Oncostatin m receptor antigen binding proteins

Abstract

To provide oncostatin M receptor antigen binding proteins.SOLUTION: The invention provides anti-oncostatin M receptor-β (OSMR) antigen binding proteins, e.g., antibodies and functional fragments, derivatives, muteins, and variants thereof. OSMR antigen binding proteins interfere with binding of OSM and / or IL-31 to OSMR. In some embodiments, anti-OSMR antigen binding proteins are useful tools in studying diseases and disorders associated with OSMR and are particularly useful in methods of treating diseases and disorders associated with OSMR and binding of OSM and / or IL-31 to OSMR.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Appl. No. 61 / 829,082, filed May 30, 2013, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Oncostatin M (OSM) and interleukin-31 (IL-31) are members of the IL-6 superfamily and share a receptor subunit, oncostatin M receptor-β (OSMR) (Dillon et al., Nat. Immunol. 5(7):752-60, 2004). All members of this family, except IL-31, share a common chain of glycoprotein 130 (gp130) in their multimeric receptor complexes. OSM signals through a heterodimeric receptor complex containing OSMR and gp130, while IL-31 utilizes a gp130-like receptor, IL-31R, in combination with OSMR (Dillon et al., supra; Drenw et al., J. Biol. Chem 279(34):36112-20, 2004). General In contrast, OSMR and gp130 are fairly ubiquitously expressed across tissues and cell types and can be induced under a variety of stimulatory conditions. IL-31R expression appears to be relatively more restricted and tightly regulated. In humans and mice alike, IL-31R mRNA expression is detectable at low levels in tissues such as the trachea, skeletal muscle, thymus, and bone marrow (Dillon et al., supra). Although the levels of expression are quite different, both IL-31R and OSMR are co-expressed on numerous tissues, including skin and intestinal epithelial cells, suggesting that these tissues are required to respond to IL-31 (Dillon et al., supra). (t al., supra; Dambacher et al., Gut 56(9);1257-65, 2007). OSMR is constitutively expressed in the lung on epithelial cells, whereas IL-31R expression is negligible to low in lung tissue but is upregulated upon various methods of airway challenge (Dillon et al., supra; Jawa et al., J. Interferon Cytokine Res. 28(4):207-19, 2008).

[0003] Secreted primarily by T lymphocytes, macrophages, and neutrophils, OSM and IL-31 are both upregulated in various disease states involving inflammation. OSM has been implicated in diverse biological roles, including bone formation, cartilage degradation, cholesterol uptake, pain, and inflammation (Cawston et al., Arthritis Rheum. 4l(10):1760-71, 1998; Hasegawa et al., Rheumatology (Oxford) 38(7);612-7, 1999; Levy et al., J. Hepatol. 32(2):218-26, 2000; Manicourt (e.g., et al., Arthritis Rheum. 43(2):281-8, 2000; de Hooge et al., Am J. Pathol. 160(5):1733-43, 2002; Luzina et al., Arthritis Rheum 48(8):2262-74, 2003; Morikawa et al., J. Neurosci. 24(8):1941-7, 2004; Kong et al., J. Lipid Res. 46(6):1163-71, 2005). OSM has been shown to be a potent regulator of the extracellular matrix (ECM) in a variety of contexts, suggesting that OSM can mediate seemingly opposing pathological outcomes, such as fibrosis (excess ECM) and cartilage degradation (ECM degradation). Both of these effects have been observed when OSM was overexpressed or exogenously administered to mouse lungs or joints, respectively, depending on the tissue type and surrounding environment (Richards et al., Biochem. Soc. Trans. 30(2):107-11, 2002; Hui et al., Arthritis Rheum, 48(12):3404-18, 2003; Rowan et al., Am. J. Pathol. 162(6):1975-84, 2003). Furthermore, OSM has previously been shown to be upregulated in human pathologies where these types of outcomes are present (Cawston et al., supra; Hasegawa et al., supra; Levy et al., supra; Manicourt et al., supra; Luzina et al., supra). OSM, a primarily locally acting cytokine, has been reported in synovial fluid from the joints of patients with rheumatoid arthritis (RA) (Cawston et al., supra; Manicourt et al. OSM is upregulated in the bronchoalveolar lavage (BAL) fluid of patients with scleroderma-associated interstitial lung disease and idiopathic pulmonary fibrosis (IPF) (Luzina et al., supra), and in the liver of patients with cirrhosis (Levy et al., supra). The proposed effect of OSM on the ECM may be attributed in part to its ability to shift the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). TIMPs bind MMPs in a 1:1 ratio with high affinity, resulting in a loss of MMP proteolytic activity. TIMP-1 and TIMP-3 have previously been shown to be differentially regulated by OSM, resulting in an increase in TIMP-1 and a decrease in TIMP-3 (Gatsios et al., Eur. J. Biochem. 241(1):56-63, 1996). In addition to regulating the digestion of extracellular matrix components, MMPs are also involved in the cleavage and subsequent activation of many proteins, such as TGF-β, a potent profibrotic cytokine (Leask et al., FASEB J. 18(7):816-27, 2004). OSM has also been reported to be able to directly induce the transcription of type I collagen in vitro. (Hasegawa et al., J. Rheumatol 25(2):308-13, 1998).

[0004] Expression of both OSM and IL-31 has been found in the skin of patients with psoriasis and atopic dermatitis, and mutations in OSMR and IL-31R have been associated with systemic cutaneous amyloidosis. System-wide transgenic overexpression of IL-31 induced a pruritic inflammatory response in mouse skin. Both OSM and IL-31 deliver signals through the neuronal OSMR, which has been suggested to promote nociceptive and pruritic responses. Taken together, these links to human disease and the ability of OSM and IL-31 to promote a diverse array of pathologies, including at least inflammation, extracellular matrix remodeling, pain, and pruritus, suggest that blockade of OSMR may be a useful target for therapeutic intervention in many diseases and disorders associated with OSMR. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Dillon et al., Nat. Immunol. 5(7):752-60, 2004 [Non-patent document 2] Drenw et al., J. Biol Chem 279(34):36112-20,2004 [Non-patent document 3] Dambacher et al.,Gut 56(9);1257-65,2007 [Non-patent document 4] Jawa et al., J. Interferon Cytokine Res.28(4):207-19,2008 [Non-patent document 5] Cawston et al.,Arthritis Rheum.4l(10):1760-71,1998 [Non-patent document 6] Hasegawa et al., Rheumatology (Oxford)38(7);612-7,1999 [Non-Patent Document 7] Levy et al., J. Hepatol, 32(2):218-26,2000 [Non-patent document 8] Manicourt et al.,Arthritis.Rheum.43(2):281-8,2000 [Non-Patent Document 9] de Hooge et al.,Am J. Pathol.160(5):1733-43,2002 [Non-Patent Document 10] Luzina et al., Arthritis Rheum 48(8):2262 - 74, 2003

Non - Patent Document 11

Non - Patent Document 12

Non - Patent Document 13

Non - Patent Document 14

Non - Patent Document 15

Non - Patent Document 16

Non - Patent Document 17

Non - Patent Document 18

Summary of the Invention

Means for Solving the Problems

[0006] The present invention provides anti-OSMR antigen binding proteins, e.g., antibodies and functional fragments thereof, with properties suitable for commercial production and therapeutic use in humans. The anti-OSMR antigen binding proteins are useful in methods of treating diseases and disorders associated with OSMR, particularly diseases and disorders associated with the binding of OSM or IL-31 to OSMR. Antigen binding proteins are described herein as antibodies that bind OSMR with high affinity and inhibit the binding of OSM and / or IL-31 to OSMR. Antibodies that bind OSMR are provided that effectively block, and thereby reduce, OSMR-mediated signaling in cells.

[0007] In a first aspect, the OSMR antigen binding protein comprises: a) a light chain variable domain having at least 90% identity, at least 95% identity, or identity to the amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29; b) a heavy chain variable domain having at least 90% identity, at least 95% identity, or identity to the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11; or c) the light chain variable domain of a) and the heavy chain variable domain of b).

[0008] Preferred antigen binding proteins of the first aspect include those comprising a light chain variable domain having at least 90% identity, at least 95% identity, or identity to the amino acid sequence set forth in SEQ ID NO:27, and a heavy chain variable domain having at least 90% identity, at least 95% identity, or identity to the amino acid sequence set forth in SEQ ID NO:9; those comprising a light chain variable domain having at least 90% identity, at least 95% identity, or identity to the amino acid sequence set forth in SEQ ID NO:28, and a heavy chain variable domain having at least 90% identity, at least 95% identity, or identity to the amino acid sequence set forth in SEQ ID NO:10; and those comprising a light chain variable domain having at least 90% identity, at least 95% identity, or identity to the amino acid sequence set forth in SEQ ID NO:29, and a heavy chain variable domain having at least 90% identity, at least 95% identity, or identity to the amino acid sequence set forth in SEQ ID NO:11. An OSMR antigen binding protein comprising a heavy chain variable domain having the above sequence relationship to SEQ ID NO:9 can optionally include an amino acid other than asparagine (e.g., aspartic acid) at the position corresponding to position 73 of SEQ ID NO:9. In such embodiments, the heavy chain variable domain optionally includes the amino acid sequence set forth in SEQ ID NO:53. An OSMR antigen binding protein comprising a heavy chain variable domain having the above sequence relationship to SEQ ID NO:10 can optionally include an amino acid other than asparagine (e.g., aspartic acid) at the position corresponding to position 73 of SEQ ID NO:10. In such embodiments, the heavy chain variable domain optionally includes the amino acid sequence set forth in SEQ ID NO:54.

[0009] In a second aspect, the OSMR antigen binding protein comprises: a) a light chain variable domain having no more than 10 or no more than 5 amino acid additions, deletions, or substitutions from the amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29; a heavy chain variable domain having no more than 10 or no more than 5 amino acid additions, deletions, or substitutions from the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:10, or b) SEQ ID NO:11; or c) the light chain variable domain of a) and the heavy chain variable domain of b).

[0010] Preferred antigen binding proteins of the second aspect include those comprising a light chain variable domain having no more than 10 or no more than 5 amino acid additions, deletions or substitutions from the amino acid sequence set forth in SEQ ID NO:27 and a heavy chain variable domain having no more than 10 or no more than 5 amino acid additions, deletions or substitutions from the amino acid sequence set forth in SEQ ID NO:9; those comprising a light chain variable domain having no more than 10 or no more than 5 amino acid additions, deletions or substitutions from the amino acid sequence set forth in SEQ ID NO:28 and a heavy chain variable domain having no more than 10 or no more than 5 amino acid additions, deletions or substitutions from the amino acid sequence set forth in SEQ ID NO:10; and those comprising a light chain variable domain having no more than 10 or no more than 5 amino acid additions, deletions or substitutions from the amino acid sequence set forth in SEQ ID NO:29 and a light chain variable domain having no more than 10 or no more than 5 amino acid additions, deletions or substitutions from the amino acid sequence set forth in SEQ ID NO:11. An OSMR antigen binding protein comprising a heavy chain variable domain having the above sequence relationship to SEQ ID NO:9 can optionally include an amino acid other than asparagine (e.g., aspartic acid) at the position corresponding to position 73 of SEQ ID NO:9. In such embodiments, the heavy chain variable domain optionally comprises the amino acid sequence set forth in SEQ ID NO:53. An OSMR antigen binding protein comprising a heavy chain variable domain having the above sequence relationship to SEQ ID NO:10 can optionally include an amino acid other than asparagine (e.g., aspartic acid) at the position corresponding to position 73 of SEQ ID NO:10. In such embodiments, the heavy chain variable domain optionally comprises the amino acid sequence set forth in SEQ ID NO:54.

[0011] In a third aspect, the OSMR antigen binding protein comprises: a) an LCDR1 having no more than three amino acid additions, deletions or substitutions from the LCDR1 sequence shown in SEQ ID NO:30, an LCDR2 having no more than three amino acid additions, deletions or substitutions from the LCDR2 sequence shown in SEQ ID NO:33, and an LCDR3 having no more than three amino acid additions, deletions or substitutions from the LCDR3 sequence shown in SEQ ID NO:36; b) an LCDR1 having no more than three amino acid additions, deletions or substitutions from the LCDR1 sequence shown in SEQ ID NO:31, an LCDR2 having no more than three amino acid additions, deletions or substitutions from the LCDR2 sequence shown in SEQ ID NO:34, and an LCDR3 having no more than three amino acid additions, deletions or substitutions from the LCDR3 sequence shown in SEQ ID NO:37; or c) an LCDR1 having no more than three amino acid additions, deletions or substitutions from the LCDR1 sequence shown in SEQ ID NO:32, an LCDR2 having no more than three amino acid additions, deletions or substitutions from the LCDR2 sequence shown in SEQ ID NO:33, and an LCDR3 having no more than three amino acid additions, deletions or substitutions from the LCDR3 sequence shown in SEQ ID NO:36. a light chain variable domain comprising an LCDR2 having not more than three amino acid additions, deletions or substitutions from the LCDR2 sequence shown in SEQ ID NO:35, and an LCDR3 having not more than three amino acid additions, deletions or substitutions from the LCDR3 sequence shown in SEQ ID NO:38; d) an HCDR1 having not more than three amino acid additions, deletions or substitutions from the HCDR1 sequence shown in SEQ ID NO:12, an HCDR2 having not more than three amino acid additions, deletions or substitutions from the HCDR2 sequence shown in SEQ ID NO:15, and an HCDR3 having not more than three amino acid additions, deletions or substitutions from the HCDR3 sequence shown in SEQ ID NO:18; e) a light chain variable domain comprising an LCDR2 having not more than three amino acid additions, deletions or substitutions from the LCDR2 sequence shown in SEQ ID NO:35, and an LCDR3 having not more than three amino acid additions, deletions or substitutions from the LCDR3 sequence shown in SEQ ID NO:38; or f) a heavy chain variable domain comprising an HCDR1 having not more than 3 amino acid additions, deletions or substitutions from the HCDR1 sequence shown in SEQ ID NO: 13, an HCDR2 having not more than 3 amino acid additions, deletions or substitutions from the HCDR2 sequence shown in SEQ ID NO: 16, and an HCDR3 having not more than 3 amino acid additions, deletions or substitutions from the HCDR3 sequence shown in SEQ ID NO: 19; or f) a heavy chain variable domain comprising an HCDR1 having not more than 3 amino acid additions, deletions or substitutions from the HCDR1 sequence shown in SEQ ID NO: 14, an HCDR2 having not more than 3 amino acid additions, deletions or substitutions from the HCDR2 sequence shown in SEQ ID NO: 17, and an HCDR3 having not more than 3 amino acid additions, deletions or substitutions from the HCDR3 sequence shown in SEQ ID NO: 20.

[0012] Preferred OSMR antigen binding proteins of the third aspect include those comprising the light chain variable domain of a) and the heavy chain variable domain of d); those comprising the light chain variable domain of b) and the heavy chain variable domain of e); and those comprising the light chain variable domain of c) and the heavy chain variable domain of f). An OSMR antigen binding protein comprising a) a light chain variable domain and d) a heavy chain variable domain can optionally comprise a heavy chain variable domain comprising an amino acid other than asparagine (e.g., aspartic acid) at a position corresponding to position 73 of SEQ ID NO:9. In such embodiments, the heavy chain variable domain optionally comprises the amino acid sequence set forth in SEQ ID NO:53. An OSMR antigen binding protein comprising the light chain variable domain of b) and the heavy chain variable domain of e) can optionally include a heavy chain variable domain comprising an amino acid other than asparagine (e.g., aspartic acid) at a position corresponding to position 73 of SEQ ID NO: 10. In such embodiments, the heavy chain variable domain optionally comprises the amino acid sequence set forth in SEQ ID NO:54.

[0013] In a fourth aspect of the invention, the OSMR antigen binding protein of the first, second, third or fourth aspect is administered at a concentration of 1 x 10 -10 It binds to human OSMR with an affinity of ≤M.

[0014] In a fifth aspect of the invention, the OSMR antigen binding protein of the first, second or third aspect inhibits binding of human OSM to human OSMR and / or human IL-31 to human OSMR.

[0015] In a sixth aspect of the invention, the OSMR antigen binding protein of the first, second, third, fourth or fifth aspect reduces human OSM-mediated and / or human IL-31-mediated OSMR signaling in human OSMR-expressing cells.

[0016] In a seventh aspect of the invention, the first, second, third, fourth, fifth or sixth OSMR antigen binding proteins reduce human OSM-mediated and / or human IL-31-mediated OSMR signaling in cynomolgus monkey OSMR-expressing cells.

[0017] In an eighth aspect of the invention, the first, second, third, fourth, fifth, sixth, or seventh OSMR antigen binding protein is an antibody, such as a human antibody. Preferred antibodies include an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO:24 and a heavy chain having the amino acid sequence set forth in SEQ ID NO:6; an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO:25 and a heavy chain having the amino acid sequence set forth in SEQ ID NO:7; and an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO:26 and a heavy chain having the amino acid sequence set forth in SEQ ID NO:8. Further antibodies include an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO:24 and a heavy chain having the amino acid sequence set forth in SEQ ID NO:50; an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO:25 and a heavy chain having the amino acid sequence set forth in SEQ ID NO:51; and an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO:26 and a heavy chain having the amino acid sequence set forth in SEQ ID NO:52.

[0018] In a ninth aspect, the present invention provides a nucleic acid or isolated nucleic acid encoding one or more polypeptide components of an OSMR antigen binding protein, such as an antibody light chain or antibody heavy chain. In a preferred embodiment, the nucleic acid encodes a polypeptide comprising:

[0019] a) a light chain variable domain having at least 95% identity to the amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29;

[0020] b) a heavy chain variable domain having at least 95% identity to the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11;

[0021] c) a light chain variable domain having five or fewer amino acid additions, deletions, or substitutions from the amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29;

[0022] d) SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11 a heavy chain variable domain having 5 or fewer amino acid additions, deletions, or substitutions from the amino acid sequence set forth in

[0023] e) a light chain variable domain comprising:

[0024] i) an LCDR1 having not more than three amino acid additions, deletions or substitutions from the LCDR1 sequence shown in SEQ ID NO:30; an LCDR2 having not more than three amino acid additions, deletions or substitutions from the LCDR2 sequence shown in SEQ ID NO:33; and an LCDR3 having not more than three amino acid additions, deletions or substitutions from the LCDR3 sequence shown in SEQ ID NO:36;

[0025] ii) an LCDR1 having not more than three amino acid additions, deletions or substitutions from the LCDR1 sequence shown in SEQ ID NO:31; an LCDR2 having not more than three amino acid additions, deletions or substitutions from the LCDR2 sequence shown in SEQ ID NO:34; and an LCDR3 having not more than three amino acid additions, deletions or substitutions from the LCDR3 sequence shown in SEQ ID NO:37; or

[0026] iii) an LCDR1 having an addition, deletion, or substitution of 3 or fewer amino acids from the LCDR1 sequence shown in SEQ ID NO:32; an LCDR2 having an addition, deletion, or substitution of 3 or fewer amino acids from the LCDR2 sequence shown in SEQ ID NO:35; and an LCDR3 having three or fewer amino acid additions, deletions, or substitutions from the LCDR3 sequence set forth in ID NO: 38; or

[0027] f) a heavy chain variable domain comprising:

[0028] i) an HCDR1 having not more than three amino acid additions, deletions or substitutions from the HCDR1 sequence shown in SEQ ID NO:12; an HCDR2 having not more than three amino acid additions, deletions or substitutions from the HCDR2 sequence shown in SEQ ID NO:15; and an HCDR3 having not more than three amino acid additions, deletions or substitutions from the HCDR3 sequence shown in SEQ ID NO:18;

[0029] ii) an HCDR1 having an addition, deletion or substitution of no more than 3 amino acids from the HCDR1 sequence shown in SEQ ID NO: 13; an HCDR2 having an addition, deletion or substitution of no more than 3 amino acids from the HCDR2 sequence shown in SEQ ID NO: 16; and an HCDR3 having an addition, deletion or substitution of no more than 3 amino acids from the HCDR3 sequence shown in SEQ ID NO: 19; or

[0030] iii) an HCDR1 having an addition, deletion, or substitution of 3 or fewer amino acids from the HCDR1 sequence shown in SEQ ID NO:14; an HCDR2 having an addition, deletion, or substitution of 3 or fewer amino acids from the HCDR2 sequence shown in SEQ ID NO:17; and An HCDR3 having three or fewer amino acid additions, deletions or substitutions from the HCDR3 sequence set forth in ID NO:20. In certain embodiments, the nucleic acid or isolated nucleic acid encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:53, or SEQ ID NO:54. In certain embodiments, the nucleic acid or isolated nucleic acid encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52.

[0031] In certain embodiments of the ninth aspect, the nucleic acid or isolated nucleic acid encodes an antibody light chain and comprises SEQ The nucleic acid or isolated nucleic acid has at least 80%, at least 90%, at least 95%, or 100% identity to the nucleotide sequence set forth in SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23. In other embodiments of the ninth aspect, the nucleic acid or isolated nucleic acid encodes an antibody heavy chain and has at least 80%, at least 90%, at least 95%, or 100% identity to the nucleotide sequence set forth in SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. In certain embodiments, the heavy chain is encoded by a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO:47, SEQ ID NO:48, or SEQ ID NO:49.

[0032] In a tenth aspect, the invention provides an expression vector comprising one or more nucleic acids or isolated nucleic acids of the ninth aspect. In certain embodiments, the expression vector encodes an antibody light chain, an antibody heavy chain, or both an antibody light chain and an antibody heavy chain.

[0033] In an eleventh aspect, the present invention provides a recombinant host cell comprising one or more nucleic acids or isolated nucleic acids of the ninth aspect operably linked to a promoter, including a recombinant host cell comprising one or more expression vectors of the tenth aspect of the invention. In a preferred embodiment, the recombinant host cell secretes an antibody that binds to OSMR. Preferred host cells are mammalian host cells, such as CHO cell lines.

[0034] In a twelfth aspect, the present invention provides a method for treating an autoimmune disease, an inflammatory disease, or a disorder associated with extracellular matrix deposition or remodeling, the method comprising administering to a patient in need thereof an effective amount of an OSMR antigen binding protein of any one of aspects 1, 2, 3, 4, 5, 6, 7, or 8. In preferred embodiments, the OSMR antigen binding protein is an antibody (e.g., Ab1) comprising a light chain variable domain amino acid sequence set forth in SEQ ID NO:27 and a heavy chain variable domain amino acid sequence set forth in SEQ ID NO:9, an antibody (e.g., Ab2) comprising a light chain variable domain amino acid sequence set forth in SEQ ID NO:28 and a heavy chain variable domain amino acid sequence set forth in SEQ ID NO:10, or an antibody (e.g., Ab3) comprising a light chain variable domain amino acid sequence set forth in SEQ ID NO:29 and a heavy chain variable domain amino acid sequence set forth in SEQ ID NO:11. In some embodiments, the OSMR antigen binding protein is an antibody (e.g., Ab1) comprising a light chain variable domain amino acid sequence set forth in SEQ ID NO:29 and a heavy chain variable domain amino acid sequence set forth in SEQ ID NO:11. an antibody comprising the light chain variable domain amino acid sequence set forth in SEQ ID NO:27 and the heavy chain variable domain amino acid sequence set forth in SEQ ID NO:53, or an antibody comprising the light chain variable domain amino acid sequence set forth in SEQ ID NO:28 and the heavy chain variable domain amino acid sequence set forth in SEQ ID NO:54. In preferred embodiments, the OSMR antigen binding protein inhibits binding of OSM to OSMR or IL-31 to OSMR. In particularly preferred embodiments, the autoimmune disease, inflammatory disease, or disorder associated with extracellular matrix deposition or remodeling is fibrosis, cartilage degradation, arthritis, rheumatoid arthritis, scleroderma, scleroderma-associated interstitial lung disease, idiopathic pulmonary fibrosis, liver cirrhosis, psoriasis, atopic dermatitis, systemic cutaneous amyloidosis, primary cutaneous amyloidosis, inflammation, pruritic inflammation, prurigo nodularis, and pain.

[0035] In a thirteenth aspect, the present invention provides a method for producing the OSMR antigen binding protein of any one of the first, second, third, fourth, fifth, sixth, seventh, or eighth aspects by culturing a recombinant host cell of the eleventh aspect and isolating the OSMR antigen binding protein from the culture.

[0036] In a fourteenth aspect, the present invention provides an OSMR antigen binding protein of any one of the first, second, third, fourth, fifth, sixth, seventh, or eighth aspects that cross-competes with an antibody selected from the group consisting of:

[0037] a) an antibody comprising a light chain comprising the amino acid sequence set forth in SEQ ID NO:24 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:6;

[0038] b) an antibody comprising a light chain comprising the amino acid sequence set forth in SEQ ID NO:25 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:7;

[0039] c) An antibody comprising a light chain comprising the amino acid sequence shown in SEQ ID NO:26 and a heavy chain comprising the amino acid sequence shown in SEQ ID NO:8. In certain embodiments, for example, the following are provided: (Item 1) a) a light chain variable domain having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29; b) a heavy chain variable domain having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11; or c) the light chain variable domain of a) and the heavy chain variable domain of b): Oncostatin M receptor (OSMR) antigen binding protein comprising: (Item 2) 2. The OSMR antigen binding protein of claim 1, wherein the light chain variable domain has at least 95% identity to the amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29. (Item 3) 3. The OSMR antigen binding protein of item 1 or 2, wherein the heavy chain variable domain has at least 95% identity to the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11. (Item 4) a) a light chain variable domain having 10 or fewer amino acid additions, deletions, or substitutions from the amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29; b) a heavy chain variable domain having 10 or fewer amino acid additions, deletions, or substitutions from the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11; or c) the light chain variable domain of a) and the heavy chain variable domain of b): Oncostatin M receptor (OSMR) antigen binding protein comprising: (Item 5) 5. The OSMR antigen binding protein of item 4, wherein the light chain variable domain has no more than five amino acid additions, deletions, or substitutions from the amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29. (Item 6) 6. The OSMR antigen binding protein of item 4 or 5, wherein the heavy chain variable domain has no more than five amino acid additions, deletions, or substitutions from the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11. (Item 7) 7. The OSMR antigen binding protein of items 1 to 6, wherein the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29. (Item 8) 8. The OSMR antigen binding protein of any one of items 1 to 7, wherein the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11. (Item 9) a) an LCDR1 having not more than three amino acid additions, deletions or substitutions from the LCDR1 sequence shown in SEQ ID NO:30; an LCDR2 having not more than three amino acid additions, deletions or substitutions from the LCDR2 sequence shown in SEQ ID NO:33; and an LCDR3 having not more than three amino acid additions, deletions or substitutions from the LCDR3 sequence shown in SEQ ID NO:36; b) an LCDR1 having no more than three amino acid additions, deletions, or substitutions from the LCDR1 sequence shown in SEQ ID NO:31; an LCDR2 having no more than three amino acid additions, deletions, or substitutions from the LCDR2 sequence shown in SEQ ID NO:34; and an LCDR3 having no more than three amino acid additions, deletions, or substitutions from the LCDR3 sequence shown in SEQ ID NO:37; or c) an LCDR1 having not more than three amino acid additions, deletions or substitutions from the LCDR1 sequence shown in SEQ ID NO: 32; an LCDR2 having not more than three amino acid additions, deletions or substitutions from the LCDR2 sequence shown in SEQ ID NO: 35; and an LCDR3 having not more than three amino acid additions, deletions or substitutions from the LCDR3 sequence shown in SEQ ID NO: 38: a light chain variable domain comprising d) an HCDR1 having an addition, deletion or substitution of 3 or fewer amino acids from the HCDR1 sequence shown in SEQ ID NO:12; an HCDR2 having an addition, deletion or substitution of 3 or fewer amino acids from the HCDR2 sequence shown in SEQ ID NO:15; and an HCDR3 having an addition, deletion or substitution of 3 or fewer amino acids from the HCDR3 sequence shown in SEQ ID NO:18; e) an HCDR1 having no more than three amino acid additions, deletions or substitutions from the HCDR1 sequence shown in SEQ ID NO: 13; an HCDR2 having no more than three amino acid additions, deletions or substitutions from the HCDR2 sequence shown in SEQ ID NO: 16; and an HCDR3 having no more than three amino acid additions, deletions or substitutions from the HCDR3 sequence shown in SEQ ID NO: 19; or f) an HCDR1 having an addition, deletion or substitution of 3 or less amino acids from the HCDR1 sequence shown in SEQ ID NO: 14; an HCDR2 having an addition, deletion or substitution of 3 or less amino acids from the HCDR2 sequence shown in SEQ ID NO: 17; and an HCDR3 having an addition, deletion or substitution of 3 or less amino acids from the HCDR3 sequence shown in SEQ ID NO: 20: and a heavy chain variable domain comprising: Oncostatin M receptor (OSMR) antigen binding protein comprising: (Item 10) 10. The OSMR antigen binding protein of item 9, comprising the light chain variable domain of a) and the heavy chain variable domain of d). (Item 11) 11. The OSMR antigen binding protein of item 10, wherein the light chain variable domain comprises an LCDR1 sequence represented by SEQ ID NO:30; an LCDR2 sequence represented by SEQ ID NO:33; and an LCDR3 sequence represented by SEQ ID NO:36; and the heavy chain variable domain comprises an HCDR1 sequence represented by SEQ ID NO:12; an HCDR2 sequence represented by SEQ ID NO:15; and an HCDR3 sequence represented by SEQ ID NO:18. (Item 12) 10. The OSMR antigen binding protein of item 9, comprising the light chain variable domain of b) and the heavy chain variable domain of e). (Item 13) 13. The OSMR antigen binding protein of item 12, wherein the light chain variable domain comprises an LCDR1 sequence represented by SEQ ID NO:31; an LCDR2 sequence represented by SEQ ID NO:34; and an LCDR3 sequence represented by SEQ ID NO:37; and the heavy chain variable domain comprises an HCDR1 sequence represented by SEQ ID NO:13; an HCDR2 sequence represented by SEQ ID NO:16; and an HCDR3 sequence represented by SEQ ID NO:19. (Item 14) 10. The OSMR antigen binding protein of item 9, comprising the light chain variable domain of c) and the heavy chain variable domain of f). (Item 15) 15. The OSMR antigen binding protein of item 14, wherein the light chain variable domain comprises an LCDR1 sequence represented by SEQ ID NO: 32; an LCDR2 sequence represented by SEQ ID NO: 35; and an LCDR3 sequence represented by SEQ ID NO: 38; and the heavy chain variable domain comprises an HCDR1 sequence represented by SEQ ID NO: 14; an HCDR2 sequence represented by SEQ ID NO: 17; and an HCDR3 sequence represented by SEQ ID NO: 20. (Item 16) 12. The OSMR antigen binding protein of any one of items 1 to 7 or 9 to 11, wherein the heavy chain variable domain comprises an amino acid other than asparagine at a position corresponding to position 73 of SEQ ID NO:9. (Item 17) 17. The OSMR antigen binding protein of item 16, wherein the heavy chain variable domain comprises an aspartic acid at a position corresponding to position 73 of SEQ ID NO:9. (Item 18) 12. The OSMR antigen binding protein of any one of items 1 to 7 or 9 to 11, wherein the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO:53. (Item 19) 14. The OSMR antigen binding protein of any one of items 1 to 7, 9, 12 or 13, wherein the heavy chain variable domain comprises an amino acid other than asparagine at a position corresponding to position 73 of SEQ ID NO:10. (Item 20) 20. The OSMR antigen binding protein of item 19, wherein the heavy chain variable domain comprises an aspartic acid at a position corresponding to position 73 of SEQ ID NO:10. (Item 21) 14. The OSMR antigen binding protein of any one of items 1 to 7, 9, 12 or 13, wherein the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO:54. (Item 22) The antigen-binding protein is 1×10 -10 22. The OSMR antigen-binding protein of any one of items 1 to 21, which specifically binds to human OSMR with an affinity of M or less. (Item 23) 23. The OSMR antigen-binding protein of any one of items 1 to 22, wherein the antigen-binding protein inhibits binding of human OSM or human IL-31 to human OSMR. (Item 24) 24. The OSMR antigen binding protein of any one of paragraphs 1 to 23, wherein the antigen binding protein reduces human OSM-mediated or human IL-31-mediated OSMR signaling in human OSMR-expressing cells. (Item 25) 25. The OSMR antigen-binding protein of any one of items 1 to 24, wherein the antigen-binding protein is an antibody. (Item 26) 26. The OSMR antigen binding protein of claim 25, wherein the antigen binding protein is a human antibody. (Item 27) The light chain comprises the amino acid sequence shown in SEQ ID:24, and the heavy chain comprises the amino acid sequence shown in SEQ ID: 27. The OSMR antigen binding protein of item 26, comprising a light chain and a heavy chain, each of which comprises the amino acid sequence set forth in NO:6. (Item 28) 27. The OSMR antigen binding protein of item 26, comprising a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO:25 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:7. (Item 29) 27. The OSMR antigen binding protein of item 26, comprising a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO:26 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:8. (Item 30) 26. The OSMR antigen binding protein of item 25, comprising a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO:24 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:50. (Item 31) 26. The OSMR antigen binding protein of item 25, comprising a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO:25 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:51. (Item 32) 26. The OSMR antigen binding protein of item 25, comprising a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO:26 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:52. (Item 33) a) a light chain variable domain having at least 95% identity to the amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29; b) a heavy chain variable domain having at least 95% identity to the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11; c) a light chain variable domain having five or fewer amino acid additions, deletions, or substitutions from the amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29; d) a heavy chain variable domain having five or fewer amino acid additions, deletions, or substitutions from the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11; e) i) an LCDR1 having three or fewer amino acid additions, deletions, or substitutions from the LCDR1 sequence shown in SEQ ID NO:30, an LCDR2 having three or fewer amino acid additions, deletions, or substitutions from the LCDR2 sequence shown in SEQ ID NO:33, and an LCDR3 having three or fewer amino acid additions, deletions, or substitutions from the LCDR3 sequence set forth in ID NO:36; ii) an LCDR1 having not more than three amino acid additions, deletions or substitutions from the LCDR1 sequence shown in SEQ ID NO:31, an LCDR2 having not more than three amino acid additions, deletions or substitutions from the LCDR2 sequence shown in SEQ ID NO:34, and an LCDR3 having not more than three amino acid additions, deletions or substitutions from the LCDR3 sequence shown in SEQ ID NO:37; or iii) an LCDR1 having an addition, deletion or substitution of 3 or less amino acids from the LCDR1 sequence shown in SEQ ID NO:32, an LCDR2 having an addition, deletion or substitution of 3 or less amino acids from the LCDR2 sequence shown in SEQ ID NO:35, and an LCDR3 having three or fewer amino acid additions, deletions, or substitutions from the LCDR3 sequence set forth in ID NO:38; a light chain variable domain comprising: f) i) an HCDR1 having an addition, deletion or substitution of 3 or less amino acids from the HCDR1 sequence shown in SEQ ID NO: 12, an HCDR2 having an addition, deletion or substitution of 3 or less amino acids from the HCDR2 sequence shown in SEQ ID NO: 15, and an HCDR3 having three or fewer amino acid additions, deletions, or substitutions from the HCDR3 sequence set forth in ID NO:18; ii) an HCDR1 having no more than three amino acid additions, deletions or substitutions from the HCDR1 sequence shown in SEQ ID NO:13, an HCDR2 having no more than three amino acid additions, deletions or substitutions from the HCDR2 sequence shown in SEQ ID NO:16, and an HCDR3 having no more than three amino acid additions, deletions or substitutions from the HCDR3 sequence shown in SEQ ID NO:19; or iii) an HCDR1 having an addition, deletion or substitution of 3 or less amino acids from the HCDR1 sequence shown in SEQ ID NO: 14, an HCDR2 having an addition, deletion or substitution of 3 or less amino acids from the HCDR2 sequence shown in SEQ ID NO: 17, and an HCDR3 having three or fewer amino acid additions, deletions, or substitutions from the HCDR3 sequence set forth in ID NO:20; A heavy chain variable domain comprising: An isolated nucleic acid encoding a polypeptide comprising: (Item 34) 34. The isolated nucleic acid of item 33, wherein the polypeptide comprises an antibody light chain. (Item 35) 35. The isolated nucleic acid of item 34, wherein the light chain is encoded by a nucleic acid comprising a nucleotide sequence that is at least 80% identical to the nucleotide sequence set forth in SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23. (Item 36) 36. The isolated nucleic acid of item 35, wherein the light chain is encoded by a nucleic acid comprising a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23. (Item 37) 37. The isolated nucleic acid of item 36, wherein the light chain is encoded by a nucleic acid comprising a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23. (Item 38) 38. The isolated nucleic acid of item 37, wherein the light chain is encoded by a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23. (Item 39) 34. The isolated nucleic acid of item 33, wherein the polypeptide comprises an antibody heavy chain. (Item 40) 40. The isolated nucleic acid of item 39, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:53 or SEQ ID NO:54. (Item 41) 40. The isolated nucleic acid of item 39, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52. (Item 42) 40. The isolated nucleic acid of paragraph 39, wherein the heavy chain is encoded by a nucleic acid comprising a nucleotide sequence that is at least 80% identical to the nucleotide sequence set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17. (Item 43) The heavy chain may be selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. 43. The isolated nucleic acid of item 42, encoded by a nucleic acid comprising a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in NO:5. (Item 44) The heavy chain may be selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. 44. The isolated nucleic acid of item 43, encoded by a nucleic acid comprising a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in NO:5. (Item 45) The heavy chain may be selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. 45. The isolated nucleic acid of item 44, encoded by a nucleic acid comprising the nucleotide sequence set forth in NO:5. (Item 46) 45. The isolated nucleic acid of item 44, wherein the heavy chain is encoded by a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO:47, SEQ ID NO:48, or SEQ ID NO:49. (Item 47) 47. An expression vector comprising the isolated nucleic acid according to any one of Items 33 to 46. (Item 48) 48. The expression vector of paragraph 47, wherein the isolated nucleic acid encodes an antibody light chain. (Item 49) 48. The expression vector of paragraph 47, wherein the isolated nucleic acid encodes an antibody heavy chain. (Item 50) 49. The expression vector of paragraph 48, further comprising an isolated nucleic acid encoding an antibody heavy chain. (Item 51) 47. A recombinant host cell comprising the isolated nucleic acid of any one of items 33 to 46 operably linked to a promoter. (Item 52) A recombinant host cell comprising the expression vector according to any one of items 47 to 50. (Item 53) 53. The recombinant host cell according to item 52, wherein the host cell comprises an expression vector according to items 48 and 49. (Item 54) 54. The recombinant host cell of paragraph 53, wherein the host cell secretes an antibody that binds to OSMR. (Item 55) 55. The recombinant host cell according to any one of items 51 to 54, wherein the cell is of mammalian origin. (Item 56) 56. The recombinant host cell of item 55, wherein the cell is a Chinese hamster ovary (CHO) cell line. (Item 57) 33. A method for treating a disease or disorder comprising administering to a patient in need thereof a therapeutically effective amount of an OSMR antigen binding protein according to any one of items 1 to 32. (Item 58) 58. The method of claim 57, wherein the OSMR antigen binding protein is an antibody. (Item 59) 59. The method of item 58, wherein the antibody comprises a light chain variable domain amino acid sequence set forth in SEQ ID NO:27 and a heavy chain variable domain amino acid sequence set forth in SEQ ID NO:9. (Item 60) The antibody according to item 59, comprising a light chain variable domain amino acid sequence shown in SEQ ID NO:24 and a heavy chain variable domain amino acid sequence shown in SEQ ID NO:6. How to post. (Item 61) 59. The method of item 58, wherein the antibody comprises a light chain variable domain amino acid sequence set forth in SEQ ID NO:27 and a heavy chain variable domain amino acid sequence set forth in SEQ ID NO:53. (Item 62) 62. The method of item 61, wherein the antibody comprises a light chain variable domain amino acid sequence set forth in SEQ ID NO:24 and a heavy chain variable domain amino acid sequence set forth in SEQ ID NO:50. (Item 63) 59. The method of item 58, wherein the antibody comprises a light chain variable domain amino acid sequence set forth in SEQ ID NO:28 and a heavy chain variable domain amino acid sequence set forth in SEQ ID NO:10. (Item 64) 64. The method of item 63, wherein the antibody comprises a light chain variable domain amino acid sequence shown in SEQ ID NO:25 and a heavy chain variable domain amino acid sequence shown in SEQ ID NO:7. (Item 65) 59. The method of item 58, wherein the antibody comprises a light chain variable domain amino acid sequence set forth in SEQ ID NO:28 and a heavy chain variable domain amino acid sequence set forth in SEQ ID NO:54. (Item 66) 66. The method of item 65, wherein the antibody comprises a light chain variable domain amino acid sequence set forth in SEQ ID NO:25 and a heavy chain variable domain amino acid sequence set forth in SEQ ID NO:51. (Item 67) 59. The method of item 58, wherein the antibody comprises a light chain variable domain amino acid sequence set forth in SEQ ID NO:29 and a heavy chain variable domain amino acid sequence set forth in SEQ ID NO:11. (Item 68) 68. The method of item 67, wherein the antibody comprises a light chain variable domain amino acid sequence set forth in SEQ ID NO:26 and a heavy chain variable domain amino acid sequence set forth in SEQ ID NO:8. (Item 69) 68. The method of item 67, wherein the antibody comprises a light chain variable domain amino acid sequence set forth in SEQ ID NO:26 and a heavy chain variable domain amino acid sequence set forth in SEQ ID NO:52. (Item 70) 70. The method of any one of paragraphs 57 to 69, wherein the antigen-binding protein inhibits binding of OSM and / or IL-31 to OSMR. (Item 71) 71. The method of item 70, wherein the disease or disorder is an autoimmune disease, an inflammatory disease, or a disorder associated with extracellular matrix deposition or remodeling. (Item 72) 72. The method of item 71, wherein the autoimmune disease, the inflammatory disease, or the disorder associated with extracellular matrix deposition or remodeling is fibrosis, cartilage degradation, arthritis, rheumatoid arthritis, scleroderma, scleroderma-associated interstitial lung disease, idiopathic pulmonary fibrosis, liver cirrhosis, psoriasis, atopic dermatitis, systemic cutaneous amyloidosis, primary cutaneous amyloidosis, inflammation, pruritic inflammation, prurigo nodularis, and pain. (Item 73) a) culturing the recombinant host cell according to any one of items 51 to 56; and b) isolating the OSMR antigen binding protein from the culture: A method for producing an oncostatin M receptor (OSMR) antigen binding protein comprising: (Item 74) The OSMR antigen binding protein comprises: a) a light chain comprising the amino acid sequence shown in SEQ ID NO:24 and a light chain comprising the amino acid sequence shown in SEQ ID NO:6 an antibody comprising a heavy chain comprising the amino acid sequence represented by: b) a light chain comprising the amino acid sequence shown in SEQ ID NO:25 and SEQ ID NO:7 an antibody comprising a heavy chain comprising an amino acid sequence represented by the formula: c) a light chain comprising the amino acid sequence set forth in SEQ ID NO:26 and SEQ ID NO:8 An antibody comprising a heavy chain comprising the amino acid sequence shown in: 33. The oncostatin M receptor (OSMR) antigen-binding protein of any one of items 1 to 32, which cross-competes with an antibody selected from the group consisting of: DETAILED DESCRIPTION OF THE INVENTION

[0040] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All references cited within the body of this specification are expressly incorporated by reference in their entirety.

[0041] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, tissue culture and transformation, protein purification, and the like. Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art, or as described herein. The following procedures and techniques can generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual 3 rded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, which is incorporated herein by reference for any purpose. Unless specific definitions are provided, the nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery and treatment of patients. OSMR

[0042] The antigen binding proteins described herein bind to OSMR. OSM and IL-31 transmit signals through OSMR. OSMR is a member of the type I cytokine receptor family. OSMR heterodimerizes with glycoprotein 130 (gp130, also known as interleukin-6 signal transducer (IL6ST), IL6-β, or CD130) to form type II OSMR. OSMR also heterodimerizes with IL-31 receptor A (IL31RA) to form the IL-31 receptor, thus transducing OSM- and IL-31-induced signaling events. In exemplary embodiments, the OSMR antigen binding protein binds to OSMR and prevents OSM- and / or IL-31-mediated signaling in cells expressing OSMR.

[0043] The human OSMR sequence is known in the art. In various embodiments, human OSMR protein sequences are provided in GenBank Accession Nos. AAI25210, AAI25211, NP_003990, and EAW55976. An exemplary human OSMR amino acid sequence (SEQ ID NO:1) is set forth in Table 1. The protein is composed of several domains: amino acids 1-27 correspond to a signal sequence that is cleaved during protein processing in mammalian cells; amino acids 28-740 correspond to the extracellular domain; and amino acids 741-761 correspond to the transmembrane domain. In preferred embodiments, the antigen binding proteins described herein bind to the extracellular domain of OSMR and prevent OSM and / or IL-31 from interacting with OSMR.

[0044] Human OSM sequences are known in the art. In various embodiments, human OSM protein sequences are provided in GenBank Accession Nos. CAG30420, CAG46504, NP_065391, P13725, AAC05173, EAW59864, and AAH11589. An exemplary human OSM amino acid sequence (SEQ ID NO:39) is set forth in Table 1. Amino acids 1-25 correspond to the signal sequence; amino acids 26-220 correspond to the mature protein; and amino acids 221-252 correspond to the propeptide sequence.

[0045] The sequence of human IL-31 is known in the art. In various embodiments, human IL-31 protein sequences are provided in GenBank Accession Nos. NP_001014358, AAS86448, AAI32999, AAI33001, Q6EBC2, and EAW98310. Exemplary human IL-31 amino acid sequences (SEQ ID NO: 1) are: ID NO:41) is provided in Table 1. Amino acids 1-23 correspond to a putative signal sequence.

[0046] The sequence of human IL31RA is known in the art. In various embodiments, the human IL31RA protein sequence is provided in GenBank Accession Nos. AAS86447, NP_001229567, and CBL94051. An exemplary human IL31RA (v4, isoform 3) amino acid sequence (SEQ ID NO:43) is provided in Table 1. Amino acids 1-32 correspond to the signal sequence; and amino acids 533-553 correspond to the transmembrane sequence.

[0047] The sequence of human gp130 is known in the art. In various embodiments, human gp130 protein sequences are provided in GenBank Accession Nos. AAI17403, AAI17405, EAW54936, NP_002175, ABK41905, and AAA59155. An exemplary human gp130 amino acid sequence (SEQ ID NO:45) is provided in Table 1. The protein is composed of several domains: amino acids 1-22 correspond to the signal sequence; amino acids 23-619 correspond to the extracellular domain; amino acids 620-641 correspond to the transmembrane domain; and amino acids 642-918 correspond to the cytoplasmic domain. Table 1 [Table 1-1] [Table 1-2] [Table 1-3]

[0048] In certain embodiments of the invention, the antigen binding proteins described herein bind both human and cynomolgus OSMR with high affinity, including those that bind cynomolgus OSM and / or IL-31 with high affinity to, and block the interaction of, cynomolgus OSMR. These properties allow for valuable toxicity studies in non-human primates.

[0049] The OSMR protein sequence for rhesus monkeys (Macaca mulatta) is known in the art and is available under GenBank Accession No. XP_0010837 45. An exemplary cynomolgus monkey (Macaca fascicularis) OSMR amino acid sequence (SEQ ID NO:2) is provided in Table 2. The protein is composed of several domains: amino acids 1-27 correspond to the signal sequence that will be cleaved during protein processing in mammalian cells; amino acids 28-737 correspond to the extracellular domain; and amino acids 738-757 correspond to the transmembrane domain. In preferred embodiments, the antigen binding proteins described herein bind to the extracellular domain of OSMR and prevent the interaction of OSM and / or IL-31 with OSMR.

[0050] The OSM protein sequence for rhesus monkeys (Macaca mulatta) is known in the art and is available under GenBank Accession No. NP_0011814 03. An exemplary cynomolgus monkey (Macaca fascicularis) OSM amino acid sequence (SEQ ID NO:40) is provided in Table 2. Amino acids 1-196 correspond to mature cynomolgus monkey OSM.

[0051] The IL-31 protein sequence of rhesus monkey (Macaca mulatta) is known in the art and is available under GenBank Accession No. XP_001096 743. An exemplary cynomolgus monkey (Macaca fascicularis) IL-31 amino acid sequence (SEQ ID NO:42) is provided in Table 2. This sequence represents mature cynomolgus monkey IL-31.

[0052] An exemplary cynomolgus monkey (Macaca fascicularis) IL31RA amino acid sequence (SEQ ID NO:44) is provided in Table 2. Amino acids 1-19 correspond to the signal sequence; and amino acids 520-540 correspond to the transmembrane domain.

[0053] The gp130 protein sequence of rhesus monkey (Macaca mulatta) is known in the art and is available under GenBank Accession No. NP_00125 2920. An exemplary cynomolgus monkey (Macaca fascicularis) gp130 amino acid sequence (SEQ ID NO:46) is provided in Table 2. The protein is composed of several domains: amino acids 1-22 correspond to the signal sequence; amino acids 23-619 correspond to the extracellular domain; amino acids 620-6 41 corresponds to the transmembrane domain; and amino acids 642-918 correspond to the cytoplasmic domain. Table 2 [Table 2-1] [Table 2-2] OSMR antigen binding protein

[0054] The present invention provides antigen binding proteins that specifically bind OSMR. Antigen binding protein embodiments include peptides and / or polypeptides that specifically bind OSMR. Such peptides or polypeptides may optionally contain one or more post-translational modifications. Antigen binding protein embodiments include antibodies and fragments thereof, variously defined herein, that specifically bind OSMR. These include antibodies that specifically bind human OSMR, including those that inhibit OSM and / or IL-31 from binding and / or activating OSMR.

[0055] The antigen binding proteins of the present invention specifically bind to OSMR. As used herein, "specifically bind" means preferentially binding to OSMR relative to other proteins. In some embodiments, "specifically bind" means that the antigen binding protein has a higher affinity for OSMR than for other proteins. An OSMR antigen binding protein that specifically binds to OSMR can be found in a concentration of 1 x 10 -7 M or less, 2 x 10 -7 M or less, 3 x 10 -7 M or less, 4 x 10 -7 M or less, 5 x 10 -7 M or less, 6 x 10 -7 M or less, 7 x 10 -7 M or less, 8 x 10 -7 M or less, 9 x 10 -7 M or less, 1 x 10 -8 M or less, 2 x 10 -8 M or less, 3 x 10 -8 M or less, 4 x 10 -8 M or less, 5 x 10 -8 M or less, 6 x 10 -8 M or less, 7 x 10 -8 M or less, 8 x 10 -8 M or less, 9 x 10 -8 M or less, 1 x 10 -9 M or less, 2 x 10 -9 M or less, 3 x 10 -9 M or less, 4 x 10 -9 M or less, 5 x 10 -9 M or less, 6 x 10 -9 M or less, 7 x 10 -9 M or less, 8 x 10 -9 M or less, 9 x 10 -9 M or less, 1 x 10 -10 M or less, 2 x 10 -10 M or less, 3 x 10 -10 M or less, 4 x 10 -10 M or less, 5 x 10 -10 M or less, 6 x 10 -10 M or less, 7 x 10 -10 M or less, 8 x 10 -10 M or less, 9 x 10 -10 M or less, 1 x 10 -11M or less, 2 x 10 -11 M or less, 3 x 10 -11 M or less, 4 x 10 -11 M or less, 5 x 10 -11 M or less, 6 x 10 -11 M or less, 7 x 10 -11 M or less, 8 x 10 -11 M or less, 9 x 10 -11 M or less, 1 x 10 -12 M or less, 2 x 10 -12 M or less, 3 x 10 -12 M or less, 4 x 10 -12 M or less, 5 x 10 -12 M or less, 6 x 10 -12 M or less, 7 x 10 -12 M or less, 8 x 10 -12 M or less, or 9 x 10 -12 The antibody may have a binding affinity for human OSMR of M or less.

[0056] Methods for measuring the binding affinity of antigen-binding proteins are known in the art. Commonly used methods for affinity determination include surface plasmon resonance (SPR) (Morton and Myszka "Kinetic analysis of macromolecular interactions using surface plasmon resonance biosensors" Methods in Enzymology (1998) 295, 268-294), biolayer interferometry (Abdiche et al "Determining Kinetics and Affinities of Protein Interactions Using a Parallel Real-time Label-free Biosensor, the Octet” Analytical Biochemistry (2008) 377, 209-217), and kinetic exclusion assay (KinExA) (Darling and Brault “Kinetic exclusion assay technology: characterization of molecular interactions” Assay and Drug Dev Tech (2004) 2, 647-657), isothermal calorimetry (Pierce et al “Isothermal Titration Calorimetry of Protein-Protein Interactions” Methods (1999) 19, 2 13-221) and analytical ultracentrifugation (Lebowitz et al. "Modern analytical ultracentrifugation in protein Science: a tutorial review” Protein Science (2002), 11:2067-2079). Example 5 provides an exemplary method for affinity determination.

[0057] When reference is made herein to various embodiments of OSMR-binding antibodies, it is understood that this also encompasses OSMR-binding fragments thereof. OSMR-binding fragments include any of the fragments or domains of antibodies described herein that retain the ability to specifically bind OSMR. OSMR-binding fragments may be within any of the scaffolds described herein.

[0058] In certain therapeutic embodiments, the OSMR antigen binding protein inhibits binding of OSM and / or IL-31 to OSMR and / or inhibits a signaling pathway associated with binding of OSM and / or IL-31 to OSMR, e.g., OSM- and / or IL-31-mediated signaling. Such antigen binding proteins are said to be "neutralizing." In certain embodiments, a neutralizing OSMR antigen binding protein specifically binds to OSMR and inhibits one or more of the biological activities of at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, Inhibits binding of OSM and / or IL-31 to OSMR by any percentage between 10% and 100%, such as by only 0, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more. For example, OSMR antigen binding proteins can be tested for neutralizing activity by determining their ability to block the binding of OSM and / or IL-31 to OSMR. See, e.g., Examples 2 and 3 for blocking assays of human OSMR and cynomolgus monkey OSMR, respectively. Alternatively, OSMR antigen binding proteins can be tested for neutralizing activity in assays measuring the effect of the presence of OSMR antigen binding proteins in assays measuring OSM- and / or IL-31-mediated biological functions. For example, the ability of OSM to induce biological responses, such as simulating plasminogen activator activity in cultured bovine aortic endothelial cells, regulating IL-6 expression in human endothelial cells, and stimulating LDL uptake and upregulating cell surface LDL receptors in HepG2 cells. Alternatively, the ability of IL-31 to induce skin inflammation.

[0059] Antigen binding protein embodiments, as variously defined herein, may comprise one or more and a scaffold having complementarity determining regions (CDRs). Embodiments further include antigen binding proteins comprising a scaffold having one or more antibody variable domains, either heavy or light chain. Embodiments include antibodies comprising a light chain variable domain selected from the group consisting of Ab1 light chain variable domain (LCv), Ab2 LCv, and Ab3 LCv (SEQ ID NOs:27-29, respectively), and / or a heavy chain variable domain selected from the group consisting of Ab1 heavy chain variable domain (HCv), Ab2 HCv, and Ab3 HCv (SEQ ID NOs:9-11, respectively), and fragments, derivatives, muteins, and variants thereof. An exemplary heavy chain variable domain variant of SEQ ID NO:9 contains an amino acid other than asparagine (e.g., aspartic acid) at the position corresponding to position 73 of SEQ ID NO:9. The amino acid sequence set forth in SEQ ID NO:53 is an example of a heavy chain variable domain variant of SEQ ID NO:9. An exemplary heavy chain variable domain variant of SEQ ID NO:10 contains an amino acid other than asparagine (e.g., aspartic acid) at the position corresponding to position 73 of SEQ ID NO:10. The amino acid sequence set forth in SEQ ID NO:54 is an example of a heavy chain variable domain variant of SEQ ID NO:10.

[0060] An exemplary light chain comprising the Ab1 LCv is a light chain comprising the amino acid sequence set forth in SEQ ID NO:24.

[0061] An exemplary light chain comprising the Ab2 LCv is a light chain comprising the amino acid sequence set forth in SEQ ID NO:25.

[0062] An exemplary light chain comprising the Ab3 LCv is a light chain comprising the amino acid sequence set forth in SEQ ID NO:26.

[0063] An exemplary heavy chain comprising the Ab1 HCv is a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 6. An exemplary heavy chain comprising a variant of Ab1 HCv is a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 50.

[0064] An exemplary heavy chain comprising the Ab2 HCv is a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 7. An exemplary heavy chain comprising a variant of the Ab2 HCv is a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 51.

[0065] An exemplary heavy chain comprising the Ab3 HCv is a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 8. An exemplary heavy chain comprising a variant of the Ab3 HCv is a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 52.

[0066] Further examples of contemplated scaffolds include, but are not limited to, fibronectin, neocarzinostatin CBM4-2, lipocalin, T-cell receptor, protein A domain (protein Z), Im9, TPR protein, zinc finger domain, pVIII, avian pancreatic polypeptide, GCN4, WW domain, Src homology domain 3, PDZ domain, TEM-1 β-lactamase, thioredoxin, staphylococcal nuclease, PHD finger domain, CL-2, BPTI, APPI, HPSTI, ecotin, LACI-DI, LDTI, MTL-II, scorpion toxin, insect defensin A peptide, EETI-II, Min-23, CBD, PBP, cytochrome b-562, receptor domain of LDL, gamma crystallin, ubiquitin, transferrin, and / or C-type lectin-like domain. Non-antibody scaffolds and their use as therapeutic agents are reviewed in Gebauer and Skerra, Curr. Opin. Chem. Biol., 13:245-255 (2009) and Binz et al., Nat. Biotech., 23(10):1257-68 (2005), which are incorporated herein by reference in their entireties.

[0067] An embodiment of the present invention is an antibody comprising the variable domain: Ab1 LCv / Ab1 HCv (SEQ ID NO:27 / SEQ ID NO:9),Ab2 LCv / Ab2 HCv(SEQ ID O:28 / SEQ ID NO:10),Ab3 LCv / Ab3 HCv(SEQ I D NO:29 / SEQ ID NO:11), and combinations thereof, as well as fragments, derivatives, muteins and variants thereof. Also included are antibodies comprising variable domains: SEQ ID NO:27 / SEQ ID NO:53; and SEQ ID NO:28 / SEQ ID NO:54.

[0068] Exemplary antibodies of the invention include Ab1 (SEQ ID NO:24 / SEQ ID NO:6), Ab2 (SEQ ID NO:25 / SEQ ID NO:7), and Ab3 (SEQ ID NO:26 / SEQ ID NO:8). Further exemplary antibodies include SEQ ID NO:24 / SEQ ID NO:50; EQ ID NO:25 / SEQ ID NO:51; and SEQ ID NO:26 / Contains SEQ ID NO:52.

[0069] Typically, each variable domain of an antibody light or heavy chain comprises three CDRs. A heavy chain variable domain comprises a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3). A light chain variable domain comprises a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3). In certain embodiments, the antigen binding protein comprises one or more CDRs contained within the preferred variable domains described herein.

[0070] Examples of such CDRs include, but are not limited to:

[0071] Ab1 LCv: CDRs of LCDR1 (SEQ ID NO: 30), LCDR2 (SEQ ID NO: 33), and LCDR3 (SEQ ID NO: 36);

[0072] Ab2 LCv: CDRs of LCDR1 (SEQ ID NO: 31), LCDR2 (SEQ ID NO: 34), and ECDR3 (SEQ ID NO: 37);

[0073] Ab3 LCv: CDR of LCDR1(SEQ ID NO:32), LCDR2(SEQ ID NO:35), and LCDR3(SEQ ID NO:38);

[0074] Ab1 HCv: CDRs of HCDR1 (SEQ ID NO:12), HCDR2 (SEQ ID NO:15), and HCDR3 (SEQ ID NO:18);

[0075] Ab2 HCv: CDRs of HCDR1 (SEQ ID NO:13), HCDR2 (SEQ ID NO:16), and HCDR3 (SEQ ID NO:19); and

[0076] Ab3 HCv: CDRs of HCDR1 (SEQ ID NO: 14), HCDR2 (SEQ ID NO: 17), and HCDR3 (SEQ ID NO: 20).

[0077] In some embodiments, the antigen binding protein comprises: A) a polypeptide that is, for example, a light chain comprising an LCDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 31, and 32; an LCDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 34, and 35; and / or an LCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 37, and 38; and / or B) a polypeptide that is, for example, a heavy chain comprising an HCDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, and 14; an HCDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16, and 17; and / or an HCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, and 20.

[0078] In a further embodiment, the antigen binding protein comprises A) a light chain amino acid sequence comprising LCDR1, LCDR2, and LCDR3 of any of Ab1 LCv, Ab2 LCv, and Ab3 LCv, and B) a heavy chain amino acid sequence comprising HCDR1, HCDR2, and HCDR3 of any of Ab1 HCv, Ab2 HCv, and Ab3 HCv.

[0079] In certain embodiments, the CDRs comprise no more than one, no more than two, no more than three, no more than four, no more than five, or no more than six amino acid additions, deletions, or substitutions from the exemplary CDRs set forth herein.

[0080] Embodiments of the invention include antibodies comprising a light chain variable domain selected from the group consisting of SEQ ID NOs: 27, 28, and 29. Embodiments of the invention include antibodies comprising a heavy chain variable domain selected from the group consisting of SEQ ID NOs: 9, 10, and 11. Further embodiments of the invention include antibodies comprising A) a light chain variable domain selected from the group consisting of SEQ ID NOs: 27, 28, and 29, and B) a heavy chain variable domain selected from the group consisting of SEQ ID NOs: 9, 10, and 11.

[0081] The antibodies of the present invention can comprise any constant region known in the art. The light chain constant region can comprise, for example, a kappa- or lambda-type light chain constant region, such as a human kappa- or lambda-type light chain constant region. The heavy chain constant region can comprise, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region, such as a human alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region. In one embodiment, the light or heavy chain constant region is a fragment, derivative, variant, or mutein of a naturally occurring constant region.

[0082] Embodiments of the invention include antibodies comprising a light chain variable region selected from the group consisting of SEQ ID NOs: 27, 28, and 29 with no more than one, two, three, four, five, six, seven, eight, nine, or ten amino acid additions, deletions, or substitutions.Embodiments of the invention include antibodies comprising a heavy chain variable region selected from the group consisting of SEQ ID NOs: 9, 10, and 11 with no more than one, two, three, four, five, six, seven, eight, nine, or ten amino acid additions, deletions, or substitutions. A further aspect of the invention includes an antibody comprising: A) a light chain variable region selected from the group consisting of SEQ ID NOs:27, 28, and 29 with no more than one, two, three, four, five, six, seven, eight, nine, or ten amino acid additions, deletions, or substitutions; and B) a heavy chain variable region selected from the group consisting of SEQ ID NOs:9, 10, and 11 with no more than one, two, three, four, five, six, seven, eight, nine, or ten amino acid additions, deletions, or substitutions.

[0083] In one variation, the antigen binding protein comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, and 29. In other variations, the antigen binding protein comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 10, and 11.In yet another embodiment, the antigen binding protein comprises: A) an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, and 29; and B) a light chain variable region amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, and 29. The light chain variable region amino acid sequence may be at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 light chain variable region amino acid sequence selected from the group consisting of NOs:9, 10, and 11. An OSMR antigen binding protein comprising a heavy chain variable domain having the sequence relationship defined above to SEQ ID NO:9 can optionally contain an amino acid other than asparagine (e.g., aspartic acid) at the position corresponding to position 73 of SEQ ID NO:9. In such embodiments, the heavy chain variable domain optionally comprises the amino acid sequence set forth in SEQ ID NO:53. An OSMR antigen binding protein comprising a heavy chain variable domain having the sequence relationship defined above to SEQ ID NO:10 can optionally contain an amino acid other than asparagine (e.g., aspartic acid) at the position corresponding to position 73 of SEQ ID NO:10. In such embodiments, the heavy chain variable domain optionally comprises the amino acid sequence set forth in SEQ ID NO:54.

[0084] In certain embodiments, the antigen binding protein comprises a light chain and / or a heavy chain CDR3. In some embodiments, the antigen binding protein comprises an amino acid sequence selected from the group of sequences set forth in SEQ ID NOs: 36, 37, 38, 18, 19, and 20. In certain embodiments, the amino acid sequence comprises no more than one, no more than two, no more than three, no more than four, no more than five, or no more than six amino acid additions, deletions, or substitutions from the exemplary sequences set forth in SEQ ID NOs: 36, 37, 38, 18, 19, and 20. Accordingly, embodiments of the present invention include antigen binding proteins comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 an amino acid sequence selected from the group of sequences set forth in SEQ ID NOs: 36, 37, 38, 18, 19, and 20.

[0085] In certain embodiments, the antigen binding protein comprises a light chain and / or a heavy chain CDR2. In some embodiments, the antigen binding protein comprises an amino acid sequence selected from the group of sequences set forth in SEQ ID NOs: 33, 34, 35, 15, 16, and 17. In certain embodiments, the amino acid sequence comprises no more than one, no more than two, no more than three, no more than four, no more than five, or no more than six amino acid additions, deletions, or substitutions from the exemplary sequences set forth in SEQ ID NOs: 33, 34, 35, 15, 16, and 17. Accordingly, embodiments of the present invention include antigen binding proteins comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 an amino acid sequence selected from the group of sequences set forth in SEQ ID NOs: 33, 34, 35, 15, 16, and 17.

[0086] In certain embodiments, the antigen binding protein comprises a light chain and / or a heavy chain CDR1. In some embodiments, the antigen binding protein comprises an amino acid sequence selected from the group of sequences set forth in SEQ ID NOs: 30, 31, 32, 12, 13, and 14. In particular embodiments, the amino acid sequence comprises no more than one, no more than two, no more than three, no more than four, no more than five, or no more than six amino acid additions, deletions, or substitutions from the exemplary sequences set forth in SEQ ID NOs: 30, 31, 32, 12, 13, and 14. Accordingly, embodiments of the present invention include antigen binding proteins comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 an amino acid sequence selected from the group of sequences set forth in SEQ ID NOs: 30, 31, 32, 12, 13, and 14.

[0087] Antigen-binding proteins of the present invention include traditional antibody scaffolds such as human and monoclonal antibodies, bispecific antibodies, diabodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and respective fragments. The above CDRs, including various combinations of CDRs, can be grafted onto any of the following scaffolds:

[0088] As used herein, the term "antibody" refers to various forms of monomeric or multimeric proteins comprising one or more polypeptide chains that specifically bind to an antigen, as variously described herein. In certain embodiments, antibodies are produced by recombinant DNA technology. In further embodiments, antibodies are produced by enzymatic or chemical cleavage of naturally occurring antibodies. In other aspects, the antibody is selected from the group consisting of: a) human antibodies; b) humanized antibodies; c) chimeric antibodies; d) monoclonal antibodies; e) polyclonal antibodies; f) recombinant antibodies; g) antigen-binding fragments; h) single-chain antibodies; i) diabodies; j) triabodies, k) tetrabodies, l) Fab fragments; m) F(ab')2 fragments, n) IgA antibodies, o) IgD antibodies, p) IgE antibodies, q) IgG1 antibodies, r) IgG2 antibodies, s) IgG3 antibodies, t) IgG4 antibodies, and u) IgM antibodies.

[0089] A variable region or domain consists of at least three heavy or light chain fragments embedded within framework regions (designated framework regions FR1, FR2, FR3, and FR4). Contains CDRs. Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, MD. Traditional antibodies The structural unit typically comprises a tetramer. Each tetramer typically consists of two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain. The amino-terminal portion of each chain contains a variable region of about 100-110 amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa or lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and correspond to IgM, IgD, and IgE, respectively. Antibody isotypes include IgG, IgA, and IgE. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2. Embodiments of the present invention include antibodies of all such classes and subclasses that incorporate the variable domains or CDRs of an antigen-binding protein as described herein.

[0090] Some naturally occurring antibodies, such as those found in camels and llamas, are dimers consisting of two heavy chains and no light chains. The present invention provides a method for detecting antibodies capable of binding to OSMR. Dimeric antibodies of two heavy chains or fragments thereof are included.

[0091] The variable regions of heavy and light chains typically exhibit the same general structure of relatively conserved framework regions (FRs) linked by three hypervariable regions, i.e., complementarity-determining regions or CDRs. The CDRs are primarily responsible for antigen recognition and binding. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From the N-terminus to the C-terminus, both light and heavy chains contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain follows the definition of Kabat.

[0092] CDRs constitute the major surface contact points for antigen binding. CDR3, or CDR3 of the light chain and, in particular, the heavy chain, may constitute the most important determinant for antigen binding in the variable regions of the light and heavy chains. In some antibodies, the heavy chain CDR3 appears to constitute the major area of contact between the antigen and the antibody. In vitro selection schemes in which only the CDR3 is varied can be used to alter the binding properties of an antibody or to determine which residues contribute to antigen binding.

[0093] Naturally occurring antibodies typically contain a signal sequence that directs the antibody into a cellular pathway for protein secretion and that is typically not present in the mature antibody. Polynucleotides encoding antibodies of the invention can encode a naturally occurring signal sequence or a heterologous signal sequence, as described below.

[0094] In one embodiment, the antigen binding protein is an antibody comprising one to six exemplary CDRs described herein. Antibodies of the invention may be of any type, including IgM, IgG (including IgG1, IgG2, IgG3, IgG4), IgD, IgA, or IgE antibodies. In a specific embodiment, the antigen binding protein is an IgG type antibody, for example, an IgG1 antibody.

[0095] In some embodiments, for example, when the antigen binding protein is an antibody with complete heavy and light chains, the CDRs are all from the same species, e.g., human. Alternatively, for example, in embodiments where the antigen binding protein contains fewer than six CDRs from the sequences outlined above, the additional CDRs may be either from another species or different human CDRs from those shown in the exemplary sequences. For example, HCDR3 and LCDR3 regions from suitable sequences identified herein can be used with HCDR1, HCDR2, LCDR1, and LCDR2, optionally selected from sequences of another species or a different human antibody, or combinations thereof. For example, the CDRs of the present invention can replace the CDR regions of a commercially relevant chimeric or humanized antibody.

[0096] Specific embodiments utilize a scaffold component of the antigen-binding protein that is a human component. However, in some embodiments, the scaffold component can be a mixture from different species. Thus, where the antigen-binding protein is an antibody, such an antibody can be a chimeric antibody and / or a humanized antibody. In general, both "chimeric antibody" and "humanized antibody" refer to antibodies that combine regions from two or more species. For example, a "chimeric antibody" traditionally contains variable regions derived from a mouse (or, in some cases, a rat) and constant regions derived from a human.

[0097] A "humanized antibody" generally refers to a non-human antibody having variable domain framework regions replaced with sequences found in a human antibody. Generally, in a humanized antibody, the entire antibody, except for one or more CDRs, is encoded by a polynucleotide of human origin or is identical to such an antibody except for one or more CDRs. Some or all of the CDRs encoded by nucleic acid from a non-human organism are grafted onto the beta-sheet framework of a human antibody variable region to create an antibody whose specificity is determined by the grafted CDRs. The creation of such antibodies is described, for example, in WO 92 / 11018, Jones 1986, Nature 321:522-525, Verhoeyen et al., 1988, Science 239:1534-1536. Humanized antibodies can also be generated using mice with genetically engineered immune systems (Roque et al., 1989). (Et al., 2004, Biotechnol. Prog. 20:639-654). In exemplary embodiments described herein, the identified CDRs are human, and therefore, in this context, both humanized and chimeric antibodies contain some non-human CDRs: for example, a humanized antibody may be generated that contains HCDR3 and LCDR3 regions along with one or more other CDR regions that are derived from a different species.

[0098] In one embodiment, the OSMR antigen-binding protein is a multispecific antibody, particularly a bispecific antibody, sometimes referred to as a "diabody." These are antibodies that bind to two or more different antigens or different epitopes on a single antigen. In certain embodiments, bispecific antibodies bind to an antigen on a human effector cell (e.g., a T cell). Such antibodies are useful for targeting effector cell responses against OSMR-expressing cells, such as OSMR-expressing tumor cells. In a preferred embodiment, the human effector cell antigen is CD3. See U.S. Pat. No. 7,235,641. Methods for producing bispecific antibodies are known in the art. One such method involves engineering the Fc portion, such as creating "knobs" and "holes," that promote heterodimerization of the heavy chains when coexpressed in cells. See U.S. Pat. No. 7,695,963. Another method involves engineering the Fc portion but using electrostatic steering to promote heterodimerization but prevent homodimerization when coexpressed in cells. WO 09 / 089,004 is incorporated herein by reference in its entirety.

[0099] In one embodiment, the OSMR antigen binding protein is a minibody, which is a minimized antibody-like protein comprising an scFv linked to a CH3 domain (Hu et al., 1996, Cancer Res. 56:3055-3061).

[0100] In one embodiment, the OSMR antigen binding protein domain is an antibody; see, e.g., US Patent No. 6,248,516. Domain antibodies (dAbs) are the derivatives of human antibodies. dABs are the functional binding domains of antibodies, corresponding to the variable regions of either the heavy chain (VH) or light chain (VL). dABs have a molecular weight of approximately 13 kDa, less than one-tenth the size of a full antibody. dABs are well expressed in a variety of hosts, including bacteria, yeast, and mammalian cell lines. dABs are also highly stable and retain activity even after exposure to harsh conditions, such as lyophilization or heat denaturation. See, e.g., U.S. Patents 6,291,158; 6,582,915; 6,593,081; 6,172,197; U.S. Serial No. 2004 / 0110941; European Patent 0368684; U.S. Patent 6,696,245; WO 04 / 058821; WO 04 / 003019; and WO 03 / 002609.

[0101] In one embodiment, the OSMR antigen-binding protein is an antibody fragment, i.e., a fragment of an antibody as outlined herein, that retains binding specificity for OSMR. In various embodiments, the antibody-binding protein includes, but is not limited to, F(ab), F(ab'), F(ab')2, Fv, or single-chain Fv fragments. At a minimum, as intended herein, an antibody includes a polypeptide capable of specifically binding to OSMR, including all or part of a light or heavy chain variable region, including one or more CDRs.

[0102] Further examples of OSMR-binding antibody fragments include, but are not limited to, (i) a Fab fragment consisting of the VL, VH, CL, and CH1 domains, (ii) a Fd fragment consisting of the VH and CH1 domains, and (iii) a fragment consisting of the VL and VH domains of a single antibody. (iv) dAb fragments consisting of a single variable fragment (Ward et al., 1989, Nature 341:544-546); (v) isolated (vi) F(ab')2 fragments, which are bivalent fragments containing two linked Fab fragments; (vii) single-chain Fv molecules (scFv), in which the VH and VL domains are linked by a peptide linker that allows the two domains to associate to form an antigen-binding site (Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883); (viii) bispecific single-chain Fv dimers (PCT / US92 / 09965); and (ix) multivalent or multispecific fragments of "diabodies" or "triabodies" constructed by gene fusion (Tomlinson et al., 2000, Methods Enzymol. 326:461-479; WO 94 / 13804; Holliger (Reiter et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448). Antibody fragments can be modified. For example, the molecules can be stabilized by the incorporation of disulfide bridges linking the VH and VL domains (Reiter et al., 1996, Nature Biotech. 14:1239-1245). Aspects of the invention include embodiments in which the non-CDR components of these fragments are human sequences.

[0103] In one embodiment, the OSMR antigen binding protein is a fully human antibody. In this embodiment, the specific structure includes the complete heavy and light chains depicted, including the CDR regions, as outlined above. Further embodiments utilize one or more CDRs of the invention along with other CDRs, framework regions, J and D regions, constant regions, etc., from other human antibodies. For example, the CDRs of the invention can replace the CDRs of any number of human antibodies, particularly commercially relevant antibodies.

[0104] Single-chain antibodies can be formed by linking heavy and light chain variable domain (Fv region) fragments via an amino acid bridge (a short peptide linker), resulting in a single polypeptide chain. Such single-chain Fvs (scFvs) consist of two variable domain polypeptides (V L and V H These polypeptides have been prepared by fusing DNA encoding a peptide linker between DNA encoding the variable domains. The resulting polypeptides can fold back on themselves to form antigen-binding monomers, or they can form multimers (e.g., dimers, trimers, or tetramers) depending on the length of the flexible linker between the two variable domains (Kortt et al., 1997, Prot. E ng.10:423;Kortt et al.,2001,Biomol.Eng.1 8:95-108). Different V L and V H By combining the containing polypeptides, multimeric scFvs that bind to different epitopes can be formed (Kriangkum (e.g., et al., 2001, Biomol. Eng. 18:31-40). Techniques developed for the production of single-chain antibodies are described in U.S. Patent No. 4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544; de Graaf et al., 2002, Methods Mol. Biol. 178:379-87. Single-chain antibodies include, but are not limited to, scFvs comprising combinations of the variable domains of antibodies Ab1 LCv / Ab1 HCv (SEQ ID NO:27 / SEQ ID NO:9), Ab2 LCv / Ab2 HCv (SEQ ID NO:28 / SEQ ID NO:10), and Ab3 LCv / Ab3 HCv (SEQ ID NO:29 / SEQ ID NO:11) provided herein. Exemplary single-chain antibodies comprise the following variable domain combinations: SEQ ID NO:27 / SEQ ID NO:53; and SEQ ID NO:28 / SEQ ID NO:54.

[0105] In one embodiment, the OSMR antigen binding protein is an antibody fusion protein (sometimes referred to herein as an "antibody conjugate"). The conjugate partner can be proteinaceous or non-proteinaceous. The latter is generally generated using functional groups on the antigen binding protein and on the conjugate partner. In certain embodiments, the antibody is conjugated to a non-proteinaceous chemical (drug) to form an antibody-drug conjugate.

[0106] In one embodiment, the OSMR antigen binding protein is an antibody analog, sometimes referred to as a "synthetic antibody." For example, various studies have utilized either alternative protein scaffolds or artificial scaffolds with grafted CDRs. Such scaffolds include, but are not limited to, fully synthetic scaffolds composed of biocompatible polymers, as well as mutations introduced to stabilize the three-dimensional structure of the binding protein. For example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, Volume 53, Issue 1:121-129. Roque et al., 2004, Biotechnol. Prog. 20:639-654. Additionally, peptide antibody mimetics ("PAMs") can be used, similar to work based on antibody mimetics that utilize fibronectin components as a scaffold.

[0107] As used herein, "protein" refers to at least two covalently attached amino acids, including proteins, polypeptides, oligopeptides, and peptides. In some embodiments, the two or more covalently attached amino acids are linked by a peptide bond. As outlined below, for example, when the protein is produced recombinantly using an expression system and host cell, the protein may be composed of naturally occurring amino acids and peptide bonds. Alternatively, the protein may be composed of synthetic amino acids (e.g., homophenylalanine, citrulline, ornithine, and norleucine), or peptidomimetic structures, i.e., peptoids (see Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89:9367, incorporated herein by reference). The term "amino acid" may include "peptide or protein analogs" such as amino acids (see references), which can withstand proteases or other physiological and / or storage conditions. Such synthetic amino acids may be incorporated, particularly when the antigen-binding protein is synthesized in vitro by conventional methods well known in the art. Also, any combination of peptidomimetic, synthetic, and naturally occurring residues / structures can be used. "Amino acid" also includes imino acid residues such as proline and hydroxyproline. The amino acid "R group" or "side chain" can be in either the (L) or (S) configuration. In certain embodiments, the amino acid is in the (L) or (S) configuration.

[0108] In certain aspects, the present invention provides recombinant antigen binding proteins that bind OSMR, and in some embodiments, recombinant human OSMR or portions thereof. In this context, a "recombinant protein" is a protein produced using recombinant techniques using any techniques and methods known in the art, i.e., by expression of a recombinant nucleic acid as described herein. Methods and techniques for the production of recombinant proteins are well known in the art. Embodiments of the present invention include recombinant antigen binding proteins that bind wild-type OSMR and variants thereof.

[0109] "Substantially consisting of" as used herein means that the sequence can vary by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15% relative to the recited SEQ ID NO: sequence and still retain biological activity. do.

[0110] In some embodiments, the antigen-binding proteins of the present invention are isolated or substantially pure proteins. An "isolated" protein is free from at least some of the materials with which it is normally associated in its natural state, e.g., comprising at least about 5% by weight, or at least about 50% by weight, of the total protein in a given sample. It is understood that an isolated protein can comprise 5-99.9% by weight of the total protein content, depending on the context. For example, proteins can be produced at significantly higher concentrations through the use of inducible or high-expression promoters such that the protein is produced at high concentration levels. This definition includes the production of antigen-binding proteins in a variety of organisms and / or host cells known in the art.

[0111] For amino acid sequences, sequence identity and / or similarity may be determined by standard techniques known in the art, such as, but not limited to, the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. USA 85:2444, computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), preferably using default settings, or by inspection, such as the BestFit sequence program described by Devereux et al., 1984, Nucl. Acid. Res. 12:387-395. Preferably, percent identity is calculated by FastDB based on the following parameters: mismatch penalty of 1; gap penalty of 1; Nalty; gap size penalty of 0.33; joining penalty of 30, “Current Methods in Sequence Comparison and Analysis,” Macromolecules Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149 (1988), Alan R. Liss, Inc.

[0112] An example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Dolittle, 1987, J. Mol. Evol. 35:351-360; this method is similar to that described by Higgins and Sharp, 1989; CABIOS 5:151-153. Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

[0113] Another example of a useful algorithm is the BLAST algorithm, which is described in Altschul et al., 1990, J. MoL. Biol. 215:403-410; Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402; and Karin et al., 1993, Proc. Natl. Acad. Sci. USA 90:5873-5787. A particularly useful BLAST program is Altschul et al., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses several search parameters, most of which are set to default values. Adjustable parameters are set to the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11. The HSP S and HSP S2 parameters are dynamic values established by the program itself depending on the composition of the specific sequence and the composition of the particular database in which the sequence of interest is searched; however, values can be adjusted to increase sensitivity.

[0114] A more useful algorithm is Gapped BLAST, reported by Altschul et al., 1993, Nucl. Acids. Res. 25:3389-3402. Gapped BLAST uses BLOSUM-62 substitution scores; the threshold T parameter is set to 9; the 2-hit method to trigger ungapped extension charges a gap length of 10+k k costs; X u is set to 16, X g is set to 40 for the database search stage and 67 for the output stage of the algorithm. Gapped alignments are triggered by scores corresponding to approximately 22 bits.

[0115] Generally, the amino acid homology, similarity, or identity between the CDRs of individual variants is at least 80% with respect to the sequences set forth herein, more typically at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and preferably increasing homology or identity to the sequences set forth herein, such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and preferably near 100%. Similarly, "percent (%) nucleic acid sequence identity" with respect to the nucleic acid sequence of a binding protein identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical with nucleotide residues in the coding sequence of the antigen binding protein. A specific method utilizes the BLASTN module of WU-BLAST-2 set to default parameters, with the overlap span and overlap fraction set to 1 and 0.125, respectively.

[0116] Generally, the nucleic acid sequence homology, similarity, or identity between the nucleotide sequence encoding each variant CDR and the nucleotide sequences set forth herein will be at least 80%, more typically at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and preferably increasing homology or identity of approximately 100%.

[0117] Thus, a "variant CDR" is one that has specific homology, similarity, or identity to a parent CDR of the invention and has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and / or activity of the parent CDR. These share biological functions, including but not limited to:

[0118] Although the site or region for introducing the amino acid sequence mutation is predetermined, the mutation itself need not be predetermined. For example, to optimize the performance of a mutation at a given site, random mutagenesis can be performed on the target codon or region and the expressed antigen-binding protein CDR mutants screened for the optimal combination of the desired activity. Techniques for making substitution mutations at a predetermined site in DNA with a known sequence are well known, such as M13 primer mutagenesis and PCR mutagenesis. Screening of the mutants is performed using an assay for antigen-binding protein activity, such as OSMR binding.

[0119] Amino acid substitutions are typically single residues; insertions will usually be on the order of about 1 to 20 amino acid residues, although significantly larger insertions can be tolerated. Deletions range from about 1 to 20 amino acid residues, although larger deletions are possible.

[0120] Substitution, deletion, insertion, or any combination thereof can be used to arrive at the final derivative or variant. Generally, these changes are made to a few amino acids to minimize changes in the molecule, especially the immunogenicity and specificity of the antigen-binding protein. However, in certain circumstances, larger changes can be tolerated. Conservative substitutions are generally made according to the following chart shown as Table 3. Table 3 [Table 3]

[0121] Substantial changes in function or immunological identity can be made by selecting substitutions that are less conservative than those shown in Table 3. For example, substitutions can be made that have a greater effect on the structure of the polypeptide backbone in the modified region, such as α-helical or β-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain. In general, the substitutions expected to produce the greatest changes in the properties of the polypeptide are those in which a hydrophilic residue, such as seryl or threonyl, is substituted for (or by) a hydrophobic residue, such as leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue with an electropositive side chain, such as lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, such as glutamyl or aspartyl; or (d) a residue with a bulky side chain, such as phenylalanine, is substituted for (or by) a residue without a side chain, such as glycine.

[0122] Mutants are also selected to optionally alter the properties of the antigen-binding protein, but typically exhibit the same qualitative biological activity and elicit the same immune response as the naturally occurring analog. Alternatively, mutants can be designed to alter the biological activity of the antigen-binding protein. For example, glycosylation sites can be altered or removed as described herein.

[0123] Other derivatives of the OSMR antibody within the scope of the present invention include covalent or aggregative conjugates of the OSMR antibody, or fragments thereof, with other proteins or polypeptides, such as by expression of recombinant fusion proteins containing heterologous polypeptides fused to the N- or C-terminus of the OSMR antibody polypeptide. For example, the conjugated peptide may be a heterologous signal (i.e., leader) polypeptide, such as the yeast α-factor leader, or a peptide such as an epitope tag. The OSMR antibody-containing fusion protein may include an additional peptide (e.g., poly-His) to facilitate purification or identification of the OSMR antibody. The OSMR antibody polypeptide may also be linked to a FLAG peptide, as described by Hopp et al., Bio / Technology 6:1204, 1988, and U.S. Patent 5,011,912. The FLAG peptide is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody (mAb), allowing for rapid assay and easy purification of the expressed recombinant protein. Reagents useful for preparing fusion proteins in which the FLAG peptide is fused to a given polypeptide are commercially available (Sigma, St. Louis, MO).

[0124] In one embodiment, oligomers are prepared using immunoglobulin-derived polypeptides. Preparation of fusion proteins comprising specific heterologous polypeptides fused to various portions of antibody-derived polypeptides (including Fc domains) is described, for example, in Ashkenazi et al., 1991, PNAS USA 88:10535; Byrn et al., 1990, Nature 344:677; and Hollenbaugh et al., 1992, "Construction of Immunoglobulin Fusion Proteins." Proteins”, in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11.

[0125] One embodiment of the present invention is directed to a dimer comprising two fusion proteins made by fusing an OSMR-binding fragment of an OSMR antibody to the Fc region of an antibody. The dimer can be made by inserting a gene fusion encoding the fusion protein into a suitable expression vector that expresses the gene fusion in a host cell transformed with the recombinant expression vector, and allowing the expressed split proteins to assemble in an antibody-like manner, with interchain disulfide bonds formed between the iron moieties to obtain a dimer.

[0126] As used herein, the term "iron polypeptide" includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Also included are truncated forms of such polypeptides containing the hinge region that promotes dimerization. Fusion proteins containing the Fc portion (and oligomers formed therefrom) offer the advantage of easy purification by affinity chromatography on Protein A or Protein G columns.

[0127] One suitable Fc polypeptide, described in PCT application WO 93 / 10151 (incorporated herein by reference), is a single-chain polypeptide extending from the N-terminal hinge region to the native C-terminus of the Fc region of a human IgG antibody. Other useful Fc polypeptides are described in US Patent 5,457,035 and Baum et al., 1994, E MBO J, 13:3992-4001. The amino acid sequence of this mutein is identical to that of the native Fc sequence shown in WO 93 / 10151, except that amino acid 19 is changed from Leu to Ala, amino acid 20 is changed from Leu to Glu, and amino acid 22 is changed from Gly to Ala. This mutein exhibits reduced affinity for Fc receptors.

[0128] In other embodiments, the variable portions of the heavy and / or light chains of an OSMR antibody may be substituted for the variable portions of the heavy and / or light chains of an antibody.

[0129] Another method for preparing oligomeric OSMR antibody derivatives involves the use of leucine zippers. Leucine zipper domains are peptides that promote oligomerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759-64, 1988), and have since been discovered in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and their derivatives that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT application WO 94 / 10308, and the leucine zipper from pulmonary surfactant protein D (SPD) is described in Hoppe et al., 1994, FEBS Letters 344:191, incorporated herein by reference. The use of a modified leucine zipper that allows stable trimerization of a fused heterologous protein is described in Fanslow et al., 1994, Semin. Immunol. 6:267-78. In one approach, a recombinant fusion protein containing an OSMR antibody fragment or derivative fused to a leucine zipper peptide is expressed in a suitable host cell, and the resulting soluble oligomeric OSMR antibody fragment or derivative is recovered from the culture supernatant.

[0130] Covalent modifications of antigen-binding proteins are included within the scope of the present invention and are generally, but not always, performed post-translationally. For example, some types of covalent modifications of antigen-binding proteins are introduced into the molecule by reacting specific amino acid residues of the antigen-binding protein with organic derivatizing agents capable of reacting with selected side chains or N- or C-terminal residues.

[0131] Cysteinyl residues are most commonly cleaved by α- Reaction with haloacetates (and corresponding amines) gives carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, α-bromoβ-(5-imidozoyl)propionic acid, chloroacetylphosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0132] Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

[0133] Lysinyl and amino-terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues are imidoesters such as methylpyroline; pyridoxal phosphate; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed derivatization. and reaction with glyoxylic acid.

[0134] Arginyl residues can be amplified by reaction with one or several conventional reagents, such as phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues is achieved by the reaction with a high pK a Therefore, these reagents must be carried out under alkaline conditions. Furthermore, these reagents can react with the lysine group and the arginine epsilon-amino group.

[0135] Specific modifications of tyrosyl residues can be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues can be labeled with chloramine T, as described above, to prepare labeled proteins for use in radioimmunoassays. 125 I or 131 It is iodinated using I.

[0136] The carboxyl side group (aspartyl or glutamyl) is optionally modified by reaction with a carbodiimide (R'-N=C=N-R'), where R and R' are optionally different, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide, Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0137] Derivatization with bifunctional reagents is useful for crosslinking antigen-binding proteins to water-insoluble support matrices or surfaces for use in a variety of methods. Commonly used crosslinking agents include N-hydroxysuccinimide esters such as 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, 4-azidosalicylic acid containing disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl propionate), N-hydroxysuccinimide esters such as esters with homobifunctional imidoesters, and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate produce photoactivatable intermediates that can form crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and their reactive substrates as described in US Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are used for protein immobilization.

[0138] Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of the invention.

[0139] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, and methylation of the α-amino group of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties,WHFreeman & Co., San Francisco, 1983, pp. 79-86), including acetylation of the N-terminal amine and amidation of any C-terminal carboxyl group.

[0140] Other types of covalent modifications of antigen binding proteins included within the scope of the present invention include altering the glycosylation pattern of the protein. As is known in the art, glycosylation patterns can depend on both the sequence of the protein (e.g., the presence or absence of particular glycosylated amino acid residues, as described below), or the host cell, or organism in which the protein is produced. Particular expression systems are described below.

[0141] Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0142] Addition of glycosylation sites to the antigen binding protein is conveniently accomplished by altering the amino acid sequence to include one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may be made by the addition of, or substitution by, one or more serine or threonine residues to the starting sequence (for O-linked glycosylation sites). To facilitate this, the antigen binding protein amino acid sequence is preferably altered by changes at the DNA level, in particular by mutating the DNA encoding the target polypeptide with preselected groups to generate codons that translate into the desired amino acids.

[0143] Another means of increasing the number of carbohydrate moieties on antigen-binding proteins is by chemical or enzymatic coupling of glycosides to the protein. These procedures are advantageous in that they do not require production of the protein in a host cell capable of N- and O-linked glycosylation. Depending on the mode of attachment used, the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as cysteine, (d) free hydroxyl groups such as serine, threonine, or hydroxyproline, (e) aromatic residues such as phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in WO 87 / 05330, published September 11, 1987, and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306.

[0144] Removal of carbohydrate moieties present on the starting antigen-binding protein can be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the polypeptide intact. Chemical deglycosylation has been described by Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and Edge et al., 1981, Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of various endo- and exo-glycosidases, as described by Thotakura et al., 1987, Meth. Enzymol. 138:350. Glycosylation at potential glycosylation sites can be achieved by the use of various endo- and exo-glycosidases, as described by Duskin et al. This can be prevented by the use of the compound tunicamycin, as described by [End Page 110] et al., 1982, J. Biol. Chem. 257:3105, which blocks the formation of protein-N-glycosidic bonds.

[0145] Another type of covalent modification of an antigen-binding protein involves conjugating the antigen-binding protein to a nonproteinaceous polymer, including, but not limited to, various polyols such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, by methods described in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192, or 4,179,337. Also, as is known in the art, amino acid substitutions can be made at various positions within the antigen-binding protein to facilitate the addition of polymers such as PEG.

[0146] In some embodiments, the covalent modification of the antigen binding proteins of the invention comprises the addition of one or more labels.

[0147] The term "labeling group" means any detectable label. Examples of suitable labeling groups include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131I), fluorescent groups (e.g., FITC, rhodamine, lanthanide fluorophores), enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups recognized by secondary reporters, biotinyl groups, or predetermined polypeptide epitopes (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal-binding domains, epitope tags). In some embodiments, the labeling group is attached to the antigen-binding protein via spacer arms of various lengths to reduce potential steric hindrance. Various methods of labeling proteins are known in the art and can be used in the practice of the present invention.

[0148] Generally, labels are divided into different classes depending on the assay in which they are to be detected: a) isotopic labels, which may be radioisotopes or heavy isotopes; b) magnetic labels (e.g., magnetic a) a redox-active particle; b) a redox-active moiety; c) an optical dye; e) an enzymatic group (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); f) a predetermined polypeptide epitope recognized by a secondary reporter (e.g., a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag, etc.). In some embodiments, the labeling group is attached to the antigen-binding protein via a spacer arm of various lengths to reduce potential steric hindrance. Various methods of labeling proteins are known in the art and can be used in the practice of the present invention.

[0149] Specific labels include optical dyes, which include, but are not limited to, chromophores, fluorophores, and fluorophores, the latter of which are often specific. Fluorophores can be either "small molecule" fluorophores or proteinaceous fluorophores.

[0150] "Fluorescent label" refers to any molecule that can be detected by its inherent fluorescent properties. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue J, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon green, Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 640), and the like. Fluor 430,Alexa Fluor 488,Alexa Fluor 546,Alexa Fluor 568,Alexa Fluor 594,Alexa Suitable optical dyes, including fluorophores, include, but are not limited to, Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow, and R-phycoerythrin (PE) (Molecular Probes, Eugene, OR), FITC, Rhodamine, and Texas Red (Pierce, Rockford, IL), Cy5, Cy5.5, and Cy7 (Amersham Life Science, Pittsburgh, PA). Suitable optical dyes, including fluorophores, are described in Molecular Probes Handbook by Richard P. Haugland, which is expressly incorporated herein by reference.

[0151] Suitable proteinaceous fluorescent labels include green fluorescent proteins, including Renilla, Ptilosarcus, or Aequorea species GFP (Chalfie et al., 1994, Science 263:802-805), EGFP (Clontech Laboratories, Inc., Genbank Accession Number U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9; Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol. 6:178-182), enhanced yellow fluorescent protein (EYFP, Clontech Laboratories, Inc. ), luciferase (Ichiki et al., 1993, J. Immunol. 150:5408-5417), β-galactosidase (Nolan et al., 1988, Proc. Natl. Acad. Sci. USA 85:2603-2607), and Renilla reniformis (WO92 / 15673, WO95 / 07463, WO98 / 14605, WO98 / 26277, WO99 / 49019, US Patent Nos. 5292658, 5418155, 5683888, 5741668, 5777079, 5804387, 5874304, 5876995, 5925558). All of the above cited references are expressly incorporated herein by reference.

[0152] The exemplary antigen binding proteins described herein have characteristics based on different epitopes on the OSMR bound by the antigen binding protein. The term "epitope" refers to the amino acids of a target molecule that are contacted by an antigen binding protein, such as an antibody, when the antigen binding protein is bound to the target molecule. Epitopes can be contiguous or discontinuous. (For example, (i) in a single-chain polypeptide, amino acid residues that are not contiguous in the polypeptide sequence but are in the context of the target molecule are bound by the antigen binding protein; or (ii) in a multimeric receptor comprising two or more individual components, e.g., OSMR and gp130 or OSMR and IL-31 receptor A, amino acid residues are present in one or more of the individual constituent components but are still bound by the antigen binding protein. Epitopic determinants can include chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics, and / or specific charge characteristics. In general, an antigen binding protein specific for a particular target molecule will preferentially recognize an epitope on the target molecule in a complex mixture of proteins and / or macromolecules.)

[0153] Methods for characterizing epitopes bound by antigen-binding proteins are well known in the art and include, but are not limited to, binning (cross-competition) (Miller et al. "Epitope binning of murine monoclonal antibodies by a multiplexed pairing assay" J Immunol Methods (2011) 365, 118-25), peptide mapping (e.g., PEPSPOT™) (Albert et al. al "The B-cel1 Epitope of the Monoclonal Anti-Factor VIII Antibody ESH8 Characterized by Peptide Array Analysis" 2008 Thromb. Haemost 99, 634-7), mutagenesis methods such as chimeras (Song et al "Epitope Mapping of Ibalizumab, a Humanized Anti-CD4 Monoclonal Antibody with Anti-HIV-1 Activity in Infected Patients" J. Virol. (2010) 84, 6935-6942), alanine scanning (Cunningham and Wells "High-resolution "Epitope mapping of HGH-receptor interactions by alanine-scanning mutagenesis" Science (1989) 244, 1081-1085), arginine scanning (Li m et al“a diversity of antibody epitopes can induce signaling through the erythropoietin receptor”Biochemistry (2010)49,3797-3804), HD exchange method (Coates et al “Epitope mapping by amide hydrogen / deuterium exchange coupled with immobilization of antibody,on-line proteolysis,liquid chromatography and mass spectrometry”Rapid Commun,mass Spectrom.(2009)23 639-647), NMR cross-saturation method (Morgan et al “Precise epitope mapping of malaria parasite inhibitory antibodies by TROSY NMR cross-saturation” Biochemistry (2005) 44, 518-23), and crystallography (Gerhardt et al. “Structure of IL-17A in complex with a potent, fully human neutralizing antibody” y" J. Mol. Biol (2009) 394, 905-21). These methods differ in the level of detail they provide regarding the amino acids that comprise the epitope. Example 4 provides an exemplary method of epitope binning.

[0154] Antigen binding proteins of the present invention include those that have an epitope that overlaps with the exemplary representative antigen binding proteins described herein, e.g., Ab1, Ab2, or Ab3. In certain embodiments, the antigen binding protein has the same epitope as the exemplary antigen binding protein. In other embodiments, the antigen binding protein binds only the same subset of amino acids as the exemplary antigen binding protein.

[0155] In certain embodiments, the OSMR antigen binding protein has an epitope identical to or overlapping with Ab1, Ab2, or Ab3 and comprises: a) a light chain variable domain having at least 90% identity, at least 95% identity, or identity to the amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29; b) a heavy chain variable domain having at least 90% identity, at least 95% identity, or identity to the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11; or c) the light chain variable domain of a) and the heavy chain variable domain of b).

[0156] In certain embodiments, the OSMR antigen binding protein has an epitope identical to or overlapping with Ab1, Ab2, or Ab3 and comprises a light chain variable domain having at least 90% identity, at least 95% identity, or identity to the amino acid sequence set forth in SEQ ID NO:27, and a heavy chain variable domain having at least 90% identity, at least 95% identity, or identity to the amino acid sequence set forth in SEQ ID NO:9; a light chain variable domain having at least 90% identity, at least 95% identity, or identity to the amino acid sequence set forth in SEQ ID NO:28, and a heavy chain variable domain having at least 90% identity, at least 95% identity, or identity to the amino acid sequence set forth in SEQ ID NO:10; and a light chain variable domain having at least 90% identity, at least 95% identity, or identity to the amino acid sequence set forth in SEQ ID NO:29, and a heavy chain variable domain having at least 90% identity, at least 95% identity, or identity to the amino acid sequence set forth in SEQ ID NO:11. A heavy chain variable domain having at least 90% identity, at least 95% identity, or identity to the amino acid sequence set forth in NO:11.

[0157] In certain embodiments, the OSMR antigen binding protein has an epitope identical to or overlapping with Ab1, Ab2, or Ab3 and comprises: a) a light chain variable domain with no more than 10 or no more than 5 amino acid additions, deletions, or substitutions from the amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29; b) a heavy chain variable domain with no more than 10 or no more than 5 amino acid additions, deletions, or substitutions from the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11; or c) the light chain variable domain of a) and the heavy chain variable domain of b).

[0158] In certain embodiments, the OSMR antigen binding protein has an epitope identical to or overlapping with Ab1, Ab2, or Ab3 and comprises a light chain variable domain having no more than 10 or no more than 5 amino acid additions, deletions, or substitutions from the amino acid sequence set forth in SEQ ID NO:27, and a heavy chain variable domain having no more than 10 or no more than 5 amino acid additions, deletions, or substitutions from the amino acid sequence set forth in SEQ ID NO:9; a light chain variable domain having no more than 10 or no more than 5 amino acid additions, deletions, or substitutions from the amino acid sequence set forth in SEQ ID NO:28, and a heavy chain variable domain having no more than 10 or no more than 5 amino acid additions, deletions, or substitutions from the amino acid sequence set forth in SEQ ID NO:10; and a light chain variable domain having no more than 10 or no more than 5 amino acid additions, deletions, or substitutions from the amino acid sequence set forth in SEQ ID NO:29, and a heavy chain variable domain having no more than 10 or no more than 5 amino acid additions, deletions, or substitutions from the amino acid sequence set forth in SEQ ID NO:11. An exemplary heavy chain variable domain variant of SEQ ID NO:9 contains an amino acid other than asparagine (e.g., aspartic acid) at the position corresponding to position 73 of SEQ ID NO:9. The amino acid sequence set forth in SEQ ID NO:53 is an example of a heavy chain variable domain variant of SEQ ID NO:9. An exemplary heavy chain variable domain variant of SEQ ID NO:10 contains an amino acid other than asparagine (e.g., aspartic acid) at the position corresponding to position 73 of SEQ ID NO:10. The amino acid sequence set forth in SEQ ID NO:54 is an example of a heavy chain variable domain variant of SEQ ID NO:10.

[0159] In certain embodiments, the OSMR antigen binding protein has an epitope that is identical to or overlaps with Ab1, Ab2, or Ab3, and has a) an LCDR1 having no more than three amino acid additions, deletions, or substitutions from the LCDR1 sequence shown in SEQ ID NO:30; an LCDR2 having no more than three amino acid additions, deletions, or substitutions from the LCDR2 sequence shown in SEQ ID NO:33; and an LCDR3 having no more than three amino acid additions, deletions, or substitutions from the LCDR3 sequence shown in SEQ ID NO:36; a light chain variable domain comprising: an LCDR1 having not more than three amino acid additions, deletions or substitutions from the LCDR1 sequence shown in SEQ ID NO:31; an LCDR2 having not more than three amino acid additions, deletions or substitutions from the LCDR2 sequence shown in SEQ ID NO:34; and an LCDR3 having not more than three amino acid additions, deletions or substitutions from the LCDR3 sequence shown in SEQ ID NO:37; or c) an LCDR1 having not more than three amino acid additions, deletions or substitutions from the LCDR1 sequence shown in SEQ ID NO:32; an LCDR2 having not more than three amino acid additions, deletions or substitutions from the LCDR2 sequence shown in SEQ ID NO:35; and an LCDR3 having not more than three amino acid additions, deletions or substitutions from the LCDR3 sequence shown in SEQ ID NO:38; and d) a light chain variable domain comprising: an LCDR1 having not more than three amino acid additions, deletions or substitutions from the LCDR1 sequence shown in SEQ ID NO:31; an LCDR2 having not more than three amino acid additions, deletions or substitutions from the LCDR2 sequence shown in SEQ ID NO:34; and an LCDR3 having not more than three amino acid additions, deletions or substitutions from the LCDR3 sequence shown in SEQ ID NO:37; an HCDR1 having not more than three amino acid additions, deletions or substitutions from the HCDR1 sequence shown in SEQ ID NO:12; an HCDR2 having not more than three amino acid additions, deletions or substitutions from the HCDR2 sequence shown in SEQ ID NO:15; and an HCDR3 having not more than three amino acid additions, deletions or substitutions from the HCDR3 sequence shown in SEQ ID NO:18; e) an HCDR1 having not more than three amino acid additions, deletions or substitutions from the HCDR1 sequence shown in SEQ ID NO:13; an HCDR2 having not more than three amino acid additions, deletions or substitutions from the HCDR2 sequence shown in SEQ ID NO:16; and an HCDR3 having not more than three amino acid additions, deletions or substitutions from the HCDR3 sequence shown in SEQ ID NO:19; or f) an HCDR1 having not more than three amino acid additions, deletions or substitutions from the HCDR1 sequence shown in SEQ ID NO:14; an HCDR2 having not more than three amino acid additions, deletions or substitutions from the HCDR2 sequence shown in SEQ ID NO:17; and a heavy chain variable domain comprising an HCDR3 having an addition, deletion or substitution of 3 or fewer amino acids from the HCDR3 sequence set forth in NO:20.

[0160] The above OSMR antigen binding proteins preferably include those comprising the light chain variable domain of a) and the heavy chain variable domain of d); those comprising the light chain variable domain of b) and the heavy chain variable domain of e); and those comprising the light chain variable domain of c) and the heavy chain variable domain of f). An OSMR antigen binding protein comprising a) a light chain variable domain and d) a heavy chain variable domain can optionally comprise a heavy chain variable domain comprising an amino acid other than asparagine (e.g., aspartic acid) at a position corresponding to position 73 of SEQ ID NO:9. In such embodiments, the heavy chain variable domain optionally comprises the amino acid sequence set forth in SEQ ID NO:53. An OSMR antigen binding protein comprising the light chain variable domain of b) and the heavy chain variable domain of e) can optionally comprise a heavy chain variable domain comprising an amino acid other than asparagine (e.g., aspartic acid) at the position corresponding to position 73 of SEQ ID NO: 10. In such embodiments, the heavy chain variable domain optionally comprises the amino acid sequence set forth in SEQ ID NO:54.

[0161] Antigen-binding proteins with identical or overlapping epitopes often cross-compete for antigen binding. Thus, in certain embodiments, an antigen-binding protein of the invention cross-competes with Ab1, Ab2, or Ab3. "Cross-compete" or "cross-competition" means competing for the same epitope or binding site on a target. Such competition can be determined by assays in which a reference antigen-binding protein (e.g., an antibody or antigen-binding portion thereof) prevents or inhibits specific binding of a test antigen-binding protein, and vice versa. Many types of competitive binding assays can be used to determine whether a test molecule competes with a reference molecule for binding. Examples of assays that can be used include solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competitive assays (see, e.g., Stahl et al. (1983) Methods in Enzymology 9:242-253), solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al. (1986) J. Immunol. 137:3614-9), solid-phase direct labeled sandwich assays, Luminex (Jia et al. "A novel method of Multiplexed Competitive Antibody Binning FOR the characterization of monoclonal antibodies” J. Immunological Methods (2004) 288, 91-98), and surface plasmon resonance (Song et al. “Epitope Mapping of Ibalizumab, a Humanized Anti-CD4 Monoclonal Antibody with Anti-HIV-1 Activity in Infected Patients, "J. Virol. (2010) 84, 6935-42." Exemplary methods for determining cross-competition are described in Example 5. Typically, when a competing antigen-binding protein is present in excess, it inhibits binding of the reference antigen-binding protein to a common antigen by at least 50%, 55%, 60%, 65%, 70%, or 75%. In some cases, binding is inhibited by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more. Polynucleotides encoding OSMR antigen-binding proteins

[0162] Nucleic acids or isolated nucleic acids encoding OSMR antigen binding proteins, including antibodies, as defined herein, are encompassed by the invention. Preferred nucleic acids include those encoding the exemplary light and heavy chains described herein.

[0163] An exemplary nucleic acid encoding Ab1 LC is the nucleic acid comprising the sequence shown in SEQ ID NO:21.

[0164] An exemplary nucleic acid encoding Ab2 LC is the nucleic acid comprising the sequence shown in SEQ ID NO:22.

[0165] An exemplary nucleic acid encoding Ab3 LC is the nucleic acid comprising the sequence shown in SEQ ID NO:23.

[0166] An exemplary nucleic acid encoding Ab1 HC is the nucleic acid comprising the sequence shown in SEQ ID NO:3.

[0167] An exemplary nucleic acid encoding Ab2 HC is the nucleic acid comprising the sequence shown in SEQ ID NO:4.

[0168] An exemplary nucleic acid encoding Ab3 HC is the nucleic acid comprising the sequence shown in SEQ ID NO:5. An exemplary nucleic acid encoding Ab1 HC is the nucleic acid comprising the sequence shown in SEQ ID NO:47. An exemplary nucleic acid encoding Ab2HC is the nucleic acid comprising the sequence shown in SEQ ID NO:48. An exemplary nucleic acid encoding Ab3HC is the nucleic acid comprising the sequence set forth in SEQ ID NO:49.

[0169] Aspects of the invention include polynucleotide variants (eg, due to degeneracy) that encode the amino acid sequences described herein.

[0170] Aspects of the present invention include various embodiments, including, but not limited to, the following exemplary embodiments.

[0171] A polynucleotide encoding one or more polypeptides comprising an amino acid sequence selected from the group consisting of:

[0172] A.1. A light chain variable domain sequence that is at least 90% identical to a light chain variable domain sequence set forth in SEQ ID NOS:27-29;

[0173] 2. A heavy chain variable domain sequence that is at least 90% identical to a heavy chain variable domain sequence set forth in SEQ ID NOS:9-11;

[0174] 3. A light chain variable domain of (1) and a heavy chain variable domain of (2); and

[0175] B. A light chain variable domain comprising CDR1, CDR2, and CDR3 and / or a heavy chain variable domain comprising CDR1, CDR2, and CDR3 that is the same as or differs by the addition, substitution, and / or deletion of no more than three amino acids in each CDR;

[0176] 1. Light chain CDR1 (SEQ ID NO: 30), CDR2 (SEQ ID NO: 31) of Ab1 NO:33), CDR3 (SEQ ID NO:36) or heavy chain CDR 1 (SEQ ID NO:12),CDR2(SEQ ID NO:15),CDR3(SEQ ID NO:18);

[0177] 2. Light chain CDR1 (SEQ ID NO: 31), CDR2 (SEQ ID NO: 32) of Ab2 NO:34), CDR3 SEQ ID NO:37) or heavy chain CDR 1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 16), CDR3 (SEQ ID NO: 19); and

[0178] 3. Light chain CDR1 (SEQ ID NO: 32), CDR2 (SEQ ID NO: 33) of Ab3 NO:35), CDR3 SEQ ID NO:38) or heavy chain CDR 1 (SEQ ID NO:14), CDR2(SEQ ID NO:17), CDR3(SEQ ID NO:20). In some embodiments, the nucleic acid encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:53 or SEQ ID NO:54. In some embodiments, the nucleic acid encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52.

[0179] In a preferred embodiment, the nucleic acid or polypeptide encoded by the isolated nucleic acid is a component of an antigen binding protein that binds OSMR.

[0180] Nucleotide sequences corresponding to the amino acid sequences described herein, to be used as probes or primers for nucleic acid isolation or for database searches of query sequences, can be obtained by "reverse translation" from the amino acid sequence or by identifying regions of amino acid identity with the identified polypeptides for which the DNA sequence codes. Well-known polymerase chain reaction (PCR) procedures can be used to isolate and amplify DNA sequences encoding desired combinations of OSMR antigen binding proteins or polypeptide fragments of OSMR antigen binding proteins. Oligonucleotides defining the desired termini of the combination of DNA fragments are used as 5' and 3' primers. The oligonucleotides can further contain recognition sites for restriction endonucleases to facilitate insertion of the amplified combination of DNA fragments into an expression vector. PCR techniques are described in Saiki et al., Science 239:487 (1988); Recombinant DNA Methodology, Wu et al., eds., Academic Press, Inc., San Diego (1989), pp. 189-196; and PCR Protocols: a Guide to Methods and Applications, Innis et al., eds., Academic Press, Inc. (1990).

[0181] The nucleic acid molecules of the present invention include DNA and RNA in both single-stranded and double-stranded forms, as well as corresponding complementary sequences. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. The nucleic acid molecules of the present invention include full-length genes or cDNA molecules, as well as combinations of fragments thereof. The nucleic acids of the present invention are preferably derived from human sources, although the present invention also includes those derived from non-human species.

[0182] In some embodiments, the nucleic acids of the invention are isolated nucleic acids. An "isolated nucleic acid," in the case of a nucleic acid isolated from a naturally occurring source, is a nucleic acid separated from contiguous gene sequences present in the genome of the organism from which the nucleic acid is isolated. In the case of a nucleic acid synthesized chemically or enzymatically from a template, such as a PCR product, a cDNA molecule, or an oligonucleotide, it is understood that the nucleic acid obtained from such a process is an isolated nucleic acid. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct. In a preferred embodiment, the nucleic acid is substantially free of contaminating endogenous material. The nucleic acid molecule is preferably derived from DNA or RNA that has been isolated at least once in substantially pure form and in an amount or concentration that permits identification, manipulation, and recovery of its component nucleotide sequences by standard biochemical methods (see Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1999). (e.g., those reviewed in "Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)"). Such sequences are preferably provided and / or constructed in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, that are typically present in eukaryotic genes. Sequences of non-translated DNA can be present 5' or 3' from the open reading frame without interfering with manipulation or expression of the coding region.

[0183] The present invention also includes nucleic acids or isolated nucleic acids that hybridize under moderately stringent conditions, and more preferably, highly stringent conditions, to multiple nucleic acids encoding OSMR antigen-binding proteins, as described herein. The basic parameters influencing the selection of hybridization conditions and guidance for designing appropriate conditions are described in Sambrook, Fritsch, and Maniatis (1989, Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11; and Current Protocols in Molecular Biology, 1995, Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4), and can be readily determined by one of skill in the art, based, for example, on the length and / or residue composition of the DNA. One method of achieving moderately stringent conditions involves the use of a pre-wash solution containing 5xSSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), a hybridization buffer containing about 50% formamide, 6xSSC, and a hybridization temperature of about 55°C (or other similar hybridization solutions, e.g., containing about 50% formamide at a hybridization temperature of about 42°C), and wash conditions in 0.5xSSC, 0.1% SDS at about 60°C. Generally, highly stringent conditions are defined as hybridization conditions as above, but with washes at about 68°C, 0.2xSSC, 0.1% SDS. SSPE (1x SSPE is 0.15 M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC hybridization and wash buffer (1x SSC is 0.15 M NaCl and 15 mM sodium citrate); washes are performed for 15 minutes after hybridization is complete.It should be understood that washing temperatures and washing salt concentrations can be adjusted as needed to achieve the desired degree of stringency by applying basic principles governing hybridization reactions and duplex stability, as known to those skilled in the art and further described below (see, e.g., Sambrook et al., 1989). When hybridizing a nucleic acid to a target nucleic acid of unknown sequence, the hybrid length is assumed to be the length of the hybridizing nucleic acid. When hybridizing a nucleic acid of known sequence, the hybrid length can be determined by aligning the sequences of the nucleic acids and identifying the region or regions of optimal sequence complementarity. The hybridization temperature for hybrids expected to be less than 50 base pairs in length should be 5-10°C lower than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following formula: For hybrids less than 18 base pairs in length, Tm (°C) = 2(# of A + T bases) + 4(# of # G + C bases). For hybrids 18 base pairs or longer, Tm(°C) = 81.5 + 16.6 (log 10 [Na + ]) + 0.41(%G+C) - (600 / N), where N is the number of groups in the hybrid and [Na + ] is the concentration of sodium ions in the hybridization buffer ([Na +]=0.165M). Preferably, each such hybridizing nucleic acid has a length of at least 15 nucleotides (or more preferably at least 18 nucleotides, or at least 20 nucleotides, or at least 25 nucleotides, or at least 30 nucleotides, or at least 40 nucleotides, or most preferably at least 50 nucleotides), or at least 25% (more preferably at least 50%, or at least 60%, or at least 70%, and most preferably at least 80%) of the length of the nucleic acid of the invention to which it hybridizes, and has at least 60% sequence identity (more preferably at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, and most preferably at least 99.5%), where sequence identity is determined by comparing the sequences of the hybridizing nucleic acids when aligned to maximize overlap and identity while minimizing sequence gaps, as described in more detail above.

[0184] The variants of the present invention are typically prepared by site-directed mutagenesis of nucleotides in DNA encoding the antigen-binding protein using cassette or PCR mutagenesis or other techniques well known in the art to generate DNA encoding the variant, followed by expression of the recombinant DNA in cell culture, as outlined herein. However, antigen-binding protein fragments containing variant CDRs with up to 100-150 residues can also be prepared by in vitro synthesis using established techniques. The variants typically exhibit the same qualitative biological activity, e.g., binding to OSMR, as the naturally occurring analog, although variants with modified properties can also be selected, as outlined in more detail below.

[0185] As will be appreciated by those skilled in the art, the degeneracy of the genetic code allows for the production of a very large number of nucleic acids, all of which encode the CDRs of the invention (and the heavy and light chains or other components of the antigen binding proteins). Thus, having identified a particular amino acid sequence, one skilled in the art could produce any number of different nucleic acids by simply modifying the sequence of one or more codons in a way that does not change the amino acid sequence of the encoded protein.

[0186] The present invention also provides expression systems and constructs in the form of plasmids, expression vectors, transcription or expression cassettes, which contain at least one polynucleotide as described above. The present invention also provides host cells containing such expression systems or constructs.

[0187] Typically, expression vectors used in any host cell will contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. In certain embodiments, such sequences, collectively referred to as "flanking sequences," will typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence including donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of a nucleic acid encoding an expressed polypeptide, and a selectable marker element. Each of these sequences is described below.

[0188] Optionally, the vector may contain a sequence encoding a "tag," i.e., an oligonucleotide molecule located at the 5' or 3' end of the sequence encoding the OSMR antigen-binding protein; the oligonucleotide sequence may encode polyHis (e.g., hexaHis) or other "tags" such as FLAG, HA (influenza virus hemagglutinin), or myc for which commercially available antibodies exist. This tag is typically fused to the polypeptide upon expression and can serve as a means for affinity purification or detection of the OSMR antigen-binding protein from host cells. Affinity purification can be performed, for example, by column chromatography using antibodies against the tag as an affinity matrix. Optionally, the tag is subsequently removed from the resulting OSMR antigen-binding protein by various means, such as using specific peptidases for cleavage.

[0189] Flanking sequences may be homologous (i.e., from the same species and / or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from multiple sources), synthetic, or naturally occurring. Thus, the source of the flanking sequences may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequences are functional in, and can be activated by, the host cell tissue.

[0190] Flanking sequences useful in the vectors of the present invention can be obtained by any of several methods well known in the art. Typically, flanking sequences useful herein have been previously identified by mapping and / or restriction endonuclease digestion and can be isolated from a suitable tissue source using the appropriate restriction endonuclease. In some cases, the complete nucleotide sequence of the flanking sequence is known. Alternatively, the flanking sequence can be synthesized using the methods described herein for nucleic acid synthesis or cloning.

[0191] If all or even just a portion of the flanking sequences are known, they can be obtained using the polymerase chain reaction (PCR) and / or by screening genomic libraries with appropriate probes, such as oligonucleotides and / or flanking sequence fragments from the same or other species. If the flanking sequences are not known, fragments of DNA containing the flanking sequences can be isolated from larger fragments of DNA that may contain, for example, coding sequences or additional genes. Isolation can be achieved by restriction endonuclease digestion to generate the appropriate DNA fragments followed by isolation using agarose gel purification, Qiagen® column chromatography (Chatsworth, CA), or other methods known to those of skill in the art. The selection of appropriate enzymes to achieve this purpose will be readily apparent to those of skill in the art.

[0192] Origins of replication are usually commercially available prokaryotic expression vectors, and the origin aids in the amplification of the vector in host cells. If the selected vector does not contain an origin of replication site, one can be synthesized based on a known chemical sequence and ligated into the vector. For example, the origin of replication from the plasmid pBR322 (New England Biolabs, Beverly, MA) is suitable for most Gram-negative bacteria, and various viral origins (e.g., SV40, polyoma, adenovirus, vesicular stomatitis virus (VSV), or papillomaviruses such as HPV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not required for mammalian expression vectors (e.g., the SV40 origin is often used simply because it also contains the viral early promoter).

[0193] Typically, a transcription termination sequence is located 3' to the end of a polypeptide coding region and serves to terminate transcription. Usually, the transcription termination in prokaryotic cells is a GC-rich fragment followed by a poly-T sequence. This sequence can be easily cloned from a library or even commercially available as part of a vector, but it can also be easily synthesized using nucleic acid synthesis methods such as those described herein.

[0194] A selectable marker gene encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium. Typical selectable marker genes are: (a) for prokaryotic host cells; (b) confer resistance to antibiotics or other toxins, such as ampicillin, tetracycline, or kanamycin; (b) complement an auxotrophic defect of the cell; or (c) supplying important nutrients not available from complex or defined media; Specific selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. Advantageously, the neomycin resistance gene can also be used for selection in both prokaryotic and eukaryotic host cells.

[0195] Other selection genes can be used to amplify the expressed gene. Amplification is the process by which a gene required for the production of a protein important for growth or cell survival is tandemly repeated in the chromosome of successive generations of recombinant cells. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and promoter-mediated thymidine kinase genes. Mammalian cell transformants are placed under selection pressure in which only transformants are uniquely adapted to survive due to the selectable gene present in the vector. Selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of the selection agent in the medium is successively increased, thereby resulting in amplification of both the selectable gene and DNA encoding other genes, such as antigen-binding protein antibodies, that bind to the OSMR polypeptide. As a result, increased amounts of a polypeptide, such as the OSMR antigen-binding protein, are synthesized from the amplified DNA.

[0196] A ribosome binding site is usually required for translation initiation of mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). The element is typically located 3' to the promoter and 5' to the coding sequence of the expressed polypeptide. In certain embodiments, one or more coding regions can be operably linked to an internal ribosome binding site (IRES), allowing translation of two open reading frames from a single RNA transcript.

[0197] In some cases, such as when glycosylation is desired in eukaryotic host cell expression systems, various pre- or pro-sequences can be engineered to improve glycosylation or yield. For example, the peptidase cleavage site of a particular signal peptide can be altered or a pro-sequence added, which can also affect glycosylation. The final protein product may have one or more additional amino acids at position -1 (relative to the first amino acid of the mature protein) that may not be completely removed upon expression. For example, the final protein product may have one or two amino acid residues found in the peptidase cleavage site attached to the amino terminus. Alternatively, the use of several enzyme cleavage sites may result in a slightly truncated form of the desired polypeptide if the enzyme cleaves at such a region within the mature polypeptide.

[0198] Expression and cloning vectors of the present invention typically contain a promoter recognized by the host organism and operably linked to the molecule encoding the OSMR antigen-binding protein. A promoter is a non-transcribed sequence located upstream (i.e., 5') of the start codon of a construct gene (generally within about 100-1000 bp) that controls transcription of the construct gene. Promoters are traditionally classified into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature. Constitutive promoters, on the other hand, transcribe operably linked genes uniformly, i.e., there is little or no control over gene expression. Numerous promoters recognized by a variety of potential host cells are well known. A suitable promoter is operably linked to heavy or light chain-encoding DNA comprising the OSMR antigen-binding protein of the present invention by removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence into the vector.

[0199] Suitable promoters for use with yeast hosts are also well known in the art. Yeast enhancers are advantageously used with yeast promoters. Promoters suitable for use with mammalian host cells are well known and include, but are not limited to, those obtained from genomic viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and most preferably, simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat shock promoters and actin promoters.

[0200] Additional promoters of interest include, but are not limited to, the SV40 early promoter (Benoist et al., 1981, Nature 290:304-310); the CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad. USA 81:659-663); the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797); the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:1444-1445); promoters and regulatory sequences from metallothionein genes (Prinster et al., 1982, Nature 296:39-42); and prokaryotic promoters such as the beta-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Also of interest are the following animal transcriptional control regions that exhibit tissue specificity and have been utilized in transgenic animals: the elastase I gene control region, which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulin gene control region, which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122); and the immunoglobulin gene control region, which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Hepatology 7:425-515). al., 1987, Mol. Cell. Biol. 7:1436-1444); mouse mammary tumor virus control region, which is active in testicular, breast, lymphoid, and mast cells (Leder et al., 1986, Cell 45:485-495) the albumin gene control region, which is active in the liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); the alpha-fetoprotein gene control region, which is active in the liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 253:53-58); the alpha-1-antitrypsin gene control region, which is active in the liver (Kelsey et al., 1987, Genes and Devel. Devel. 1:161-171); the beta-globin gene control region, which is active in bone marrow cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94); the myelin basic protein gene control region, which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); the myosin light chain-2 gene control region, which is active in skeletal muscle (Sani, 1985, Nature 314:283-286); and the gonadotropin-releasing hormone gene control region, which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

[0201] Enhancers can be inserted into vectors to increase transcription of DNA encoding the light or heavy chain, including the OSMR antigen binding protein of the present invention, by higher eukaryotes. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp in length, that act on a promoter to increase transcription. Enhancers are relatively orientation- and position-independent and have been found at both 5' and 3' positions of transcription units. Several enhancers are known from mammalian genes (e.g., globin, elastase, albumin, α-fetoprotein, and insulin). However, enhancers of viral origin are typically used. SV40 enhancers, cytomegalovirus early promoter enhancers, polyoma enhancers, and adenovirus enhancers, known in the art, are exemplary enhancing elements for the activation of eukaryotic promoters. Enhancers can be positioned either 5' or 3' to the coding sequence in a vector, but are typically located 5' from the promoter. A sequence encoding an appropriate native or heterologous signal sequence (leader sequence or signal peptide) can be incorporated into an expression vector to promote extracellular secretion of the antibody. The choice of signal peptide or leader depends on the type of host cell in which the antibody is to be produced, and a heterologous signal sequence can replace the native signal sequence. Examples of signal peptides functional in mammalian host cells include the interleukin-7 (IL-7) signal sequence described in U.S. Patent No. 4,965,195; the interleukin-2 receptor signal sequence described in Cosman et al., 1984, Nature 312:768; the interleukin-4 receptor signal peptide described in EP Patent No. 0367566; the type I interleukin-1 receptor signal peptide described in U.S. Patent No. 4,968,607; and the type II interleukin-1 receptor signal peptide described in EP Patent No. 0460846.

[0202] A vector may contain one or more elements that facilitate expression when the vector is integrated into a host cell genome. Examples include EASE elements (Aldrich et al. 2003 Biotechnol Prog. 19:1433-38) and matrix attachment regions (MARs). MARs mediate the structural organization of chromatin and can shield integrated vectors from "position" effects. Therefore, MARs are particularly useful when the vector is used to generate stable transfectants. Numerous natural and synthetic MAR-containing nucleic acids are known in the art, e.g., U.S. Pat. Nos. 6,239,328; 7,326,567; 6,177,612; 6,388,066; 6,245,974; 7,259,010; 6,037,525; 7,422,874; 7,129,062, etc.

[0203] The expression vector of the present invention can be constructed from a starting vector, such as a commercially available vector. Such a vector may or may not contain all of the desired flanking sequences. If one or more of the flanking sequences described herein are not already present in the vector, they can be individually obtained and ligated into the vector. The methods used to obtain each flanking sequence are well known to those skilled in the art.

[0204] After a vector has been constructed and a nucleic acid molecule encoding a light chain, heavy chain, or light and heavy chains containing an OSMR antigen-binding sequence has been inserted into the appropriate site of the vector, the completed vector can be inserted into a suitable host cell for amplification and / or polypeptide expression. Transformation of the OSMR antigen-binding protein expression vector into a selected host cell can be carried out by well-known methods, including transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-dextran-mediated transfection, or other known techniques. The method selected will, in part, be a function of the type of host cell used. These and other suitable methods are well known to those of skill in the art and are described, for example, in Sambrook et al., 2001, supra.

[0205] When cultured under appropriate conditions, host cells synthesize an OSMR antigen binding protein that can be subsequently recovered from the culture medium (if the host cells secrete it into the medium) or directly from the host cells that produce it (if it is not secreted). The selection of an appropriate host cell will depend on various factors, such as the desired expression level, modifications of the polypeptide desired or required for activity (such as glycosylation or phosphorylation), and the ease of folding into a biologically active molecule. Host cells can be eukaryotic or prokaryotic.

[0206] Mammalian cell lines available as expression hosts are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC). Any cell line used in an expression system known in the art can be used to produce the recombinant polypeptides of the present invention. Generally, host cells are transformed with a recombinant expression vector containing DNA encoding the desired anti-OSMR antibody polypeptide. Among the host cells that can be used are prokaryotes, yeast, or higher eukaryotic cells. Prokaryotes include gram-negative or gram-positive organisms, such as Escherichia coli or Bacillus. Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include the COS-7 strain of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, and 3T3 cells (A). ATCC CCL 163), Chinese hamster ovary (CHO) cells or their derivatives such as vegetative CHO and related cell lines grown in serum-free medium (Rasmussen et al., 1998, Cytotechnology 28:31), HeLa cells, BHK (ATCC CRL 10) cell lines, and CVI / EBNA cell lines derived from the African green monkey kidney cell line CVI (ATCC CCL 70) as described by McMahan et al., 1991, EMBO J. 10:2821, human embryonic kidney cells such as 293, 293 EBNA or MSR 293, human epithelial A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell lines obtained from in vitro culture of primary tissues, primary explants, HL-60, U937, HaK, or Jurkat cells. If it is desired to use the polypeptide in various signal transduction or reporter assays, mammalian cell lines such as HepG2 / 3B, KB, NIH 3T3, or S49 can optionally be used to express the polypeptide. Alternatively, the polypeptide can be produced in lower eukaryotes such as yeast or in prokaryotes such as bacteria. Suitable yeasts include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous polypeptides. Suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous polypeptides. If the polypeptide is produced in yeast or bacteria, it may be desirable to modify the polypeptide produced therein, for example, by phosphorylation or glycosylation of appropriate sites, to obtain a functional polypeptide. Such covalent linkages can be achieved using known chemical or enzymatic methods. Polypeptides can also be produced in one or more insect expression vectors and using insect expression systems by operably linking the nucleic acid or isolated nucleic acid of the invention to appropriate control sequences.Materials and methods for baculovirus / insect cell expression systems are available, for example, in kit form from Invitrogen, San Diego, CA, USA (the MaxBac® kit). Such methods are commercially available in various forms, and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), and Luckow and Summers, Bio / Technology 6:47 (1988). Cell-free translation systems can also be used to produce polypeptides using RNA derived from the nucleic acid constructs disclosed herein. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described by Pouwels et al. (Cloning Vectors: a Laboratory Manual, Elsevier, New York, 1985). A host cell containing a nucleic acid or isolated nucleic acid of the invention, preferably operably linked to at least one expression control sequence, is a "recombinant host cell."

[0207] In certain embodiments, cell lines can be selected by determining which cell lines have high expression levels and constitutively produce antigen binding proteins with OSMR-binding properties. In other embodiments, cell lines derived from the B cell lineage that do not make their own antibodies but have the ability to make and secrete heterologous antibodies can be selected. Cell-depleted OSMR antigen-binding protein

[0208] In preferred embodiments, the OSMR antigen binding protein binds OSMR and inhibits OSM and / or IL-31 binding, thereby reducing OSM- and / or IL-31-mediated signaling in OSMR-expressing cells. However, in specific embodiments, the OSMR antigen binding protein binds OSMR and targets OSMR-expressing cells for depletion. In various aspects, the OSMR antigen binding protein inhibits OSM and / or IL-31 binding and targets OSMR cells for depletion.

[0209] Cell-depleted OSMR antigen binding proteins are particularly useful for treating diseases or disorders associated with overexpression of OSMR, such as, for example, autoimmune diseases, inflammatory diseases, diseases or disorders associated with extracellular matrix deposition or remodeling, or OSMR-expressing tumors. Methods for targeting cells bearing antigen binding proteins, such as antibodies, are well known in the art. Exemplary embodiments are described below. Antibody-drug conjugates

[0210] Embodiments of the present invention include antibody-drug conjugates (ADCs). Generally, ADCs comprise an antibody conjugated to a chemotherapeutic agent, such as a cytotoxic agent, a cytostatic agent, a toxin, or a radioactive substance. Linker molecules can be used to conjugate the drug to the antibody. A variety of linkers and drugs useful in ADC technology are known in the art and can be used in embodiments of the present invention. (See US20090028856; US2009 / 0274713; US2007 / 0031402; WO2005 / 084390; WO2009 / 099728; US5208020; US5416064; US5475092; 5585499; 6436931; 6372738; and 6340701, all of which are incorporated herein by reference.) Linker

[0211] In certain embodiments, the ADCs comprise a linker composed of one or more linker components. Exemplary linker components include 6-maleimidocaproyl, maleimidopropanoyl, valine-citrulline, alanine-phenylalanine, p-aminobenzyloxycarbonyl, and those resulting from conjugation with linker reagents including, but not limited to, N-succinimidyl-4-(2-pyridylthio)pentanoate (“SPP”), N-succinimidyl 4-(n-maleimidomethyl)cyclohexane-1-carboxylate (“SMCC,” also referred to herein as “MCC”), and N-succinimidyl(4-iodoacetyl)aminobenzoate (“SIAB”).

[0212] The linker can be a "cleavable" linker or a "non-cleavable" linker (Ducry and Stump, Bioconjugate Chem. 2010, 21 ,5-13; the entire contents of which are incorporated herein by reference). Cleavable linkers are designed to release the drug upon exposure to a specific environmental trigger, such as upon internalization into target cells. Cleavable linkers include acid-labile linkers, protease-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers. Non-cleavable linkers tend to remain covalently bound to at least one amino acid of the antibody and drug upon internalization by and degradation within the target cells. An exemplary non-cleavable linker is MCC. Drugs

[0213] In certain embodiments, the antibody is conjugated to a chemotherapeutic agent. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; azirines such as benzodopa, carboquone, meturedopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide, and trimethylolmelamine. Ethylenimines and methylamelamines, including ethylenimines (especially ethylenimines and ethylenimines); acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictycin n); spongistatins; nitrogen mustards such as chlorambucil, chlomaphazine, colofosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; (e.g., calicheamicin, particularly calicheamicin gamma 1 and calicheamicin theta 1, e.g., Agnew Chemical Company, See Intl. Ed. Engl. 33:183-186 (1994); enediyne antibiotics (such as dynemicin A, esperamicin, and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores). Antibiotics such as aclacinomycins, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin (m arcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folates such as denopterin, methotrexate, pteropterin, and trimetrexate purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluricin, enocitabine, floxuridine, and 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane; florinic acid Folic acid replenishers such as aceglatone, aldophosphamide glycosides, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, epothilone, etoglucid, gallium nitrateHydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; Trichothecenes (especially T-2 toxin, verracurin A, loricin ( roridine A and anguidine; urethane; vindesi methadone; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; paclitaxel (TAXOL™, Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel (TAXOTERE®, Rhone-Poulenc Taxoids such as cyclosporine (Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylolnithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. This definition includes, for example, tamoxifen, raloxifene, the aromatase-inhibiting 4(5)-imidazoles, droloxifene, 4-hydroxytamoxifene, Also included are antiestrogens, including phen, trioxifene, keoxifene, LY117018, onapristone, and toremifene; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and antihormonal agents that act to regulate or suppress hormone action on tumors, such as siRNA and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Other chemotherapeutic agents that can be used in the present invention are disclosed in US Publication No. 20080171040 or US Publication No. 20080305044, each of which is incorporated herein by reference in its entirety.

[0214] It is contemplated that the antibody may be conjugated to two or more different chemotherapeutic agents, or the pharmaceutical composition may be characterized as comprising a mixture of antibodies in which the antibody components are identical except that they are conjugated to different chemotherapeutic agents. Such embodiments may be useful for targeting multiple biological pathways in a target cell.

[0215] In a preferred embodiment, the ADC comprises an antibody conjugated to one or more maytansinoid molecules, which are mitotic inhibitors that act to inhibit tubulin polymerization. Maytansinoids containing such modifications are described in U.S. Pat. Nos. 3896111; 4151042; 4137230; 4248870; 4256746; 4260608; 4265814; 4294757; 4307016; 4308268; 4309428; 4313946; 4315929; 4317821; 4322348; 4331598; 4361650; 4364866; 4424219; 4450254; 4362663; 4371533; and WO 2009 / 099728. Maytansinoid drug moieties can be isolated from natural sources, produced using recombinant technology, or prepared synthetically. Exemplary maytansinoids are C-19-dechloro (U.S. Pat. No. 4,256,746), C-20-hydroxy (or C-20-demethyl) + / - C-19-de Chloro (U.S. Pat. Nos. 4,307016 and 4,361650), C-20-demethoxy (or C-20-acyloxy (-OCOR) + / -dechloro (U.S. Pat. No. 4,294757), C-9-SH (U.S. Pat. No. 4,424,219), C-14-alkoxymethyl (demethoxy / CH2OR) (U.S. Pat. No. 4,331,598), C-14 hydroxymethyl or acyloxymethyl (CH2OH or CHOAc) (U.S. Pat. No. 4,450,254), C-15-hydroxy / acyloxy (U.S. Pat. No. 4,364,866), C-15-methoxy C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348), and 4,5-deoxy (U.S. Pat. No. 4,371,533).

[0216] Various positions on the maytansinoid compound can be used as the attachment position, depending on the type of bond desired. For example, the C-3 position bearing a hydroxyl group, the hydrozymethyl-modified C-14 position, the hydroxyl-modified C-15 position, and the C-20 position bearing a hydroxyl group are all suitable for forming an ester bond (U.S. Pat. Nos. 5208020, RE39151, and 6913748; U.S. Patent Appl. Pub. Nos. 20060167245 and 20070037972, and WO 2009099728).

[0217] Preferred maytansinoids are those known in the art as DM1, DM3, and DM4 (US Pat. Appl. Pub. Nos. 2009030924 and 20050276812, incorporated herein by reference).

[0218] ADCs containing maytansinoids, methods for making such ADCs, and their therapeutic uses are described in U.S. Patent Nos. 5,208,020 and 5,416,064, U.S. Pat. Appl. Pub. No. 20050276812, and WO 2009099728 (all incorporated herein by reference). Linkers useful for preparing ADCs of maytansinoids are known in the art (U.S. Pat. No. 5208020 and U.S. Pat. Appl. Pub. Nos. 2005016993 and 20090274713; all incorporated herein by reference). ADCs of maytansinoids containing an SMCC linker can be prepared as disclosed in U.S. Pat. Pub. No. 2005 / 0276812. Antibodies with enhanced effector functions

[0219] One function of the Fc portion of an antibody is to communicate with the immune system when the antibody binds to its target. This is considered an "effector function." Communication can result in antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). ADCC and ADCP are mediated through binding of Fc to Fc receptors on the surface of cells of the immune system. CDC is mediated through binding of Fc to complement system proteins, such as C1q.

[0220] IgG subclasses vary in their ability to mediate effector functions. For example, IgG1 is much better than IgG2 and IgG4 at mediating ADCC and CDC. Therefore, in embodiments in which cells expressing OSMR are targeted for destruction, anti-OSMR IgG1 antibodies may be preferred.

[0221] The effector function of an antibody can be increased or decreased by introducing one or more mutations into the Fc. Embodiments of the present invention include antigen-binding proteins, such as antibodies, with Fc engineered to increase effector function (US Pat. No. 7,317,091 and Strohl, Curr. Opin. Biotech., 20:685-691, 2009; both are incorporated herein by reference in their entireties). An exemplary IgG1 Fc molecule engineered to increase effector function contains the following substitutions (based on the Kabat numbering scheme):

[0222] S239D / I332E

[0223] S239D / A330S / I332E

[0224] S239D / A330L / I332E

[0225] S298A / D333A / K334A

[0226] P247I / A339D

[0227] P247I / A339Q

[0228] D280H / K290S

[0229] D280H / K290S / S298D

[0230] D280H / K290S / S298V

[0231] F243L / R292P / Y300L

[0232] F243L / R292P / Y300L / P396L

[0233] F243L / R292P / Y300L / V305I / P396L

[0234] G236A / S239D / I332E

[0235] K326A / E333A

[0236] K326W / E333S

[0237] K290E / S298G / T299A

[0238] K290N / S298G / T299A

[0239] K290E / S298G / T299A / K326E

[0240] K290N / S298G / T299A / K326E

[0241] Further embodiments of the present invention include antigen binding proteins, e.g., antibodies, having an Fc engineered to reduce effector function. Exemplary Fc molecules with reduced effector function include the following substitutions (based on the Kabat numbering scheme):

[0242] N297A(IgG1)

[0243] L234A / L235A(IgG1)

[0244] V234A / G237A(IgG2)

[0245] L235A / G237A / E318A(IgG4)

[0246] H268Q / V309L / A330S / A331S(IgG2)

[0247] C220S / C226S / C229S / P238S(IgG1)

[0248] C226S / C229S / E233P / L234V / L235A(IgG1)

[0249] L234F / L235E / P331S(IgG1)

[0250] S267E / L328F(IgG1)

[0251] Another method for increasing the effector function of IgG Fc-containing proteins is by reducing Fc fucosylation. Removal of core fucose from biantennary complex oligosaccharides attached to Fc significantly increased ADCC effector function without altering antigen binding or CDC effector function. Several methods are known for reducing or eliminating fucosylation of Fc-containing molecules, such as antibodies. These include recombinant expression in certain mammalian cell lines, including FUT8 knockout cell lines, the mutant CHO line Lec13, the rat hybridoma cell line YB2 / 0, cell lines containing small interfering RNA specific for the FUT8 gene, and cell lines coexpressing β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II. Alternatively, Fc-containing molecules can be expressed in non-mammalian cells, such as plant cells, yeast, or prokaryotic cells, such as E. coli. In certain embodiments of the invention, the composition comprises an antibody, such as, for example, Ab1, Ab2, or Ab3, that has reduced fucosylation or is completely devoid of fucosylation. Pharmaceutical Composition

[0252] In some embodiments, the present invention provides pharmaceutical compositions comprising a therapeutically effective amount of one or more antigen binding proteins of the present invention together with a pharmaceutically effective diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant. In certain embodiments, the antigen binding protein is an antibody. Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0253] Preferably, formulation materials are non-toxic to recipients at the dosages and concentrations employed. In certain embodiments, pharmaceutical compositions are provided that include a therapeutically effective amount of an OSMR antigen binding protein, e.g., an OSMR-binding antibody.

[0254] In certain embodiments, pharmaceutical compositions may contain formulation materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. In such embodiments, suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine, proline, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite, sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and (glycols). other carbohydrates (such as cellulose, mannose, or dextrins); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; These include, but are not limited to, surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, Triton, tromethamine, lecithin, cholesterol, tyloxapol, etc.); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol, sorbitol, etc.); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. See REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company.

[0255] In certain embodiments, optimal pharmaceutical compositions can be determined by those of skill in the art, depending, for example, on the intended route of administration, delivery format, and desired dosage. See, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In certain embodiments, such compositions may influence the physical state, stability, in vivo release rate, and in vivo clearance rate of the antigen-binding proteins of the invention. In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition may be aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, saline, or artificial cerebrospinal fluid, possibly supplemented with other substances common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin is a further exemplary vehicle. In specific embodiments, the pharmaceutical composition comprises Tris buffer of about pH 7.0-8.5 or acetate buffer of about pH 4.0-5.5, and may further include sorbitol or a suitable substitute thereof. In certain embodiments of the invention, OSMR antigen binding protein compositions can be prepared for storage in the form of a lyophilized cake or aqueous solution by mixing selected compositions of the desired purity with any prescription drug (REMINGTON'S PHARMACEUTICAL SCIENCES, supra.) Additionally, in certain embodiments, OSMR antigen binding protein products can be formulated as lyophilizates using appropriate excipients, such as sucrose.

[0256] The pharmaceutical compositions of the present invention can be selected for parenteral administration. Alternatively, the compositions can be selected for administration via inhalation or the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of those in the art. The formulation components are preferably in concentrations acceptable to the site of administration. In certain embodiments, a buffering agent is used to maintain the composition at physiological pH or at a slightly lower pH, typically within the pH range of about 5 to about 8.

[0257] When parenteral administration is intended, the therapeutic compositions used in the present invention can be provided in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired OSMR antigen-binding protein in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, in which the OSMR antigen-binding protein is formulated as a sterile, isotonic solution and properly stored. In certain embodiments, the preparation can involve formulation of the desired molecule with substances such as injectable microspheres, biodegradable particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, which can provide controlled or sustained release of the product that can be delivered via depot injection. In certain embodiments, hyaluronic acid, which has the effect of promoting duration in the circulation, can also be used. In certain embodiments, implantable drug delivery devices can be used to introduce the desired antigen-binding protein.

[0258] Pharmaceutical compositions of the invention can be formulated for inhalation. In these embodiments, the OSMR antigen binding protein is advantageously formulated as a dry, inhalable powder. In specific embodiments, an OSMR antigen binding protein inhalation solution can be formulated with a propellant for aerosol delivery. In certain embodiments, the solution can be nebulized. Thus, methods of pulmonary administration and formulation are further described in International Patent Application No. PCT / US94 / 001875, which is incorporated by reference and describes pulmonary delivery of chemically modified proteins.

[0259] It is also contemplated that the formulation may be administered orally. OSMR antigen binding protein administered in this manner may be formulated with or without carriers typically used in compounding solid dosage forms such as tablets and capsules. In certain embodiments, capsules may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be included to enhance absorption of the OSMR antigen binding protein. Diluents, flavorings, low-melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be used.

[0260] Additional pharmaceutical compositions, including formulations containing OSMR antigen binding proteins in sustained- or controlled-delivery formulations, will be apparent to those skilled in the art. Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and depot injections, are also well known to those skilled in the art. See, for example, International Patent Application No. PCT / US93 / 00829, which is incorporated by reference and describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. Sustained-release formulations can include semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices include polyester hydrogels, polylactides (disclosed in U.S. Pat. No. 3,773,919 and European Patent Application Publication No. EP 058481, each incorporated by reference), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinyl acetate (Langer et al., 1981, supra) or poly-D(-)-3-hydroxybutyric acid (EUROPEAN Patent Application Publication No. EP 133,988). Sustained-release compositions may also include liposomes, which may be prepared by any of several methods known in the art. See, for example, Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-3692; European Patent Application Publication Nos. EP 133,988, incorporated by reference. See EP 036,676; EP 088,046 and EP 143,949.

[0261] Pharmaceutical compositions for in vivo administration are typically provided as sterile preparations.Sterilization can be achieved by filtration through a sterile filtration membrane.If the composition is lyophilized, sterilization using this method can be carried out either before or after lyophilization and remodeling.Compositions for parenteral administration can be stored in lyophilized form or in solution.Parenteral compositions are generally placed in a container with a sterile access port, such as an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0262] Aspects of the present invention include self-buffering OSMR antigen binding protein formulations, as described in International Patent Application WO 06138181A2 (PCT / US2006 / 022599), which is incorporated herein by reference in its entirety.

[0263] As noted above, certain embodiments provide protein compositions of OSMR antigen binding proteins, particularly pharmaceutical OSMR antigen binding proteins, that include, in addition to the OSMR antigen binding protein, one or more excipients, such as those illustratively described in this section and elsewhere herein. Excipients can be used in this regard for a variety of purposes, such as adjusting the physical, chemical, or biological properties of the formulation, such as adjusting viscosity, and / or for the processes of the invention to improve the efficiency and / or stabilize such formulations and processes against deterioration and damage due to stresses encountered during and after manufacture, transportation, storage, preparation prior to use, and management.

[0264] Regarding protein stabilization and formulation materials and methods useful in this regard, see Arakawa et al., "Solvent interactions in pharmaceutical formulations," Pharm Res. 8(3):285-91 (1991); Kendrick et al., "Physical stabilization of proteins in aqueous solution," in: Rational Design of Stable Protein Formulations: Theory and Practice, Carpe Various disclosures are available, such as, for example, John Deacon and Manning, eds. Pharmaceutical Biotechnology. 13: 61-84 (2002), and Randolph et al., "Surfactant-protein interactions," Pharm Biotechnol. 13: 159-75 (2002), each of which is incorporated herein by reference in its entirety, particularly in relation to protein pharmaceuticals and processes for veterinary and / or human medical use, as well as portions relating to the same excipients and processes for self-buffering protein formulations according to the present invention.

[0265] For example, salts may be used in accordance with certain embodiments of the invention to adjust the ionic strength and / or tonicity of the formulation and / or to improve the solubility and / or physical stability of proteins or other components of compositions in accordance with the invention.

[0266] As is well known, ions can stabilize the native state of proteins by binding to charged residues on the surface of the protein and by shielding charged and polar groups in the protein, thereby reducing the strength of electrostatic, attractive, and repulsive interactions. Ions can also stabilize the denatured state of proteins, particularly by binding to the denatured peptide bond (--CONH) of proteins. Furthermore, ionic interactions with charged and polar groups in proteins can also reduce intermolecular electrostatic interactions, thereby preventing or reducing protein aggregation and insolubility.

[0267] Ionic species vary greatly in their effects on proteins. Many categorical rankings of ions and their effects on proteins have been developed that can be used to formulate pharmaceutical compositions according to the present invention. One example is the Hofmeister series, which ranks ionic and polar nonionic solutes by their effect on the conformational stability of proteins in solution. Stabilizing solutes are called "kosmotropics." Destabilizing solutes are called "chaotropics." Kosmotropes are commonly used at high concentrations (e.g., >1 molar ammonium sulfate) to precipitate proteins from solution ("salting out"). Chaotropes are commonly used in dentures and / or to solubilize proteins ("salting in"). The relative effectiveness of an ion for "salting out" and "salting in" defines its position within the Hofmeister series.

[0268] Free amino acids can be used in OSMR antigen binding protein formulations according to various embodiments of the invention as bulking agents, stabilizers, and antioxidants, as well as for other standard uses. Lysine, proline, serine, and alanine can be used to stabilize proteins in the formulation. Glycine is useful for lyophilization to ensure proper cake structure and properties. Arginine can be useful in inhibiting protein aggregation in both liquid and lyophilized formulations. Methionine is useful as an antioxidant.

[0269] Polyols include sugars such as mannitol, sucrose, and sorbitol, as well as polyhydric alcohols such as glycerol and propylene glycol, and, for purposes of this specification, polyethylene glycol (PEG) and related substances. Polyols are kosmotropic. They are useful stabilizers in both liquid and lyophilized formulations to protect proteins from physical and chemical degradation processes. Polyols are also useful for adjusting the tonicity of the formulation.

[0270] Among the polyols useful in selected embodiments of the present invention is mannitol, which is commonly used in lyophilized formulations to ensure cake structural stability. Mannitol provides structural stability to the cake. Mannitol is typically used in conjunction with a lyoprotectant, such as sucrose. Sorbitol and sucrose are among the preferred stabilizers for adjusting tonicity and protecting against freeze-thaw stress during bulk transport or preparation during the manufacturing process. For example, reducing sugars (containing free aldehyde or ketone groups), such as glucose and lactose, can glycate surface lysine and arginine residues. Therefore, they are generally not among the preferred polyols for use in accordance with the present invention. Furthermore, sugars that hydrolyze to fructose and glucose under acidic conditions, thereby forming such reactive species as sucrose that result in glycation, are not among the preferred polyols of the present invention in this regard. PEG is useful for stabilizing proteins and as a lyoprotectant, and in this regard can be used in the present invention.

[0271] Embodiments of OSMR antigen binding protein formulations further comprise a surfactant. Protein molecules may be susceptible to adsorption on surfaces and to denaturation and resulting aggregation at air-liquid, solid-liquid, and liquid-liquid interfaces. These effects generally scale inversely with protein concentration. These adverse interactions generally scale inversely with protein concentration and are typically exacerbated by physical agitation, such as that generated during shipping and handling.

[0272] Surfactants are commonly used to prevent, minimize, or reduce surface adsorption. In this regard, surfactants useful in the present invention include polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan polyethoxylate, and poloxamer 188.

[0273] Detergents are also commonly used to control the conformational stability of proteins. In this respect, the use of detergents is subsequently protein-specific, with any given detergent typically stabilizing some proteins and destabilizing others.

[0274] Polysorbates are susceptible to oxidative degradation and, as supplied, often contain sufficient amounts of peroxide to cause oxidation of protein residue side chains, particularly methionine. For this reason, polysorbates should be used with caution and, if used, at their lowest effective concentrations. In this respect, polysorbates exemplify the principle that excipients should be used at their lowest effective concentrations.

[0275] Embodiments of OSMR protein formulations further include one or more antioxidants. Harmful oxidation can be prevented to some extent in pharmaceutical formulations by maintaining adequate levels of ambient oxygen and temperature and avoiding exposure to light. Antioxidant excipients can also be used to prevent oxidative degradation of proteins. Useful antioxidants in this regard include reducing agents, oxygen / free radical scavengers, and chelating agents. Antioxidants for therapeutic protein formulations of the present invention are preferably water-soluble and maintain protein activity throughout the shelf life of the product. EDTA is a preferred antioxidant in this regard.

[0276] Antioxidants can damage proteins. For example, reducing agents, particularly glutathione, can disrupt intramolecular disulfide bonds. Therefore, the antioxidants used in the present invention are selected to eliminate or significantly reduce their own potential to damage proteins in the formulation.

[0277] Formulations according to the present invention may contain metal ions, which are protein cofactors and required to form protein coordination complexes, such as zinc, which is necessary to form certain insulin suspensions. Metal ions can also inhibit some processes that degrade proteins. However, metal ions also catalyze the physical and chemical processes that degrade proteins.

[0278] Magnesium ions (10-120 mM) can be used to inhibit the isomerization of aspartate to isoaspartate. +2 ions (up to 100 mM) can increase the stability of human deoxyribonuclease. +2 ,Mn +2 , and Zn +2 can destabilize rhDNase. +2 and Sr + 2 can stabilize factor VIII, which is +2 ,Mn +2 and Zn +2 ,Cu +2 and Fe +2 and the aggregation can be destabilized by Al +3 It can be augmented by ions.

[0279] Embodiments of OSMR antigen binding protein formulations further include one or more preservatives. Preservatives are necessary when developing multi-dose parenteral formulations involving multiple extractions from the same container. Their primary function is to inhibit microbial growth and ensure product sterility throughout the shelf life or use period of the formulation. Commonly used preservatives include benzyl alcohol, phenol, and m-cresol. While preservatives have a long history of use in parenteral administration of small molecules, developing protein formulations containing preservatives presents significant challenges. Preservatives almost always have a destabilizing effect on proteins (aggregation), which has been a major factor limiting their use in multi-dose protein formulations. To date, most protein drugs have been formulated for single-use only. However, when multi-dose formulations are possible, they offer the added benefit of allowing patient convenience and increased marketability. A good example is that of human growth hormone (hGH), where the development of a preserved formulation led to the commercialization of a more convenient, multi-use injection pen. At least four such pen devices containing a preserved formulation of hGH are currently on the market. Norditropin (liquid, Novo Nordisk) rdisk), Nutropin AQ (liquid, Genentech) & Genotropin (lyophilized-dual chamber cartridge, Pharmacia & Upjohn) contain phenol, while Somatrope (Eli Lilly) contains m-cresol. It is formulated using

[0280] Several aspects need to be considered during the formulation and development of a preserved formulation. The effective preservative concentration in the pharmaceutical product must be optimized. This requires testing a given preservative in a dosage form with a range of concentrations that provide antimicrobial efficacy without compromising protein stability.

[0281] As expected, developing a liquid formulation containing a preservative is more challenging than a lyophilized formulation. Lyophilized products can be lyophilized without preservatives and then remodeled with a preservative-containing diluent at the time of use. This reduces the time the preservative is in contact with the protein, significantly minimizing the stability risks associated with this exposure. For liquid formulations, the preservative's effectiveness and stability must be maintained over the entire product shelf life (approximately 18-24 months). It is important to note that preservative effectiveness must be demonstrated in the final formulation, which contains the active drug and all excipient components.

[0282] OSMR antigen binding protein formulations will generally be designed for a particular route and method of administration, a particular dosage and frequency of administration, a particular treatment of a particular disease, and with a particular range of bioavailability and duration. Thus, formulations can be designed in accordance with the present invention for delivery by any suitable route, including, but not limited to, oral, otic, ocular, rectal, and vaginal, as well as parenteral routes, including intravenous and intraarterial, intramuscular, and subcutaneous injection.

[0283] Once the pharmaceutical composition has been formulated, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, crystal, or dehydrated or lyophilized powder. Such formulations can be stored either in a ready-to-use form or in a form that is remodeled (e.g., lyophilized) prior to administration. The present invention also provides kits for producing single-dose administration units. Each kit of the present invention can include both a first container with a dried protein and a second container with an aqueous formulation. In certain embodiments of the present invention, single-chamber and multi-chamber pre-filled syringes (e.g., liquid syringes and A kit is provided that includes the syringe and a lyophilized syringe.

[0284] The therapeutically effective amount of a pharmaceutical composition containing OSMR antigen binding protein used will depend, for example, on the context and purpose of the treatment. Those skilled in the art will appreciate that appropriate dosage levels for treatment will vary, in part, depending on the molecule being delivered, the indication for which OSMR antigen binding protein is being used, the route of administration, and the patient's size (weight, body surface, or organ size) and / or condition (age and general health). In certain embodiments, clinicians can titrate dosages and modify the route of administration to obtain optimal therapeutic effects. Typical doses can range from about 0.1 μg / kg to about 30 mg / kg or more, depending on the factors described above. In specific embodiments, doses can range from 1.0 μg / kg to about 20 mg / kg, alternatively from 10 μg / kg to about 10 mg / kg, or from 100 μg / kg to about 5 mg / kg.

[0285] A therapeutically effective amount of an OSMR antigen binding protein preferably results in a decrease in the severity of disease symptoms, an increase in the frequency or duration of disease symptom-free periods, or prevention of disability resulting from the infliction of injury or disease.

[0286] The pharmaceutical composition can be administered using a medical device. Examples of medical devices for administering pharmaceutical compositions are described in U.S. Patent Nos. 4,475,196; 4,439,196; 4,447,224; 4,447,233; 4,486,194; 4,4 87,603; 4,596,556; 4,790,824; 4,941,880; 5,064,413; 5,312,335; 5,312,335; 5,383,851; and 5,399,163, all of which are incorporated herein by reference. Methods for diagnosing or treating OSMR-related diseases or disorders

[0287] The OSMR antigen binding protein of the present invention is particularly useful for detecting OSMR in biological samples. In certain embodiments, a biological sample obtained from a patient is contacted with the OSMR antigen binding protein. The binding of OSMR to the OSMR antigen binding protein is then detected to determine the presence or relative amount of OSMR in the sample. Such a method can be useful for diagnosing or determining whether a patient is suitable for treatment with the OSMR antigen binding protein.

[0288] In specific embodiments, the OSMR antigen binding proteins of the invention are used to diagnose, detect, or treat autoimmune disorders, inflammatory disorders, or disorders associated with extracellular matrix deposition or remodeling.

[0289] In treating these disorders, OSMR antigen binding proteins can target OSMR-expressing cells of the immune system for destruction and / or inhibit the expression of OSMR, OSM, and / or Alternatively, the interaction with IL-31 can be blocked.

[0290] A disease or disorder associated with OSMR-mediated signaling is one of the diseases or disorders disclosed herein. These are particularly suitable for treatment with OSMR antigen binding proteins.Such disorders include inflammation, pain, pruritus, prurigo nodularis, dermatitis, asthma, autoimmune diseases, paraneoplastic autoimmune diseases, cartilage inflammation, fibrosis (including, but not limited to, pulmonary fibrosis and dermal fibrosis), fibro, fibrotic diseases, chronic obstructive pulmonary disease (COPD), interstitial pneumonia, abnormal collagen deposition, systemic cutaneous amyloidosis, primary cutaneous amyloidosis, Behcet's disease, nasal polyposis, cirrhosis of the liver, cartilage degradation, bone degradation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, total rheumatoid arthritis, ... Juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic rheumatoid arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic-onset rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), dermatomyositis, psoriatic arthritis, scleroderma, scleroderma pulmonary disease, vasculitis, myelitis , polymyositis, dermatomyositis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, scleroderma, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, rhinosinusitis, polyps These conditions include, but are not limited to, sinusitis associated with rhinosinusitis, eosinophilic esophagitis, eosinophilic bronchitis, bronchitis, Guillain-Barré disease, type 1 diabetes, thyroiditis (e.g., Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, GVHD, transplant rejection, nephropathy, cardiovascular disease, infection, sepsis, HIV infection, trauma, kidney transplant nephropathy, IgA nephropathy, diabetic nephropathy, diabetic retinopathy, macular degeneration, biliary atresia, congestive heart failure, atherosclerosis, restenosis, radiation-induced fibrosis, chemotherapy-induced fibrosis, burns, surgical trauma, glomerulosclerosis, and the like.

[0291] In a preferred embodiment, the autoimmune disorder, inflammatory disorder, or disorder associated with impaired extracellular matrix deposition or remodeling is fibrosis, cartilage degradation, arthritis, rheumatoid arthritis, scleroderma, scleroderma-associated interstitial lung disease, idiopathic pulmonary fibrosis, liver cirrhosis, psoriasis, atopic dermatitis, systemic cutaneous amyloidosis, primary cutaneous amyloidosis, inflammation, pruritus, inflammation, prurigo nodularis, and pain.

[0292] In certain embodiments, the OSMR antigen binding proteins of the present invention are used to diagnose, detect, or treat cancer or tumorigenic disorders. In treating cancer or tumorigenic disorders, the OSMR antigen binding proteins target OSMR-expressing cells of the immune system for destruction. and / or can block the interaction of OSMR with OSM and / or IL-31, thereby reducing OSMR-mediated signaling. OSMR antigen binding proteins that block OSM- and / or IL-31-mediated signaling are expected to be useful in promoting improved survival in cancer patients. Cancers or tumorigenic disorders that can be diagnosed, detected, or treated with OSMR antigen binding proteins include, but are not limited to, general solid tumors, lung cancer, ovarian cancer, breast cancer, prostate cancer, endometrial cancer, kidney cancer, esophageal cancer, pancreatic cancer, squamous cell carcinoma, uveal melanoma, cervical cancer, colorectal cancer, bladder cancer, brain, pancreas, head, neck cancer, liver cancer, leukemia, lymphoma, and Hodgkin's disease, multiple myeloma, melanoma, gastric cancer, astrocytic carcinoma, stomach, and lung adenocarcinoma.

[0293] Antigen binding proteins can be used to inhibit tumor growth, progression, and / or metastasis. Such inhibition can be monitored using various methods. For example, inhibition can result in a decrease in tumor size and / or a decrease in metabolic activity within the tumor. Both of these parameters can be measured, for example, by MRI or PET scan. Inhibition can also be monitored by biopsy to determine the level of necrosis, tumor cell death, and intratumoral vascularity. The extent of metastasis can be monitored using known methods.

[0294] The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to further clarify embodiments of the invention and does not limit the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. [Example]

[0295] The following examples, both actual and prophetic, are provided for the purpose of illustrating specific embodiments and features of the present invention and are not intended to limit its scope. Example 1: Generation of anti-OSMR antibodies using the XENOMOUSE® platform

[0296] Fully human antibodies directed against human OSMR have been developed using XENOMOUSE® technology (United States Patent Nos. 6,114,598; 6,162,963; 6,833,268; 7,049,426; 7,064,244, which and Green et al., Nat. ure Genetics 7:13-21, 1994; Mendez et al., Nature Genetics 15:146-56; 1997; Green et al., J.Ex.Med. 188:483-95, 1998; and Kellerman n et al., Current Opinion in Biotechnology, 13:593-7, 2002).

[0297] To generate antibodies to OSMR, two strains of XENOMOUSE® animals, i.e., XMG2-KL and XMG4-KL mice, were immunized with human OSMR-Fc soluble protein (prepared by Amgen, Seattle, Wash.). An appropriate amount of immunogen (i.e., 10 μg / mouse of soluble human OSMR-Fc protein) was used for the initial immunization of XENOMOUSE® animals, as disclosed in U.S. Patent Application Serial No. 08 / 759,620, filed December 3, 1996, and International Patent Applications WO 98 / 24893, published June 11, 1998, and WO 00 / 76310, published December 21, 2000. The antibodies were used according to the methods described in the literature, the disclosures of which are incorporated herein by reference. After the initial immunization, subsequent boosts of immunogen (5 μg / mouse of soluble human OSMR-Fc protein) were administered according to a schedule for a period necessary to induce adequate titers of anti-OSMR antibodies in the mice.

[0298] Serum was collected approximately 4 weeks after the first injection, and specific titers were determined by ELISA. The protocol used to titer XENOMOUSE® animals was as follows: Costar 3368 medium-binding plates were coated with neutravidin at 8 μg / mL (50 μL / well) and incubated overnight at 4°C in 1X PBS / 0.05% azide. Plates were washed with Titertek 3-cycle wash with RO water. Plates were blocked with 250 μL of 1X PBS / 1% milk and incubated at room temperature for at least 30 minutes. The block was rinsed with RO water using Titertek 3-cycle wash. Biotinylated huOSMR-FNFH (prepared at Amgen, Seattle, WA) was then captured at 2 μg / mL in 50 μL / well of 1X PBS / 1% milk / 10 mM Ca2+ (assay diluent) and incubated at room temperature for 1 hour. The plates were then washed with RO water using a Titertek 3-cycle wash. For the primary antibody, sera were titrated 1:3 in duplicate starting at 1:100. This was done in 50 μL / well of assay diluent and incubated for 1 hour at room temperature. Then, the plates were washed with RO water using a Titertek 3-cycle wash. The secondary antibody was 400 ng / mL goat anti-human IgG Fc HRP in 50 μL / well of assay diluent. This was incubated for 1 hour at room temperature. Then, the plates were washed with RO water using a Titertek 3-cycle wash and tapped dry on a paper towel. The substrate was developed using a one-step TMB solution (Neogen, Lexington, Kentucky) (50 μL / well) for 30 minutes at room temperature.

[0299] Animals showing appropriate titers were identified. 5XMG2KL animals were identified with IgG immune responses specific to OSMR. Spleens and draining lymph nodes were harvested from these animals and pooled together for hybridoma generation. 5XMG4KL animals with specific immune responses were similarly collected and carried forward as separate fusion screening campaigns. Enriched B cells from immunized animals were fused to non-secretory myeloma P3x63AG8.653 cells (American Type Culture Collection CRL-1580; Kearney et al., J. Immunol. 123:1548-50, 1979) to generate hybridomas using standard techniques (Kohler et al., Nature 256, 495-7, 1975).

[0300] Hybridomas were then seeded at high density (multiple distinct hybridoma clones per well) onto 96-well tissue culture plates and grown for 4 weeks. Supernatants from hybridoma lines were screened for binding to full-length human and cynomolgus OSMR expressed on transiently transfected 293T cells using fluorescence microvolume assay technology (FMAT) (Applied Biosystems, Foster City, CA). Briefly, in a 384-well FMAT plate, a 40 μl mixture of 3,000 OSMR 293T transfected cells and 15,000 parental 293T cells was mixed with 15 μL of hybridoma supernatant and 10 μL of anti-human light chain (hukappa / hulambda) Alexa647 (Invitrogen, Carlsbad, CA) labeled secondary antibody (final concentration of 1.0 μg / mL). Plates were then incubated at room temperature for 3 hours, and fluorescence was read using an FMAT reader. These screens identified 885 hybridoma lines that bound to human and cynomolgus OSMR. Example 2: Human OSMR Blocking Assay

[0301] The ability of OSMR antibodies to block signaling through human OSMR was determined using two assays with either human oncostatin M (OSM) or human interleukin-31 (IL-31) as the ligand. In combination, the assays were used to determine whether antibodies could inhibit OSMR signaling triggered through binding of OSM and / or IL-31.

[0302] In the initial screen, antibodies were evaluated for their ability to block OSM signaling through the OSMR. Stimulation of primary normal human lung fibroblasts with OSM induces STAT3 phosphorylation and subsequent nuclear translocation. Cells were seeded at 3,000 cells per well in Costar 384-well plates and allowed to adhere overnight. Cells were pretreated with antibody supernatant for 20 minutes and then stimulated with 80 pM human OSM for 30 minutes. Cells were then washed three times with PBS, fixed with 3.5% formaldehyde solution, washed three times with PBST, and permeabilized with 0.5% Triton X-100 solution. Cells were then stained with anti-phospho-STAT3 antibody for 1 hour, washed, and stained with AlexaFluor-conjugated antibodies (all included in the HitKit from Cellomics). Plates were covered and read on an ArrayScan instrument using Cellomics' proprietary algorithm to generate nuclear and cytoplasmic intensity values. Results were reported as the difference between these two values and were further normalized to control for data including maximally stimulated and medium-treated cells (POC).

[0303] In a second assay, antibodies were evaluated for their ability to inhibit IL-31 growth signaling through OSMR in a stable cell line overexpressing human IL-31RA4 and OSMR. BaF3 cells were stably transfected with two plasmids: pcDNA3.1+huOSMRb(NeoR) and pcDNA3.1+huIL31RA4(ZeoR). In the absence of mouse IL-3, this cell line was able to proliferate in response to human IL-31 and could therefore be used to specifically evaluate the blocking ability of anti-OSMR antibodies. BaF3 cells were seeded into 96-well plates at a density of 20,000 cells per well. Antibodies and ligands (huIL-31, Peprotech) were added to the wells in a final volume of 100 μL, and the plates were incubated in a humidified chamber at 37°C with 5% CO2 for 72 hours. After incubation, 20 μL of Alamar Blue was added to each well, and the plates were returned to the incubator. Plates were incubated with Molecular Devices Vmax™ at various time points after addition of Alamar Blue. The plate was read using a plate reader (570-600 nm).

[0304] The results of the two assays are shown in Table 4 below. Over 3,000 hybridoma supernatants were screened for blocking ability in these two assays; the top 200 blockers were further tested in four-point titrations, and 14 were selected for recombinant protein production and further testing. The IC50s of three exemplary antibodies (Antibodies 1-3) are shown for both assays. Some antibodies inhibit OSM-induced STAT3 translocation more completely than they inhibit IL-31-induced proliferation, and vice versa. However, all three antibodies were potent inhibitors of OSM- and IL-31-mediated signaling. [Table 4] Example 3: Cynomolgus Macaque OSMR Blocking Assay

[0305] The ability of OSMR antibodies to block signaling through cynomolgus OSMR was investigated using two assays with either human OSM or human IL-31 as the ligand.

[0306] In the initial screening, antibodies were evaluated for their ability to block OSM signaling through cynomolgus monkey OSMR using a primary kidney epithelial cell line. Stimulation of these cells with cynomolgus monkey (cyno) OSM induces STAT3 phosphorylation and subsequent nuclear translocation. Cells were seeded at 3,000 cells per well in Costar 384-well plates and allowed to adhere overnight. Cells were pretreated with antibody supernatant for 20 minutes and then stimulated with 80 pM cynomolgus monkey OSM for 30 minutes. Cells were then washed three times with PBS, fixed with 3.5% formaldehyde solution, washed three times with PBST, and permeabilized with 0.5% Triton X-100 solution. Cells were then stained with anti-phospho-STAT3 antibody for 1 hour, washed, and stained with AlexaFluor-conjugated antibodies (all included in the HitKit from Cellomics). Plates were covered and read on an ArrayScan instrument using Cellomics' proprietary algorithm to generate nuclear and cytoplasmic intensity values. Results were reported as the difference between these two values and further normalized to control for data including maximally stimulated and medium-treated cells (POC).

[0307] In a second assay, antibodies were evaluated for their ability to inhibit IL-31 growth signaling through cynomolgus OSMR in a stable cell line overexpressing cynomolgus IL-31RA and OSMR. As in Example 2, BaF3 cells were seeded into 96-well plates at a density of 20,000 cells per well. Antibodies and ligands (cynomolgus IL-31, in-house, Amgen, Seattle, WA) were added to the wells in a final volume of 100 μL, and the plates were incubated in a humidified chamber at 37°C with 5% CO for 72 hours. After incubation, 20 μL of Alamar Blue was added to each well, and the plates were returned to the incubator. Plates were read on a Molecular Devices Vmax Plate reader (570-600 nm) at various time points after addition of Alamar Blue.

[0308] The results of the two assays are shown below in Table 5, along with the IC50 for each antibody shown for both assays. The results confirm that antibodies 1, 2, and 3 are each potent inhibitors of OSM- and IL-31-mediated signaling. [Table 5] Example 4: Epitope binning of anti-OSMR antibodies

[0309] Antibody competition studies were performed to characterize the epitopes of anti-OSMR xenomouse antibodies. Antibodies that compete with each other can be considered to bind the same site on the target. In these experiments, OSMR or an unrelated antibody was captured on streptavidin-coated Luminex beads (biotinylated monovalent mouse anti-human IgG Fc antibody) pre-bound to the capture antibody. OSMR antigen or buffer (no antigen) was added to the wells, and a probe antibody was added to each well and detected with a PE-labeled monovalent mouse anti-human IgG Fc antibody. The mean fluorescence intensity of each well was measured. For a complete reference, see Jia et al., J. Immunol. Methods 288:91-8, 2018. See Table 04. Detection of fluorescence in a given well suggested that the probe antibody was capable of binding to OSMR even in the presence of other OSMR antibodies, demonstrating that it was binding to a distinct epitope. A minimum of three bins was found, as shown in Table 6 below. [Table 6] Example 5: Determination of affinity of anti-OSMR antibodies

[0310] Determination of the affinity of anti-OSMR antibodies. Kinetic constant determinations were performed to investigate the interaction of antibodies 1–3 (Abs1–3) with human OSMR.

[0311] Biosensor analysis was performed using a Biacore 3000 optical biosensor equipped with a CM5 sensor chip in a (HBS-EP + (1X) buffer system (10 mM HEPES The assay was performed in pH 7.4, 150 mM NaCl, 3.0 mM EDTA, 0.05% surfactant P20 at 25°C. All reagents were kept at 8°C prior to injection. Goat anti-human IgG (Jackson ImmunoResearch, #109-005-098) was immobilized to the sensor chip via standard amine coupling to flow cells 1 and 2 (~3000 RU) and then blocked with ethanolamine. hOSMR.FH was prepared in running buffer at 150 nM and diluted 3-fold to 0.617 nM. Antibodies 1-3 were diluted in running buffer (0.25-0.5 μg / mL). Antibodies were injected (15 μL) into flow cell 2 at a flow rate of 10 μL / min. Approximately 50 RU of antibody was captured. The surface was allowed to stabilize (90 seconds), and then various concentrations of hOSMR (150, 50.0, 16.7, 5.56, 1.85, and 0.617) were passed through flow cells 1 and 2 at a flow rate of 50 μL / min to observe association (5 minutes) and dissociation (5 minutes). Samples were run in duplicate and in random order.

[0312] A buffer sample blank (0 nM hOSMR) was injected before, during, and after sample injection. Antibody was injected (15 μL) into flow cell 2 at a flow rate of 10 μL / min. Approximately 50 RU of antibody was captured. After allowing the surface to stabilize (90 seconds), various concentrations of hOSMR (150 nM) were then passed through flow cells 1 and 2 at a flow rate of 50 μL / min to observe association (5 minutes) and dissociation (1–2 hours). Samples were run in triplicate.

[0313] A buffer analyte blank (0 nM hOSMR) was injected before and after sample injection. The surface was regenerated with two injections of 10 mM glycine (pH 1.5, 50 μL) at a flow rate of 50 μL / min, followed by a buffer blank injection (15 s).

[0314] Data were analyzed using Scrubber 2.0 software as follows: Data from flow cell 2 were subtracted from data from flow cell 1 (blank reference). The reference-subtracted data (2-1) were then subtracted from the nearest 0 nM concentration data (double reference). The double-referenced long dissociation data were fitted to a 1:1 binding model to determine the dissociation rate constant (kd). This dissociation rate constant was used as a fixed parameter to fit the double-referenced short dissociation data to a 1:1 binding model to determine the association rate constant (ka) and equilibrium dissociation constant (kd).

[0315] The reagents behaved quite well and the data (see Table 7 below) fit fairly well to a 1:1 binding model. [Table 7] Example 6: Anti-OSMR antibodies

[0316] Fully human antibodies directed against human OSMR were generated using the XENOMOUSE® technology described in Example 1 above. Antibodies 1, 2, and 3 were each shown to be potent inhibitors of OSM- and IL-31-mediated signaling. The sequences of antibodies 1, 2, and 3 (i.e., Ab1, Ab2, and Ab3) were determined and are shown in Table 8 below. vinegar. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] Example 7: Modified anti-OSMR antibodies

[0317] Modified versions of Ab1, Ab2, and Ab3 were generated. In all three modified forms of the antibodies, the C-terminal lysine of the heavy chain was removed. For Ab1 and Ab2, the glycosylation site at position 73 was removed by substituting aspartic acid for asparagine at position 73. These variants are called Ab1-N73D and Ab2-N73D. The sequences of the modified antibodies are listed in Table 9 below (modified nucleotides and amino acids are underlined). [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4]

[0318] ELISA experiments were performed in various formats (Capture ELISA for avidity-free format; Sandwich ELISA for solution-phase format; Direct ELISA for solid-binding format) using antibodies containing Ab1 or Ab2 variable regions (or the N73D mutant of Ab1 or Ab2) with different Fc regions.

[0319] Each of Ab1 and Ab2 contains the CH1, CH2, and CH3 domains derived from human IgG2. As used herein, the terms "Ab1" and "Ab1 IgG2 WT" refer to the same antibody. Similarly, the terms "Ab2" and "Ab2 IgG2 WT" refer to the same antibody.

[0320] The antibody identified as "IgG4P agly / IgG1" contains a CH1 domain from human IgG4 with a Ser to Pro mutation (at position 228) to reduce shuffling, a hinge from human IgG4, and an Asn to remove an N-linked glycosylation site. The "IgG4P agly / IgG1" framework comprises the variable region of Ab1 or Ab2 (or the N73D mutant of Ab1 or Ab2) fused to a CH2 domain from human IgG4 with a N73D to Gln mutation (at position 297) and a CH3 domain from human IgG1. The "IgG4P agly / IgG1" framework is described in U.S. Published Patent Application No. US2012 / 0100140.

[0321] ELISA results showed that removal of the glycosylation site via the N73D substitution did not affect the binding of the modified antibody to OSMR. See Table 10. [Table 10]

[0322] Binding studies were performed using BIAcore technology. Antibodies containing Ab1 or Ab2 variable regions with different Fc regions (or the N73D mutant of Ab1 or Ab2) were immobilized on a CM4 chip (GE Lifesciences) according to the manufacturer's protocol. Soluble OSMR was used as the analyte. Removal of the glycosylation site on Ab1 and Ab2 via the N73D substitution improved binding affinity. For Ab1, the substitution improved the K on rate, while for Ab2, the substitution improved the K off rate. See Table 11. [Table 11]

[0323] The stability of the Fab fragments was determined by assessing the unfolding temperature of the antibodies. A higher melting temperature of the Fab fragment directly correlates with increased stability. Removal of the glycosylation site on Ab2 via the N73D substitution did not affect the thermal stability of the Fab fragments, as assessed by differential scanning fluorimetry experiments, and showed a small effect on Ab1. See Table 12. [Table 12]

[0324] The ability of the modified anti-OSMR antibodies to block signaling through human OSMR was evaluated. The modified antibodies were evaluated for their ability to inhibit proliferation of the BaF_hu-IL31R / OSMR / gp130 cell line in the presence of IL31, OSM, or IL31 and OSM. The results are shown in Tables 13 and 14 below, along with the IC50 for each antibody indicated. The results confirm that the modified versions of Ab1 and Ab2 are potent inhibitors of OSM- and IL-31-mediated signaling. [Table 13] [Table 14]

[0325] The present disclosure has been described in terms of specific embodiments that have been discovered or proposed to include specific modes for carrying out the disclosure. Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Claims

[Claim 1] A composition or method as described in the specification.

Images