joint swelling in this animal model. e 3.3. In Vivo ty of a Murine B DVD-lg Binding Protein in Osteoarthritis Models The above study showed that the blockade of both IL—lu and IL-ll?) with a ation of anti-mouse IL-lu and lL-IB dies as well as a murine anti-mouse IL-la/B DVD-lg binding proteins was significantly more efficacious than either single antibody alone in the mouse collagen- induced arthritis (CIA) model for rheumatoid arthritis (see, also, Wu et al., Nature Biotech, 25(11): 12901297 (2007)). The efficacy of anti—IL—l therapy in osteoarthritis (0A) was studied two animal models of OA, i.e., the joint ility model ("JIM") and the destabilization of medial meniscus ("DMM") in mice.

Example 3.3.1. In Vivo Activity of Mouse Anti-Murine IL-1oc and Anti-Murine IL-1[3 Monoclonal Antibodies in Joint Instability Model (JIM) of Osteoarthritis in Mice Example 3.3.1.A. Study Design The design of study of the in viva activity of a mlL—loc/B DVD—Ig molecule in the joint instability model (JIM) of osteoarthritis in mice is shown in Table 19. All groups contained 25 mice (n = 25), ng imately 30 grams (BW = 30 g). The study was terminated after 21 days.

Two and a half month old, male Swiss Webster mice (25fgr0up/time point), housed 5/cage, were anesthetized with isoflurane anesthesia. Anterior cruciate ligament (ACL) trauma was administered on the right and left knees on day 0 in an t to induce OA lesions. Intraperitoneal (ip) treatments with IL-1 inhibitory dies IL-loc (9H 10) and IL-ll3 (10G11), alone and in combination, were initiated the day of ACL trauma and continued every 4th day (q4d) for 20 days.

The vehicle for the non-treatment control was PBS. Right and left knees were harvested on day 21, and histopathologic tions were scored and characterized. Data were analyzed using only joints that had proliferative responses indicative of successful instability induction. The proliferative response/instability was scored 0-3, and only unstable joints (scores of 1, 2, or 3) were used in the 2O final is. Medial and lateral femoral and tibial cartilage degeneration was scored for severity of cartilage degeneration on a scale 0-5. Four days after the last antibody dose, blood was drawn from all animals from each group for serum harvest. Body weights were recorded weekly. On the final day, s (10 animals in Groups 1, 2, 3, 4) were dosed with Zymosan A and blood drawn 4 hours later (at termination) for serum analysis of IL—6 TNF dependent). Animals were killed on day 21 and right and left knee joints collected into formalin.

Table 19. Design of Study in Joint Instability Animal Model for Osteoarthritis Treatment Group Compound Route Dosing Regimen Group 1 Vehicle 1 ip Q4D (PBS) (2 times per week) Group 2 anti-IL— 1 (x ip Q4D (9H10) (2 times per week) Group 3 anti-lL-ll3 ip Q4D (10G11) (2 times per week) Group 4 L— 1 (x ip Q4D (9H10) (2 times per week) anti-lL-l [3 (10G1 1 ) Example 3.3.1.B. Tissue Preparation and Analysis Tissue preparation: Following 2-3 days in a decalcifying e (Surgipath Decalifier, Surgipath Medical Ind, Inc, Richmond VA), both lmee joints were trimmed of extraneous tissue, embedded in the frontal plane and sectioned. One section was taken from each animal (2 joints/block) at the approximate mid point of the frontal plane. All sections were 8 pm and were stained with ine blue. Slides were examined by a board-certified veterinary pathologist blinded to study group designation, and were scored according to the method below. Statistical is was performed using Microsoft Excel, and included a 2-tailed t-test comparing all treatment groups to the control group using a confidence level of 95%.

Histopathologic scoring of joints: Medial and lateral femoral and tibial cartilage degeneration were scored for severity of cartilage degeneration using the following system: Depth/extent 0f chondrocyte and proteoglycan loss with fibrillation : 1= superficial damage, tangential layer of collagen absent over 50% or greater of the zone surface or up to 10% loss of proteoglycan and/0r chondrocytes in focal or diffuse distribution in zone 2=matrix loss extends into upper 1/4 of 50% or greater area of the zone or up to 25% loss of glycan and/0r chondrocytes in focal or diffuse bution in zone 3: matrix loss extends through 1/2 of cartilage thickness over 50% or greater of the zone or up to 50% loss of proteoglycan and/or chondrocytes in focal or e distribution in zone 2O 4: matrix loss extends through 3/4 of cartilage thiclmess over 50% or greater of the zone or up to 75% loss of proteoglycan and/or chondrocytes in focal or diffuse distribution in zone : matrix loss extends through entire cartilage thiclmess over 50% or greater of the zone or up to 100% loss of proteoglycan and/or chondrocytes in focal or diffuse distribution in zone Scores were assigned with attention to zonal (inside, middle and outside) distribution of lesions, and the scores (0-5) for each third were summed for each area of the joint and then for the whole joint.

Osteophytes st on tibial or femoral surface under evaluation) were measured using digital software (NIS-Elements version 3.0).

Osteophyte tion = 1, 2, or 3 for small, medium or large depending on size Small osteophytes = 1 (up to 150 um) Medium hytes = 2 (151-300 pm) Large hytes = 3 (>301 um) Synovial reaction was described and characterized with respect to inflammation type and degree if present was not included in the score.

The mean i SE for each of the various parameters for each animal was determined and summed to arrive at a total joint score.

Joints with histologic evidence of instability ion were identified by the presence of proliferative s in the medial synovium and collateral ligaments. In addition, an instability score was ed for each joint according to the following criteria. 0=No instability 1=Mild instability (minimal to mild proliferative changes in ligaments and marginal zones) 2=Moderate instability (moderate proliferative changes in ligaments and marginal zones) re instability (severe proliferative s in ligaments and al zones) Results: Only unstable joints with scores of 1, 2, or 3 (0r 2, 3) were used in the final data analysis.

Data were ultimately analyzed on a total affected joint basis rather than affected animal basis. The results of this study indicated that joints of animals treated with either anti-IL-loc or IL-lB monoclonal antibodies exhibited cartilage degeneration similar to that seen in vehicle-treated control joints. However, a combination therapy with both a mouse anti-mouse IL-lu mAb (9H10) and a mouse anti-mouse IL-lB mAb (10G11) significantly decreased medial tibial cartilage degeneration scores. Figure 2 shows bar graphs of cartilage degeneration scores in the joints of the animals in the 2O various treatment .

Example 3.3.2. In Vivo Activity of mIL-lu/B DVD-lg Binding Protein in Joint Instability Model (JIM) 0f Osteoarthritis in Mice The joint ility model for osteoarthritis as described above in Example 3.3.1, was also used to compare the efficacy of anti-IL—l therapy as provided by a combination of a mouse anti-mIL- la monoclonal antibody (9H10) and a mouse anti-mIL-IB monoclonal antibody (lOGl 1) with that provided by a mIL-loL/B DVD-Ig binding protein generated from the two monoclonal antibodies.

Example A. Study Design The design of this study was r to that described above in Example 3.3.1. Two and a half month old, male Swiss r mice oup/time point), housed 5/cage, were anesthetized 3O with isoflurane anesthesia and the right and left knee area clipped and prepared for intra-articular trauma to the te ligaments on day 0. Dosing (initiated on day 0, ip) was carried out once every 4 days (Q4D) with animals terminated on day 21 (for Group 2). Dosing (initiated on day 0, ip) was carried out three times weekly with animals terminated on day 21 (for Groups 1, 3 and 4). Four days after the last antibody dose, all animals from each group had blood drawn for serum harvest. Body weights were recorded weekly. On the final day, animals (10 animals in Groups 1, 2, 3, 4) were dosed with Zymosan A and blood drawn 4 hours later (at termination) for serum analysis of IL-6 (IL- l/TNF ent). Animals were ized on day 21 and right and left knee joints ted into in. See the study design table below.

Table 20. Design of Study of Joint Instability Animal Model for Osteoarthritis Treatment Compound Route Dosing Regimen Dose Level Group (mg/kg) Group 1 Vehicle 1 ’ 3 times per week 6.7 ml/kg (PBS) Group 2 anti-IL-loe Q4D anti-IL-l on at 6 mg/kg (9H 10) (3 times per (180 ug/mouse) and week) and anti-IL- 1|3 anti-IL-IB at 6 mg/kg ) (180 pig/mouse) Group 3 3 times per week 12 mg/kg (500 ttg/mouse) Group 4 3 times per week 6 mg/kg (250 ttg/mouse) Example 3.3.2.B. Tissue Preparation and Analysis Tissue preparation and histological scoring of articular cartilage from joints of animals in the various treatments groups were carried out as described above in e 3.3.1.

Results: The results indicated that treatment of mice with the mIL-la/B 10Gl l-9H10 DVD-lg binding protein significantly inhibited the progression of osteoarthritis (P < 0.05 vs. vehicle) with comparable efficacy to the combination of the parental mAbs 9H10 and 10Gl 1. Figure 3 shows bar graphs of cartilage degeneration scores in the joints of the animals in the various treatment groups.

As shown in Figure 3, results of ent with 6 or 12 mgi’kg the DVD—1g binding protein were similar in their efficacy in terms of preventing osteoarthritic lesions. (A treatment using 3 mg/kg DVD-lg binding protein had not effect on cartilage ration and results were similar to vehicle treated joints. See, Example 3.3.3, below.) The results of this study indicated that treatment with a combination of anti-IL—loc and anti-lL-1|3 monoclonal antibodies or with an IL-la/B DVD-lg binding protein had significant beneficial s on histopathological parameters in the mouse model of 2O osteoarthritis.

Example 3.3.3. Follow Up Study in Joint Instability Model of Osteoarthritis to Compare Treatment with Anti-IL-IB Monoclonal Antibody and Other g Proteins A follow up study was performed using the joint instability model of rthritis to more closely ine whether or not there may be any significant effect on medial tibial cartilage degeneration scores in animals treated with an anti-lL-IB monoclonal antibody alone compared to other treatments. The effect of treatment with an L—lB monoclonal antibody was evaluated in a 21 day study.

Example 3.3.3.A. Study Design The design of this study was similar to that described above in Example 3.3.1.

Table 21. Design Of 21 Day Joint Instability Animal Model For Osteoarthritis Treatment Compound Route Dosing Regimen Dose Level Group (mg/kg) Group 1 Vehicle 1 ' Q4D 6.7 ml/kg (PBS) Group 2 ' anti-IL—loc L-l Ct at 6 mg/kg (9H10) (180 ug/mouse) and and anti-IL—ll3 anti-IL-IB at 6 mg/kg (10G11) (180 se) Group 3 anti-IL-IB ' 12 mg/kg ) (360 ug/mouse) Group 4 anti-IL-IB ‘ 6 mg/kg (10G11) (180 ug/mouse) Group 5 anti-IL-IB 3 mg/kg (10G11) (90 ug/mouse) Example 3.3.3.B. Tissue Preparation and Analysis Tissue preparation and histological scoring of articular cartilage from joints of animals in the various treatments groups were carried out as described above in Example 3.3.1.

Results: As shown in the Figure 4, the results confirmed those of previous studies where similar positive treatment effects were noted using a combination of anti-IL-loc and anti-IL-lB monoclonal antibodies and that treatment with an anti-IL—lB monoclonal antibody alone was not ive in preventing cartilage degradation at any dose tested.

Example 3.3.4. Zymosan-Induced Interleukin-6 (IL-6) Production in a Joint Instability Model Study As a pharmacodynamic readout to determine if anti-IL-loc and anti-IL-ll?) monoclonal antibodies as well as mIL-lot/B DVD-Ig g protein were functional and capable of lizing IL-loc and IL-1[3 in viva, zymosan-induced c and IL—lB dependent) production of eukin 6 (IL-6) was measured in a joint instability model study of OA.

Briefly, on day 21 in the model, zymosan was ded in PBS vehicle and placed in boiling water for half an hour, then allowed to cool before using. The mice were then injected ip with 50 mg/kg zymosan in suspension (mixed well before injection) in 0.5 ml PBS. The mice were then bled 4 hours after zymosan injection. Serum was collected for lL-6 ements.

Results: As shown in Figure 5, a combination of anti-IL—loc monoclonal antibody (6 mg/kg) and anti-IL-ll3 onal antibody (6 mgikg) monoclonal antibodies as well as mJL-loc/B DVD-lg binding protein (at 6 mg/kg or at 12 mgikg) significantly neutralized zymosan-induced IL-6 production as ed to vehicle-treated control animals. Accordingly, the antibodies exhibited functional ty in viva.

Example 3.3.5. Effect Of Cytokine Inhibitory Antibody Treatments on Osteoarthritis Induced by Destabilization of Medial Meniscus (DMM) Model in SV129 Mice An alternative animal model was also employed to study the affect of anti-IL-loc and anti-IL- 1[3 activity on slowing progression of cartilage ration in osteoarthritis. The mouse destabilization of medial meniscus (DMM) model for osteoarthritis is less invasive and inflammatory than the JIM procedure (see, above) and results in s primarily on the central weight-bearing region of the medial tibial plateau and medial femoral condyle. The severity and location of lesions following DMM are more consistent when compared to JIM.

Example 3.3.5.A. Study Design Male SV129 mice weighing approximately 30 grams, housed 5/cage, were anesthetized on Day 0 with ketamine/xylazine cocktail, administered eritoneally (ip), and then the medial eral ligament was exposed by blunt dissection, and transected to reflect the meniscus toward the femur. The joint space was closed with 8—0 vicryl suture. The skin was closed with wound glue.

For sham surgeries, a vertical skin incision was made over the medial aspect of the knee and the joint capsule was opened. The capsule was closed with 8-0 vicryl suture and the skin was closed with wound glue. Groups of animals were then treated with intraperitoneal (ip) injections of antibody or vehicle according to the study design table below. Two sex-matched, age-matched, and strain-matched naive mice were also included in the study. Animals were euthanized after 8 weeks of treatment, and knee joints were harvested in 10% neutral buffered formalin.

Table 22. Design of Study Using Destabilization of Medial Meniscus (DMM) Model of Osteoarthritis Treatment Com oundp N11233:]? Dose, Route, and Sacrifice Group Regimen Time Point sham A 8 weeks (none) B vehicle (PBS) 8 weeks anti-IL-la mAb 180 pig/mouse for C and 11 8 weeks each antibody ip anti-IL-IB mAb every 4th day mlL-l d/B 250 se, ip, D 10 8 weeks DVD-1g 3x/week Example 3.3.5.B. ation of Tissues in-fixed samples were decalcified, and specimens were sectioned in the frontal plane into 15 step sections each. Each section was approximately 6 pm thick, and step sections were separated by 70 um. All slides were stained with toluidine blue (T. Blue). Slides were assessed according to the Chambers method (Chambers et al. (2001) Arthritis Rheum 44: 1455-65) for cartilage ration (see below) by a board-certified veterinary pathologist. Each of the four age surfaces, the medial femoral condyle (MFC), the medial tibial u (MTP), the lateral femoral condyle (LFC), and the lateral tibial plateau (LTP), was scored from 0-6 according to the scale in the table below.

Table 23. Chambers Scoring System for Cartilage Degeneration Grade rthritic Damage 0 No damage 1 Roughened articular surface and small fibrillations Fibrillation down to the layer immediately below the superficial layer (zone 2) and some loss of surface lamina Loss of surface lamina and fibrillations extending down to the calcified cartilage Major fibrillations and cartilage erosion down to the subchondral bone Major fibrillations and erosion of up to 80% of the cartilage —Morethan 80% loss of cartilage tical analysis was performed using GraphPad Prism software version 4.03 using Mann- Whitney U test.

Example C. Results As shown in Figure 6, a combination therapy with both anti-mouse IL-la monoclonal antibody (9H10, 6 mg/kg) and anti-mouse lL-lB onal antibody (10G11, 6 mg/kg) or with lL-la/B DVD-Ig binding protein (6 mg/kg) significantly d cartilage degeneration in the mouse DMM model of osteoarthritis. e 3.3.6. Titration of Anti-IL-loe and Anti-IL-lB Activities in Mice in a Destabilization of Medial Meniscus (DMM) Model for Osteoarthritis (8 Week Study) An 8 week study was conducted on the effects on cartilage degeneration in response to various doses of anti-mIL-loc monoclonal antibody (9H10 mAb) and anti-mIL-IB monoclonal antibody (10G11 mAb), alone or in combination, and of mlL-lu/B 10G11-9H10 DVD-lg binding protein in the DMM model of osteoarthritis.

Example 3.3.6.A. Study Design Male SV129 mice ng approximately 30 grams, housed 5/cage, were anesthetized on Day 0 with ketamine/xylazine cocktail, administered intraperitoneally (ip), and then the medial collateral ligament was exposed by blunt dissection, and transected to reflect the meniscus toward the femur. The joint space was closed with 8—0 vicryl suture. The skin was closed with wound glue.

For sham surgeries, a al skin incision was made over the medial aspect of the knee and the joint capsule was opened. The capsule was closed with 8-0 vicryl suture, and the skin was closed with wound glue. Groups of s were then treated with intraperitoneal (ip) injections of anti- mIL-la monoclonal antibody, anti-mlL-l B (alone or in combination), mIL-la/B DVD-lg binding protein, or vehicle according to the study design table below (Table 24). Animals were euthanized after 8 weeks of treatment and lmee joints were harvested in 10% l buffered in.

Table 24. Design of Study to Test Various Treatments in DMM Model of Osteoarthritis ent Number Of Dose, Route and Sacrifice Compound Animals Group Regimen. Time Pomt. .

| A | Sham (None) 5 None | 8 weeks | | B | Vehicle (PBS) ip, 3X/week | 8 weeks | anti-IL-lo. 180 ug/mouse, ip, C 10 (5) 8 weeks (9H10) every 4th day anti-IL—lB 180 ug/mouse, ip, D 10 (8) 8 weeks (10G11) every 4th day L-lo. (9H10) 180 pg/mouse for each E + antibody, ip, every 4th 8 weeks anti-IL— 1[3 day (10G1 1) 62.5 ug/mouse, ip, F mIL-IOLB DVD-1g 8 weeks 3x/week 125 ug/mouse, ip, G mIL—lotB DVD-1g 10 8 weeks 3x/week 250 ug/mouse, ip, H LB DVD-lg 10 8 weeks 3x/week The numbers in parentheses in column 3 of the above table (Number of Animals) are the actual numbers of animals included in the data set. A brief description of exclusions s: Group B: Only 9 animals were included in Group B. One animal was excluded from the data set because at ogic examination the medial meniscus appeared intact and there was essentially no osteoarthritic change.

Group C: Only 5 animals were included in Group C. Five animals were excluded from the data set because at ogic examination the medial meniscus appeared mostly intact and there was little osteoarthritic change.

Group D: Only 8 animals were included in Group D. Slides for one animal were not present.

Another animal was excluded from the data set because at histologic examination the medial meniscus appeared mostly intact and there was little osteoarthritic .

Group E: Only 8 animals were included in Group E. Two s were excluded because they were considered outliers.

Group F: Only 9 animals were included in Group F. One animal was excluded from the data set because at histologic examination the medial meniscus appeared intact and there was ially no osteoarthritic change.

Example 3.3.6.B. Tissue Preparation and Examination Tissues were prepared and scored as described above in Example 3.3.5.

Example 3.3.6.C. Results The results are shown in Figure 7. Similar to the results obtained in the joint ility model (JIM) for osteoarthritis (above), treatment with either L-loc monoclonal antibody (9H10) or L-IB monoclonal antibody ) alone resulted in a decrease in cartilage degeneration that was not statistically significant from that observed in vehicle control joints. r, a combination therapy with both monoclonal antibodies or with IL-1 a/B DVD-lg binding protein (except for IL—lu/B DVD at 1.5 mgikg) icantly reduced the mean sum Chambers scores ating overall cartilage degeneration) and mean maximum Chambers scores (most affected area in the joint). A statistically significant dose effect was observed for mean sum Chambers scores in the mIL-la/B DVD-1g treatment groups. Accordingly, in this study, 8 weeks of treatment with both anti-mIL-lu monoclonal antibody and anti-mIL-IB monoclonal antibody (6 mg/kg, each) and with mIL-lu/B DVD-1g binding protein (at 3 and 6 mg/kg) ameliorated progression of cartilage degeneration induced in the DMM model of osteoarthritis.

Example 3.3.7. Effects of Anti-IL-loe and Anti-IL-lB Activities and Small Molecule Treatment on Cartilage Degeneration in Animals in Destabilization 0f Medial Meniscus (DMM) Model of Osteoarthritis Doxycycline has been shown to prevent cartilage degradation in the DMM model of osteoarthritis (Effects of Disease-modifying Osteoarthritis Drugs in an in-vivo Animal Model 1Silva et al., Univ. Mass. Med. School, Worcester, MA, USA, Trans ORS 2009 — need full on) 2O and also in a human clinical trial (Brandt et a1. (2005) Arthrit. Rheum. 52(7):2015-2025). In this study, the effect of treating animals in the DMM model with doxycycline was compared to the effect of treating animals with a combination of L-loc and anti-IL-lB monoclonal antibodies.

Example 3.3.7.A. Study Design Male SV129 mice weighing approximately 30 grams had the medial eral nt exposed by blunt dissection, and transected to reflect the meniscus toward the femur. The joint space was closed with 8-0 vicryl suture. The skin was closed with wound glue. For sham surgeries, a vertical skin incision was made over the medial aspect of the knee and the joint capsule was opened. The capsule was closed with 8-0 vicryl suture, and the skin was closed with wound glue.

Groups of animals were then treated per os (po) or Via intraperitoneal (ip) injections of vehicle or treatments according to the study design table below. Three sex-matched, age-matched, and strain- matched naive mice were also included in the study. Animals were ized after 4 weeks of treatment, and knee joints were ted in 10% neutral buffered formalin.

Table 25. Design of Study to Test Various Anti-IL-l Antibodies and Doxycycline Treatments in DMM Model of Osteoarthritis Sacrifice Time Grou Com oundp N11231:]? Dose, Route and Regimen Point A Sham (None) None 4 weeks Vehicle B (0.02% Tween80/ p0, BID 4 weeks 0.5% HPMC) . 30 mg/kg, po, C Doxycycline 10 4 weeks L-l (x (9H10) 180 ug/mouse D + 10 each, ip, every 4 weeks anti-IL-IB (10G11) 4th day Example 3.3.7.B. Tissue Preparation And ation Tissues were prepared and scored as described above in e 3.3.5.

Example 3.3.7.C. Results The s are shown in Figure 8. Under the conditions of the study, 4 weeks of treatment with a ation of anti-IL-lo. and anti—IL—lB monoclonal antibodies, closed at 180 ug/mouse each, resulted in significant amelioration of the osteoarthritic changes induced by ilization of the medial meniscus in male SV129 mice. Mean sum Chambers scores and mean maximum Chambers scores were icantly reduced, and mean total summed Chambers scores were notably reduced by this combination ent compared to the vehicle control group. Doxycycline treatment at 30 mg/kg also resulted in a similar treatment , but the antibody combination therapy was more effective.

Example 4. Pain Efficacy of Anti-IL-lou’B Therapy The DMM model also generates measurable pain in the mice. Recently, Malfait et a1. (see, Malfait et a1. (2010) Osteoarthritis Cartilage, 18: 572-580) showed robust decrease in paw withdrawal threshold (mechanical allodynia indication of pain) in hind paws after DMM but not sham surgery. The efficacy of anti-IL-lw’fi therapy was studied in the DMM animal model. As shown in this series of studies, mice with DMM surgery were allodynic as early as day 7 after the surgery and exhibited a decrease in paw withdrawal threshold till day 35.

The potential of anti-IL-l therapy to ameliorate pain was tested in the DMM mouse model of 0A in terms of mechanical allodynia. Dosing of the combination of IL-loc and IL-1[3 mAbs was initiated on the day of the surgery and was repeated twice a week for 5 weeks. Animals were treated with PBS e alone, with an IgG isotype control, or with a combination of anti-IL-loc and anti- IL-lB mAbs (at a dose of 6 mg/kg for each mAb). Animals were tested weekly for paw withdrawal threshold. The ipsilateral (surgical) limb of groups treated with PBS or IgG isotype control was allodynic (painful) compared to the contra lateral (non—surgical) limb or compared to s that had undergone sham surgery. See Figure 9. In contrast, the group treated with the combination of anti-IL-IOL and anti-IL-ll?) mAbs demonstrated a cantly higher paw withdrawal threshold indicating reduction in development of allodynia (pain) as compared to PBS vehicle or IgG isotype treated groups at week 5 (day 35). See Figure 10. This reduction in pain was seen as early as week 1 following treatment (data not shown).

To evaluate the effect of lL-l y in established pain, animals in the IgG control group with established OA and mechanical allodynia were treated on day 35 with the combination of anti- IL-la and anti-lL-l B monoclonal antibodies and the paw withdrawal threshold was tested 24 hours later (day 36). The data demonstrated that combination therapy with anti-IL-l 0c and anti-lL-lB mAbs reversed nia ed OA pain) in animals with established disease. These data indicate that anti-IL-l therapy provides an analgesic effect independent of a potential disease modifying effect. See Figure 11. 2O Example 5. Further Study of Pain cy of Anti-IL-lu/B Combination Therapy The potential of anti-IL-l therapy to ameliorate pain in the DMM mouse model of OA (see, above) in terms of mechanical allodynia was further evaluated. Dosing of the combination of IL-la and IL-lB mAbs was ted on the day of the surgery and was repeated twice a week for 4 weeks.

DMM animals were treated with PBS vehicle only, with anti-IL-lu mAb (6 mg/kg), with anti-lL-lB mAb (6 mg/kg), or with a combination of anti-IL—la mAb (6 mg/kg) and anti-IL-lB mAb (6 mg/kg), administered eritoneally (ip) twice per week for four weeks. s were tested for allodynia on day 28. The teral cal) limb of groups treated with vehicle was allodynic ul) compared to the contra lateral (non—surgical) limb or compared to s that had undergone sham surgery. See Figure 12. Similarly, a group d with either anti-IL-la or anti-IL-IB mAbs alone was allodynic (painful). In contrast, a group treated with the combination of anti-IL-la and anti-IL- IB mAbs demonstrated a significantly higher pain withdrawal threshold ting reduction in development of allodynia (pain) as compared to vehicle treated groups (Figure 12).

Example 6. Dose-Dependent Pain Efficacy of Anti-IL-lw’B Combination y Dosing of the combination of anti-IL—la and anti-IL—lB mAbs was initiated on the day on which surgery was performed on animals in the DMM mouse model and was repeated twice a week for four weeks. Animals were treated with PBS vehicle only or with a combination of anti-IL-la mAb and anti-IL-lB mAb, both stered intraperitoneally (ip) every four days for four weeks at 1 mg/kg, at 3 mg/kg, or at 6 mg/kg. s were tested on Day 28 for paw withdrawal threshold.

The ipsilateral (surgical) limb of groups treated with vehicle showed icant reduction in the paw withdrawal threshold indicating allodynia (pain) compared to the contra lateral (non-surgical) limb or compared to animals that had undergone sham surgery (Figure 13). In contrast, the group treated with the combination of anti-IL—lo. and L—lB mAbs demonstrated a dose-dependent se in paw withdrawal threshold indicating reduction in development of allodynia (pain) as compared to vehicle treated groups (Figure 13).

Example 7. Anti-IL-la/B Combination Therapy in ished Pain To evaluate the effect of anti-11.40013 combination therapy in established pain, animals in the DMM mouse model with established OA and mechanical allodynia were treated at day 27 with a combination of anti-IL-ld mAb and anti-lL-l B mAb, both administered intraperitoneally (ip) at 1 mg/kg, at 3 mg/kg, or at 6 mg/kg at 24 hours prior to testing for allodynia on day 28. Results are shown in Figure 14. The data demonstrated that combination therapy with anti-IL-lu and L-lB mAbs reversed mechanical allodynia (reduced OA pain) in animals with established disease in a dose-related manner (Figure 14). e 8. Anti-Hyperalgesic Effect of L—loz/B Combination y To evaluate whether neutralization of IL—loc and IL-lB produces antinociceptive effects in 2O inflammatory pain conditions, anti-IL—loc and anti-IL—lB mAbs were evaluated for the ability to reverse established pain in the mouse carrageenan paw model of nociceptive pain.

The efficacy of the anti-lL—loc and anti—IL-ll3 mAbs was evaluated when administered alone or in combination in the mouse carrageenan model (Figure 15). At 30 hours after intraplantar eenan injection: 900 ug of either anti-IL-loc mAb, anti-IL-lB mAb, or their combination were administered in different groups of mice. Thermal hyperalgesia testing was performed 48 and 96 hours after carrageenan. As seen in the previous study, the 900 ug dose combination significantly attenuated l hyperalgesia (carrageenan paw 8.11 i 0.62 3 compared to 4.98 i 0.34 s in PBS group at 48 hours, p<0.01, Figure 15A; carrageenan paw 7.62 i 0.45 s compared to 4.45 i 0.55 s in PBS group at 96 hours, p<0.01, Figure ISB). The antinociceptive effect in 900 ug combination 3O group was 53 i 7% at 48 hours, and 82 i 11% at 96 hours. The diclofenac (non-steroidal, anti- atory analgesic) positive control group again exhibited a significant attenuation of l hyperalgesia (p<0.01 at both time points). In contrast, no icant antinociceptive effect was seen at both time points when anti-IL—loc and anti-IL-IB mAbs were administered alone indicating that simultaneous neutralization of both lL-loc and IL-1|3 is required for antinociceptive efficacy in the mouse carrageenan inflammatory pain model. e 9. Dose-Dependent Anti-Hyperalgesic Effect of Anti-IL-la/B Combination Therapy As shown in Figure 16, lantar eenan injection in mice (female BALB/c) produces a long lasting thermal hyperalgesia as evidenced by a decrease in paw withdrawal latency to a radiant heat us ng performed 48 and 96 hours after eenan injection; p<0.01 for l uninjured versus carrageenan paws in PBS vehicle group at both time points). Different groups of animals were treated either with vehicle or a ation of anti-IL-loc and anti-IL-ll?) mAbs (100, 300, or 900 Mg /mouse of each mAb) 30 hours after carrageenan injection. A dose- related effect was seen, with the 900 ttg dose combination of anti-IL-loc and anti-IL-ll?) mAbs significantly attenuating thermal hyperalgesia (carrageenan paw 3.23 i 0.34 s in PBS compared to 7.88 i 0.93 s in 900 11g groups at 48 hours, p<0.01, Figure 16A; eenan paw 4.45 i 0.55 s in PBS compared to 7.62 i 0.45 s in 900 ug groups at 96 hours, p<0.01, Figure 16B). The antinociceptive effect in 900 ug treatment group was 70 i 9% at 48 hours, and 62 i 4% at 96 hours.

Diclofenac (30 mg/kg), a non-steroidal, anti-inflammatory analgesic, was included as positive control in the study, and it increased the mean paw withdrawal latency of the injured paw to 7.01 i 0.58 s at 48 hours and 7.48 i 0.34 s at 96 hours when stered p.o. 60 minutes before testing (p<0.01 at both time points). The IgG control and low dose 100 ug mAb groups did not attenuate l hyperalgesia at either time point. The mAbs and diclofenac did not significantly change the paw withdrawal latency values in the control uninjured paw.

Example 10. Efficacy of Anti-IL-lu/B Combination Therapy in CFA Pain Model 2O The CFA (Complete Freund's Adjuvant) inflammatory pain model was used to test whether the anti-IL-loc and L-IB mAb combination would produce efficacy against a mechanical endpoint. Therefore, mechanical allodynia was assessed using von Frey monofilaments in animals (female BALB/c mice) 48 hours after they were injected intraplantar with CFA. As seen in Figure 17A, intraplantar CFA produced robust mechanical nia as evidenced by a decreased paw withdrawal threshold in the CFA injected paw treated with PBS vehicle only (0.052 i 0.028 g versus 0.860 i 0.090 g in contralateral uninjured paw, p<0.01). Separate groups of animals were treated either with IgG isotype control or a combination of anti—IL—loc and anti—IL—ll3 mAbs (900 Mg /mouse of each mAb) 30 hours after CFA injection. The mAb treated group showed a significant attenuation of mechanical allodynia (0.419 t 0.101 g, p<0.01 versus PBS vehicle, Figure 17A). The 3O magnitude of efficacy (% MPE) was 65.51 i 12.35% (Figure 17B). The anti-inflammatory, analgesic positive control diclofenac (30 mg/kg, 1 hour pretreatment) also attenuated mechanical nia by increasing paw withdrawal thresholds (0.359 1 0.088 g, p<0.01 versus PBS vehicle) with %MPE of 64.51 i 10.20%.

Example 11. Efficacy of Anti-IL-ltx/B Combination Therapy in Neuropathic Pain Whether an L-10t and anti—IL—IB mAb ation therapy would produce efficacy in neuropathic pain ions was examined in an L5/L6 spinal nerve ligation (SNL) model in mice (male CD1). Testing was performed six days after SNL surgery, 24 and 72 hours after animals were treated with mAb combination (900 ttg [mouse of each mAb). ical nia was ed in mice treated with PBS vehicle only after SNL surgery as evidenced by a decreased paw withdrawal threshold to von Frey monofilament stimulation (0.073 i 0.032 g versus 0.735 i 0.109 g for contralateral paw at 24 hours, Figure 18A; 0.128 1 0.048 g versus 0.732 1 0.132 g for contralateral paw at 72 hours, Figure 18B; p<0.01 at both time points). The mAb treated group showed a significant attenuation of mechanical allodynia at both time points (0.477 1 0.131 g, p<0.01 versus PBS vehicle at 24 hours, Figure 18A; 0527 i 0.111 g, p<0.01 versus PBS vehicle at 72 hours, Figure 18B). The magnitude of cy (% MPE) was 72.20 i 14.39% at 24 hours and 80.62 i 12.33% at 72 hours (Figure 19). The positive control gabapentin (100 mg/kg, 1 hour pretreatment) was fully efficacious in attenuating mechanical allodynia by significantly increasing paw withdrawal thresholds (0.858 i 0.077 g at 24 hours and 1.138 i 0.096 g at 72 hours, p<0.01 versus PBS vehicle at both time points). In contrast, the IgG control group did not produce any effect on mechanical allodynia at both time .

Conclusion of Pain Treatment Studies The results of the pain studies described herein te that an anti-IL-ltx/B combination 2O y according to the invention is effective as a treatment for any form of pain and not just pain associated with osteoarthritis. Such anti—IL—loc/B combination therapy can be used to treat pain in an individual suffering from any type of pain condition including, but not limited to, the pain conditions allodynia, hyperalgesia, and a combination of allodynia and hyperalgesia. Anti-IL-lot/B combination therapy can be provided to an individual by administering to the individual a combination (for example, a mixture, concurrent administration, or successive administration) of IL-lu and IL-IB g ns, such as a combination of anti-IL-la and anti-IL-IB monoclonal antibodies, or by administering to the individual a protein that binds both IL-oc and IL-lB, such as a bispecific antibody or an lL-l (x/B DVD-1g binding protein that binds both IL—oc and IL—1 [3. [near oration 1) Re erence The contents of all cited references (including literature references, patents, patent applications, and websites) that maybe cited throughout this application are hereby sly incorporated by reference in their entirety, as are the references cited therein. The practice of the t invention will employ, unless otherwise indicated, conventional techniques of pharmaceutical science, immunology, molecular y, and cell biology, which are well known in the art. lents The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all ts illustrative rather than limiting of the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced herein.

Claims (29)

We claim:

1. Use of a binding protein that binds both IL-1α and IL-1β for the manufacture of a ment for treating pain in an individual.

2. The use according to claim 1, wherein the individual suffers from a pain condition selected from the group consisting of allodynia, hyperalgesia, and a combination of allodynia and hyperalgesia.

3. The use ing to claim 1, wherein said g protein that binds both IL-1and IL-1 is a bispecific antibody.

4. The use according to claim 1, wherein said binding protein that binds both IL-1 and IL-1 is a dual variable domain immunoglobulin (DVD-Ig) binding protein.

5. The use ing to any one of claims 1 to 4, wherein said binding protein that binds both IL-1 and IL-1 is formulated in a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

6. The use according to any one of claims 1 to 4, wherein said binding protein that binds both IL-1 and IL-1 is llized.

7. The use according to claim 6, wherein said crystallized binding protein that binds both IL-1 and IL-1 is formulated in a ition further comprising: optionally, an ingredient; and a ric carrier.

8. The use according to claim 7, wherein said polymeric carrier is a polymer selected from one or more of the group consisting of a poly (acrylic acid), a poly (cyanoacrylate), a poly (amino acids), a poly (anhydride), a poly (depsipeptide), a poly (ester), a poly (lactic (10721333_1):GGG acid), a poly (lactic-co-glycolic acid) or PLGA, a poly (b-hydroxybutryate), a poly (caprolactone), a poly (dioxanone); a poly (ethylene glycol), a poly ((hydroxypropyl) methacrylamide, a poly [(organo)phosphazene], a poly (ortho esters), a poly (vinyl alcohol), a poly (vinylpyrrolidone), a maleic anhydride- alkyl vinyl ether copolymer, a pluronic polyol, an albumin, an alginate, a ose, a cellulose derivative, a collagen, a fibrin, a gelatin, a onic acid, an oligosaccharide, a glycaminoglycan, a sulfated polysaccharide, a blend, and a copolymer thereof.

9. The use according to claim 7, wherein said ingredient, when present, is selected from the group consisting of albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl- dextrin , methoxypolyethylene glycol, and polyethylene .

10. The use according to any one of claims 1 to 4, further comprising stering to the individual a second agent, n said second agent is one or more compounds in the group consisting of budenoside, epidermal growth factor, corticosteroids, cyclosporin, sulfasalazine, aminosalicylates, 6-mercaptopurine, oprine, metronidazole, lipoxygenase inhibitors, mesalamine, olsalazine, balsalazide, antioxidants, thromboxane inhibitors, IL-1 receptor antagonists, anti-IL-1β monoclonal antibodies, anti-IL-6 monoclonal antibodies, growth factors, elastase inhibitors, nyl-imidazole compounds, antibodies of TNF, LT, IL-2, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-16, IL-18, IL-23, EMAP-II, , FGF, and PDGF, antibodies of CD2, CD3, CD4, CD8, CD-19, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands, methotrexate, cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, ibuprofen, corticosteroids, prednisolone, phosphodiesterase tors, adenosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, IRAK, NIK, IKK, p38, MAP kinase inhibitors, IL-1β converting enzyme inhibitors, TNFα converting enzyme inhibitors, T-cell signalling tors, metalloproteinase tors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine ors, soluble p55 TNF receptor, soluble p75 TNF receptor, sIL-1RI, sIL-1RII, sIL-6R, nflammatory cytokines, IL-4, IL-10, IL-11, IL-13, and TGFβ. (10721333_1):GGG

11. The use according to any one of claims 1 to 4, wherein the dual is ing from a disease or disorder selected from the group comprising osteoarthritis, toid arthritis, juvenile chronic tis, septic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflammatory bowel e, insulin dependent diabetes mellitus, thyroiditis, asthma, allergic diseases, sis, dermatitis, scleroderma, graft versus host disease, organ lant rejection, acute immune disease associated with organ transplantation, chronic immune disease ated with organ transplantation, dosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki's disease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schoenlein purpurea, microscopic vasculitis of the kidneys, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, infectious diseases, parasitic diseases, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignancies, heart e, myocardial infarction, Addison's disease, sporadic andular ency type I, polyglandular deficiency type II (Schmidt's syndrome), adult (acute) respiratory distress syndrome, alopecia, alopecia areata, seronegative arthropathy, arthropathy, Reiter's e, psoriatic arthropathy, ulcerative colitic arthropathy, enteropathic synovitis, dia-associated arthropathy, ia-associated arthropathy, Salmonella-associated arthropathy, spondyloarthropathy, atheromatous disease/arteriosclerosis, atopic allergy, mune bullous disease, gus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune haemolytic anaemia, Coombs positive haemolytic anaemia, ed pernicious anaemia, le pernicious anaemia, myalgic encephalitis/Royal Free disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sing hepatitis, cryptogenic autoimmune hepatitis, acquired immunodeficiency syndrome, acquired immunodeficiency related diseases, hepatitis B, hepatitis C, common variable immunodeficiency (common variable hypogammaglobulinaemia), dilated cardiomyopathy, female infertility, ovarian failure, premature ovarian e, fibrotic lung disease, cryptogenic fibrosing alveolitis, postinflammatory interstitial lung disease, interstitial pneumonitis, connective tissue disease associated interstitial lung disease, mixed connective tissue disease associated lung disease, systemic sclerosis associated interstitial lung disease, rheumatoid arthritis associated (10721333_1):GGG interstitial lung disease, systemic lupus erythematosus associated lung e, omyositis/polymyositis associated lung disease, Sjögren's disease associated lung disease, ankylosing spondylitis associated lung disease, itic diffuse lung disease, haemosiderosis associated lung disease, drug-induced interstitial lung disease, fibrosis, radiation fibrosis, bronchiolitis rans, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease, postinfectious titial lung disease, gouty arthritis, autoimmune hepatitis, type-1 autoimmune hepatitis (classic autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune mediated hypoglycemia, type B insulin resistance with acanthosis ans, hypoparathyroidism, osteoarthrosis, y sclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathic leucopaenia, autoimmune neutropaenia, renal disease NOS, glomerulonephritides, Lyme disease, discoid lupus erythematosus, male infertility idiopathic or NOS, sperm autoimmunity, multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertension secondary to tive tissue e, Goodpasture's syndrome, pulmonary manifestation of polyarteritis nodosa, acute rheumatic fever, toid spondylitis, Still's disease, systemic sclerosis, Sjögren's me, Takayasu's disease/arteritis, autoimmune thrombocytopaenia, idiopathic ocytopaenia, autoimmune thyroid disease, hyperthyroidism, goitrous autoimmune yroidism (Hashimoto's e), ic autoimmune hypothyroidism, primary myxoedema, phacogenic s, primary vasculitis, vitiligo, acute liver disease, c liver diseases, alcoholic cirrhosis, alcohol-induced liver injury, cholestasis, idiosyncratic liver disease, drug-induced hepatitis, non-alcoholic steatohepatitis, allergy, group B streptococci (GBS) infection, mental disorders (e.g., depression and schizophrenia), Th2 Type and Th1 Type mediated diseases, acute and chronic pain (different forms of pain), cancers such as lung, breast, stomach, bladder, colon, pancreas, ovarian, prostate and rectal cancer. hematopoietic malignancies (leukemia and lymphoma), abetalipoproteinemia, acrocyanosis, acute and chronic parasitic or infectious processes, acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute or c bacterial ion, acute pancreatitis, acute renal failure, adenocarcinomas, atrial ectopic beats, AIDS dementia complex, alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis, allograft rejection, alpha-l- antitrypsin deficiency, amyotrophic lateral sclerosis, , angina pectoris, anterior horn cell degeneration, anti-CD3 therapy, antiphospholipid (10721333_1):GGG syndrome, anti-receptor hypersensitivity reactions, aortic and peripheral aneurysms, aortic dissection, arterial ension, arteriosclerosis, arteriovenous fistula, , atrial fibrillation (sustained or paroxysmal), atrial r, atrioventricular block, B cell lymphoma, bone graft rejection, bone marrow transplant (BMT) rejection, bundle branch block, Burkitt's lymphoma, burns, cardiac hmias, cardiac stun syndrome, cardiac tumors, cardiomyopathy, pulmonary bypass inflammation se, age transplant rejection, cerebellar cortical degenerations, cerebellar disorders, chaotic or multifocal atrial tachycardia, chemotherapy associated disorders, chronic myelocytic leukemia (CML), c alcoholism, chronic inflammatory pathologies, chronic cytic leukemia (CLL), chronic obstructive pulmonary disease (COPD), chronic salicylate intoxication, colorectal carcinoma, congestive heart failure, conjunctivitis, contact dermatitis, cor pulmonale, coronary artery disease, Creutzfeldt-Jakob disease, culture negative sepsis, cystic is, cytokine therapy associated disorders, dementia pugilistica, demyelinating diseases, dengue hemorrhagic fever, dermatologic conditions, diabetes, ic arteriosclerotic disease, diffuse Lewy body disease, dilated congestive cardiomyopathy, disorders of the basal ganglia, Down's syndrome in middle age, drug- induced movement disorders induced by drugs which block CNS dopamine receptors, drug sensitivity, eczema, encephalomyelitis, endocarditis, endocrinopathy, epiglottitis, Epstein-Barr virus infection, erythromelalgia, extrapyramidal and cerebellar disorders, familial hemophagocytic lymphohistiocytosis, fetal thymus implant rejection, Friedreich's ataxia, functional peripheral arterial disorders, fungal , gas gangrene, gastric ulcer, glomerular nephritis, graft rejection of any organ or tissue, gram negative sepsis, gram positive sepsis, granulomas due to intracellular organisms, hairy cell leukemia, Hallervorden-Spatz disease, Hashimoto's thyroiditis, hay fever, heart transplant rejection, hemochromatosis, hemodialysis, hemolytic uremic syndrome/thrombolytic ocytopenic purpura, hage, hepatitis A, His bundle arrhythmias, HIV infection/HIV neuropathy, n's disease, hyperkinetic movement disorders, hypersensitivity reactions, hypersensitivity pneumonitis, hypertension, hypokinetic movement disorders, hypothalamic-pituitary-adrenal axis evaluation, idiopathic Addison's disease, idiopathic pulmonary fibrosis, antibody mediated xicity, asthenia, infantile spinal muscular y, inflammation of the aorta, influenza A, ng radiation exposure, iridocyclitis/uveitis/optic neuritis, ischemia- reperfusion injury, ischemic stroke, juvenile (10721333_1):GGG rheumatoid arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma, kidney transplant rejection, ella, leishmaniasis, leprosy, lesions of the corticospinal system, lipedema, liver lant rejection, lymphedema, a, malignant lymphoma, malignant histiocytosis, malignant melanoma, meningitis, meningococcemia, metabolic migraine he, idiopathic migraine he, mitochondrial multisystem disorder, mixed connective tissue disease, monoclonal gammopathy, multiple myeloma, multiple systems degenerations (Menzel, Dejerine-Thomas, Shy-Drager, and Machado-Joseph), enia , cterium avium intracellulare, mycobacterium tuberculosis, myelodysplastic syndrome, myocardial ischemic disorders, nasopharyngeal carcinoma, neonatal chronic lung disease, nephritis, nephrosis, neurodegenerative diseases, neurogenic muscular atrophies, neutropenic fever, non- n's lymphoma, occlusion of the abdominal aorta and its branches, ive arterial disorders, OKT therapy, orchitis/epididymitis, orchitis/vasectomy reversal procedures, organomegaly, osteoporosis, pancreas transplant rejection, pancreatic carcinoma, paraneoplastic syndrome/hypercalcemia of malignancy, parathyroid transplant rejection, pelvic matory disease, perennial rhinitis, pericardial disease, peripheral atherosclerotic disease, peripheral vascular disorders, peritonitis, pernicious , pneumocystis carinii pneumonia, pneumonia, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes syndrome), post perfusion syndrome, post pump syndrome, post-MI cardiotomy syndrome, preeclampsia, progressive supranucleo palsy, primary ary hypertension, radiation therapy, d's phenomenon, Raynaud's disease, Refsum's disease, regular narrow QRS tachycardia, renovascular hypertension, usion injury, restrictive cardiomyopathy, sarcomas, senile chorea, senile dementia of Lewy body type, seronegative arthropathies, shock, sickle cell anemia, skin allograft rejection, skin changes syndrome, small bowel transplant rejection, solid , specific arrhythmias, spinal ataxia, spinocerebellar degenerations, streptococcal myositis, structural lesions of the cerebellum, subacute sing panencephalitis, syncope, syphilis of the cardiovascular , systemic anaphylaxis, systemic inflammatory response syndrome, systemic onset juvenile rheumatoid arthritis, T- cell or FAB ALL, telangiectasia, thromboangiitis obliterans, thrombocytopenia, toxicity, transplants, trauma/hemorrhage, type III hypersensitivity reactions, type IV hypersensitivity, unstable , uremia, urosepsis, ria, valvular heart diseases, varicose veins, vasculitis, venous diseases, venous (10721333_1):GGG thrombosis, ventricular fibrillation, viral and fungal ions, viral encephalitis/aseptic meningitis, viral-associated hemophagocytic syndrome, Wernicke- Korsakoff syndrome, 's disease, xenograft rejection of any organ or tissue, acute coronary syndromes, acute idiopathic polyneuritis, acute matory demyelinating polyradiculoneuropathy, acute ischemia, adult Still’s disease, alopecia , anaphylaxis, anti-phospholipid antibody syndrome, aplastic anemia, arteriosclerosis, atopic eczema, atopic dermatitis, autoimmune dermatitis, autoimmune disorder associated with streptococcus infection, autoimmune enteropathy, autoimmune hearing loss, autoimmune lymphoproliferative me (ALPS), autoimmune myocarditis, autoimmune premature ovarian failure, blepharitis, bronchiectasis, bullous pemphigoid, vascular disease, catastrophic antiphospholipid syndrome, celiac disease, cervical spondylosis, chronic ischemia, icial pemphigoid, clinically isolated syndrome (CIS) with risk for multiple sclerosis, childhood onset atric disorder, dacryocystitis, dermatomyositis, diabetic retinopathy, disk herniation, disk prolapse, drug induced immune hemolytic anemia, endocarditis, endometriosis, endophthalmitis, episcleritis, erythema orme, erythema multiforme major, gestational pemphigoid, Guillain-Barré syndrome (GBS), hay fever, Hughes syndrome, idiopathic Parkinson’s disease, thic interstitial pneumonia, IgE-mediated allergy, immune hemolytic anemia, inclusion body myositis, infectious ocular inflammatory disease, inflammatory demyelinating disease, inflammatory heart e, inflammatory kidney disease, P, iritis, keratitis, junctivitis sicca, Kussmaul disease or Kussmaul-Meier disease, Landry's paralysis, Langerhan's cell histiocytosis, livedo reticularis, r degeneration, microscopic polyangiitis, morbus Bechterew, motor neuron disorders, mucous membrane pemphigoid, multiple organ failure, myasthenia gravis, myelodysplastic me, myocarditis, nerve root ers, neuropathy, non-A non-B tis, optic neuritis, osteolysis, ovarian cancer, pauciarticular JRA, peripheral artery ive disease (PAOD), peripheral vascular disease (PVD), peripheral artery e (PAD), phlebitis, polyarteritis nodosa (or periarteritis nodosa), polychondritis, polymyalgia rheumatica, poliosis, polyarticular JRA, polyendocrine deficiency syndrome, polymyositis, polymyalgia rheumatica (PMR), post-pump syndrome, primary Parkinsonism, prostatitis, pure red cell aplasia, primary adrenal insufficiency, recurrent neuromyelitis , restenosis, rheumatic heart disease, SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis), secondary amyloidosis, shock lung, scleritis, sciatica, (10721333_1):GGG secondary adrenal insufficiency, silicone associated connective tissue e, Sneddon- Wilkinson dermatosis, spondylitis ankylosans, Stevens-Johnson syndrome (SJS), systemic inflammatory response syndrome, temporal arteritis, toxoplasmic retinitis, toxic epidermal necrolysis, transverse myelitis, TRAPS (tumor-necrosis factor receptor type 1 (TNFR)- associated periodic syndrome), type 1 allergic reaction, type II diabetes, usual interstitial pneumonia (UIP), vernal ctivitis, viral retinitis, Vogt-Koyanagi-Harada syndrome (VKH syndrome), wet macular degeneration, and wound healing.

12. The use according to any one of claims 1 to 4, n the individual is ing from a disease selected from the group consisting of a primary cancer, a metastatic cancer, breast cancer, colon cancer, rectal cancer, lung cancer, oropharynx cancer, hypopharynx cancer, esophagus cancer, h cancer, pancreatic cancer, liver cancer, gallbladder , bile duct cancer, small ine cancer, colon cancer, urinary tract cancer, kidney cancer, bladder , urothelium cancer, female genital tract cancer, cervical cancer, uterine cancer, ovarian , carcinoma, gestational trophoblastic disease, male l tract cancer, prostate cancer, seminal vesicle cancer, testicular cancer, germ cell tumor, endocrine gland cancer, thyroid cancer, l cancer, pituitary gland , skin cancer, hemangioma, melanoma, sarcoma, bone cancer, soft tissue cancer, Kaposi’s sarcoma, tumor of the brain, nerve cancer, eye cancer, cancer of the meninges, ytoma, glioma, glioblastoma, retinoblastoma, neuroma, lastoma, Schwannoma, meningioma, a solid tumor arising from hematopoietic malignancy, leukemia, Hodgkin's ma, and non -Hodgkin's lymphoma.

13. Use of a binding protein that binds both IL-1α and IL-1β for the manufacture of a medicament of treating osteoarthritis in an individual.

14. The use according to claim 13, wherein said binding protein that binds both IL-1and IL-1 is a bispecific antibody.

15. The use according to claim 13, wherein said binding protein that binds both IL-1and IL-1 is a dual variable domain immunoglobulin (DVD-Ig) binding protein. (10721333_1):GGG

16. The use according to any one of claims 13 to 15, wherein said binding protein that binds both IL-1 and IL-1 is formulated in a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

17. The use according to any one of claims 13 to 15, wherein said binding protein that binds both IL-1 and IL-1 is crystallized.

18. The use ing to claim 17, wherein said crystallized binding protein is formulated in a composition further comprising an optional ingredient and a polymeric carrier.

19. The use according to claim 18, wherein said polymeric carrier is a r selected from one or more of the group consisting of a poly (acrylic acid), a poly (cyanoacrylate), a poly (amino acid), a poly (anhydride), a poly (depsipeptide), a poly (ester), a poly (lactic acid), a poly (lactic-co-glycolic acid) or PLGA, a poly (b-hydroxybutryate), a poly (caprolactone), a poly none); a poly ene glycol), a poly ((hydroxypropyl) methacrylamide, a poly [(organo)phosphazene], a poly (ortho ester), a poly (vinyl alcohol), a poly (vinylpyrrolidone), a maleic anhydride- alkyl vinyl ether copolymer, a ic , an albumin, an alginate, a cellulose and a cellulose tive, a collagen, a fibrin, a gelatin, a hyaluronic acid, an oligosaccharide, a glycaminoglycan, a sulfated polysaccharide, a blend, and a copolymer thereof.

20. The use according to claim 18, wherein said optional ingredient is selected from the group consisting of albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-βcyclodextrin , methoxypolyethylene glycol and polyethylene glycol.

21. The use according to claim 13 further comprising administering to the individual a second agent, wherein said second agent is one or more nds in the group consisting of budenoside, epidermal growth factor, corticosteroids, cyclosporin, alazine, aminosalicylates, 6-mercaptopurine, azathioprine, idazole, lipoxygenase inhibitors, (10721333_1):GGG mesalamine, olsalazine, balsalazide, antioxidants, thromboxane inhibitors, IL-1 receptor antagonists, anti-IL-1β monoclonal antibodies, anti-IL-6 monoclonal antibodies, growth factors, elastase inhibitors, pyridinyl-imidazole compounds, antibodies of TNF, LT, IL-2, IL- 6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-16, IL- 18, IL-23, EMAP-II, GM-CSF, FGF, and PDGF, antibodies of CD2, CD3, CD4, CD8, CD-19, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands, methotrexate, cyclosporin, FK506, rapamycin, mycophenolate l, leflunomide, NSAIDs, ibuprofen, corticosteroids, prednisolone, phosphodiesterase tors, adenosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, IRAK, NIK, IKK, p38, MAP kinase inhibitors, IL- 1β ting enzyme inhibitors, TNFα converting enzyme tors, T-cell signalling inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine ors, soluble p55 TNF receptor, soluble p75 TNF receptor, sIL-1RI, sIL-1RII, , anti-inflammatory cytokines, IL-4, IL-10, IL-11, IL-13, and TGFβ.

22. The use ing to any one of claims 13 to 21, wherein said step of administering to said individual is by at least one mode selected from parenteral, subcutaneous, intramuscular, enous, intra-articular, intrabronchial, intraabdominal, apsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and ermal.

23. The use ing to claim 13, wherein the osteoarthritis comprises an osteoarthritis lesion.

24. The use according to claim 13, wherein the osteoarthritis comprises an injury to an anterior cruciate ligament.

25. The use according to claim 13, wherein osteoarthritis comprises cartilage (10721333_1):GGG ration and sion of IL-6.

26. The use according to claim 25, wherein the cartilage degeneration ses femoral cartilage degeneration or tibial cartilage degeneration.

27. The use according to claim 13, wherein administering the binding protein produces an antinociceptive effect.

28. The use according to claim 13, wherein the binding protein further treats pain.

29. The use according to claim 28, wherein the pain is selected from the group consisting of allodynia, hyperalgesia, and a combination of allodynia and hyperalgesia.