WO2017210598A1

WO2017210598A1 - Compositions and methods for treating an articular disorder

Info

Publication number: WO2017210598A1
Application number: PCT/US2017/035745
Authority: WO
Inventors: John E. Sims; Chris GABEL; Roy Black; Warren D'souza; Peng Li; Alice Bakker; Benjamin M. Alba; Maria Rosalyn DAYAO; Amy DUGUAY; Kevin Moore
Original assignee: Amgen Inc.
Priority date: 2016-06-03
Filing date: 2017-06-02
Publication date: 2017-12-07

Abstract

The bioavailability of drugs in articular joints and their ability to target chondrocytes is limited by the avascular nature of cartilage, rapid clearance from the joint space, and constraints with entry into cartilage. To overcome these limitations and aid in the development of osteoarthritis drugs, the disclosure provides strategies to prolong the retention and facilitate cartilage penetration of intra-articularly delivered protein therapeutics.

Description

COMPOSITIONS AND METHODS FOR TREATING AN ARTICULAR DISORDER CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62/345,533, filed June 3, 2016, the disclosure of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present disclosure is directed to compositions comprising a polypeptide that is retained in the joint of a patient.

INCORPORATION BY REFERENCE

[0003] Incorporated by reference in its entirety is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: ASCII text file named "50288_Seqlisting.txt,"; 171,512 bytes; created May 31, 2017.

BACKGROUND

[0004] Osteoarthritis (OA), the most common form of arthritis, is characterized by the progressive loss of articular cartilage, development of bony overgrowths (osteophytes), pain, and loss of joint function [Goldring et ah, J Cell Physiol 213: 626-634 (2007)]. It is estimated that symptomatic OA affects 12.1% of US adults ages 25-74 [Lawrence et ah, Arthritis Rheum 58: 26-35 (2008)], and 34% of those over age 65 [Scanzello et al, Bone 51: 249-257 (2012)].

Current treatment options include analgesics to treat pain, mechanical devices (including joint replacements) to aid function, and supportive care. Current pharmacological treatments are limited, provide modest pain relief, and chronic use is often associated with toxicity [Hunter, Nat Rev Rheumatol 7: 13-22 (2011)]. More importantly, there is no approved therapeutic that can reverse, halt, or even slow down cartilage loss. Hence, there is a pressing need for safe and efficacious disease modifying OA drugs (DMO ADs).

[0005] Recent studies have suggested that OA is a disease affecting all components of the joint, including the synovium. However, in the case of knee and hip OA, it is the pain that ultimately drives patients to seek joint replacement therapy. Cartilage is avascular but bathed in synovial fluid present within the joint cavity. Intra- articular (IA) injection of potential therapeutics is an attractive delivery option for maximizing efficacy at the diseased site, while concurrently reducing the risk of systemic liabilities. However, IA injections require physician assistance and carry a risk of infection and these restrictions limit the number of IA injections which can be administered. Moreover, drug concentrations achieved post-injection rapidly dissipate as a result of the synovial fluid being in equilibrium with the systemic circulation

[Edwards et al, Vet J 190: 15-21 (2011); Gerwin et al, Adv Drug Deliv Rev 58: 226-242 (2006)]. An additional complication exists for therapeutics that target chondrocytes; the highly crosslinked and negatively charged cartilage extracellular matrix imposes restrictions on the types of therapeutics which can penetrate and access the cellular constituents [Foy et al., J Magn Reson 148: 126-134 (2001); van Lent et al., J Rheumatol 14, 798-805 (1987); van Lent et al., Rheumatol Int 8: 145-152 (1988)]. Thus, the physical nature of cartilage and other unique aspects of the joint environment pose challenges for therapeutic development.

[0006] Interleukin-1 (IL-1) has emerged as a promising target for the treatment of OA. IL-1 is an inflammatory cytokine that encompasses two separate molecules, IL-la and IL-Ιβ. IL-IRa is a related protein that binds the IL-1 receptor (IL-IR) but unlike IL-la and IL-Ιβ, lacks biological activity and functions as an inhibitor of IL-1 signaling. IL-la, IL-Ιβ and IL-IR are up-regulated in osteoarthritic chondrocytes and cartilage [Attur et al, Proc Assoc Am Physicians 110: 65-72 (1998); LeGrand et al, Arthritis Rheum 44: 2078-2083 (2001); Martel-Pelletier et al, Arthritis Rheum 35: 530-540 (1992); Melchiorri et al, Arthritis Rheum 41: 2165-2174 (1998); Sadouk et al, Lab Invest 73: 347-355 (1995); Tetlow et al, Arthritis Rheum 44: 585-594 (2001); Towle et al, Osteoarthritis Cartilage 5: 293-300 (1997)], and elevated IL-1 levels can also be observed in synovial fluid [Farahat et al, Ann Rheum Dis 52: 870-875 (1993); Martel-Pelletier et al, Semin Arthritis Rheum 18: 19-26 (1989); Wood et al, Arthritis Rheum 26: 975-983 (1983)]. The effect of IL-1 on cartilage mimics the catabolic changes observed in OA such as induction of metalloproteinases, aggrecanases, nitric oxide and prostaglandin E2, and the inhibition of collagen and proteoglycan synthesis [Attur et al, Proc Assoc Am Physicians 110: 65-72 (1998); LeGrand et al, Arthritis Rheum 44: 2078-2083 (2001); Tetlow et al, Arthritis Rheum 44: 585- 594 (2001); Attur et al, J Biol Chem 275: 40307-40315 (2000); Goldring et al, Clin Orthop Relat Res 427 Suppl: S27-36 (2004); Tan et al, J Biol Chem 278: 35678-35686 (2003);

Majumdar et al, Arthritis Rheum 56: 3670-3674 (2007); Pratta et al, J Biol Chem 278: 45539- 45545 (2003)]. Furthermore, an inhibitor of IL-1 blocked the spontaneous breakdown of collagen and proteoglycan observed in cartilage explants from OA cartilage [Kobayashi et al, Arthritis Rheum 52: 128-135 (2005)], and IL-1 inhibition was shown to be efficacious in rat, rabbit, dog, and horse models of OA [Calich et al, Clin Rheumatol 29: 451-455 (2010); Caron et al, Arthritis Rheum 39: 1535-1544 (1996); Fernandes et al. , Am J Pathol 154: 1159-1169 (1999); Frisbie et al, Gene Ther 9, 12-20 (2002); Zhang et al, J Orthop Res 22: 742-750 (2004)]. In addition, genetic studies have associated IL-Ιβ and IL-1R1 with hand OA, and IL- lRa with the severity of knee OA [Attur et al, Ann Rheum Dis 69: 856-861 (2010); Kerkhof et al, Osteoarthritis Cartilage 19: 265-271 (2011); Moxley et al, Osteoarthritis Cartilage 18: 200- 207 (2010); Nakki et al, BMC Med Genet 11: 50 (2010); Solovieva et al, J Rheumatol 36: 1977-1986 (2009)]. OA patients with elevated levels of IL-1 and IL-l-induced genes in blood progress substantially more rapidly than those with "normal" blood expression of these genes [Attur et al, Arthritis Rheum 63: 1908-1917 (2011)]. This large body of evidence implicating IL- 1 as a mediator of OA prompted several clinical trials of IL- 1 antagonists but these trials to date have been largely unsuccessful [Chevalier et al, Arthritis Rheum 61: 344-352 (2009);

Cohen et al, Arthritis Res Ther 13: R125 (2011); Chevalier et al Arthritis Res Ther 13: R124 (2011)]. Based on evidence that chondrocytes are both producers of IL-1 as well as responders [Konttinen et al, Arthritis Rheum 64: 613-616 (2012)], failure of the tested anti-IL-lRl antibody to provide benefit to OA patients may reflect its inability to access chondrocytes, whereas lack of success of IL-IRa can be attributed to inadequate target coverage due to its rapid clearance. As such, IL-1 antagonism remains an attractive target for the treatment of OA.

[0007] As a result, several strategies are currently being evaluated to overcome the challenges involved in maintaining adequate concentrations of therapeutics within the joint space. The majority of these involve the use of specialized formulations such as liposomes, hydrogels, micelles, microspheres and nanoparticles to prolong the retention of IA administered drugs

[Edwards et al, Vet J 190: 15-21 (2011); Gerwin et al, Adv Drug Deliv Rev 58: 226-242 (2006); Kang et al., Expert Opin Drug Deliv 11: 269-282 (2014)]. In addition, amino acid encoding sequences have been described that can act as tethers to facilitate binding of proteins to cartilage and synovial tissues [Rothenfluh et al., Nat Mater 7: 248-254 (2008); Wythe et al., Ann Rheum Dis 72: 129-135 (2013); Loffredo et al., Arthritis Rheumatol 66: 1247-1255 (2014)].

SUMMARY OF THE INVENTION

[0008] The present disclosure provides a polypeptide comprising a domain that binds to a protein found in cartilage. In some embodiments, the domain is a non-naturally-occurring domain consisting of 30 to 50 amino acids and comprises at least one disulfide bond.

[0009] Accordingly, in some aspects the disclosure provides a polypeptide comprising a domain that binds to a protein found in cartilage wherein the domain is a non-naturally-occurring domain comprising 30 to 50 amino acids and comprises at least one disulfide bond. In some embodiments, the polypeptide comprises at least two domains that bind the protein. In further embodiments, the domain binds to aggrecan (SEQ ID NO: 143; GenBank Accession No.

P16112), decorin (SEQ ID NO: 144; GenBank Accession No. P07585), biglycan (SEQ ID NO: 145; GenBank Accession No. P21810), or fibromodulin (SEQ ID NO: 146; GenBank Accession No. Q06828). Preferably, the domain binds to human aggrecan, human decorin, human biglycan, or human fibromodulin.

[0010] In some embodiments, the domain binds to collagen-2. Preferably, the domain binds to human collagen. In related embodiments, the domain comprises an amino acid sequence at least 75% identical to SEQ ID NO: 1. In further embodiments, the amino acid sequence is a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. In some embodiments, the amino acid sequence is set out in SEQ ID NO: 5.

[0011] In additional embodiments, the domain binds horse collagen. In some embodiments, the domain binds dog collagen. In further embodiments, the domain binds cat, goat, sheep, pig, bovine, camel, or elephant collagen. In still further embodiments, the domain binds horse, dog, cat, goat, sheep, pig, bovine, camel, or elephant collagen-II.

[0012] In some embodiments, the polypeptide further comprises an additional domain having a binding specificity for a target molecule. In further embodiments, the additional domain has a binding specificity for interleukin-1 receptor (IL-1R). In some embodiments, the IL-1R is human IL-1R, equine IL-1R, or canine IL-1R.

[0013] In some embodiments, the additional domain comprises an amino acid sequence as set out in SEQ ID NO: 140.

[0014] In further embodiments, the domain or the additional domain comprises an anti- catabolic agent. In related embodiments, the anti-catabolic agent is an scFv fragment, a complement inhibitory molecule, or an inhibitor of a cartilage-degrading protease. In some embodiments, the inhibitor of a cartilage-degrading protease is Tissue Inhibitor of

Metalloproteinase 2, an inhibitor of matrix metalloproteinase 13 (MMP-13), or an inhibitor of aggrecanase. In further embodiments, the scFv targets tumor necrosis factor (TNF), a matrix metalloproteinase, an aggrecanase, nerve growth factor, a complement component, or interleukin 6 (IL-6). In some embodiments, the inhibitor of matrix metalloproteinase 13 (MMP-13) is proteinaceous. In further embodiments, the inhibitor of matrix metalloproteinase 13 (MMP-13) is a small molecule inhibitor.

[0015] The disclosure also provides embodiments wherein the additional domain comprises an anabolic agent. In related embodiments, the anabolic agent is fibroblast growth factor 18 (FGF- 18), insulin-like growth factor 1 (IGF-1), a transforming growth factor beta (TGFP) family member, a Wnt inhibitor, or a chondrogenic peptide. In further embodiments, the TGFP family member is growth and differentiation factor 5 (GDF5) or bone morphogenetic protein 7 (BMP7). In still further embodiments, the Wnt inhibitor is dickkopf 1 (Dkk-1), Frizzled Related Protein B (FrzB), or sclerostin. In additional embodiments, the chondrogenic peptide is TPX-100. In some embodiments, the additional domain is a non-protein domain to which a metal ion or metal- containing compound is attached for use in imaging (X-ray or MRI), or an isotopically labeled molecule such as 18-F is attached for use in PET scans. In further embodiments, a metal ion or metal-containing compound is attached directly to the collagen-binding domain for use in imaging (X-ray or MRI), or an isotopically labeled molecule such as 18-F is attached for use in PET scans.

[0016] The disclosure also provides embodiments in which a small molecule drug is attached, directly or via a linker, to any of the polypeptides disclosed herein. Thus, in some embodiments, a small molecule inhibitor is attached directly, or via a linker, to a collagen-binding domain as disclosed herein. In some embodiments, the small molecule drug is a metalloproteinase inhibitor. Small molecule drugs contemplated for use according to the disclosure include, but are not limited to, inhibitors of matrix metalloproteinase (MMP)-3, MMP-9, and MMP-13; inhibitors of ADAMTS-4 and ADAMTS-5; a complement inhibitory protein; an inhibitor of a

prostaglandin-synthesizing enzyme; a caspase-1 inhibitor; an inhibitor of cathepsin-K; inhibitors of neutrophil proteases capable of activating pro-ILla and pro-ILiP; an inhibitor of the

Adenosine A3 receptor; an inhibitor of p38 kinase; and an inhibitor of inducible nitric oxide synthase (iNOS).

[0017] In some embodiments, a polypeptide of the disclosure comprises two disulfide bonds. In further embodiments, a polypeptide of the disclosure comprises three disulfide bonds.

[0018] In any of the embodiments of the disclosure, a polypeptide disclosed herein further comprises a purification peptide such as, without limitation, a poly-histidine tag or a HA tag. [0019] In some embodiments, the domain and the additional domain are linked by a linker. In further embodiments, the linker is GGGGSGGGGS (SEQ ID NO: 139).

[0020] In some embodiments, the polypeptide is M26 (SEQ ID NO: 5). In some

embodiments, the polypeptide further comprises IL-lRa. In related embodiments, the IL-lRa is mammalian IL-lRa. In further embodiments, the mammalian IL-lRa is canine IL-lRa, equine IL-lRa, or human IL-lRa. In some embodiments, the mammalian IL-lRa is human IL-lRa (SEQ ID NO: 140).

[0021] In various embodiments, a polypeptide of the disclosure is less than about 50 kilodaltons.

[0022] The disclosure further provides, in various aspects, a polynucleotide encoding any of the polypeptides disclosed herein. In some embodiments, the polynucleotide comprises or consists of a sequence as set out in SEQ ID NO: 142.

[0023] In some aspects, a polypeptide is provided that comprises a domain that binds to interleukin- 1 receptor (IL-IR) wherein the domain is a non-naturally-occurring domain comprising 30 to 50 amino acids and comprises at least one disulfide bond. In further embodiments, the polypeptide comprises at least two domains that bind IL- IR. In some embodiments, the domain comprises an amino acid sequence at least 75% identical to SEQ ID NO: 114 or SEQ ID NO: 119. In additional embodiments, the amino acid sequence is a sequence as set out in any of SEQ ID NOs: 96-138. In some embodiments, the amino acid sequence is set out in SEQ ID NO: 114. In some aspects, the disclosure provides a

polynucleotide encoding a polypeptide that comprises a domain that binds to interleukin- 1 receptor (IL-IR). In some embodiments, the polynucleotide comprises or consists of a sequence as set out in SEQ ID NO: 153.

[0024] In some aspects, the disclosure provides a polypeptide comprising an amino acid sequence as set out in SEQ ID NO: 5 linked by a linker to an amino acid sequence as set out in SEQ ID NO: 114. In some embodiments, the linker is GGGGSGGGGS (SEQ ID NO: 139). In some embodiments, the amino acid sequence as set out in SEQ ID NO: 5 is amino terminal to the linker. In further embodiments, the amino acid sequence as set out in SEQ ID NO: 5 is carboxy terminal to the linker. [0025] In some aspects, the disclosure provides a vector comprising a polynucleotide of the disclosure operably linked to a promoter. In further aspects, the disclosure provides a host cell comprising a vector of the disclosure. In some embodiments, the host cell is an Escherichia coli cell. In further embodiments, the host cell is a mammalian cell. In related embodiments, the host cell is a Chinese Hamster Ovary (CHO) cell. In still further embodiments, the host cell is a yeast cell.

[0026] In further aspects of the disclosure, a method of producing any of the polypeptides disclosed herein is provided, the method comprising the step of culturing a host cell of the disclosure under conditions appropriate to induce expression of the polypeptide. In some aspects, a method of producing any of the polypeptides disclosed herein is provided, the method comprising the step of performing in vitro transcription and translation of a vector comprising a polynucleotide of the disclosure operably linked to a promoter under conditions appropriate to induce expression of the polypeptide. In related embodiments of producing a polypeptide of the disclosure, the polypeptide is isolated.

[0027] In some aspects, the disclosure provides a pharmaceutical composition comprising a polypeptide of the disclosure and a pharmaceutically acceptable carrier, adjuvant, or diluent.

[0028] In further aspects, a method of treating or preventing osteoarthritis (OA) in a patient is provided comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition disclosed herein. In some embodiments, the OA is primary OA. In further embodiments, the OA is idiopathic. In still further embodiments, the OA is a

consequence of obesity, old age, malalignment, occupational stress on a joint, or genetic bone malformations such as, e.g., femoral-tibial malalignment or femeroacetabular impingement. In some embodiments, the OA develops following a ligament tear, a sprain, an articular fracture, or a meniscus tear. In some embodiments, the administration results in faster healing of the ligament tear, sprain, or meniscus tear relative to a patient that is not administered the pharmaceutical composition. The disclosure also provides, in various aspects, a method of treating or preventing a repetitive use injury in a patient comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition disclosed herein. In still further aspects, a method of treating or preventing intervertebral disc (IVD) degeneration in a patient is provided comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition of the disclosure. [0029] In various embodiments, at least one pharmaceutical composition is administered to the patient. In further embodiments, two pharmaceutical compositions are administered to the patient. In related embodiments, the two pharmaceutical compositions are administered separately. In further embodiments, the two pharmaceutical compositions are administered together as a single formulation.

[0030] In further aspects of the disclosure, a method of treating or preventing an interleukin- 1 (IL-1) mediated disease is provided comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition of the disclosure. In some embodiments, at least one pharmaceutical composition is administered to the patient. In further embodiments, two pharmaceutical compositions are administered to the patient. In some embodiments the two pharmaceutical compositions are administered separately, while in still further embodiments the two pharmaceutical compositions are administered together as a single formulation. In some embodiments, the interleukin- 1 (IL-1) mediated disease is Stills Disease, gout, rheumatoid arthritis, juvenile rheumatoid arthritis, or calcium pyrophosphate deposition disease (CPPD).

[0031] In any of the embodiments of the disclosure, administration of a pharmaceutical composition or pharmaceutical compositions is intra-articular, sub-cutaneous, parenteral, intravenous, or a combination thereof.

BRIEF DESCRIPTION OF THE FIGURES

[0032] Figure 1 depicts the sequences of the type II collagen avimers (domains) that were generated. The underlined sequences represent the A domains; the double underlined sequences represent the linkers; and the bolded and italicized sequences represent sequences inserted for cloning purposes.

[0033] Figure 2 depicts the sequences of the IL-1R avimers (domains) that were generated. The underlined sequences represent the A domains; the double underlined sequences represent the linkers; and the bolded and italicized sequences represent sequences inserted for cloning purposes.

[0034] Figure 3 depicts a characterization of M18 and M26 Avimer specificities. Purified collagens and aggrecan (preadsorbed to plates) or pulverized cartilage were incubated with the indicated concentrations of CII Avimers. Bound Avimers subsequently were detected using an anti-HA-HRP antibody. Binding curves indicate observed OD₄so values as a function of avimer concentration. From the binding curves EC₅₀ values were calculated and are indicated in the table, n.d. = not detectable.

[0035] Figure 4 depicts the specificity of CII binding avimers.

[0036] Figure 5 shows that avimers Ml 8 and M26 bind to human C-II at comparable affinities. CPS = counts per second.

[0037] Figure 6 shows that avimer M26 accumulates within the pericellular matrix of human cartilage ex vivo, (a) Representative images comparing in vitro binding of biotinylated M26 and a negative control avimer (M07), also biotinylated, to human cartilage sections. The biotinylated probes were detected using fluorescently-tagged streptavidin (red), (b) Representative SHG and fluorescent (MPM) images of human cartilage explants following 5 day incubation in the absence or presence of FITC-labeled M26 or M07 avimers. Differential interference contrast (DIC) images are also shown, (c) Quantification of the mean fluorescence intensity (MFI) of the FITC signal observed in the 3 different samples. Binding of M26 was significantly enhanced over M07 using a two-tailed unpaired t test. *** p<0.001

[0038] Figure 7 shows that the AF680-labeled M26 avimer persists in the rat knee joint following IA injection, (a) Representative bioluminescence images of rat knee joints at the indicated times following IA injection of AF680-labeled M26 or M07 (NC) avimers. The inset shows fluorescence intensity corresponding to a region of interest (ROI) positioned over the injection site as the mean + SEM (n= 3) for each time point. The ROI employed immediately after injection (time 0) was used to collect all subsequent images for each individual animal allowing comparison of the joint fluorescence values (y-axis) across time-points (x-axis). (b) Ex vivo imaging of dissected tibia and femur one day post-IA injection of AF680-labeled CII Avimer M18 or the NC avimer M07 into four rat knee joints. Circles in the background of the image were printed on the dissection board and have no significance, (c) Confocal imaging of articular cartilage isolated from rat knee joints 28 days post-IA injection of AF647-labeled M26 or M07 avimers. DAPI nuclear staining is also seen.

[0039] Figure 8 demonstrates the prolonged joint retention and cartilage penetration of a therapeutic warhead using the avimer strategy disclosed herein. The figure shows that CII avimers provide a mechanism for tethering IL-IRa to cartilage. (8a & 8b) IL-IRa containing M18 or M26 fused at either its N- or C-termini were coated on plates, blocked and incubated with biotinylated targets. Bound targets subsequently were assessed with Streptavidin-HRP. Uniform immobilization of fusion constructs was confirmed via anti-His Ab-HRP detection. (8c) The indicated concentrations of IL-lRa_M26 fusion construct, IL-lRa-M07 (NC), or native IL- lRa were incubated with CH-coated plates after which IL-IR-Fc was added and the amount of Fc bound to the plates was detected using anti-Fc-HRP. (8d) Human chondrocytes were stimulated with IL-Ιβ in the presence of the indicated IL-IRa construct. The amount of IL-8 produced is indicated as a % of the level observed in the absence of inhibitor. (8e, 8f)

Representative SHG and multiphoton images of human cartilage explants incubated with AF647- labeled avimer constructs for 24 hours, followed by extensive washing and then imaged either immediately (Day 0) or after incubating for an additional 72 hours (Day 3). Bar = 20 μιη.

[0040] Figure 9 shows additional characterization of the IL-lRa_M26 fusion construct, (a) Binding of the indicated construct to IL-1R as assessed in an AlphaScreen format by evaluating the inhibition of ILIR/Fc binding to biotinylated-ILl-beta. The indicated constructs (starting at 100 nM) were serially diluted in assay buffer (40 mM sodium HEPES, pH 7.5, 100 mM NaCl, 1 mM CaCl₂, 0.1% BSA, 0.05% Tween-20) and 2 uL were added into wells of a 384-well Greiner microtiter plate. 2 uL of humanlL-lRI/Fc (0.3 nM; Amgen) was added to the microtiter plate followed by the addition of 2 uL of a mixture containing biotinylated huIL-lbeta (12 nM;

Amgen), and AlphaScreen 'donor' streptavidin- and 'acceptor' Protein A-beads (10 μg/ml each; PerkinElmer). The microtiter plate was sealed and incubated overnight at 20°C. Inhibition of complex formation subsequently was measured as a reduction in chemiluminescent signal (excitation at 680 nm and emission at 520-620 nm using a PerkinElmer Fusion Plate Reader). The observed counts per second (CPS) are indicated as a function of construct concentration, (b) Side-by-side comparison of the binding of M26 Avimer and IL-lRa_M26 fusion to type 2 collagen was assessed in an AlphaScreen format. Unlabeled M26 or IL-lRa_M26 constructs were serially diluted (starting at 1000 nM) into wells of a 384-well Greiner microtiter plate. Human type II collagen (2 nM) was subsequently introduced along with murine anti-CII antibody (1 nM). Each well then received an equivalent volume of a mix of biotinylated-M26 (1 nM), AlphaScreen "donor" streptavidin beads (10 μg/ml), and AlphaScreen "acceptor" anti- murine-IgG beads (10 μg/ml). Plates were incubated overnight at 20oC then read. The resulting chemiluminescent signal (CPS) is indicated as a function of the concentration of unlabeled construct. Derived IC₅₀ values are indicated in the inset, (c) Demonstration that IL-lRa_M26, but not IL-IRa fused to a negative control avimer (IL-lRa_M07) can bind simultaneously to pulverized human cartilage and IL-1R. [0041] Figure 10 shows that IL-lRa_M26 retains its ability to inhibit IL-1 signaling in an in vivo rat IL-Ιβ challenge model, (a) IL-6 levels recovered in synovial lavage 4 hours after IA injection of PBS or IL-Ιβ alone or with the indicated molar excess of IL-lRa. (b) IL-IRa or IL- lRa_M26 were co-administered with IL-Ιβ (Day 0) or injected into the rat knee joint 7 days prior to the IL-Ιβ challenge (Day -7). IL-6 levels in the synovial lavage fluid were assessed 4 hours post-IA injection of 30 ng IL-Ιβ or PBS. One-Way ANOVA, Dunnett's Multiple

Comparison. * p<0.05, ** p<0.01, *** p<0.001

[0042] Figure 11 shows that IL-lRa_M26 fusion inhibits IL-6 release that is induced by Ιίΐβ.

[0043] Figure 12 depicts the amino acid sequence of key constructs. The entire sequences of the Coll2 M26 and IL-lRa_Coll2 M26 constructs are indicated and annotated as to the origin of various segments.

DETAILED DESCRIPTION OF THE INVENTION

[0044] The present disclosure provides a joint-retention strategy based upon modification of therapeutics using avimer scaffolds. Avimers are derived from the A-domains of extracellular receptors, and are involved in mediating protein-protein interactions [Silverman et al., Nat Biotechnol 23: 1556-1561 (2005)]. High affinity specific avimers were generated that bound the major component of articular cartilage, type II collagen (CII avimers) and their ability to persist within the rat knee following IA injection was tested. Given that articular cartilage is highly anionic and largely impermeable to several molecules based upon size and charge [Foy et al. , J Magn Reson 148: 126-134 (2001); van Lent et al, J Rheumatol 14: 798-805 (1987); van Lent et al, Rheumatol Int 8: 145-152 (1988)], the ability of the avimers to penetrate cartilage was also tested. Lastly, to demonstrate functional activity of potential therapeutic warheads that could be fused to avimers, an IL-lRa_CII avimer bi-specific molecule was generated and its function examined in vitro and in vivo. Results presented herein demonstrate that IA delivered CII avimers are retained in the rat joint for at least 28 days post-injection, and have the ability to penetrate cartilage, and accumulate around chondrocyte lacunae. The IL-lRa_CII avimer fusion protein was able to bind type II collagen while concurrently retaining its ability to block IL-Ιβ - induced responses. Furthermore, pre-injection of a single dose of the IL-lRa_CII avimer fusion, but not unmodified IL-IRa, at one week prior to IL-lb challenge, significantly inhibited IL- lb- induced pro-inflammatory cytokine production. Thus, this approach provides an effective targeting and retention strategy for therapeutics in the treatment of diseases such as osteoarthritis. [0045] The terms "type II collagen" and "collagen-2" are used interchangeably herein.

[0046] The term "domain" as used herein refers to a discrete region found in a protein or polypeptide. A domain forms a native three-dimensional structure in solution in the absence of flanking native amino acid sequences. Domains of the disclosure will often bind to a target molecule. For example, a polypeptide that forms a three-dimensional structure that binds to a target molecule is a domain. As used herein, the term "domain" does not encompass the complementarity determining region (CDR) of an antibody.

[0047] The term "loop" refers to that portion of a monomer domain that is typically exposed to the environment by the assembly of the scaffold structure of the domain protein, and which is involved in target binding. The present disclosure provides three types of loops that are identified by specific features, such as, potential for disulfide bonding, bridging between secondary protein secondary structures, and molecular dynamics (i.e., flexibility). The three types of loop sequences are a cysteine-defined loop sequence, a structure-defined loop sequence, and a B -factor-defined loop sequence.

[0048] As used herein, the term "cysteine-defined loop sequence" refers to a subsequence of a naturally occurring domain-encoding sequence that is bound at each end by a cysteine residue that is conserved with respect to at least one other naturally occurring domain of the same family. Cysteine-defined loop sequences are identified by multiple sequence alignment of the naturally occurring monomer domains, followed by sequence analysis to identify conserved cysteine residues. The sequence between a consecutive pair of conserved cysteine residues that form a disulfide bond is a cysteine-defined loop sequence. The cysteine-defined loop sequence does not include the cysteine residues adjacent to each terminus. Domains having cysteine- defined loop sequences include the LDL receptor A-domains, EGF-like domains, sushi domains, Fibronectin type 1 domains, and the like. Thus, for example, in the case of LDL receptor A- domains represented by the consensus sequence, CXeCX jCXeCXsCXgC, wherein X₆, X₄, X₅, and X₈ each represent a cysteine-defined loop sequence comprising the designated number of amino acids.

[0049] As used herein, the term "structure-defined loop sequence" refers to a subsequence of a domain-encoding sequence that is bound at each end to subsequences that each form a secondary structure. Secondary structures for proteins with known three dimensional structures are identified in accordance with the algorithm STRIDE for assigning protein secondary structure as described in Frishman, D. and Argos, P. (1995) "Knowledge-based secondary structure assignment," Proteins, 23(4):566-79 (see also //hgmp. mrc.ac.uk/Registered/Option/stride.html at the World Wide Web). Secondary structures for proteins with unknown or uncharacterized three dimensional structures are identified in accordance with the algorithm described in Jones, D.T. (1999), "Protein secondary structure prediction based on position- specific scoring matrices," J. Mol. Biol.. 292: 195-202 (see also McGuffin, L. J., Bryson, K., Jones, D.T. (2000) "The

PSIPRED protein structure prediction server," Bioinformatics. 16:404-405, and

//bioinf. cs.ucl.ac.uk/psipred/ at the World Wide Web). Secondary structures include, for example, pleated sheets, helices, and the like. Examples of monomer domains having structure- defined loop sequences are the C2 domains, Ig domains, Factor 5/8 C domains, Fibronectin type 3 domains, and the like.

[0050] The terms "polypeptide," "peptide," and "protein" are used herein interchangeably to refer to an amino acid sequence of two or more amino acids.

[0051] The term "multimer" is used herein to indicate a polypeptide comprising at least two monomer domains. The separate monomer domains in a multimer can be joined together by a linker. A multimer is also known as a combinatorial mosaic protein or a recombinant mosaic protein.

[0052] The term "family" and "family class" are used interchangeably to indicate proteins that are grouped together based on similarities in their amino acid sequences. These similar sequences are generally conserved because they are important for the function of the protein and/or the maintenance of the three dimensional structure of the protein. Examples of such families include the collagen family, LDL Receptor A-domain family, the EGF-like family, and the like. Additionally, related sequences that bind to the same target molecule can be divided into families based on common sequence motifs.

[0053] The term "ligand," also referred to herein as a "target molecule," encompasses a wide variety of substances and molecules, which range from simple molecules to complex targets. Target molecules can be proteins, nucleic acids, lipids, carbohydrates or any other molecule capable of recognition by a polypeptide domain. For example, a target molecule can include a chemical compound (i.e. , non-biological compound such as, e.g. , an organic molecule, an inorganic molecule, or a molecule having both organic and inorganic atoms, but excluding polynucleotides and proteins), a mixture of chemical compounds, an array of spatially localized compounds, a biological macromolecule, a bacteriophage peptide display library, a polysome peptide display library, an extract made from a biological materials such as bacteria, plants, fungi, or animal (e.g. , mammalian) cells or tissue, a protein, a toxin, a peptide hormone, a cell, a virus, or the like. Other target molecules include, e.g. , a whole cell, a whole tissue, a mixture of related or unrelated proteins, a mixture of viruses or bacterial strains or the like. Target molecules can also be defined by inclusion in screening assays described herein or by enhancing or inhibiting a specific protein interaction (i.e. , an agent that selectively inhibits a binding interaction between two predetermined polypeptides). As disclosed herein, and in various aspects, ligands of the disclosure are human type II collagen (SEQ ID NO: 147; GenBank Accession No. P02458) and human IL- 1R (SEQ ID NO: 148; GenBank Accession No. P14778).

[0054] The term "linker" is used herein to indicate a moiety or group of moieties that joins or connects two or more discrete separate monomer domains. The linker can be of the appropriate size to allow the discrete separate monomer domains to cooperate. The linker moiety is typically a substantially linear moiety. Suitable linkers include polypeptides, polynucleic acids, peptide nucleic acids and the like. Suitable linkers also include optionally substituted alkylene moieties that have one or more oxygen atoms incorporated in the carbon backbone. Typically, the molecular weight of the linker is less than about 2000 daltons. More typically, the molecular weight of the linker is less than about 1500 daltons and usually is less than about 1000 daltons. The linker can be small enough to allow the discrete separate monomer domains to cooperate, e.g. , where each of the discrete separate domains in a multimer binds to the same target molecule via separate binding sites. Exemplary linkers include a polynucleotide encoding a polypeptide, or a polypeptide of amino acids or other non-naturally occurring moieties. The linker can be a portion of a native sequence, a variant thereof, or a synthetic sequence. Linkers can comprise, e.g. , naturally occurring, non-naturally occurring amino acids, or a combination of both.

[0055] The term "separate" is used herein to indicate a property of a moiety that is

independent and remains independent even when complexed with other moieties, including for example, other domains. A domain is a separate domain in a protein because it has an independent property that can be recognized and separated from the protein. For instance, the ligand binding ability of the type II collagen domain is an independent property. Other examples of separate include the separate domains in a multimer that remain separate independent domains even when complexed or joined together in the multimer by a linker. Another example of a separate property is the separate binding sites in a multimer for a ligand. [0056] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g. , hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e. , an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g. , homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g. , norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimetics" refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0057] "Conservative amino acid substitution" refers to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic - hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine - leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine, and asparagine- glutamine.

[0058] The phrase "nucleic acid sequence" refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end or an analog thereof.

[0059] The term "encoding" refers to a polynucleotide sequence encoding one or more amino acids. The term does not require a start or stop codon. An amino acid sequence can be encoded in any one of six different reading frames provided by a polynucleotide sequence.

[0060] The term "promoter" refers to regions or sequence located upstream and/or

downstream from the start of transcription that are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. [0061] A "vector" refers to a polynucleotide, which when independent of the host chromosome, is capable of replication in a host organism. Examples of vectors include plasmids. Vectors typically have an origin of replication. Vectors can comprise, e.g. , transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular nucleic acid.

[0062] The term "recombinant' when used with reference, e.g. , to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (nonrecombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all.

[0063] The phrase "specifically (or selectively) binds" to a polypeptide, when referring to a monomer or multimer, refers to a binding reaction that can be determinative of the presence of the polypeptide in a heterogeneous population of proteins (e.g. , a cell or tissue lysate) and other biologies. Thus, under standard conditions used in antibody binding assays, the specified monomer or multimer binds to a particular target molecule above background (e.g. , 2X, 5X, 10X or more above background) and does not bind in a significant amount to other molecules present in the sample.

[0064] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same. "Substantially identical" refers to two or more nucleic acids or polypeptide sequences having a specified percentage of amino acid residues or nucleotides that are the same (i.e. , 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum

correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition also refers to the complement of a test sequence. Optionally, the identity or substantial identity exists over a region that is at least about 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides or amino acids in length.

[0065] A polynucleotide or amino acid sequence is "heterologous to" a second sequence if the two sequences are not linked in the same manner as found in naturally-occurring sequences. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence which is different from any naturally-occurring allelic variants which normally follow it. The term "heterologous linker," when used in reference to a multimer, indicates that the multimer comprises a linker and a monomer that are not found in the same relationship to each other in nature (e.g. , they form a non-naturally occurring fusion protein).

[0066] A "non-naturally-occurring amino acid" in a protein sequence refers to any amino acid other than the amino acid that occurs in the corresponding position in an alignment with a naturally-occurring polypeptide with the lowest smallest sum probability where the comparison window is the length of the monomer domain queried and when compared to a naturally- occurring sequence in the non-redundant ("nr") database of Genbank using BLAST 2.0 as described herein.

[0067] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e. , gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0068] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0069] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well- known in the art. Optimal alignment of sequences for comparison can be conducted, e.g. , by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sd. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g. , Ausubel et ah , Current Protocols in Molecular Biology (1995 supplement)).

[0070] One example of a useful algorithm is the BLAST 2.0 algorithm, which is described in Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information ( htt : //www . nc hirtlm. nih . gov/) . This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et ah , supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11 , an expectation (E) or 10, M~5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. [0071] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g. , Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0072] It is noted here that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.

Domains

[0073] Domains can be polypeptide chains of any size. In some embodiments, domains have about 25 to about 500, about 30 to about 200, about 30 to about 100, about 35 to about 50, about 35 to about 100, about 90 to about 200, about 30 to about 250, about 30 to about 60, about 9 to about 150, about 100 to about 150, about 25 to about 50, or about 30 to about 150 amino acids. Similarly, a domain of the present disclosure can comprise, e.g. , from about 30 to about 200 amino acids; from about 25 to about 180 amino acids; from about 40 to about 150 amino acids; from about 50 to about 130 amino acids; or from about 75 to about 125 amino acids. Domains can typically maintain a stable conformation in solution, and are often heat stable, e.g. , stable at 95° C for at least 10 minutes without losing binding affinity. Sometimes, domains can fold independently into a stable conformation. In some embodiments, the stable conformation is stabilized by ions {e.g. , such as metal or calcium ions). The stable conformation can optionally contain disulfide bonds {e.g. , at least one, two, or three or more disulfide bonds). The disulfide bonds can optionally be formed between two cysteine residues. In some embodiments, domains, or domain variants, are substantially identical to the sequences exemplified.

Collagen binders

[0074] In some aspects, the disclosure provides a domain that binds to a type II collagen polypeptide or a portion thereof. A portion of a polypeptide can be, e.g. , at least 5, 10, 15, 20, 30, 50, 100, or more contiguous amino acids of the polypeptide.

[0075] A multitude of type II collagen binding polypeptide sequences were generated. As described in detail herein, several domains that bind to type II collagen have been identified. The consensus sequence below indicates common amino acid residues between type II collagen binders. In some embodiments, the type II collagen domain comprises the following sequence: C(L/M)(A/P)NQFKCRSSRTCLLPEWVCDG(I/V)DDCPDGSDESP(A/V/T)(N/T)CPTPTSLQK ASGALE (SEQ ID NO: 1). In further embodiments, the type II collagen domain comprises any one of the following sequences:

CLANQFKCSSSRTCLLRRWVCDGVDDCPDGSDESPANCPTPTSLQKASGALE (CII-M05 -

- SEQ ID NO: 2),

CMPNQFKCRSSRTCLLPGWVCDGIDDCPDGSDESPANCPTPTSLQKASGALE (CII-M13 -

- SEQ ID NO: 3),

CMANQFKCRSSRTCLLPGWVCDGIDDCPDGSDESPVTCPTPTSLQKASGALE (CII-M18 -

- SEQ ID NO: 4), or

CMPNQFKCRSSRTCLLPEWVCDGIDDCPDGSDESPTNCPTPTSLQKASGALE (CII-M26 -- SEQ ID NO: 5).

[0076] In still further embodiments, the type II collagen domain comprises any one of the sequences set out in SEQ ID NOs: 6-95, each as listed in Figure 1.

[0077] In some embodiments, the type II collagen domain comprises a sequence that is less than 100% identical to a sequence set out in SEQ ID NOs: 1-95. In various embodiments, the disclosure contemplates type II collagen domain sequences that are at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence set out in SEQ ID NOs: 1-95. In still further embodiments, the disclosure contemplates type II collagen domain sequences that are 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence set out in SEQ ID NOs: 1-95.

[0078] Accordingly, in some embodiments the disclosure provides type II collagen domain sequences that comprise an amino acid substitution, addition, or deletion relative to a sequence set out in SEQ ID NOs: 1-95. Exemplary deletions or substitutions include those wherein one or more, including all, of the amino acids of the linker and/or other non-A domain regions are deleted or substituted for another amino acid. In various embodiments, the disclosure contemplates a type II collagen domain sequence that has 10, 9, 8, 7, 6, 5, 4, 3, 1, or 1 amino acid substitution, addition, deletion, or a combination thereof relative to a sequence set out in SEQ ID NOs: 1-95. [0079] In some embodiments, the polypeptide comprises at least one and no more than six domains that bind type II collagen. In some embodiments, the polypeptide comprises at least two domains that bind type II collagen.

[0080] In some embodiments, the polypeptide comprises at least one and no more than six domains. In some embodiments, the polypeptide comprises at least two domains and the domains are linked by a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is from about 4 to about 12 amino acids long. In some embodiments, the domains are each from about 35 to 45 amino acids.

[0081] In some embodiments, each domain comprises two disulfide bonds. In some embodiments, each domain comprises three disulfide bonds. In some embodiments, the domain comprises an amino acid sequence in which at least 10% of the amino acids in the sequence are cysteine; and/or at least 25% of the amino acids are non-naturally-occurring amino acids.

[0082] In some embodiments, domain binds an ion. In some embodiments, the ion is a metal ion. In further embodiments, the ion is a calcium ion.

IL-1R binders

[0083] The disclosure further provide domains that bind to IL- 1R. In some embodiments, the domains bind to an IL- 1R polypeptide or a portion thereof. A portion of a polypeptide can be, e.g. , at least 5, 10, 15, 20, 30, 50, 100, or more contiguous amino acids of the polypeptide.

[0084] A multitude of IL- 1R binding sequences were generated. As described in detail herein, several domains that bind to IL-1R have been identified. In some embodiments, the IL-1R domain comprises the sequence

CPPNEFRCNSGQCIPPHWLCDGDDDCRDGSDETDCTELTCRADEFPCDNGSCVPLRWRC DGVNDCGDSSDESPSHCEARTSLQKASGALE (SEQ ID NO: 114) or

CPANEFRCNSGQCVPPHWLCDGDDDCGDGSDETDCTERRCRSDEFPCDNGNCVPLRWR CDGVDDCGDRSDEAPEHCEARTSLQKASGALE (SEQ ID NO: 119). In further

embodiments, the IL- 1R domain comprises any one of the sequences as set out in SEQ ID NOs: 96-138, each as listed in Figure 2.

[0085] In some embodiments, the IL-1R domain comprises a sequence that is less than 100% identical to a sequence set out in SEQ ID NOs: 96- 138. In various embodiments, the disclosure contemplates IL-1R domain sequences that are at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence set out in SEQ ID NOs: 96-138. In still further embodiments, the disclosure contemplates IL-IR domain sequences that are 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence set out in SEQ ID NOs: 96-138.

[0086] Accordingly, in some embodiments the disclosure provides IL-IR domain sequences that comprise an amino acid substitution, addition, or deletion relative to a sequence set out in SEQ ID NOs: 96-138. Exemplary deletions or substitutions include those wherein one or more, including all, of the amino acids of the linker and/or other non-A domain regions are deleted or substituted for another amino acid. In various embodiments, the disclosure contemplates an IL- IR domain sequence that has 10, 9, 8, 7, 6, 5, 4, 3, 1, or 1 amino acid substitution, addition, deletion, or a combination thereof relative to a sequence set out in SEQ ID NOs: 96-138.

[0087] In some embodiments, the polypeptide comprises at least one and no more than six domains that bind IL-IR. In some embodiments, the polypeptide comprises at least two domains that bind IL-IR. In some embodiments, the polypeptide comprises at least one and no more than six domains. In some embodiments, the polypeptide comprises at least two domains and the domains are linked by a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is from about 4 to about 12 amino acids long. In some embodiments, the domains are each from about 35 to 45 amino acids.

[0088] In some embodiments, each domain comprises two disulfide bonds. In some embodiments, each domain comprises three disulfide bonds. In some embodiments, the domain comprises an amino acid sequence in which at least 10% of the amino acids in the sequence are cysteine; and/or at least 25% of the amino acids are non-naturally-occurring amino acids.

[0089] In some embodiments, domain binds an ion. In some embodiments, the ion is a metal ion. In further embodiments, the ion is a calcium ion.

Discussion of Domains

[0090] Domains that are particularly suitable for use in the practice of the present disclosure are cysteine-rich domains comprising disulfide bonds. Cysteine-rich domains employed in the practice of the present disclosure typically do not form an a helix, a β sheet, or a β-barrel structure. Typically, the disulfide bonds promote folding of the domain into a three-dimensional structure. Usually, cysteine-rich domains have at least two disulfide bonds, more typically at least three disulfide bonds. In some embodiments, at least 5, 10, 15, or 20% of the amino acids in a domain are cysteines.

[0091] Domains can have any number of characteristics. For example, in some embodiments, the domains have low or no immunogenicity in an animal (e.g., a human). Domains can have a small size. In some embodiments, the domains are small enough to penetrate skin or other tissues. Domains can have a range of in vivo half-lives or stabilities.

[0092] Illustrative domains suitable for use in the practice of the present disclosure include, e.g., an EGF-like domain, a Kringle-domain, a fibronectin type I domain, a fibronectin type II domain, a fibronectin type III domain, a PAN domain, a Gla domain, a SRCR domain, a Kunitz/Bovine pancreatic trypsin Inhibitor domain, a Kazal-type serine protease inhibitor domain, a Trefoil (P-type) domain, a von Willebrand factor type C domain, an Anaphylatoxin- like domain, a CUB domain, a thyroglobulin type I repeat, LDL-receptor class A domain, a Sushi domain, a Link domain, a Thrombospondin type I domain, an Immunoglobulin-like domain, a C-type lectin domain, a MAM domain, a von Willebrand factor type A domain, a Somatomedin B domain, a WAP -type four disulfide core domain, a F5/8 type C domain, a Hemopexin domain, an SH2 domain, an SH3 domain, a Laminin-type EGF domain, a C2 domain, an ankyrin domain, a lipocalin domain, a knottin domain and other such domains known to those of ordinary skill in the art, as well as derivatives and/or variants thereof.

[0093] In some embodiments, suitable domains (e.g., domains with the ability to fold independently or with some limited assistance) can be selected from the families of protein domains that contain β-sandwich or β-barrel three dimensional structures as defined by such computational sequence analysis tools as Simple Modular Architecture Research Tool

(SMART), see Shultz et al., SMART: a web-based tool for the study of genetically mobile domains, (2000) Nucleic Acids Research 28(1):231-234) or CATH (see Pearl et al, Assigning genomic sequences to CATH, (2000) Nucleic Acids Research 28(l):277-282).

[0094] In further embodiments, domains of the present invention include domains other than a fibronectin type III domain, an anticalin domain and a Ig-like domain from CTLA-4. Some aspects of these domains are described in WO 01/64942 entitled "Protein scaffolds for antibody mimics and other binding proteins" by Lipovsek et al, published on September 7, 2001, WO 99/16873 entitled "Anticalins" by Beste et al, published April 8, 1999 and WO 00/60070 entitled "A polypeptide structure for use as a scaffold" by Desmet et ah , published on October 12, 2000.

[0095] As described supra, domains are optionally cysteine rich. Suitable cysteine rich monomer domains include, e.g. , the LDL receptor class A domain ("A-domain") or the EGF domain. The domains can also have a cluster of negatively charged residues.

[0096] Other features of domains can include the ability to bind ligands or the ability to bind an ion {e.g. , Ca²⁺ binding by the LDL receptor A-domain). Domains that bind ions to maintain their secondary structure include, e.g. , A domain, EGF domain, EF Hand {e.g. , such as those found in calmodulin and troponin C), Cadherin domain, C-type lectin, C2 domain, Annexin, Gla- domain, Thrombospondin type 3 domain, all of which bind calcium, and zinc fingers {e.g. , C2H2 type C3HC4 type (RING finger), Integrase Zinc binding domain, PHD finger, GATA zinc finger, FYVE zinc finger, B-box zinc finger), which bind zinc. Without intending to limit the disclosure, it is believed that ion-binding provides stability of secondary structure while providing sufficient flexibility to allow for numerous binding conformations depending on primary sequence.

[0097] As described herein, domains may be selected for the ability to bind to targets other than the target that a homologous naturally occurring domain may bind. Thus, in some embodiments, the disclosure provides domains (monomers and multimers comprising such monomers) that do not bind to the target or the class or family of target proteins that a substantially identical naturally occurring domain may bind.

[0098] Characteristics of a domain can include the ability to fold independently and the ability to form a stable structure. Thus, the structure of the domain is often conserved, although the polynucleotide sequence encoding the monomer need not be conserved. For example, the A- domain structure is conserved among the members of the A-domain family, while the A-domain nucleic acid sequence is not. Thus, for example, a domain is classified as an A-domain by its cysteine residues and its affinity for calcium, not necessarily by its nucleic acid sequence.

[0099] Specifically, the A-domains (sometimes called "complement-type repeats" or "LDL receptor type or class A domains") contain about 30-50 or 30-65 amino acids. In some embodiments, the domains comprise about 35-45 amino acids and in some cases about 40 amino acids. Within the 30-50 amino acids, there are about 6 cysteine residues. Of the six cysteines, disulfide bonds typically are found between the following cysteines: C I and C3, C2 and C5, C4 and C6. The cysteine residues of the domain are disulfide linked to form a compact, stable, functionally independent moiety. Clusters of these repeats make up a ligand binding domain, and differential clustering can impart specificity with respect to the ligand binding.

[0100] Polynucleotides encoding the domains are typically employed to make domains via expression. Nucleic acids that encode domains can be derived from a variety of different sources. Libraries of domains can be prepared by expressing a plurality of different nucleic acids encoding naturally occurring domains, altered domains (i.e. , domain variants), or a combination thereof. For example, libraries may be designed in which a scaffold of amino acids remain constant (e.g. , an LDL A receptor domain, EGF domain) while the intervening amino acids in the scaffold comprise randomly generated amino acids.

[0101] Domains can be naturally-occurring or altered (non-natural variants). The term

"naturally occurring" is used herein to indicate that an object can be found in nature. For example, natural monomer domains can include human monomer domains or optionally, domains derived from different species or sources, e.g. , mammals, primates, rodents, fish, birds, reptiles, plants, etc. The natural occurring monomer domains can be obtained by a number of methods, e.g. , by PCR amplification of genomic DNA or cDNA.

[0102] Domains of the present disclosure can be naturally-occurring domains or non-naturally occurring variants. Libraries of domains employed in the practice of the present disclosure may contain naturally-occurring monomer domain, non-naturally occurring domain variants, or a combination thereof.

[0103] Domain variants can include ancestral domains, chimeric domains, randomized domains, mutated domains, and the like. For example, ancestral domains can be based on phylo genetic analysis. Chimeric domains are domains in which one or more regions are replaced by corresponding regions from other domains of the same family. For example, chimeric domains can be constructed by combining loop sequences from multiple related domains of the same family to form novel domains with potentially lowered immunogenicity relative to randomly generated domains. Those of skill in the art will recognized the immunologic benefit of constructing modified binding domain monomers by combining loop regions from various related domains of the same family rather than creating random amino acid sequences. For example, by constructing variant domains by combining loop sequences or even multiple loop sequences that occur naturally in human LDL receptor class A-domains, the resulting domains may contain novel binding properties but may not contain any immunogenic protein sequences because all of the exposed loops are of human origin. The combining of loop amino acid sequences in endogenous context can be applied to all of the monomer constructs of the invention. Thus the present disclosure provides a method for generating a library of chimeric monomer domains derived from human proteins, the method comprising: providing loop sequences corresponding to at least one loop from each of at least two different naturally occurring variants of a human protein, wherein the loop sequences are polynucleotide or polypeptide sequences; and covalently combining loop sequences to generate a library of at least two different chimeric sequences, wherein each chimeric sequence encodes a chimeric monomer domain having at least two loops. Typically, the chimeric domain has at least four loops, and usually at least six loops. As described above, the present disclosure provides three types of loops that are identified by specific features, such as, potential for disulfide bonding, bridging between secondary protein structures, and molecular dynamics (i.e., flexibility). The three types of loop sequences are a cysteine-defined loop sequence, a structure-defined loop sequence, and a B -factor-defined loop sequence.

[0104] Randomized domains are domains in which one or more regions are randomized. The randomization can be based on full randomization, or optionally, partial randomization based on natural distribution of sequence diversity.

[0105] The present disclosure also provides recombinant nucleic acids encoding one or more polypeptides comprising one or a plurality of domains that bind collagen and/or IL-1R. For example, the polypeptide can be selected to comprise a non-naturally occurring domain from the group consisting of: an EGF-like domain, a Kringle-domain, a fibronectin type I domain, a fibronectin type II domain, a fibronectin type III domain, a PAN domain, a Gla domain, a SRCR domain, a Kunitz/Bovine pancreatic trypsin Inhibitor domain, a Kazal-type serine protease inhibitor domain, a Trefoil (P-type) domain, a von Willebrand factor type C domain, an

Anaphylatoxin-like domain, a CUB domain, a thyroglobulin type I repeat, LDL-receptor class A domain, a Sushi domain, a Link domain, a Thrombospondin type I domain, an Immunoglobulin- like domain, a C-type lectin domain, a MAM domain, a von Willebrand factor type A domain, a Somatomedin B domain, a WAP -type four disulfide core domain, a F5/8 type C domain, a Hemopexin domain, an SH2 domain, an SH3 domain, a Laminin-type EGF-like domain, a C2 domain, an ankyrin domain, a lipocalin domain, a knottin domain and variants of one or more thereof. In another embodiment, the naturally occurring polypeptide encodes a monomer domain found in the Pfam database and/or the SMART database.

[0106] All the compositions of the present disclosure, including the compositions produced by the methods of the present disclosure, e.g. , domains and/or immunoglobulin-type domains, as well as multimers and libraries thereof can be optionally bound to a matrix of an affinity material. Examples of affinity material include beads, a column, a solid support, a microarray, other pools of reagent-supports, and the like.

Multimers

[0107] Methods for generating multimers are a feature of the present disclosure. Multimers comprise at least two domains. For example, multimers of the disclosure can comprise, in various embodiments, from 2 to about 10 domains, from 2 to about 8 domains, from about 3 to about 10 domains, about 7 domains, about 6 domains, about 5 domains, or about 4 domains. In some embodiments, the multimer comprises 3 or at least 3 domains. In some embodiments, the multimers have no more than 2, 3, 4, 5, 6, 7, or 8 domains. In view of the possible range of domain sizes, the multimers of the invention may be, e.g. , less than 100 kD, less than 90kD, less than 80 kD, less than 70 kD, less than 60 kD, less than 50 kD, less than 40 kD, less than 30 kD, less than 25kD, less than 20 kD, less than 15 kD, less than 10 kD or may be smaller or larger. In some cases, the domains have been pre- selected for binding to the target molecule of interest (e.g. , collagen).

[0108] In some embodiments, each domain specifically binds to one target molecule (e.g. , collagen). In some of these embodiments, each domain binds to a different position (analogous to an epitope) on a target molecule. Multiple domains that bind to the same target molecule results in an avidity effect resulting in improved affinity of the multimer for the target molecule compared to the affinity of each individual monomer. In some embodiments, the multimer has an avidity of at least about 1.5, 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, or 1000 times the avidity of a single domain alone. In some embodiments, at least one, two, three, four or more (e.g. , all) domains of a multimer bind an ion such as calcium or another ion. Multimers can comprise a variety of combinations of domains. For example, in a single multimer, the selected domains can be identical or different. In addition, the selected domains can comprise various different domains from the same domain family (i.e. , collagen binding domains or IL- 1R binding domains), or various domains from different domain families, or optionally, a combination of both. For example, the domains may be selected from the collagen binding domains disclosed herein and the IL- IR binding domains as disclosed herein. In some embodiments, at least one of the domains is selected from the collagen binding domains disclosed herein. Exemplary multimers (comprised of one collagen-binding domain and one IL- IR binding domain) are listed herein.

[0109] Multimers that are generated in the practice of the present disclosure may be any of the following:

(1) A homo-multimer (a multimer of the same domain, i.e. , A1-A1-A1-A1);

(2) A hetero-multimer of different domains of the same domain class, e.g. , A1-A2-A3- A4. For example, hetero-multimer include multimers where Al, A2, A3 and A4 are different non-naturally occurring variants of particular collagen-binding domains, or where some of Al, A2, A3, and A4 are naturally-occurring variants of a collagen-binding domain.

(3) A hetero-multimer of domains from different monomer domain classes, e.g. , A1-B2- A2-B 1. For example, where Al and A2 are two different domains (either naturally occurring or non-naturally-occurring) from the collagen-binding domains disclosed herein, and B 1 and B2 are two different domains (either naturally occurring or non- naturally occurring) from the IL-IR binding domains disclosed herein).

[0110] In another embodiment, the multimer comprises monomer domains with specificities for different targets (e.g. , a blood factor such as serum albumin or immunoglobulin, or a cell type such as an erythrocyte). For example, in some embodiments, the multimers of the disclosure comprises 1, 2, 3, or more domains that bind to collagen and at least one domain that binds to a second target molecule. In further embodiments, the multimers of the disclosure comprises 1, 2, 3, or more domains that bind to collagen, 1, 2, 3, or more domains that bind IL- IR, and at least one domain that binds to an additional target molecule. Exemplary additional target molecules include, e.g. , an integrin, a complement molecule, a Wnt signaling component, an aggrecanase, a metalloproteinase, a molecule that positively regulates the foregoing enzymes or pathways, or a combination thereof. Further exemplary additional target molecules include those that negatively regulate signaling by insulin-like growth factor (IGF), platelet-derived growth factor (PDGR), or one or more activin family members. In some embodiments, a domain is a IGF or a TGFP family member rather than an avimer. An exemplary multimer will include a monomer domain from the list of collagen-binding domains and a monomer or dimer domain from the list of IL-IR binding domains. A further exemplary multimer includes a monomer domain from the list of collagen-binding domains and IL- lRa. Thus, in further embodiments, a multimer of the disclosure comprises a fusion of a collagen-binding avimer to a non-avimer molecule such as IL- lRa or a growth factor.

[0111] Multimer libraries employed in the practice of the present invention may contain homo-multimers, hetero-multimers of different monomer domains (natural or non-natural) of the same monomer class, or hetero-multimers of monomer domains (natural or non-natural) from different monomer classes, or combinations thereof.

[0112] Domains, as described herein, are also readily employed in an immunoglobulin-type domain-containing heteromultimer (i.e. , a multimer that has at least one immunoglobulin-type domain variant and one domain variant). Thus, multimers of the present disclosure may have at least one immunoglobulin-type domain such as a minibody, a single-domain antibody, a single chain variable fragment (ScFv), or a Fab fragment; and at least one monomer domain, such as, for example, an EGF-like domain, a Kringle-domain, a fibronectin type I domain, a fibronectin type II domain, a fibronectin type III domain, a PAN domain, a Gla domain, a SRCR domain, a Kunitz/Bovine pancreatic trypsin Inhibitor domain, a Kazal-type serine protease inhibitor domain, a Trefoil (P-type) domain, a von Willebrand factor type C domain, an Anaphylatoxin- like domain, a CUB domain, a thyroglobulin type I repeat, LDL-receptor class A domain, a Sushi domain, a Link domain, a Thrombospondin type I domain, an Immunoglobulin-like domain (e.g. , an scFv), a C-type lectin domain, a MAM domain, a von Willebrand factor type A domain, a Somatomedin B domain, a WAP -type four disulfide core domain, a F5/8 type C domain, a Hemopexin domain, an SH2 domain, an SH3 domain, a Laminin-type EGF-like domain, a C2 domain, an ankyrin domain, a lipocalin domain, a knottin domain or variants thereof.

[0113] Domains need not be selected before the domains are linked to form multimers. On the other hand, the domains can be selected for the ability to bind to a target molecule before being linked into multimers. Thus, for example, a multimer can comprise two domains that bind to one target molecule and a third domain that binds to a second target molecule.

[0114] The multimers of the present disclosure may have the following qualities: multivalent, multispecific, single chain, heat stable, extended serum and/or shelf half-life. Moreover, at least one, more than one or all of the domains may bind an ion (e.g. , a metal ion or a calcium ion), at least one, more than one or all monomer domains may be derived from LDL receptor A domains and/or EGF-like domains, at least one, more than one or all of the monomer domains may be non-naturally occurring, and/or at least one, more than one or all of the monomer domains may comprise 1, 2, 3, or 4 disulfide bonds per monomer domain. In some embodiments, the multimers comprise at least two (or at least three) domains, wherein at least one domain is a non- naturally occurring domain and the domains bind calcium. In some embodiments, the multimers comprise at least 4 domains, wherein at least one domain is non-naturally occurring, and wherein: a. each domain is between 30- 100 amino acids and each of the domains comprise at least one disulfide linkage; or b. each domain is between 30- 100 amino acids and is derived from an extracellular protein; or c. each domain is between 30- 100 amino acids and binds to a protein target.

[0115] In some embodiments, the multimers comprise at least 4 domains, wherein at least one domain is non-naturally occurring, and wherein: a. each domain is between 35- 100 amino acids; or b. each domain comprises at least one disulfide bond and is derived from a human protein and/or an extracellular protein.

[0116] In some embodiments, the multimers comprise at least two domains, wherein at least one domain is non-naturally occurring, and wherein each domain is: a. 25-50 amino acids long and comprises at least one disulfide bond; or b. 25-50 amino acids long and is derived from an extracellular protein; or c. 25-50 amino acids and binds to a protein target; or d. 35-50 amino acids long.

[0117] In some embodiments, the multimers comprise at least two domains, wherein at least one monomer domain is non-naturally-occurring and: a. each monomer domain comprises at least one disulfide bond; or b. at least one monomer domain is derived from an extracellular protein; or c. at least one monomer domain binds to a target protein.

[0118] The domains and/or multimers identified can have biological activity, which is meant to include at least specific binding affinity for a selected or desired ligand, and, in some instances, will further include the ability to block the binding of other compounds, to stimulate or inhibit metabolic pathways, to act as a signal or messenger, to stimulate or inhibit cellular activity, and the like. Domains can be generated to function as ligands for receptors where the natural ligand for the receptor has not yet been identified (orphan receptors). These orphan ligands can be created to either block or activate the receptor top which they bind.

[0119] A single ligand can be used, or optionally a variety of ligands can be used to select the domains and/or multimers. A domain of the present disclosure can bind a single ligand or a variety of ligands. A multimer of the present disclosure can have multiple discrete binding sites for a single ligand, or optionally, can have multiple binding sites for a variety of ligands.

[0120] In some embodiments, the multimer comprises domains with specificities for different proteins. The different proteins can be related or unrelated.

[0121] In some embodiments, one or more domains of the disclosure is linked to a molecule (e.g. , a protein, nucleic acid, synthetic small molecule, etc.) useful as a pharmaceutical.

Exemplary pharmaceutical proteins include, e.g. , cytokines, antibodies, chemokines, growth factors, interleukins, cell-surface proteins, extracellular domains, cell surface receptors, cytotoxins, corticosteroids (e.g. , triamcinolone acetonide), etc. Exemplary small molecule pharmaceuticals include toxins or therapeutic agents. In some embodiments, the small molecule drug is a metalloproteinase inhibitor. Small molecule drugs contemplated for use according to the disclosure include, but are not limited to, inhibitors of the following: matrix

metalloproteinase (MMP)-3, MMP-9, and MMP- 13; ADAMTS-4 and ADAMTS-5; a complement protein; a prostaglandin-synthesizing enzyme; caspase-1 ; cathepsin-K; a neutrophil protease capable of activating pro-ILla and pro-ILiP; the Adenosine A3 receptor; p38 kinase; inducible nitric oxide synthase (iNOS), and a combination thereof. Further small molecule pharmaceuticals contemplated by the disclosure include an aggrecanase inhibitor and a nonsteroidal anti-inflammatory drug (NSAID) such as aspirin, a Cox2 inhibitor (e.g. , Celebrex®), ibuprofen, and other propionic acid derivatives (alminoprofen, benoxaprofen, bucloxic acid, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, suprofen, tiaprofenic acid, and tioxaprofen), acetic acid derivatives (indomethacin, acemetacin, alclofenac, clidanac, diclofenac, fenclofenac, fenclozic acid, fentiazac, fuirofenac, ibufenac, isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidometacin, and zomepirac), fenamic acid derivatives (flufenamic acid, meclofenamic acid, mefenamic acid, niflumic acid and tolfenamic acid), biphenylcarboxylic acid derivatives (diflunisal and flufenisal), oxicams (isoxicam, piroxicam, sudoxicam and tenoxican), salicylates (acetyl salicylic acid, sulfasalazine) and the pyrazolones (apazone, bezpiperylon, feprazone, mofebutazone, oxyphenbutazone, phenylbutazone). In some embodiments, a metal can be bound to the polypeptides of the invention. This can be useful, e.g. , as a contrast agent, e.g. , for X-ray or MRI. In some embodiments, a small molecule is appended to the CII binding avimer via a "cleavable" linker. In such embodiments, the small molecule is a derivative of any of the drugs listed herein.

[0122] According to the present disclosure, the domain or multimer is selected to bind to a tissue- or disease- specific target protein. Tissue-specific proteins are proteins that are expressed exclusively, or at a significantly higher level, in one or several particular tissue(s) compared to other tissues in an animal. As type II collagen is expressed at significant levels in articular cartilage, domains that bind to type II collagen are used to target other molecules, including other domains (e.g. , an IL- 1R binding domain as disclosed herein), to the articular cartilage. This is used to target cartilage- specific diseases, for example, by targeting therapeutic or toxic molecules to the cartilage. As disclosed herein, an example of such a cartilage disease is osteoarthritis.

[0123] In some embodiments, the domain or multimer that binds to the target protein is linked to the pharmaceutical protein or small molecule such that the resulting complex or fusion is targeted to the specific tissue or disease-related cell(s) where the target protein (e.g. , type II collagen) is expressed. Domains or multimers for use in such complexes or fusions can be initially selected for binding to the target protein and may be subsequently selected by negative selection against other cells or tissue where it is desired that binding be reduced or eliminated in other non-target cells or tissues. By keeping the pharmaceutical away from sensitive tissues, the therapeutic window is increased so that a higher dose may be administered safely. In another alternative, in vivo panning can be performed in animals by injecting a library of domains or multimers into an animal and then isolating the domains or multimers that bind to a particular tissue or cell of interest. [0124] The fusion proteins described above may also include a linker peptide between the pharmaceutical protein and the domains or multimers. A peptide linker sequence may be employed to separate, for example, the polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Fusion proteins may generally be prepared using standard techniques, including chemical conjugation. Fusion proteins can also be expressed as recombinant proteins in an expression system by standard techniques.

[0125] Multimers or domains of the disclosure can be produced according to any methods known in the art. In some embodiments, E. coli comprising a pET-derived plasmid encoding the polypeptides are induced to express the protein. After harvesting the bacteria, they may be lysed and clarified by centrifugation. Polypeptides containing a Histidine tag (His-tag) may be purified using Ni-NTA agarose elution and refolded by dialysis. Misfolded proteins may be neutralized by capping free sulfhydrils with iodoacetic acid. Q sepharose elution, butyl sepharose flow-through, SP sepharose elution, DEAE sepharose elution, and/or CM sepharose elution may be used to purify the polypeptides. Equivalent anion and/or cation exchange purification steps may also be employed, as well as affinity chromatography {e.g., selection for properly folded CII domains by binding to and eluting from CII).

[0126] In some embodiments, the polypeptide comprising a domain or multimer of the disclosure is linked to itself (C-terminus to N-terminus), e.g., for protein stability.

Linkers

[0127] Domains can be joined by a linker to form a multimer. For example, a linker may be positioned between each separate discrete domain in a multimer.

[0128] Joining the selected monomer domains via a linker can be accomplished using a variety of techniques known in the art. For example, combinatorial assembly of polynucleotides encoding selected monomer domains can be achieved by restriction digestion and re-ligation, by PCR-based, self -priming overlap reactions, or other recombinant methods. The linker can be attached to a domain before the domain is identified for its ability to bind to a target multimer or after the domain has been selected for the ability to bind to a target multimer.

[0129] The linker can be naturally-occurring, synthetic or a combination of both. For example, the synthetic linker can be a randomized linker, e.g., both in sequence and size. In one aspect, the randomized linker can comprise a fully randomized sequence, or optionally, the randomized linker can be based on natural linker sequences. The linker can comprise, e.g. , a non-polypeptide moiety, a polynucleotide, a polypeptide or the like.

[0130] A linker can be rigid, or flexible, or a combination of both. Linker flexibility can be a function of the composition of both the linker and the domains with which the linker interacts. The linker joins two selected domains, and maintains the domains as separate discrete domains. The linker can allow the separate discrete monomer domains to cooperate yet maintain separate properties such as multiple separate binding sites for the same ligand in a multimer, or e.g. , multiple separate binding sites for different ligands in a multimer.

[0131] Choosing a suitable linker for a specific case where two or more domains (i.e. , polypeptide chains) are to be connected may depend on a variety of parameters including, e.g. , the nature of the domains, the structure and nature of the target to which the polypeptide multimer should bind and/or the stability of the peptide linker towards proteolysis and oxidation.

[0132] The present disclosure provides methods for optimizing the choice of linker once the desired domains/variants have been identified. Generally, libraries of multimers having a composition that is fixed with regard to domain composition, but variable in linker composition and length, can be readily prepared and screened. In some embodiments, the linker is

GGGGSGGGGS (SEQ ID NO: 139).

[0133] A more detailed discussion of linkers can be found in, e.g. , U.S. Patent Publication No. 2005/0048512, which is incorporated by reference herein in its entirety.

Identifying Monomers or Multimers with Affinity for a Target Molecule

[0134] Those of skill in the art can readily identify domains with a desired property (e.g. , binding affinity). For those embodiments, any method resulting in selection of domains with a desired property (e.g. , a specific binding property) can be used. For example, the methods can comprise providing a plurality of different nucleic acids, each nucleic acid encoding a monomer domain; translating the plurality of different nucleic acids, thereby providing a plurality of different monomer domains; screening the plurality of different monomer domains for binding of the desired ligand or a mixture of ligands; and, identifying members of the plurality of different monomer domains that bind the desired ligand or mixture of ligands. [0135] In addition, any method of mutagenesis, such as site-directed mutagenesis and random mutagenesis (e.g. , chemical mutagenesis) can be used to produce monomer domains, e.g. , for a monomer domain library. In some embodiments, error-prone PCR is employed to create variants. Additional methods include aligning a plurality of naturally occurring domains by aligning conserved amino acids in the plurality of naturally occurring domains; and, designing the non-naturally occurring domain by maintaining the conserved amino acids and inserting, deleting or altering amino acids around the conserved amino acids to generate the non-naturally occurring domain. In some embodiments, the conserved amino acids comprise cysteines. In further embodiments, the inserting step uses random amino acids, or optionally, the inserting step uses portions of the naturally occurring domains. The portions could ideally encode loops from domains from the same family. Amino acids are inserted or exchanged using synthetic oligonucleotides, or by shuffling, or by restriction enzyme based recombination. Human chimeric domains of the present disclosure are useful for therapeutic applications where minimal immunogenicity is desired. The present disclosure provides methods for generating libraries of human chimeric domains. Human chimeric domain libraries can be constructed by combining loop sequences from different variants of a human domain, as described above. The loop sequences that are combined may be sequence-defined loops, structure-defined loops, B-factor- defined loops, or a combination of any two or more thereof.

[0136] Alternatively, a human chimeric domain library can be generated by modifying naturally-occurring human domains at the amino acid level, as compared to the loop level. In some embodiments, to minimize the potential for immunogenicity, only those residues that naturally occur in protein sequences from the same family of human domains are utilized to create the chimeric sequences. This can be achieved by providing a sequence alignment of at least two human domains from the same family of domains, identifying amino acid residues in corresponding positions in the human domain sequences that differ between the human domains, generating two or more human chimeric domains, wherein each human chimeric monomer domain sequence consists of amino acid residues that correspond in type and position to residues from two or more human domains from the same family of domains. Libraries of human chimeric domains can be employed to identify human chimeric domains that bind to a target of interest by: screening the library of human chimeric domains for binding to a target molecule, and identifying a human chimeric domain that binds to the target molecule. Suitable naturally- occurring human domain sequences employed in the initial sequence alignment step include those corresponding to any of the naturally-occurring domains described herein.

[0137] Domains of human monomer variant libraries of the present disclosure (whether generated by varying loops or single amino acid residues) can be prepared by methods known to those having ordinary skill in the art. Methods particularly suitable for generating these libraries are split-pool format and trinucleotide synthesis format as described in WO 01/23401.

[0138] In some embodiments, domains of the disclosure are screened for potential

immunogenicity by: providing a candidate protein sequence; comparing the candidate protein sequence to a database of human protein sequences; identifying portions of the candidate protein sequence that correspond to portions of human protein sequences from the database; and determining the extent of correspondence between the candidate protein sequence and the human protein sequences from the database.

[0139] In general, the greater the extent of correspondence between the candidate protein sequence and one or more of the human protein sequences from the database, the lower the potential for immunogenicity is predicted as compared to a candidate protein having little correspondence with any of the human protein sequences from the database. A database of human protein sequences that is suitable for use in the practice of the methods for screening candidate proteins can be found online at NCBI's BLAST website.

[0140] The disclosure also includes compositions that are produced by methods of the present disclosure. For example, the present disclosure includes domains selected or identified from a library and/or libraries comprising domains produced by the methods of the present disclosure.

[0141] The present disclosure also provides libraries of domains and libraries of nucleic acids that encode domains. The libraries can include, e.g., about 100, 250, 500 or more nucleic acids encoding domains, or the library can include, e.g., about 100, 250, 500 or more polypeptides that encode domains. Libraries can include monomer domains containing the same cysteine frame, e.g., A-domains or EGF-like domains.

Therapeutic and Prophylactic Treatment Methods

[0142] The present disclosure also includes methods of therapeutically or prophylactically treating a disease or disorder by administering in vivo or ex vivo one or more nucleic acids or polypeptides of the disclosure described above (or compositions comprising a pharmaceutically acceptable excipient and one or more such nucleic acids or polypeptides) to a subject, including, e.g. , a mammal, including a human, primate, dog, cat, mouse, pig, cow, goat, rabbit, rat, guinea pig, hamster, horse, sheep; or a non-mammalian vertebrate such as a bird (e.g. , a chicken or duck), fish, or invertebrate.

[0143] Type II collagen-binding peptides, including type II collagen -binding domains or multimers of the disclosure, are useful in treatment of disorders found in the joint or cartilage of a subject. More particularly, type II collagen-binding peptides are useful in treating or preventing osteoarthritis (OA) by anchoring appended payloads (e.g. , an IL- 1R domain as disclosed herein) in the joint or cartilage of a subject.

[0144] Individuals can be treated, for example, by once weekly intravenous injections of a soluble formulation of a type II collagen-binding peptide composed of type II collagen-binding domains or multimers of the disclosure, optionally in combination with one or more additional therapeutic entities, for example either biologic or chemo therapeutic.

[0145] IL- 1R antagonists, including IL- lR-binding domains or multimers of the disclosure, are useful in treatment of disorders including Stills Disease, gout, rheumatoid arthritis, juvenile rheumatoid arthritis, or calcium pyrophosphate deposition disease (CPPD). Individuals can be treated, for example, by once weekly intravenous injections of a soluble formulation of a IL-1R antagonist composed of IL-lR-binding domains or multimers of the disclosure, optionally in combination with one or more additional therapeutic entities, for example either biologic or chemotherapeutic .

[0146] In some aspects, in ex vivo methods, one or more cells or a population of cells of interest of the subject (e.g. , a chondrocyte) are obtained or removed from the subject and contacted with an amount of a selected domain and/or multimer of the disclosure that is effective in prophylactically or therapeutically treating the disease, disorder, or other condition. The contacted cells are then returned or delivered to the subject to the site from which they were obtained or to another site of interest in the subject to be treated. If desired, the contacted cells can be grafted onto a tissue, organ, or system site of interest in the subject using standard and well-known grafting techniques or, e.g. , delivered to the blood or lymph system using standard delivery or transfusion techniques.

[0147] The disclosure also provides in vivo methods in which one or more cells or a population of cells of interest of the subject are contacted directly or indirectly with an amount of a selected domain and/or multimer of the disclosure effective in prophylactically or therapeutically treating the disease, disorder, or other condition. In direct contact/administration formats, the selected domain and/or multimer is typically administered or transferred directly to the cells to be treated or to the tissue site of interest (e.g., cartilage) by any of a variety of formats, including topical administration, injection (e.g., by using a needle or syringe), or vaccine or gene gun delivery, pushing into a tissue, organ, or skin site. The selected domain and/or multimer can be delivered, for example, via intra- articular, sub-cutaneous, parenteral, or intravenous delivery, or placed within a cavity of the body (including, e.g., during surgery).

[0148] In in vivo indirect contact/administration formats, the selected domain and/or multimer is typically administered or transferred indirectly to the cells to be treated or to the tissue site of interest, including those described above, by contacting or administering the polypeptide of the disclosure directly to one or more cells or population of cells from which treatment can be facilitated.

[0149] In each of the in vivo and ex vivo treatment methods of the disclosure, a composition comprising an excipient and the polypeptide or nucleic acid of the invention can be administered or delivered. In one aspect, a composition comprising a pharmaceutically acceptable excipient and a polypeptide or nucleic acid of the disclosure is administered or delivered to the subject as described above in an amount effective to treat the disease or disorder.

FURTHER MANIPULATING MONOMER DOMAINS AND/OR MULTIMER

NUCLEIC ACIDS AND POLYPEPTIDES

[0150] Further aspects of the disclosure include the cloning and expression of domains, selected domains, multimers and/or selected multimers coding nucleic acids. Thus, multimer domains can be synthesized as a single protein using expression systems well known in the art. General texts which describe molecular biological techniques useful herein, including the use of vectors, promoters and many other topics relevant to expressing nucleic acids such as monomer domains, selected domains, multimers and/or selected multimers, include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et ah, Molecular Cloning - A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 and Current Protocols in Molecular Biology, F.M. Ausubel et ah, eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 1999). Examples of techniques sufficient to direct persons of skill through in vitro amplification methods, useful in identifying, isolating and cloning monomer domains and multimers coding nucleic acids, including the polymerase chain reaction (PCR) the ligase chain reaction (LCR), Q-replicase amplification and other RNA polymerase mediated techniques {e.g., NASBA), are known in the art. One of skill will appreciate that essentially any RNA can be converted into a double stranded DNA suitable for restriction digestion, PCR expansion and sequencing using reverse transcriptase and a polymerase.

[0151] The present disclosure also relates to the introduction of vectors of the invention into host cells, and the production of monomer domains, selected domains, multimers and/or selected multimers of the invention by recombinant techniques. Host cells are genetically engineered {i.e., transduced, transformed or transfected) with the vectors of this invention, which can be, for example, a cloning vector or an expression vector. The vector can be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the domain, selected domain, multimer and/or selected multimer gene(s) of interest. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art and in the references cited herein, including, e.g., Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley-Liss, New York and the references cited therein.

[0152] Polypeptides of the disclosure can also be produced in non-animal cells such as plants, yeast, fungi, bacteria and the like. Indeed, phage display is an especially relevant technique for producing such polypeptides. In addition to references cited above, details regarding cell culture can be found in Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, NY; Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer- Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL.

[0153] The present disclosure also includes alterations of domains, immunoglobulin-type domains and/or multimers to improve pharmacological properties, to reduce immunogenicity, or to facilitate the transport of the multimer and/or monomer domain into a cell or tissue {e.g., through the skin). These types of alterations include a variety of modifications (e.g. , the addition of sugar-groups or glycosylation), the addition of PEG, the addition of protein domains that bind a certain protein (e.g. , HSA or other serum protein), the addition of proteins fragments or sequences that signal movement or transport into, out of and through a cell, and/or a slight amino acid sequence change to reduce immunogenicity. Additional components can also be added to a multimer and/or domain to manipulate the properties of the multimer and/or domain. A variety of components can also be added including, e.g. , a domain that binds a known receptor (e.g. , a Fc-region protein domain that binds a Fc receptor), a toxin(s) or part of a toxin, a prodomain that can be optionally cleaved off to activate the multimer or domain, a reporter molecule (e.g. , green fluorescent protein), a component that binds a reporter molecule (such as a radionuclide for radiotherapy, biotin or avidin) or a combination of modifications. In some embodiments of the disclosure in which treatment OA, other cartilage diseases, or intervertebral disc degeneration, it is advantageous to keep the overall size of the composition to a minimum so that the composition can migrate into the cartilage or nucleus pulposus matrix. Accordingly, it is contemplated that in some embodiments the composition does not comprise a large moiety such as PEG, extra carbohydrate or green fluorescent protein (GFP).

Kits

[0154] Kits comprising the components needed in the methods (typically in an unmixed form) and kit components (packaging materials, instructions for using the components and/or the methods, one or more containers (reaction tubes, columns, etc.)) for holding the components are a feature of the present disclosure. Kits of the present disclosure may contain a multimer library, or a single type of domain or multimer. Kits can also include reagents suitable for promoting target molecule binding, such as buffers or reagents that facilitate detection, including detectably-labeled molecules. Standards for calibrating a ligand binding to a domain or the like, can also be included in the kits of the disclosure.

[0155] The present disclosure also provides commercially valuable binding assays and kits to practice the assays. In some of the assays of the disclosure, one or more ligand is employed to detect binding of a domain, immunoglobulin-type domains and/or multimer. Such assays are based on any known method in the art, e.g. , flow cytometry, fluorescent microscopy, plasmon resonance, and the like, to detect binding of a ligand(s) to the domain and/or multimer. [0156] Kits based on the assay are also provided. The kits typically include a container, and one or more ligand. The kits optionally comprise directions for performing the assays, additional detection reagents, buffers, or instructions for the use of any of these components, or the like. Alternatively, kits can include cells, vectors, (e.g., expression vectors, secretion vectors comprising a polypeptide of the invention), for the expression of a domain and/or a multimer of the disclosure.

[0157] In further aspects, the present disclosure provides for the use of any composition, domain, immunoglobulin-type domain, multimer, cell, cell culture, apparatus, apparatus component or kit herein, for the practice of any method or assay herein, and/or for the use of any apparatus or kit to practice any assay or method herein and/or for the use of cells, cell cultures, compositions or other features herein as a therapeutic formulation. The manufacture of all components herein as therapeutic formulations for the treatments described herein is also provided.

EXAMPLES

[0158] The following examples describe the identification of high affinity avimers that bound to type II collagen, penetrated cartilage, and persisted in rat knee joint cartilage for at least a month following intra- articular injection. A fusion protein constructed with a cartilage binding avimer and IL-IRa bound to type II collagen and inhibited IL-1 -induced responses in vitro. Moreover, in contrast to unmodified IL-IRa, the fusion protein retained neutralizing activity in vivo for at least a week following a single intra- articular injection, thus demonstrating the utility of this strategy in the treatment of osteoarthritis.

Example 1

Materials and methods

Generation of Type II collagen binding avimers

[0159] Avimer libraries, based on human A-domain scaffolds, were constructed in the fUSE5 M13 phage vector and panned and screened against immobilized protein targets as previously described [Silverman et al., Nat Biotechnol 23: 1556-1561 (2005)]. After three rounds of panning the avimer libraries against human and rat type II collagen, three monomers of interest were isolated. To increase their affinities to type II collagen, two avimer monomers (M02 and M05) underwent affinity maturation via biased random mutagenesis for which each monomer was encoded by two overlapping oligonucleotides. Avimer scaffold residues [Silverman et al., Nat Biotechnol 23: 1556-1561 (2005)] were fixed and variable residues were encoded by 70% base doping (i.e., 70% parental base, 10% for each of the other three bases). One resultant mutant avimer was chosen for an additional round of random mutagenesis where two

mutagenesis libraries were constructed, with 85% and 91% base doping, respectively. The overlapping oligonucleotides were annealed via a fixed nine base pair overlap at 30°C, and extended to create double-stranded DNA by PCR with LA Taq polymerase (Takara). The assembled double- stranded library DNA was then cloned into the fUSE5HA phage vector, and the ligated vector was purified and transformed into EC 100 E. coli. Each library was subjected to three rounds of panning against human and rat type II collagen, and pulverized human cartilage. Selected phage-derived avimers were cloned into a pEVE expression plasmid, transformed into BL21(DE3) Gold cells (Stratagene) and purified as previously described

[Silverman et ah, Nat Biotechnol 23: 1556-1561 (2005)] for characterization of their affinity and specificity by ELISA and AlphaScreen against target proteins and pulverized human cartilage.

[0160] Expression of all individual avimer domains disclosed herein utilized the same construct format shown for Coll2 M26 (Figure 12); only residues within the A domain segment (excluding scaffold residues) were subjected to mutation during the optimization phase. Within the Avimer libraries employed for screening, non-redundant linker domains were present which correspond to natural linkers found in native A domain-containing proteins; in the case of M26, the linker is PTPT. During mutagenesis linker domains also were held constant. See Figure 12. The constructs referred to in the Examples below include the additional amino acids depicted in Figure 12. Any of the sequences or constructs described herein are contemplated to contain an A-domain, and optionally also contain a linker, and/or a tag for protein purification, and/or amino acids inserted for cloning/expression.

Generation of IL-lRa_avimer Bi-specific Fusion Proteins

[0161] CII binding avimer and human IL-IRa were linked as fusion proteins via

GGGGSGGGGS ((G4S)₂) linker (SEQ ID NO: 139) with IL-IRa either at N-terminus or C- terminus of the fusion protein. A negative control (NC) fusion protein was constructed by fusing an Avimer (M07) that binds to the extracellular domain of an irrelevant protein to human IL- IRa in the same manner. To facilitate purification, the fusion proteins contained an N-terminal 8-His tag. For imaging studies and in vitro characterization, avimers or fusion proteins were labeled with Biotin, FITC, Alexa Fluor® 647 or 680 (AF647 or AF680) using Pierce conjugation kits. The average degree of labeling was 7.5.

Binding of CII avimers to collagen

[0162] To assess Avimer binding to CII and other molecules, target proteins were directly immobilized in a 96-well Maxisorp ELISA plate (20 nM; BD Bioscience, Chondrex, R&D) using coating buffer (20 mM Tris, pH 7.5, 150 mM NaCl) while pulverized human cartilage was resuspended in blocking buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM CaCl₂, 1% BSA) and added to a 96-well PCR microplate (0.5 mg per well; Articular Engineering, Chicago, IL). Both plates were incubated at room temperature (RT) with shaking for 1 hour. After each incubation and wash step, the plates containing pulverized cartilage were briefly vortexed and then centrifuged at 4000 rpm for 5 minutes to pellet the cartilage. Following the coating period, unbound supernatants were decanted, blocking buffer was added to each well, and the plates incubated for 60 minutes at RT. Wells subsequently were washed 3 times with wash buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM CaCl₂ and 0.05% Tween-20). Serial 3-fold dilutions of avimer (starting at 1 uM) in assay buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM CaCl₂, 0.1% BSA, and 0.05% Tween-20) were added to allocated wells and the plates were incubated at RT with shaking for 1.5 hours. Plates were then washed 3 times with wash buffer. Bound avimers were detected using either an anti HA-HRP antibody (clone 3F10; Roche; diluted 2000-fold in assay buffer and incubated at RT for 1 hour) or anti His-HRP antibody (clone His-1 ; Sigma; diluted 2000-fold in assay buffer). Plates were then washed 3 times with wash buffer after which TMB/H₂0₂ substrate mix was added (Thermo Scientific Cat #34028). Reactions were stopped by addition of 2N H₂S0₄. At this point, PCR plates containing pulverized cartilage were centrifuged at 4000 rpm for 5 minutes and the resulting supernatants were transferred to a 96- well Maxisorp ELISA plate. Plates were read at OD₄so nm using a SpectraMax Plus. huIL-lRa_M26 avimer Dual Binding ELISA

[0163] All assay incubations were carried out at RT with shaking. 96-well Maxisorp ELISA plates were first coated with human type II collagen (20 nM; Chondrex). After a 1.5 hour incubation, binding solutions were decanted and the wells incubated for 1 hour with blocking buffer. Dose-response curves (starting at 1 μΜ) were generated by serially diluting huIL- lRa_M26 fusion, M26 alone, or hulL-IRa in assay buffer. The diluted proteins were added to CH-coated wells and the plates were incubated for 1.5 hours, and then washed 3 times with wash buffer. HuIL-lRI-Fc fusion protein (20 nM; Amgen) in assay buffer was added to each well of the washed plates and incubated for 1 hour. After washing 3 times with wash buffer, anti-huIgG- HRP (0.67 nM; BioSource International) was added to each well, and the plates were incubated for 1 hour and then washed three times with wash buffer prior to addition of the HRP substrate mix (TMB/H₂O₂; reaction stopped with 2N H₂S0₄). Plates were read at OD₄so nm using

SpectraMax Plus.

[0164] Pulverized human cartilage (0.5 mg per well; Articular Engineering) was resuspended in blocking buffer and with constant mixing was added to wells of a 96-well PCR microplate and incubated with blocking buffer for 1 hour. After each incubation and wash step the plates were briefly vortexed then centrifuged at 4000 rpm for 5 minutes to pellet the cartilage. The plate was then washed three times with washing buffer. Serial 3-fold titrations of IL-lra_M26, M26, or IL-lra (starting at 300 nM) were added to allocated wells and incubated for 1.5 hours. The plate was then washed three times with wash buffer. Biotinylated IL-IRI-Fc fusion protein was added (12 nM; Amgen) and incubated for 1 hour. The plate subsequently was washed 3 times with wash buffer and bound biotin was detected by addition of streptavidin-HRP (0.67 nM; Jackson ImmunoResearch). After a lhour incubation, the plate was washed 3 times with wash buffer prior to addition of the HRP substrate mix (TMB/H₂0₂; reaction stopped with 2N H₂S0₄). The plate was centrifuged at 4000 rpm for 5 minutes to sediment insoluble cartilage and the supernatants were transferred to a 96-well Maxisorp ELISA plate prior to reading at OD450 (nm) using a SpectraMax Plus.

SW982 IL-6 Assay

[0165] Human synovial sarcoma SW982 cells were maintained in RPMI 1640 media supplemented with 10%FBS/1%PSG. One day before assay, cells were seeded at 2.5xl0⁴

in low serum growth media (1%FBS) in 96-well tissue culture plate. The next day, cells were washed once with PBS, followed by addition of 80 μΐ_^ IL-IRa or IL-lRa_avimer fusions diluted in low serum growth media to appropriate wells. After 0.5 hour incubation at room temperature, 20 μΐ_^ of low serum growth medium containing 1 ng/ml rhIL-Ιβ was added to each well. Cells were then incubated at 37°C for 4 hours. After a brief centrifugation, supernatants were transferred to a new plate for IL-6 analysis using IL-6 ELISA kit (BioSource International). Human Chondrocyte IL-8 assay

[0166] Human chondrocyte monolayers (96 well plate; 50, 000 cells/cm ) were purchased from Articular Engineering (Chicago, IL). Chondrocyte monolayers were maintained in

Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L- glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C. Twenty four hours prior to IL-Ιβ challenge, cells were incubated in low serum growth media (1% FBS). Chondrocytes were incubated with IL-Ra or IL-lRa_CII Avimer fusion for 30 minutes prior to stimulation with 1 ng/ml rhuIL-Ιβ. Cells were incubated with the cytokine stimulus at 37°C for 24 hours after which the plates were subjected to a brief centrifugation, and the resulting supernatants were transferred to a new plate. IL-8 levels in the supernatants were measured using an IL-8

Fluorokine Assay (R&D Systems).

Animal care

[0167] Rats were cared for in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council (U.S.). Committee for the Update of the Guide for the Care and Use of Laboratory Animals., Institute for Laboratory Animal Research (U.S.), and National Academies Press (U.S.) 2011. Guide for the care and use of laboratory animals. 8th ed. National Academies Press, Washington, D.C. xxv, 220 p.). Rats were pair housed at an AAALAC, Intl- accredited facility in non-sterile ventilated micro-isolator housing on corn cob bedding. All research protocols were approved by the Institutional Animal Care and Use Committee. Animals had ad libitum access to pelleted feed (Harlan 2020X, Madison, WI) and water (reverse osmosis- purified with the addition of .20-.50ppm chlorine added) via an automatic watering system. Animals were maintained on a 12: 12 hour light: dark cycle in rooms held at 68-79° F with 20- 70% relative humidity. Animals had access to enrichment opportunities. All rodents were determined specific pathogen-free for standard Level 1 pathogens, including viruses, bacteria, enteric protozoa, arthropod ectoparasites, and helminth endoparasites.

In Vivo Imaging

[0168] Animal protocols and procedures were approved by the Institutional Animal Care and Use Committee of Amgen Inc. Female Lewis rats (120-140 grams; Harlan Labs, USA) were housed in filter-top cages with food and water ad libitum on a 12 hour light dark cycle. Rats were anesthetized using 4% isoflurane in oxygen (lL/min) during induction and 2% isoflurane in oxygen (0.5 L/min) delivered via nosecone during intra- articular injection and imaging. Prior to intra- articular injection, animals were shaved under anesthesia from the right leg to the inguinal area including the surrounding abdomen. Shaving a larger region around the injection site was important for later region of interest (ROI) placement as the fluorescent signal decreased over time allowing easier demarcation of the injection site from background fluorescence in the neighboring fur. Animals that were held for more than 7 days post-injection were shaved again prior to imaging while under anesthesia. The injection area was cleaned with 3 alcohol wipes prior to injection. Using a 28 gauge needle, 30 ul of fluorescently labeled material at

concentrations between 75-100 μg/ml diluted in HEPES (N-2-hydroxyethylpiperazine-N-2- ethane sulfonic acid) containing buffer was injected through the patellar ligament and into the articular space. Animals were kept warm during imaging sessions using the warmed platform in the IVIS Spectrum (Perkin Elmer, Waltham, MA) imaging system. Each rat was positioned in the imaging system on its left side and curled slightly, allowing the right knee to drape naturally for imaging and keeping the knee within the field of view (FOV) of the system. Having the animal maintain its natural body position aided in consistent positioning between imaging sessions. All images were acquired using planar epi-illumination and the following settings on the IVIS Spectrum (Bin:M(8); FOV 13mm; f stop2; 1 second) using Living Image software version 3.0 (PerkinElmer). For AF680 labeled probes the excitation filter was set to 675nm with the emission filter at 720nm. For AF647 labeled probes the excitation filter was set to 640nm and the emission filter at 680nm. All images were subjected to the following adjustments: flat- field correction, cosmic ray correction and read bias subtraction. Fluorescence intensity is reported in Radiant Efficiency (p/sec/cm /steridian) using a ROI positioned over the injection site of the first image acquired (Time 0) immediately after injection. The ROI was used on all subsequent images for each individual animal to compare values across time-points. The values were graphed using GraphPad Prism version 5.0 (GraphPad software, La Jolla, CA). For ex vivo imaging and comparisons to in vivo images, Radiance values are reported (p/sec/cm /steridian).

Multiphoton Imaging

[0169] Human non-diseased cartilage explants (5 mm in diameter) were obtained from

Articular Engineering, LLC and maintained in 50/50 DMEM/F12 with 10% FBS in 48 well plates (1 explant per well). Serum was lowered to 1% for 24 hours prior to the start of each study. To assess binding and penetration of CII Avimers, human cartilage explants were incubated with 2.5 μΜ FITC-labeled Avimers for 5 days at 37°C in low serum growth media. At the end of culture period, the explants were placed in a 35 mm glass-bottom dish (MatTek Corporation, Ashland, MA) and Z-stack imaging was performed using a Zeiss LSM 510 META NLO imaging system equipped with a Coherent Chameleon Ti-sapphire tunable multiphoton laser. An 800 nm laser line was used as excitation wavelength and appropriate band-pass filters were used as emission filters for second harmonic generation (SHG) signal and FITC signal.

[0170] To assess penetration and retention of the IL-lRa_M26 fusion, cartilage explants were incubated with 2.5 μΜ of AF647 labeled construct. For comparison, similar concentrations of AF647-labeled M26, AF647-labeled M07 (negative control avimer), and AF647-labeled IL-lRa were incubated with separate explants. The cultures were incubated 24 hours at 37°C in low serum growth media followed by extensive washing with PBS. Cartilage explants were then incubated with fresh media at 37°C for an additional 3 days. At Day 0 or Day 3 post-avimer incubation, explants were placed in a 35 mm glass-bottom dishes and three-dimensional image acquisition and data acquisition/processing conducted as above.

Immunohistochemistry

[0171] In vitro binding of CII avimers to human cartilage: OCT-embedded human cartilage was sectioned at 4 μιη on a cryostat with the use of the Cryojane tape-transfer system. Sections were mounted onto 4X-adhesive slides and "flashed" 2X with UV light to promote adherence of the tissues to the slides. This was necessary to overcome the charge repulsion of the cartilage for the glass slides. Slides were air-dried and then hydrated with ELISA Wash Buffer. Blocking was performed for endogenous proteins (ELISA blocking buffer), avidin, biotin and endogenous peroxidase. Sections were washed with ELISA wash buffer in-between each blocking step. Biotinylated Avimers (M26 and M07) were added to the sections at 10 μg/ml (diluted in ELISA Blocking Buffer) and incubated overnight at 4°C. Slides were removed from the refrigerator, washed and allowed to acclimate to RT. Slides were incubated with Streptavidin-AlexaFluor647 (Invitrogen #S32357 at a 1:200 dilution in ELISA Blocking Buffer) for 30 minutes at RT. Slides were washed with ELISA Wash Buffer, rinsed in DI Water and covered with a coverslip using Prolong Gold Mounting Medium containing DAPI (Invitrogen #P36935). Images were acquired using NIS elements v3.0 software at the 647 nM wavelength on a Nikon Eclipse 50i fluorescent microscope illuminated by an EXFO Excite 120 light source and equipped with a Cool Snap EZ digital camera. Positive and negative control images were collected using an identical exposure time. [0172] Direct fluorescence detection of CII avimers in rat cartilage following IA injection of AF647-labeled CII avimers: Female Lewis Rats (120-140 grams) were injected IA with 30 μΐ of AF647-labeled Avimers as described above and euthanized at 28 days post-injection. The fluorescent label was changed from AF680 to AF647 for the ex vivo imaging experiments in order to accommodate the optimal excitation and emission configurations of the imaging system used for the respective experiments.

[0173] Duplicate samples of cartilage were carefully dissected from both the right and left tibial plateaus to minimize the inclusion of underlying bone. The cartilage was then embedded in OCT for frozen sectioning and OCT-embedded rat cartilage was sectioned at 4 μιη on a cryostat with the use of the Cryojane tape-transfer system. Sections were mounted onto 4X- adhesive slides and "flashed" 3X with UV light to promote adherence of the tissues to the slides. Slides were air-dried overnight and coverslips were then mounted using Prolong Gold Mounting Medium containing DAPI. Samples were reviewed using the 647 nM fluorescence cube on a Nikon fluorescent microscope controlled by NIS Elements software. Each test sample was reviewed using the "autoexposure" feature of the software to determine the optimal exposure time for that sample. The sample with the brightest signal had an optimal exposure time of 500 ms. Negative control samples were subsequently reviewed, and the exposure time adjusted so that background fluorescence was just barely visible (3 sec). Subsequently, photomicrographs of one representative animal from each group were collected using the optimal exposure time of the brightest sample (500 ms). This approach maximized the opportunity to observe the presence of the fluorescent constructs while minimizing autofluorescence.

IA Rat IL-Ιβ Challenge Model

[0174] Male Sprague-Dawley rats (250-275 grams; Harlan Labs, USA) were housed in filter- top cages with food and water ad libitum on a 12-hr light dark cycle. For intra-articular (IA) injections, rats were anesthetized with 1.5 - 4% isoflurane + 0₂ and were maintained under a nose cone for the duration of the procedure. The area of the patella was shaved free of hair, swabbed with antiseptic, and 30 ng of rat IL-Ιβ in 50 μΐ_^ of phosphate buffer saline (PBS) was injected into the knee joint using a 28 gauge needle. Rats injected with 50 μΐ_^ of PBS served as vehicle controls. Animals were sacrificed 4 hours post IL-Ιβ injection for synovial lavage collection. Knee joints were lavaged with 100 μΐ saline and lavage fluid was digested with 500 units/mL hyaluronidase (Sigma #3884) for 30 minutes. IL-6 levels were detected using an ELISA kit (R&D Systems; SR6000B). For the inhibitor studies, 146 μg IL-IRa or 247 μg IL- lRa_M26 fusion was co-administered with 30 ng IL-Ιβ to give a 5000 molar excess compared to IL-Ιβ (pilot studies utilized different amounts of IL-IRa). To assess whether the activity of IL- lRa_M26 fusion persisted in the joint, IL-IRa or IL-lRa_M26 were administered 7 days prior to IL-Ιβ challenge.

Example 2

Generation of Type II Collagen binding avimer domains

[0175] In order to generate type II collagen binding avimers (CII avimers), libraries constructed in the fUSE5 M13 phage vector [Silverman et al., Nat Biotechnol 23: 1556-1561 (2005)] were panned and screened against purified human and rat type II collagen. After three rounds of panning, three monomers that displayed weak binding to human and rat type II collagen, and no detectable binding to human cartilage were isolated. An initial round of affinity maturation yielded avimers with sub-nM affinities to type II collagen, and detectable binding to pulverized human cartilage (Table 1). Additional rounds of affinity maturation led to the identification of the CII-M18 (SEQ ID NO: 4) and CII-M26 (SEQ ID NO: 5) avimers that bound type II collagen at sub-nM EC₅₀, and pulverized human cartilage at low single digit nM EC₅₀ (Table 1). Despite achieving a 1700-fold increase in binding affinity for type II collagen, only 7 of the 52 amino acids required substitution to move from M05 (EC₅₀ = 306 nM) to M26 (EC₅₀ = 0.18 nM; Table 1). The CII avimers were specific to type II collagen and showed no binding to closely related collagen family members type VI, type IX and human aggrecan. The M18 and M26 avimers did bind to rat type I collagen, but binding was 178-fold and 780-fold weaker, respectively, than that observed with rat type II collagen, and they displayed negligible binding to human type I collagen (Figure 3).

Table 1. Affinity maturation of CII binding avimers: binding of selected avimers to human and rat type II collagen and pulverized human cartilage. Binding affinities (EC₅₀ values) are indicated. The corresponding amino acid sequences are indicated in the lower panel. Panning of a naive Avimer library led to identification of three leads (exemplified by M05) possessing weak binding affinity for human and rat CII but no detectable binding to pulverized human cartilage. Following an initial round of affinity maturation avimers with sub-nM affinities for human and rat CII were identified which demonstrated binding to human cartilage (exemplified by Ml 3). An additional round of affinity maturation further increased the affinities for human and rat CII as well as pulverized human cartilage (exemplified by M18 and M26).

[0176] Next, additional experiments were conducted to investigate the specificity of the CII binding avimers. Briefly, target proteins were directly immobilized in a 96- well Maxisorp ELISA plate (20 nM; BD Bioscience, Chondrex, R&D) and incubated at room temperature for 1 hr. Next, the ELISA was blocked with (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM CaCl₂ 1% BSA) and incubated at room temperature for 1 hour. The plate was then washed 3X with (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM CaCl₂ + 0.05% Tween-20). Following the wash step, a 3-fold titration of avimer (starting at 300 nM) was added to each well and incubated at room

temperature for 1.5 hours. The plate was then washed 3X with (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM CaCl₂ + 0.05% Tween-20). Bound avimers were detected with an anti HA-HRP detection antibody (0.67 nM; clone 3F10 Roche) at room temperature for 1 hour. The plate was washed 3X with (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM CaCl₂ + 0.05% Tween-20). ELISA was developed with TMB/H₂0₂ (Thermo Scientific) and stopped with 2N H₂S0₄. Plates were read at OD450nm using a SpectraMax Plus. The results showed that C-II binding avimers Ml 8 and M26 bound to C-II, but not to human and rat Aggrecan, and human CD 163 (Figure 4).

[0177] An alpha screen competition assay was then performed to determine the relative affinities of M18 and M26. Dose-response curves were generated by serially diluting unlabeled avimers (starting at 1 mM) in assay buffer (40 mM sodium HEPES pH 7.5, 100 mM NaCl, 1 mM CaCl₂, 0.1% BSA, 0.05% Tween-20) in a 384-well Greiner microtiter plate. Human C-II (2 nM; Chondrex) plus a non-avimer blocking mu-X-C-II Ab (1 nM; Chondrex) were added to the microtiter plate followed by the addition of a mixture containing a tracer amount of biotinylated M26 avimer (1 nM), and AlphaScreen 'donor' strepavidin and 'acceptor' X-muIgG beads (10 μg/ml each; PerkinElmer) all diluted in assay buffer. The microtiter plate was then sealed and incubated overnight at 20°C. Inhibition of complex formation was measured as a reduction in chemiluminescent signal (CPS) as measured on the Fusion Plate Reader (PerkinElmer) using excitation at 680 nm and emission at 520-620 nm (Figure 5). Derived IC50 values are indicated in the inset to Figure 5.

[0178] To further characterize the binding properties of CII avimers to native human cartilage in vitro, biotinylated CII avimer and negative control avimer, CD163-M07 (NC avimer) were incubated with human cartilage sections overnight at 4 °C. M26 bound to human cartilage yielding a diffuse and pericellular staining pattern (Figure 6a); no significant binding of the NC avimer M07 was observed (Figure 6a). To assess the ability of avimers to penetrate and localize within cartilage, FITC-labeled M26 and M07 were incubated with human cartilage explants for 5 days at 37°C and subsequently imaged using multiphoton microscopy (MPM). Representative multiphoton-induced second harmonic generation (SHG) images originating from fibrillar collagen are depicted, as are the FITC-labeled avimers (Figure 6b). The SHG image shows that there are abundant intact type II collagen fibrils in the cultured explants. Specific diffuse and pericellular accumulation of the M26 avimer, but not the M07 avimer, was observed around lacunae where chondrocytes reside (Figure 6b). To quantify the FITC signal for the two Avimers, mean fluorescence intensity was measured from Z-stack images. The quantification result (Figure 6c) confirms accumulation of the M26 avimer relative to M07.

AF680-labeled CII avimers Persist in the Rat Knee joint Following a Single IA

Administration

[0179] To assess the joint retention and cartilage binding properties of CII avimers in vivo, AF680-labeled avimers were injected IA into rat knee joints and imaged over the course of 28 days. AF680-labeled M26 signals persisted at levels well above baseline values throughout the 28 day observation period (Figure 7). The highest intensity signal was observed immediately post-injection after which the intensity declined over the next 72 hours and then remained near a plateau value throughout the observation period. In contrast, the majority of AF680-labeled NC Avimer M07 was cleared from the joint within 24 hours and no signal was detected beyond 144 hours (Figure 7a, 7b). In joint half-life estimates for AF680-labeled M26 and M07 were 215 and 6.2 hours, respectively. Ex vivo imaging of dissected tibia and femur revealed that the fluorescence signal from AF680-labeled Ml 8 was located on the surfaces of the tibia and femur and not the surrounding ligaments (Figure 7c). Moreover, confocal imaging of articular cartilage isolated from rat knee joints 28 days following a single IA injection of CII Avimer confirmed that AF647-labeled M26 had penetrated well into articular cartilage (Figure 7d(3c)). Taken together, these data demonstrate that type II collagen binding avimer domains are able to bind and to penetrate cartilage both in vitro and in vivo.

Example 3

Generation and characterization of IL-lRa_CII avimer Fusions

[0180] There is substantial evidence illustrating the central role played by IL-1 in animal models of OA via the induction of proteolytic enzymes and pro-inflammatory cytokines, and the inhibition of collagen and proteoglycan synthesis [Chevalier et al., Nat Rev Rheumatol 9: 400- 410 (2013)]. Hence, to demonstrate prolonged joint retention and cartilage penetration of a therapeutic warhead using the avimer strategy, IL-lRa was chosen as a proof of concept molecule. To this end, the CII-M18 and CII-M26 avimers were fused to IL-lRa in two orientations, at the N-or C-terminus. The fusion of the CII avimer to IL-lRa in either orientation had minimal impact on the binding of IL-lRa to IL-1R1 (Figure 8 A and Figure 9A). However, the fusion of IL-lRa to the C-termini of CII avimers greatly diminished binding to human type II collagen (Figure 8B). In contrast, the fusion of IL-lRa to the N-terminus of the CII avimer allowed the latter to bind CII (Figure 8B), although binding was slightly reduced relative to the CII avimer alone (Figure 9B). A dual ELISA format was then used to determine whether the IL- lRa_CII-M26 avimer fusion could bind IL-1R and CII simultaneously, and it was observed that this was indeed the case. Plates coated with CII were incubated with IL-lRa_avimer constructs and then exposed to biotinylated soluble IL-1R1. With IL-lRa_M26, a high amount of biotinylated receptor was captured on the plate (Figure 8C). As expected, the control avimer IL- lRa_NC did not show any binding in this format. Similar results were obtained on testing the ability of the fusion protein to simultaneously bind IL-1R and pulverized human cartilage (Figure 9C). These data demonstrate that both IL-lRa and CII avimer maintain their ability to bind their respective targets when fused in this format. To confirm that the IL-lRa_CII avimer fusion was capable of inhibiting IL-1 -induced responses, the effects on IL-Ιβ induced IL-8 production by primary human chondrocytes was assessed. Consistent with the results of the binding studies, the IL-lRa_M26 avimer and IL- lRa were equipotent at inhibiting IL-8 production, and the IL-lRa_NC avimer also exhibited similar activity (Figure 8D). To compare the retention properties of the IL-1R_CII avimer fusion, human cartilage explants were incubated with AF647-labeled CII avimer, IL-lRa_CII avimer or IL-lRa_NC fusions for 24 hours and imaged using MPM. Similar to the CII M26 avimer, the IL-lRa_CII M26 avimer fusion penetrated and remained associated with the cartilage explants for at least 72 hours (Figure 8E, 8F). Again, it is noted that the strongest accumulation was observed in regions of the lacunae where the chondrocytes reside.

Example 4

IA Rat IL-Ιβ Challenge Model

[0181] Lastly, a pharmacodynamic model was developed to compare the effects of IL-lRa with the IL-lRa_M26 avimer on IL-ip-induced cytokine levels in the synovial fluid in vivo. It was observed that injection of 30 ng of IL-Ιβ into the rat knee joint was sufficient to lead to a significant increase in IL-6 levels in the synovial lavage four hours later. The amount of IL-lRa that was required for inhibition of IL-Ιβ induced IL-6 production was then determined.

Consistent with previous reports, a large molar excess of IL-lRa was needed to inhibit IL-Ιβ induced responses [Arend et al, J Clin Invest 85: 1694-1697 (1990); Gabay et al, Nat Rev Rheumatol 6: 232-241 (2010)] and complete inhibition was seen using a 5000-fold molar excess of IL-lRa but not at 500- or 100-fold excess (Figure 10a). Comparison of IL-lra and IL- lra_M26 (at 5000-fold molar excess) simultaneously administered with the IL-1 challenge indicated that both constructs effectively suppressed IL-6 output (Figure 10b). On the other hand, when IL-lRa and IL-lRa_M26 were administered 7 days in advance of the IL-1 challenge, only IL-lRa_M26-treated animals showed reduced IL-6 output relative to control challenged animals (Figure 10b). Thus, when fused with a CII avimer, IL-lRa retained biological activity in vivo and the CII avimer extended the duration of biological impact of this therapeutic molecule.

[0182] An additional experiment was performed to show that the IL-lRa_M26 fusion inhibits IL-6 release that is induced by ILip. Briefly, a SW982 Cell Bioassay was used to determine the inhibition of huIL-Ιβ induced IL-6 production by human synovial sarcoma SW982

(ATCC#HTB-93) cells. SW982 cells were maintained in RPMI 1640 media supplemented with 10%FBS/1%PSG. One day before assay, cells were seeded at 2.5xl0⁴ cells/ ΙΟΟμίΛνεΙΙ in low serum growth media (1%FBS) in 96-well tissue culture plate. The next day, cells were washed once with PBS, followed by addition of 80 μΐ_^ IL-lRa or IL-lRa fusions diluted in low serum growth media to appropriate wells. After a 0.5 hr. incubation at room temperature, 20 μΐ_^ of low serum growth medium containing 1 ng/ml rhIL-Ιβ was added to each well. Cells were then incubated at 37°C for 4 hours. After a brief centrifugation, supernatants were transferred to a new plate for IL-6 analysis using IL-6 ELISA kit from BioSource following manufacturer's instruction. Results shown in Figure 11 demonstrate that IL-lRa_M26 is equally effective as IL- lRa at blocking IL-6 output.

Discussion

[0183] Disease modifying strategies in OA are largely focused on curtailing joint damage or promoting repair, and include several large molecule candidates that target chondrocytes.

However, the avascular nature of cartilage and size-dependent restriction of macromolecule entry to the joint space makes achieving the desired drug concentration in the joint a challenging proposition [Evans et al., Nat Rev Rheumatol 10: 11-22 (2014)]. Intra- articular injection overcomes some of these challenges by ensuring local bioavailability at the disease site. IA injection also reduces exposure and toxicity at distant sites which is critical given the chronic nature of the disease and the presence of co-morbidities in aged OA patients. However, IA injection does not guarantee a durable pharmacological response as the synovial fluid is not a closed compartment. Hence, several strategies have been employed to prolong joint retention and these primarily involve the use of specialized formulations such as liposomes, hydrogels, micelles, microspheres and nanoparticles [Edwards et al., Vet J 190: 15-21 (2011); Gerwin et al., Adv Drug Deliv Rev 58: 226-242 (2006); Kang et al., Expert Opin Drug Deliv 11: 269-282 (2014)].

[0184] Some of these formulations have proven to be very successful at prolonging joint retention of drugs. Self-assembling nanoparticles were shown to prolong the retention of IL-lRa within the joint space [Whitmire et al., Biomaterials 33: 7665-7675 (2012)]. However, most formulation strategies typically do not address the hurdle faced when using biologies that target chondrocytes; viz. the need to traverse an avascular, anionic matrix that is not very permeable to large molecules (e.g., those greater than 50 kD), and persist there for extended periods of time [Foy et al., J Magn Reson 148: 126-134 (2001); van Lent et al., J Rheumatol 14: 798-805 (1987); van Lent et al, Rheumatol Int 8: 145-152 (1988)]. [0185] The CII binding avimers described in this disclosure provide a means for developing durable cartilage-penetrating biological therapeutics. After just two rounds of optimization, leads from an original avimer library screen were transformed to species possessing high binding affinity and selectivity for CII. Amongst the optimized avimers, M26 possessed the highest affinity for human CII (0.18 nM) and for human cartilage (1.54 nM). M26 also bound selectively to rat CII (0.05 nM affinity); cross reactivity with rat CII is not surprising given the highly conserved nature of type II collagens. In contrast, M26 demonstrated no detectable binding to human types I, VI, IX collagen or to human aggrecan. When incubated with human cartilage explants, fluorescently-tagged M26 appeared to accumulate preferentially within pericellular regions surrounding chondrocytes. In contrast, a fluorescently tagged negative control Avimer (M07) showed no accumulation within cartilage explants. Remarkably, following injection of fluorescently-tagged M26 into rat knee joints, the avimer remained detectable for up to 28 days post-injection. In contrast, the residence time of a fluorescently- tagged negative control avimer within the joint was much shorter. Ex vivo imaging of knee joints harvested from animals injected with CH-binding avimers provided evidence that they accumulated in both tibial and femoral cartilage. Moreover, confocal imaging of cartilage sections isolated from animals 28 days post-injection demonstrated that fluorescently-tagged M26 avimer remained detectable within cartilage, with the highest levels appearing near the surface.

[0186] When fused to IL-lRa, Cll-binding avimers did not impact IL-lRa's activity as an antagonist of IL-1. However, the ability of Cll-binding avimers to bind to collagen was affected by their positioning. When appended to the N-terminus of IL-lRa, both M26 and Ml 8 showed weak binding. However, when attached at the C-terminus of IL-lRa, both avimers demonstrated high binding affinities to human CII, though in the case of IL-lRa_M26 the binding affinity was reduced approximately 7 -fold relative to the free avimer. Importantly, the IL-lRa_M26 construct was capable of binding to both CII and IL-1R simultaneously, indicating that the two binding domains function independently of the other's state of occupancy. Like the M26 avimer itself, fluorescently-tagged IL-lRa_M26 bound selectively to human cartilage explants with the highest accumulation occurring in pericellular locations.

[0187] As a proof-of-concept that Cll-binding Avimers can extend the therapeutic utility of IL-lRa, native human IL-lRa and IL-lRa_M26 were compared as antagonists of an in vivo IL-1 response. Following IA injection of IL-Ιβ into rat knee joints, levels of IL-6 within the synovial fluid increased. As expected, co-injection of native IL-IRa could suppress the IL-6 response, provided the antagonist was present at a 5000-fold excess over the cytokine; this is consistent with previous studies [Arend et al., J Clin Invest 85: 1694-1697 (1990); Gabay et al., Nat Rev Rheumatol 6: 232-241 (2010)]. Likewise, IL-lRa_M26 co-injected with the IL-Ιβ stimulus at a 5000-fold excess inhibited IL-6 production. However, when injected 1 week prior to the IL-Ιβ challenge, only IL-lRa_M26-treated animals showed evidence of a pharmacological response. Failure of native IL-IRa to provide a durable response is consistent with its well documented short half-life. In contrast, tethering of the IL-lRa_M26 fusion construct to cartilage prolonged the pharmacological effectiveness of the antagonist. Therefore, conjugates including but not limited to IL-lRa_M26 represent a therapeutic modality for the treatment of OA.

[0188] The CH-binding Avimers thus provide a mechanism for targeting therapeutic payloads to cartilage and for prolonging residence time within joints. CH-binding Avimers represent a versatile option for tethering other payloads to cartilage and extending pharmacodynamic durability.

Claims

WHAT IS CLAIMED IS:

1. A polypeptide comprising a domain that binds to a protein found in cartilage wherein the domain: is a non-naturally-occurring domain comprising 30 to 50 amino acids and comprises at least one disulfide bond.

2. The polypeptide of claim 1, wherein the polypeptide comprises at least two domains that bind the protein.

3. The polypeptide of claiml or claim 2, wherein the domain binds to aggrecan, decorin, biglycan, or fibromodulin.

4. The polypeptide of claim 1 or claim 2, wherein the domain binds to collagen-2.

5. The polypeptide of claim 4, wherein the domain comprises an amino acid sequence at least 75% identical to SEQ ID NO: 1.

6. The polypeptide of claim 5, wherein the amino acid sequence is a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

7. The polypeptide of claim 6, wherein the amino acid sequence is set out in SEQ ID

NO: 5.

8. The polypeptide of claim 5, wherein the domain binds horse collagen.

9. The polypeptide of claim 5, wherein the domain binds dog collagen.

10. The polypeptide of claim 5, wherein the domain binds cat, goat, sheep, pig, bovine, camel, or elephant collagen.

11. The polypeptide of any one of claims 1-10, wherein the polypeptide further comprises an additional domain having a binding specificity for a target molecule.

12. The polypeptide of claim 11, wherein the additional domain has a binding specificity for interleukin-1 receptor (IL-IR).

13. The polypeptide of claim 12, wherein the IL-IR is human IL-IR, equine IL-IR, or canine IL-IR.

14. The additional domain of claim 12, wherein the additional domain comprises an amino acid sequence as set out in SEQ ID NO: 140.

15. The polypeptide of claim 11, wherein the additional domain comprises an agent that interferes with the degradation of cartilage or the loss of chondrocyte viability and function.

16. The polypeptide of claim 15, wherein the agent that interferes with the

degradation of cartilage or the loss of chondrocyte viability and function is an scFv fragment, a complement inhibitory molecule, or an inhibitor of a cartilage-degrading protease.

17. The polypeptide of claim 16, wherein the inhibitor of a cartilage-degrading protease is Tissue Inhibitor of Metalloproteinase 2, an inhibitor of MMP-13, an inhibitor of aggrecanase, or a small molecular inhibitor of a metalloproteinase.

18. The polypeptide of claim 16, wherein the scFv targets tumor necrosis factor (TNF), a complement component, or interleukin 6 (IL-6).

19. The polypeptide of claim 11, wherein the additional domain comprises an agent to promote chondrocyte viability and function.

20. The polypeptide of claim 19, wherein the anabolic agent is fibroblast growth factor 18 (FGF-18), insulin-like growth factor 1 (IGF-1), a transforming growth factor beta (TGFP) family member, a Wnt inhibitor, or a chondrogenic peptide.

21. The polypeptide of claim 20, wherein the TGFP family member is growth and differentiation factor 5 (GDF5) or bone morphogenetic protein 7 (BMP7).

22. The polypeptide of claim 20, wherein the Wnt inhibitor is dickkopf 1 (Dkk-1), Frizzled Related Protein B (FrzB), or sclerostin.

23. The polypeptide of claim 20, wherein the chondrogenic peptide is TPX-100.

24. The polypeptide of any one of claims 1-23 which comprises two disulfide bonds.

25. The polypeptide of any one of claims 1-24 which comprises three disulfide bonds.

26. The polypeptide of any one of claims 1-25, wherein the domain and the additional domain are linked by a linker.

27. The polypeptide of claim 26, wherein the linker is GGGGSGGGGS (SEQ ID NO:

139).

28. The polypeptide of claim 1 which is M26 (SEQ ID NO: 5).

29. The polypeptide of claim 28 wherein the polypeptide further comprises IL-lRa.

30. The polypeptide of claim 29 wherein the IL-lRa is mammalian IL-lRa.

31. The polypeptide of claim 30 wherein the mammalian IL-lRa is canine IL-lRa, equine IL-lRa, or human IL-lRa.

32. The polypeptide of claim 31 wherein the mammalian IL-lRa is human IL-lRa (SEQ ID NO: 140).

33. The polypeptide of claim 1 which is less than about 50 kilodaltons.

34. A polynucleotide encoding the polypeptide of any one of claims 1-32.

35. The polynucleotide of claim 34 which is SEQ ID NO: 151.

36. A polypeptide comprising a domain that binds to interleukin-1 receptor (IL-1R) wherein the domain: is a non-naturally-occurring domain comprising 30 to 50 amino acids and comprises at least one disulfide bond.

37. The polypeptide of claim 36, wherein the polypeptide comprises at least two domains that bind IL-1R.

38. The polypeptide of claim 36 or claim 37, wherein the domain comprises an amino acid sequence at least 75% identical to SEQ ID NO: 114 or SEQ ID NO: 119.

39. The polypeptide of claim 36 or claim 37, wherein the amino acid sequence is a sequence as set out in any of SEQ ID NOs: 96-138.

40. The polypeptide of claim 39, wherein the amino acid sequence is set out in SEQ ID NO: 114.

41. A polynucleotide encoding the polypeptide of any one of claims 36-40.

42. The polynucleotide of claim 41 which is SEQ ID NO: 153.

43. A polypeptide comprising an amino acid sequence as set out in SEQ ID NO: 5 linked by a linker to an amino acid sequence as set out in SEQ ID NO: 114.

44. The polypeptide of claim 43, wherein the linker is GGGGSGGGGS (SEQ ID NO:

139).

45. The polypeptide of claim 44 wherein the amino acid sequence as set out in SEQ ID NO: 5 is amino terminal to the linker.

46. The polypeptide of claim 44 wherein the amino acid sequence as set out in SEQ ID NO: 5 is carboxy terminal to the linker.

47. A polynucleotide encoding the polypeptide of any one of claims 43-46.

48. A vector comprising the polynucleotide of any one of claims 34, 41, or 47 operably linked to a promoter.

49. A host cell comprising the vector of claim 48.

50. The host cell of claim 49 which is an Escherichia coli cell.

51. The host cell of claim 49 which is a mammalian cell.

52. The host cell of claim 49 or claim 51 which is a Chinese Hamster Ovary (CHO) cell.

53. The host cell of claim 49 which is a yeast cell.

54. A method of producing the polypeptide of any one of claims 1-33, 36-40, or 43- 46 comprising the step of culturing the host cell of any one of claims 49-53 under conditions appropriate to induce expression of the polypeptide.

55. The method of claim 54 wherein the polypeptide is isolated.

56. A pharmaceutical composition comprising the polypeptide of any one of claims 1- 33, 36-40, or 43-46 and a pharmaceutically acceptable carrier, adjuvant, or diluent.

57. A method of treating or preventing osteoarthritis (OA) in a patient comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition of claim 56.

58. The method of claim 57, wherein the OA develops following a ligament tear, a sprain, an articular fracture, or a meniscus tear.

59. The method of claim 58, wherein the administration results in faster healing of the ligament tear, sprain, or meniscus tear relative to a patient that is not administered the pharmaceutical composition.

60. A method of treating or preventing a repetitive use injury in a patient comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition of claim 56.

61. A method of treating or preventing intervertebral disc (IVD) degeneration in a patient comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition of claim 56.

62. The method of any one of claims 57-61 wherein at least one pharmaceutical composition is administered to the patient.

63. The method of claim 62, wherein two pharmaceutical compositions are administered to the patient.

64. The method of claim 63, wherein the two pharmaceutical compositions are administered separately.

65. The method of claim 63, wherein the two pharmaceutical compositions are administered together as a single formulation.

66. A method of treating or preventing an interleukin- 1 (IL-1) mediated disease comprising administering to a patient in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 56.

67. The method of claim 66 wherein at least one pharmaceutical composition is administered to the patient.

68. The method of claim 67, wherein two pharmaceutical compositions are administered to the patient.

69. The method of claim 68, wherein the two pharmaceutical compositions are administered separately.

70. The method of claim 68, wherein the two pharmaceutical compositions are administered together as a single formulation.

71. The method of any one of claims 66-70, wherein the interleukin-1 (IL-1) mediated disease is Stills Disease, gout, rheumatoid arthritis, juvenile rheumatoid arthritis, or calcium pyrophosphate deposition disease (CPPD).

72. The method of any one of claims 57-71 wherein administration of the pharmaceutical composition or pharmaceutical compositions is intra-articular, sub-cutaneous, parenteral, intravenous, or a combination thereof.