EA041171B1 - ANTIBODIES TO ONCOSTATIN M RECEPTOR AND THEIR USE - Google Patents

Description

Cross-reference to related applications

This application claims priority in US Provisional Application No. 61/829082, filed May 30, 2013, the contents of which are incorporated herein by reference in their entirety.

State of the art

Oncostatin M (OSM) and interleukin-31 (IL-31) are members of the IL-6 superfamily and share a common receptor subunit, oncostatin M receptor-β (OSMR) (Dillon et al., Nat. Immunol. 5(7):752 -60, 2004). All members of this family, with the exception of IL-31, contain a common chain of glycoprotein 130 (gp130) in their multimeric receptor complexes. OSM signaling occurs via a heterodimeric receptor complex containing OSMR and gp130, while IL-31 uses the gp130-like receptor IL-31R in combination with OSMR (Dillon et al., supra; Dreuw et al., J. Biol Chem 279(34):36112-20, 2004). In general, OSMR and gp130 are expressed in virtually all tissues and cell types and can be induced under various stimulation conditions. The expression of IL-31R is more limited and highly regulated. In both humans and mice, 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 their level of expression differs sharply, both IL-31R and OSMR are co-expressed in a variety of tissues, including skin and intestinal epithelial cells, suggesting that these tissues must respond to IL-31 (Dillon et al., supra; Dambacher et al., Gut 56(9):1257-65, 2007). While OSMR is constitutively expressed on lung epithelial cells, IL-31R expression levels in lung tissues are negligible or low, but can be increased by various airway stimulation methods (Dillon et al., supra; Jawa et al., J Interferon Cytokine Res. 28(4):207-19, 2008).

Since OSM and IL-31 are secreted primarily by T lymphocytes, macrophages, and neutrophils, both are upregulated in many disease states associated with inflammation. OSM is involved in a variety of biological processes including bone formation, cartilage breakdown, cholesterol uptake, pain and inflammation (Cawston et al., Arthritis Rheum. 41(10):1760-71, 1998; Hasegawa et al., Rheumatology ( Oxford, 38(7):612-7, 1999; Levy et al., J. Hepatol. 32(2):218-26, 2000; Micourt 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 an effective extracellular matrix (ECM) modulator under different circumstances, suggesting that OSM is able to mediate seemingly opposite pathological outcomes, including fibrosis (excess ECM) and cartilage destruction (deficiency of ECM). Depending on tissue type and environment, both of these effects were observed in cases of overexpression or exogenous administration of OSM to the lungs or joints of mice, respectively (Richards et al., Biochem. Soc. Trs. 30(2): 107-11, 2002; Hui et al., Arthritis Rheum 48(12):3404-18, 2003; Row et al., Am J. Pathol 162(6):1975-84, 2003). In addition, human pathologies in which these types of outcomes are observed have previously been shown to be upregulated by OSM (Cawston et al., supra; Hasegawa et al., supra; Levy et al., supra; Micourt et al. , supra; Luzina et al., supra). In most cases, there is upregulation of the locally acting cytokine OSM in the synovial fluid from the joints of patients with rheumatoid arthritis (PA) (Cawston et al., supra; Micourt et al., supra), in the bronchoalveolar lavage fluid (BAL) of patients with associated scleroderma interstitial lung disease (Luzina et al., supra), idiopathic pulmonary fibrosis (IPF), and in the liver of cirrhotic patients (Levy et al., supra). The possible effect on the ECM by OSM may be partly due to the ability of OSM to change the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). TIMPs bind with high affinity to MMPs in a 1:1 ratio, resulting in the loss of the proteolytic activity of MMPs. It has previously been shown that regulation of TIMP-1 and TIMP-3 by OSM occurs differently, 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 breakdown of extracellular matrix components, MMPs are involved in the breakdown and subsequent activation of a wide range of proteins, including TGF-β, an effective profibrotic cytokine (Leask et al., FASEB J. 18(7):816-27, 2004). It has also been reported that OSM is able to directly induce collagen type I transcription in vitro (Hasegawa et al., J. Rheumatol. 25(2):308-13, 1998).

Expression of OSM and IL-31 has been found in the skin tissues of patients with psoriasis and atopic dermatitis, and mutations in OSMR and IL-31R have been associated with systemic cutaneous amyloidosis. Systemic transgenic overexpression of IL-31 induced an pruritic inflammatory response in the skin tissues of mice. Both OSM and IL-31 signaling occurs via OSMR on neurons where stimulation of nociceptive and pruritic responses is thought to occur.

Taken together, this is related to human disease and the ability of OSM and IL-31 to stimulate a variety of pathologies, including at least inflammation, extracellular matrix remodeling, pain, and pruritus, suggesting that blocking OSMR is a suitable target for therapeutic intervention in the case of many diseases and disorders associated with

OSMR.

The essence of the invention

The invention provides anti-OSMR antigen-binding proteins, such as antibodies and functional fragments thereof, having properties suitable for commercial production and therapeutic use in humans. Anti-OSMR antigen binding proteins are useful in methods of treating diseases and disorders associated with OSMR, and in particular those associated with the binding of OSM or IL-31 to OSMR. This document provides OSMR binding antibodies that bind OSMR with high affinity and effectively block the binding of OSM and/or IL-31 to OSMR, thus reducing OSMR mediated signaling in the cell.

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 identical to the amino acid sequence shown 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 identical to the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11; or c) a light chain variable domain according to a) and a heavy chain variable domain according to b).

Preferred antigen-binding proteins of the first aspect include those comprising a light chain variable domain having at least 90%, at least 95% or identical amino acid sequence as set forth in SEQ ID NO: 27 and a heavy chain variable domain having at least at least 90%, at least 95% or identical to the amino acid sequence shown in SEQ ID NO: 9; those containing a light chain variable domain having at least 90%, at least 95% or identical to the amino acid sequence shown in SEQ ID NO: 28, and a heavy chain variable domain having at least 90%, at least 95% or identical to the amino acid sequence shown in SEQ ID NO: 10; and those comprising a light chain variable domain having at least 90%, at least 95% or identical amino acid sequence set forth in SEQ ID NO: 29, and a heavy chain variable domain having at least 90%, at least at least 95% or identical to the amino acid sequence shown in SEQ ID NO: 11.

OSMR antigen-binding proteins comprising a heavy chain variable domain related to SEQ ID NO: 9 as defined above may 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 shown in SEQ ID NO: 53.

OSMR antigen-binding proteins comprising a heavy chain variable domain related to SEQ ID NO: 10 as defined above may 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 shown in SEQ ID NO: 54.

In a second aspect, the OSMR antigen-binding protein comprises a) a light chain variable domain containing no more than ten or no more than five amino acid additions, deletions, or substitutions compared to the amino acid sequence shown in SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 29; b) a heavy chain variable domain containing no more than ten or no more than five amino acid additions, deletions or substitutions compared to the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11; or c) a light chain variable domain according to a) and a heavy chain variable domain according to b).

Preferred antigen-binding proteins from the second aspect include those that contain a light chain variable domain containing no more than ten or no more than five amino acid additions, deletions or substitutions compared to the amino acid sequence shown in SEQ ID NO: 27, and a heavy chain variable domain, containing no more than ten or no more than five amino acid additions, deletions or substitutions compared to the amino acid sequence shown in SEQ ID NO: 9; those containing a light chain variable domain containing no more than ten or no more than five amino acid additions, deletions or substitutions compared to the amino acid sequence shown in SEQ ID NO: 28, and a heavy chain variable domain containing no more than ten or no more five amino acid additions, deletions or substitutions compared to the amino acid sequence shown in SEQ ID NO: 10; and those containing a light chain variable domain containing no more than ten or no more than five amino acid additions, deletions or substitutions compared to the amino acid sequence shown in SEQ ID NO: 29, and a heavy chain variable domain containing no more than ten or no more than five amino acid additions, deletions or substitutions compared to the amino acid sequence shown in SEQ ID NO: 11.

OSMR antigen-binding proteins containing a heavy chain variable domain having

- 2 041171 the above degree of relationship with SEQ ID NO: 9 may optionally contain an amino acid other than asparagine (for example, aspartic acid) at the position corresponding to position 73 in

SEQ ID NO: 9. In such embodiments, the heavy chain variable domain optionally comprises the amino acid sequence shown in SEQ ID NO: 53.

OSMR antigen-binding proteins comprising a heavy chain variable domain related to SEQ ID NO: 10 as defined above may 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 shown in SEQ ID NO: 54.

In a third aspect, the OSMR antigen-binding protein comprises a light chain variable domain comprising a) an LCDR1 containing no more than three amino acid additions, deletions, or substitutions compared to the LCDR1 sequence shown in SEQ ID NO: 30; An LCDR2 containing no more than three amino acid additions, deletions or substitutions compared to the LCDR2 sequence shown in SEQ ID NO: 33; and LCDR3 containing no more than three amino acid additions, deletions or substitutions compared to the LCDR3 sequence shown in SEQ ID NO: 36; b) LCDR1 containing no more than three amino acid additions, deletions or substitutions compared to the LCDR1 sequence shown in SEQ ID NO: 31; LCDR2 containing no more than three amino acid additions, deletions or substitutions compared to the LCDR2 sequence shown in SEQ ID NO: 34; and LCDR3 containing no more than three amino acid additions, deletions or substitutions compared to the LCDR3 sequence shown in SEQ ID NO: 37; or c) an LCDR1 containing no more than three amino acid additions, deletions or substitutions compared to the LCDR1 sequence shown in SEQ ID NO: 32; LCDR2 containing no more than three amino acid additions, deletions or substitutions compared to the LCDR2 sequence shown in SEQ ID NO: 35; and LCDR3 containing no more than three amino acid additions, deletions or substitutions compared to the LCDR3 sequence shown in SEQ ID NO: 38; and a heavy chain variable domain containing d) HCDR1 containing no more than three amino acid additions, deletions or substitutions compared to the HCDR1 sequence shown in SEQ ID NO: 12; HCDR2 containing no more than three amino acid additions, deletions or substitutions compared to the HCDR2 sequence shown in SEQ ID NO: 15; and HCDR3 containing no more than three amino acid additions, deletions or substitutions compared to the HCDR3 sequence shown in SEQ ID NO: 18; e) HCDR1 containing no more than three amino acid additions, deletions or substitutions compared to the HCDR1 sequence shown in SEQ ID NO: 13; HCDR2 containing no more than three amino acid additions, deletions or substitutions compared to the HCDR2 sequence shown in SEQ ID NO: 16; and HCDR3 containing no more than three amino acid additions, deletions or substitutions compared to the HCDR3 sequence shown in SEQ ID NO: 19; or f) HCDR1 containing no more than three amino acid additions, deletions or substitutions compared to the HCDR1 sequence shown in SEQ ID NO: 14; HCDR2 containing no more than three amino acid additions, deletions or substitutions compared to the HCDR2 sequence shown in SEQ ID NO: 17; and HCDR3 containing no more than three amino acid additions, deletions or substitutions compared to the HCDR3 sequence shown in SEQ ID NO: 20.

Preferred OSMR antigen-binding proteins from the third aspect include those comprising a light chain variable domain according to a) and a heavy chain variable domain according to d); those containing the light chain variable domain according to b) and the heavy chain variable domain according to e); and those containing a light chain variable domain according to c) and a heavy chain variable domain according to f).

OSMR antigen-binding proteins comprising a light chain variable domain according to a) and a heavy chain variable domain according to d) may 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 shown in SEQ ID NO: 53.

OSMR antigen-binding proteins comprising a light chain variable domain according to b) and a heavy chain variable domain according to e) may 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 shown in SEQ ID NO: 54.

In a fourth aspect of the invention, the OSMR antigen-binding protein of the first, second or third aspect binds to human OSMR with an affinity less than or equal to 1x10'1° M.

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

In a sixth aspect of the invention, the OSMR antigen-binding protein of the first, second, third, fourth or fifth aspect reduces human OSM and/or human IL-31 mediated

- 3 041171 OSMR signaling in human cells expressing OSMR.

In a seventh aspect of the invention, the OSMR antigen-binding protein of the sixth aspect reduces OSM and/or cynomolgus IL-31 mediated OSMR signaling in cynomolgus monkey cells expressing OSMR.

In an eighth aspect of the invention, the OSMR antigen-binding protein of the first, second, third, fourth, fifth, sixth, or seventh aspect is an antibody, such as a human antibody. Preferred antibodies include those containing a light chain having the amino acid sequence shown in SEQ ID NO: 24 and a heavy chain having the amino acid sequence shown in SEQ ID NO: 6; those containing a light chain having the amino acid sequence shown in SEQ ID NO: 25 and a heavy chain having the amino acid sequence shown in SEQ ID NO: 7; and those containing a light chain having the amino acid sequence given in SEQ ID NO: 26 and a heavy chain having the amino acid sequence given in SEQ ID NO: 8.

Additional antibodies include those antibodies that contain a light chain having the amino acid sequence shown in SEQ ID NO: 24 and a heavy chain having the amino acid sequence shown in SEQ ID NO: 50; those containing a light chain having the amino acid sequence shown in SEQ ID NO: 25 and a heavy chain having the amino acid sequence shown in SEQ ID NO: 51; and those containing a light chain having the amino acid sequence given in SEQ ID NO: 26 and a heavy chain having the amino acid sequence given in SEQ ID NO: 52.

In a ninth aspect, the invention provides methods for treating an autoimmune disorder, an inflammatory disorder, or an extracellular matrix accumulation or remodeling disorder, said method comprising administering a therapeutically effective amount of an OSMR antigen-binding protein from any of the first, second, third, fourth, fifth, sixth, seventh or eighth aspect to a patient in need. In preferred embodiments, the OSMR antigen-binding protein is an antibody comprising the light chain variable 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: 9 (e.g., Ab1), an antibody comprising a light chain variable amino acid sequence as set forth in SEQ ID NO: 28 and a heavy chain variable amino acid sequence as set forth in SEQ ID NO: 10 (e.g., Ab2), or an antibody comprising a light chain variable amino acid sequence, set forth in SEQ ID NO: 29 and the amino acid sequence of the heavy chain variable domain set forth in SEQ ID NO: 11 eg Ab3). In some embodiments, the OSMR antigen-binding protein is an antibody comprising the light chain variable 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 amino acid the light chain variable sequence set forth in SEQ ID NO: 28; and the heavy chain variable 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 disorder, inflammatory disorder, or extracellular matrix accumulation or remodeling disorder is fibrosis, cartilage destruction, arthritis, rheumatoid arthritis, scleroderma, scleroderma-associated interstitial lung disease, idiopathic pulmonary fibrosis, cirrhosis, psoriasis, atopic dermatitis, systemic cutaneous amyloidosis, primary cutaneous amyloidosis, inflammation, pruritic inflammation, nodular pruritus and pain.

Detailed description of the invention

The section titles used in this document are used solely for structuring purposes and should not be taken as limiting the subject matter being described. All references cited in the text of this specification are incorporated by reference in their entirety.

For recombinant DNA production, oligonucleotide synthesis, tissue culture and transformation, protein purification, etc. standard methods can be used. Enzymatic reactions and purification can be carried out according to the manufacturer's instructions or as is conventional in the art or as described herein. The following procedures and methods can generally be carried out in accordance with conventional methods well known in the art and described in many of the general and more specialized references given and discussed in this description. See, for example, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manuel, ^3rd ed., Cold Spring Harbor Laboratory Press, cold Spring Harbor, NY, which is incorporated herein by reference for any purpose. Although specific definitions are given in the text, the nomenclature used in connection with and laboratory procedures

- 4 041171 and the methods of analytical chemistry, organic chemistry and medicinal and pharmaceutical chemistry described in this document are well known and widely used in the art. For chemical synthesis, chemical analysis, pharmaceutical formulation, formulation, and patient delivery and treatment, standard methods can be used.

OSMR.

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

Human OSMR sequences are known in the art. In various aspects, human OSMR protein sequences are listed in GenBank under accession numbers AAI25210, AAI25211, NP_003990 and EAW55976. Typical amino acid sequence of human OSMR (SEQ ID NO: 1) is shown in table. 1. The protein consists of several domains: amino acids 1-27 correspond to a signal sequence that can be cleaved off 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 interaction of OSM and/or IL-31 with OSMR.

Human OSM sequences are known in the art. In various aspects, human OSM protein sequences are listed in GenBank under accession numbers CAG30420, C7AG46504, NP_065391, P13725, AAC05173, EAW59864 and AAH11589. Typical amino acid sequence of human OSM (SEQ ID NO: 39) is shown 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.

The sequences of human IL-31 are known in the art. In various aspects, the protein sequences of human IL-31 are listed in GenBank under accession numbers NP_001014358, AAS86448, AAI32999, AAI33001, Q6EBC2, and EAW98310. Typical amino acid sequence of human IL-31 (SEQ ID NO: 41) is shown in table. 1. Amino acids 1-23 correspond to the putative signal sequence.

The sequences of human IL31RA are known in the art. In various aspects, the protein sequences of human IL31RA are listed in GenBank under accession numbers AAS86447, NP_001229567 and CBL94051. Typical amino acid sequence of human IL31RA (v4, isoform 3) (SEQ ID NO: 43) is shown in table. 1. Amino acids 1-32 correspond to the signal sequence; and amino acids 533-553 correspond to the transmembrane sequence.

Human gp130 sequences are known in the art. In various aspects, the human gp130 protein sequences are listed in GenBank under accession numbers AAI17403, AAI17405, EAW54936, NP_002175, ABK41905 and AAA59155. Typical amino acid sequence of human gp130 (SEQ ID NO: 45) is shown in table. 1. The protein consists 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.

- 5 041171 _____________________________________________________________Table 1

Amino acid sequence of human OSMR (SEQ ID no: 1)

MALFAVFQTTFFLTLLSLRTYQSEVLAERLPLTPVSLKVSTNSTRQSLHLQWTVHNLPYHQEL KMVFQIQISRIETSWIWVGNYSTTVKWNQVLHWSWESELPLECATHFVRIKSLVDDAKFPEP NFWSNWSSWEEVEVQDSTGQDILFVFPKDKLVEEGTm^TICYVSRNIQNNVSCYLEGKQIHGE QLDPHVTAFNLNSVPEIRNKGTNIYCEASQGNVSEGMKGIVLFVSKVLEEPKDFSCETEDFKT LHCTWDPGTDTALGWSKQPSQSYTLFESFSGEKKLCTHKNWCNWQITQDSQETYNFTLIAENY LRKRSVNILFNLTHRVYLMNPFSVNFENVNATMAIMTWKVHSTRNNFTYLCQIELHGEGKMMQ YNVSIKVNGEYFLSELEPATEYMARVRCADASHFWKWSEWSGQNFTTLEAAPSEAPDVWRIVS LEPGNHTVTLFWKPLSKLHANGKILFYNWVENLDKPSSSELHSIPAPARSTKLILDRCSYQI CVIANWSVGASPASVIVISADPENKEVEEERIAGTEGGFSLGWKPQPGDVIGYWDWCDHTQD VLGDFQWKNVGPKTTSTVISTDAFRPGVRYDFRIYGLSTKRIACLLEKKTGYSQELAPSDNPH VLVDTLTSHSFTLSWKDYSTESQPGFIQGYHVYLKSKARQCHPRFEKAVLSDGSECCKYKTDN PEEKALIVDNLKPESFYEFFITPFTSAGEGPSATFTKVTTPDEHSSMLIHILLPMVFCVLLIM VMCYLKSQWIKETCYPDIPDPYKSSILSLIKFKENPHLIIMNVSDCIPDAIEVVSKPEGTKIQ FLGTRKSLTETELTKPNYLYLLPTEKNHSGPGPCICFENLTYNQAASDSGSCGHVPVSPKAPS MLGLMTSPENVLKALEKNYMNSLGEIPAGETSLNYVSQLASPMFGDKDSLPTNPVEAPHCSEY KMQMAVSLRLALPPPTENSSLSSITLLDPGEHYC

Amino acid sequence of human OSM (SEQ ID no: 39'

MGVLLTQRTLLSLVLALLFPSMASMAAIGSCSKEYRVLLGQLQKQTDLKQDTSRLLDPYIRIQ GLDVPKLREHCRERPGAFPSEETLRGLGRRGFLQTLNATLGCVLHRLADLEQRLPKAQDLERS GLNIEDLEKLQMARPNILGLRNNIYCMAQLLDNSDTAEPTKAGRGASQPPTPTPASDAFQRKL EGCRFLHGYHRFKHSVGRVFSKWGESPNRSRRHSPHQALRKGVRRTRPSRKGKRLMTRGQLPR Аминокислотная последовательность человеческого IL-31 (SEQ ID №: 41J

MASHSGPSTSVLFLFCCLGGWLASHTLPVRLLRPSDDVQKIVEELQSLSKMLLKDVEEEKGVL VSQNYTLPCLSPDAQPPNNIHSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIFQDAPEYNISV PTDTHECKRFILTISQQFSECMDLALKSLTSGAQQATT

Amino acid sequence of human IL31RA (SEQ ID no: 4 3

MKLSPQPSCVNLGMMWTWALWMLPSLCKFSLAALPAKPENISCVYYYRKNLTCTWSPGKETSY TQYTVKRTYAFGEKHDNCTTNSSTSENRASCSFFLPRITIPDNYTIEVEAENGDGVIKSHMTY WRLENIAKTEPPKIFRVKPY ^T LGIKRMIQIEWIKPELAPVSSDLKYTLRFRTWSTSWYEWFA KNRKDKNQTYNLTGLQPFTEYVIALRCAVKESKFWSDWSQEKMGMTEEEAPCGLELWRVLKPA EADGRRPVRLLWKKARGAPVLEKTLGYNIWYYPESNTNLTETMNTTNQQLELHLGGESFWVSM ISYNSLGKSPVATLRIPAIQEKSFQCIEVMQACVAEDQLWKWQSSALDVNTWMIEWFPDVDS EPTTLGWESVSQATNWTIQQDKLKPFWCYNISVYPMLHDKVGEPYSIQAYAKEGVPSEGPETK VENIGVKTVTITWKEIPKSERKGIICNYTTFYQAEGGKGFSKTVNSSILQYGLESLKRKTGYI VQVMASTSAGGTNGTSINFKTLSFSVFEIILITSLIGGGLLILIILTVAYGLKKPNKLPHLCW PTVPNPAESSIATWHGDDFKDKLNLRESDDSVNTEDRILKPCSTPSDKLVIDKLWNFGNVLQ EIFTDEARTGQEKNLGGEKNGTRILSSCPTSI

- 6 041171

Amino acid sequence of human gp!30 (SEQ ID no: 4 5 ।

MLTLQTWLVQALFIFLTTESTGELLDPCGYISPESPVVQLHSNFTAVCVLKEKCMDYjHVNAN

YIVWKTNNHFTIPKEQYTIINRTASSVTFIDIASLN1QLTCNILTFGQLEQWYGITIISGLPP

IEKPKNLSCIVNEGKKMRCEWDGGRETHLETNFTLKSEWATHKFADCKAKRDTPTSCTVDYSTV

YFVNIEVWVEAENALGKVTSDHINFDPVYKVKPNPPHNLSVINSEELSSILKLTWTNPSIKSV IILKYKIQYRTKDASTWSQIPPEDTASTRSSFTVQDLKPFTEYVFRIRCMKEDGKGYWSDWSE EASGITYEDRPSKAPSEWYKIDPSHTQGYRTVQLVWKTLPPFEANGKILDYEVTLTRWKSHLQ NYTVNATKLTVNLTNDRYLATLTVRNLVGKSDAAVLTIPACDFQATHPVMDLKAFPKDNMLWV EWTTPRESVKKYILEWCVLSDKAPCITDWQOEDGTVHRTYLRGKLAESKCYLITVTPVYADGP GSPESIKAYLKQAPPSKGPTVRTKKVGKNEAVLEWDQLPVDVQKGFTRNYUFYRTIIGNETA VNVDSSHTEYTLSSLTSDTLYMVRMAAYTDEGGKDGPEFTFTTPKFAQGEIEAIWPVGLAFL LTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDSNFTDVSV VEIVARDKKPFPEDLKGLDLFKKEKINTEGHSSGIGGSSCMGSERPSISSSDENESGQNTSGT VQYSTWHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQNC SQHESSPDTSHFERSKQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAFGPGTE GQVERFETVGMEAATDEGMPKSYEPQrVRQGGYMFQ

In specific embodiments, the antigen binding proteins described herein bind both human and cynomolgus OSMR with high affinity, including those that bind with high affinity and block the interaction of cynomolgus monkey OSM and/or IL-31 with cynomolgus OSMR. These characteristics allow for informative studies in monkeys.

The rhesus monkey (Macaca mulatta) OSMR protein sequence is known in the art and listed in GenBank under accession number XP_001083745. Typical amino acid sequence OSMR cynomolgus macaque (Macaca fascicularis) (SEQ ID NO: 2) is shown in table. 2. The protein consists of several domains: amino acids 1-27 correspond to a signal sequence that can be cleaved off 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 interaction of OSM and/or IL-31 with OSMR.

The OSM protein sequence of the rhesus monkey (Macaca mulatta) is known in the art and listed in GenBank under accession number NP_001181403. The exemplary amino acid sequence of the OSM cynomolgus macaque (Macaca fascicularis) (SEQ ID NO: 40) is shown in Table 1. 2. Amino acids 1-196 correspond to mature cynomolgus monkey OSM.

The IL-31 protein sequence of the rhesus monkey (Macaca mulatta) is known in the art and listed in GenBank under accession number XP_001096743. Typical amino acid sequence of IL-31 cynomolgus monkey (Macaca fascicularis) (SEQ ID NO: 42) is shown in table. 2. This sequence represents mature cynomolgus monkey IL-31.

Typical amino acid sequence of IL31RA cynomolgus monkey (Macaca fascicularis) (SEQ ID NO: 44) is shown in table. 2. Amino acids 1-19 correspond to the signal sequence; and amino acids 520-540 correspond to the transmembrane domain.

The gp130 protein sequence of the rhesus monkey (Macaca mulatta) is known in the art and listed in GenBank under accession number NP_001252920. Typical amino acid sequence of gp130 cynomolgus monkey (Macaca fascicularis) (SEQ ID NO: 46) is shown in table. 2. The protein consists 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 2

Cynomolgus monkey OSMR amino acid sequence (SEQ ID no: 2 5

MALFWFQTTFFLILLSLRTYQSEVLAERLPLTPVSLKVSTNSIHQSLHLQWTVHNLPYHQEL

KMVFQIQISRIETSNWWVGNYSTPVKWNQVLHWSWESELPLECATHFVRIKSVIDDASFPEP

NFWSNWSSWEEVSVQDYLGRGTLFVFPRDKLVEEGSWTICYVSRNIQNNVSCYLEGKQIHGE

- 7 041171

QLDPHVTAFNLNSVPFIRNRGTNIYCEASQGNVSKGIEGIVLFVSKVLEEPKDFSCESQDFKT LHCTWDPGTDTALGWSKQPSQSYTLFESFSGEKKLCTHKKWCNWQITQDSQEMYNFTLIAENY LRKRSVNILFNLTnRVYLMNPFSVNFENVNATNAIMTWKVHSMRNNFTYLCQIELnGEGKMMQ YDVSINWGEYFLSELEPATEYMARVRCADASHFWKWTEWSGQNFTTLEAAPSEAPDVWRSVN sepgnhtvtlfwkplsklhangkilfynwvenldkpsrselrsipapanstklildrcsyq: CVTANNSVGASPAS IIVISADPENKEVEEERIAGTEGGFSLSWKPQPGDVIGYWDWCDHPQD VLQWKWGPNTTGTVISTDAFRPGVRYDFRIYGLSTKRIACLLEKKTGYSQELAPSDNPHVLV DMLTSHSFTLSWKDYSTESQPGFIQGYHVYLKSKARQCHPRFQKAVLSDGSECCRYKIDNPEE KALIVDNLKPESFYEFFVTPFTSAGEGPNATFTKVTTPDEHSSMLIRILLPMVFCVLLIMIVC YLKSQWIKETCYPDIPDPYKSSILSLIKFKENPHLTIMNVSDCIPDAIEWSKPEGTKIQLLG TRKSLTETELTKPNYLYLLPTEKNHSGPGPCICFENFTYKQAASDAGSCGHVPVPPKAPPSML GLMTSPENVLKALEKNYMNSLGEVPAGETSLNYVSQLASPMSGDKDSLPTNPVEPPHCSEYKM QMAVPLRLALPPPTENSSLGSITLLDPGEHYR

Amino acid sequence of cynomolgus macaque OSM (SEQ ID no: 40)

AAMGSCSKEYRMLLGQLQKQTDLMQDTSRLLDPYIRIQGLDIPKLREHCRESPGAFPSEETLR GLGRRGFLQTLNATLGRILHRLADLEQHLPKAQDLERSGLNIEDLEKLQMARPNVLGLRNNVY CEIAQLLDNSDMTEPTKAGRGTPQPPTPTPTSDVFQRKLEGCSFLRGYHRFMHSVGRVFSKWGE SPNRSRR

Amino acid sequence of cynomolgus monkey IL-31 (SEQ ID NG 42)

TLPVHFLQPSDIQKIVEELQSLSKMLLKDVKEDKGVLVSQNYTLPCLTPDAQPPNIIHSPAIR AYLKTIRQLDNK5VIDEIIEHLDKLIFQDAPETKISVPTDTHECKRFILTISQQFSECMDLAL KSLTSGAQQATT

Amino acid sequence of cynomolgus monkey IL31RA (SEQ ID NO:44)

MMWTWALWMFPLLCKFGLAALPAKPENISCVYYYRKKLTCTWSPGKETSYTQYTAKRTYAFGK KnDNCTTSSSTSENRASCSFFLPRITIPDNYTIEVEAENGDGVIKSDMTCWRLEDIAKTEPPE IFSVKPVLGIKK4IRIEWIKPELAPVSSDLKYALRFRTVKSTSWMEVNEAKNRKDTNQTYNLM GLQAFTEYWALRCAVKESKFWSDWSQEKMGMTEEEAPCGLELWRVLKPTEVDGRRPVRLLWK KARGAPVLEKTLGYNIWYFPENNTNLTETVNTTKQQLELHLGGESYWVSMISYNSLGKSPVT ¹ ? LRIPAIQEKSFRCIEVMQACLAEDQLWKWQSSALDVNTWMIEWFPDMDSEHPTLSWES\ESQA TNWTIQQDKLKPFHCYNISVYPMLHDKVGEPYSIQAYAKEGIPSKGPETKVENIGVKTVTITW KEIPKSERKGIICNYTIFYQAEGGTGFSKTVNSSILQYGLESLKRKTSYTVRVMASTSAGGIN GTSINFKTLSFSVFEIILITSLIGGGLLILIILTVAYGLKKPNKLTHLCWPSVPNPAESSIAT

- 8 041171

WRGDDFKDKLNLKESDDSVNTEDRILKPCSTPSDKLVIDKSWNFGNVLQEMFTDEARTGQEN NLGGEKNENRILSSCPTSI

Cynomolgus macaque gpl30 amino acid sequence (SEQ ID NO: 4 6)

MLTLQTWWQALFIFLTTESIGELLDPCGYISPESPWQLHSNFTAVCVLKEKCMDYFHVNAN YIVWKTNHFTIPKEQYTIINRTASGVTFTDISSLNIQLTCNILTFGQLEQNVYGITIISGLPP EKPKNLSCTVNEGKKMRCEWNRGRETHLETNFTLKSEWATHKFADCKAKRDTPTSCTVDYSTV YFVNIEVWVEAENALGKVTSDHINFDPVYKVKPFPPHNLSVINSEELSSILKLTWTNPSIKSV IRLKYN1QYRTKDASTWSQIPREDTASTRSSFTVQDLKPFTEYVFRICCMKEDGKGYWSDWSE EANGITYEDRPSKAPSFWYKIDPSHAQGYRTVQLMWKTLPPFEANGKILDYEVTLTRWKSHLQ NYTVNDTKLTVNLTNDRYVATLTARNLVGKSDAAVLTIPACDFQATHPVMDLKAFPKDNMLWV EWTTPRESVKKYTLEWCVLSDKAPCIADWQQEDGTVHRTHLRGNLAESKCYLITVTPVYADGP GSPESIKAYLKQAPPSKGPTVRTKKVGKNEAVLEWDQLRVDVQNGFIRNYTIFYRTIIGNETA VNVDSSHTEYTLSSLTSDTLYMVRMAAYTDEGGKDGPEFTFTTPKFAQGEIEAIWPVCLAFL LTTLLGVLFCFNKRDLIKKHIWPWPDPSKSHIAQWSPHTPPRHNFSSKDQMYSDGNFTDVSV VEIEAWDKKPFPEDLKSLDLFKKEKINTEGHSSGTGGSSCMSSSRPSTSSSDENESSQN ^m SS ^m VQYSTWHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDGSDDILPRQQYFKQNC SQHESSFDISHFERSKQVSSVNEEDFVRLKQQIGDHISQGCGSGEMKMFQEVSAADPFGPGTE GQVERFETIGMEAAIDEGMPKSYLPQTVRQGGYEPQ

Antigen-binding proteins to OSMR.

The present invention provides antigen-binding proteins that specifically bind OSMR. Embodiments of antigen-binding proteins include peptides and/or polypeptides that specifically bind OSMR. Such peptides or polypeptides may optionally contain one or more post-translational modifications. Embodiments of antigen-binding proteins include antibodies and fragments thereof, as comprehensively defined herein, that specifically bind OSMR. These include antibodies that specifically bind human OSMR, including those that inhibit OSMR binding and/or activation of OSM and/or IL-31.

The antigen binding proteins of the invention bind specifically to OSMR. In the context of this document, specifically binds means that the antigen-binding protein preferentially binds OSMR over other proteins. In some embodiments, specific binding means that the OSMR antigen-binding protein has a higher affinity for OSMR than for other proteins. OSMR antigen-binding proteins that specifically bind OSMR may have a binding affinity for human OSMR less less less less less less less less less less less less less less less less less less or or or or or or or or or or or or or or or or equal equal equal equal equal equal equal equal equal equal equal equal equal equal equal equal equal equal equal

1x10 4x10 7x10 1x10 4x10

7x10 1x10 4x10 7x10 >-8

1x10 4x10 7x10' 1x10 4x10 7x10 1x10 4x10^- .-10 >-12

M, M, M, M, M, M, M, M, M, M, M, M, M, M, M, M, M, less less less less less less less less less less less less less less less less less less less less or or or or or or or or or or or or or or or or or equal equal equal equal equal equal equal equal equal equal equal equal equal equal equal equal equal equal

2x10 5x10 8x10 2x10 5x10 8x10

2x10 5x10 8x10 >-8 >-9

2x10' 5x10' 8x10' 2x10' 5x10 8x10

2x10 5x10^- .-10 >-12

M, M, M, M, M, M, M, M, M, M, M, M, M, M, M, M, M, less less less less less less less less less less less less less less less less less less less less or or or or or or or or or or or or or or or or or equal equal equal equal equal equal equal equal equal equal equal equal equal equal equal equal equal equal

3x10

6x10

9x10

3x10

6x10

9x10

3x10

6x10

9x10 >-8 >-9

^3x10-10

^6x10-10

9x10 3x10' 6x10 9x10 3x10' 6x10

M, M, M, M, M, M, M, M, M, M, M, M, M, M, M, M, M, less than or equal to 7x10 ^-12 M, less than or equal to 8x10 ^-12 M or less than or equal to 9x10 ^-12 M.

Methods for measuring the binding affinity of an antigen binding protein are well known in the art. Methods that are commonly used to determine affinity 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), analysis

- 9 041171 kinetic exclusion (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, 213221) and analytical ultracentrifugation (Lebowitz et al. Modern analytical ultracentrifugation in protein science: A tutorial review, Protein Science (2002), 11:2067-2079). Example 5 shows typical methods for determining affinity.

It should be understood that, within the scope of this document, referring to various embodiments of OSMR-binding antibodies also includes their OSMR-binding fragments. An OSMR binding fragment contains any fragments or domains of the antibodies described herein that retain the ability to specifically bind to OSMR. The OSMR binding fragment may be in any of the scaffolds described herein.

In certain therapeutic embodiments, the OSMR antigen-binding protein inhibits the binding of OSM and/or IL-31 to OSMR and/or inhibits one or more biological activities associated with binding of OSM and/or IL-31 to OSMR, such as OSM- and/or or IL-31 mediated signaling. Such antigen-binding proteins are said to be neutralizing. In certain embodiments, the OSMR neutralizing antigen-binding protein specifically binds the OSMR and inhibits the binding of OSM and/or IL-31 to the OSMR by about 10-100%, such as 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, 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 ability by determining the ability of the antigen-binding protein to block the binding of OSM and/or IL-31 to OSMR, see e.g. human and OSMR and cynomolgus OSMR blocking assays of Examples 2 and 3, respectively. Alternatively, antigen-binding proteins to OSMR can be assayed for neutralizing capacity by a method that determines the effect of the presence of an antigen-binding protein to OSMR in an assay that determines OSM and/or IL-31 mediated biological function, e.g., the ability of OSM to induce a biological response, such as stimulation of plasminogen activator activity in cultured bovine aortic endothelial cells, regulation of IL-6 expression in human endothelial cells, and stimulation of LDL uptake and upregulation of cell surface LDL receptors in HepG2 cells, alternatively the ability of IL-31 to induce inflammation in skin tissues.

Embodiments of antigen-binding proteins include a framework structure, comprehensively defined herein, with one or more complementarity-determining regions (CDRs). Embodiments further include antigen-binding proteins comprising a scaffold structure with one or more antibody variable domains, both heavy and light. Embodiments include antibodies that comprise a light chain variable domain selected from the group consisting of Ab1 (LCv), Ab2 LCv, and Ab3 LCv light chain variable domain (SEQ ID NOs: 27-29 respectively) and/or a heavy chain variable domain , selected from the group consisting of the heavy chain variable domain Ab1 (HCv), Ab2 HCv and Ab3 HCv (SEQ ID NO: 9-11, respectively), as well as fragments, mutein derivatives and variants.

An exemplary heavy chain variable domain variant of SEQ ID NO: 9 contains an amino acid other than asparagine (e.g., aspartic acid) at a position corresponding to position 73 of SEQ ID NO: 9. The amino acid sequence shown 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 a position corresponding to position 73 of SEQ ID NO: 10. The amino acid sequence shown in SEQ ID NO: 54 is an example of a heavy chain variable domain variant of SEQ ID NO: 10.

An exemplary light chain containing Ab1 LCv is a light chain containing the amino acid sequence shown in SEQ ID NO: 24.

An exemplary light chain containing Ab2 LCv is a light chain containing the amino acid sequence shown in SEQ ID NO: 25.

An exemplary light chain containing Ab3 LCv is a light chain containing the amino acid sequence shown in SEQ ID NO: 26.

An exemplary heavy chain containing Ab1 HCv is a heavy chain containing the amino acid sequence shown in SEQ ID NO: 6.

An exemplary heavy chain containing the HCv Ab1 variant is a heavy chain containing the amino acid sequence shown in SEQ ID NO: 50.

An exemplary heavy chain containing Ab2 HCv is a heavy chain containing the amino acid sequence shown in SEQ ID NO: 7.

An exemplary heavy chain containing the HCv Ab2 variant is a heavy chain containing the amino acid sequence shown in SEQ ID NO: 51.

- 10 041171

An exemplary heavy chain containing Ab3 HCv is a heavy chain containing the amino acid sequence shown in SEQ ID NO: 8.

An exemplary heavy chain containing the Ab3 HCv variant is a heavy chain containing the amino acid sequence shown in SEQ ID NO: 52.

Additional examples of scaffolds considered include, but are not limited to, fibronectin, CBM4-2 neocarzinostatin, lipocalins, T cell receptor, protein A (protein Z) domain, Im9, TPR proteins, zinc finger domains, pVIII, avian pancreatic polypeptide, GCN4, domain WW, Src homology domain 3, PDZ domains, TEM-1 beta-lactamase, thioredoxin, staphylococcal nuclease, PHD finger domains, CL-2, BPTI, APPI, HPSTI, ecotin, LACI-D1, LDTI, MTI-II, scorpion toxins, insect defensin-A peptide, EETI-II, Min-23, CBD, PBP, cytochrome b-562, LDL receptor domains, gamma-crystallin, ubiquitin, transferrin and/or C-type lectin-like domains. For a review of non-antibody scaffolds and their use as therapeutic agents, see 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 entirety.

Aspects of the invention include antibodies containing the following variable domains: Ab1 LCv/Ab1 HCv (SEQ ID NO: 27/SEQ ID NO: 9), Ab2 LCv/Ab2 HCv (SEQ ID NO: 28/SEQ ID NO: 10), Ab3 LCv /Ab3 HCv (SEQ ID NO: 29/SEQ ID NO: 11) and combinations thereof, as well as fragments, derivatives, muteins and variants thereof.

Also included are antibodies containing the following variable domains: SEQ ID NO: 27/SEQ ID NO: 53 and SEQ ID NO: 28/SEQ ID NO: 54.

Representative 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).

Additional generic antibodies include SEQ ID NO: 24/SEQ ID NO: 50;

SEQ ID NO: 25/SEQ ID NO: 51 and SEQ ID NO: 26/SEQ ID NO: 52.

Typically, each antibody light or heavy chain variable domain contains three CDRs. The heavy chain variable domain comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3). The light chain variable domain comprises light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3). In certain embodiments, the antigen binding protein comprises one or more CDRs located in the preferred variable domains described herein.

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

CDRs belonging to Ab1 LCv: LCDR1 (SEQ ID NO: 30), LCDR2 (SEQ ID NO: 33) and LCDR3 (SEQ ID NO: 36);

CDRs belonging to Ab2 LCv: LCDR1 (SEQ ID NO: 31), LCDR2 (SEQ ID NO: 34) and LCDR3 (SEQ ID NO: 37);

CDRs belonging to Ab3 LCv: LCDR1 (SEQ ID NO: 32), LCDR2 (SEQ ID NO: 35) and LCDR3 (SEQ ID NO: 38);

CDRs belonging to Ab1 HCv: HCDR1 (SEQ ID NO: 12), HCDR2 (SEQ ID NO: 15) and HCDR3 (SEQ ID NO: 18);

CDRs belonging to Ab2 HCv: HCDR1 (SEQ ID NO: 13), HCDR2 (SEQ ID NO: 16) and HCDR3 (SEQ ID NO: 19); And

CDRs belonging to Ab3 HCv: HCDR1 (SEQ ID NO: 14), HCDR2 (SEQ ID NO: 17) and HCDR3 (SEQ ID NO: 20).

In some embodiments, the antigen-binding protein comprises:

A) a polypeptide, eg a light chain, which contains an LCDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 31 and 32; 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, eg a heavy chain, which contains HCDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13 and 14; HCDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16 and 17; and/or HCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19 and 20.

In additional embodiments, the antigen-binding protein comprises:

A) a light chain amino acid sequence that contains LCDR1, LCDR2 and LCDR3 from any of Ab1 LCv, Ab2 LCv and Ab3 LCv; And

B) a heavy chain amino acid sequence that contains HCDR1, HCDR2, and HCDR3 from any of HCv Ab1, HCv Ab2, and HCv Ab3.

In certain embodiments, the CDR contains 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 compared to the exemplary CDR provided herein.

- 11 041171

Aspects of the invention include antibodies comprising a light chain variable domain selected from the group consisting of SEQ ID NOs: 27, 28, and 29. Aspects of the invention include antibodies comprising a heavy chain variable domain selected from the group consisting of SEQ ID NO: 9, 10 and 11. Additional aspects 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 NO: 9, 10 and 11.

Antibodies of the invention may contain any constant region known in the art. The light chain constant region may be, for example, a kappa or lambda type light chain constant region, eg a human kappa or lambda type light chain constant region. The heavy chain constant region may be, 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. type. In one embodiment, the light or heavy chain constant region is a fragment, derivative, variant, or mutein of a naturally occurring constant region.

Aspects of the invention include antibodies containing a light chain variable region selected from the group consisting of SEQ ID NOs: 27, 28 and 29, containing no more than one, no more than two, no more than three, no more than four, no more than five, no more six, no more than seven, no more than eight, no more than nine, or no more than ten amino acid additions, deletions or substitutions. Aspects of the invention include antibodies comprising a heavy chain variable region selected from the group consisting of SEQ ID NOs: 9, 10 and 11, containing no more than one, no more than two, no more than three, no more than four, no more than five, no more than six, no more than seven, no more than eight, no more than nine, or no more than ten amino acid additions, deletions or substitutions.

Additional aspects of the invention include antibodies containing:

A) a light chain variable region selected from the group consisting of SEQ ID NOs: 27, 28 and 29, containing no more than one, no more than two, no more than three, no more than four, no more than five, no more than six, no more seven, no more than eight, no more than nine, or no more than 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, containing no more than one, no more than two, no more than three, no more than four, no more than five, no more than six, no more seven, no more than eight, no more than nine, or no more than ten amino acid additions, deletions or substitutions.

In one variation, the antigen-binding protein contains 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 the amino acid sequence of the light chain variable region selected from the group consisting of SEQ ID NOS: 27, 28 and 29.

In another variation, the antigen-binding protein contains 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 the amino acid sequence of the heavy chain variable region selected from the group consisting of SEQ ID NOS: 9, 10 and 11.

In a further embodiment of the invention, 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 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 the amino acid sequence of the variable region a light chain selected from the group consisting of SEQ ID NOs: 27, 28 and 29; And

B) 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 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% amino acid sequence identical 041171 heavy chain variable region selected from the group consisting of SEQ ID NOs: 9 and 11.

OSMR antigen-binding proteins comprising a heavy chain variable domain related to SEQ ID NO: 9 as defined above may 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 contains the amino acid sequence shown in SEQ ID NO: 53.

OSMR antigen-binding proteins comprising a heavy chain variable domain related to SEQ ID NO: 10 as defined above may 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 contains the amino acid sequence shown in SEQ ID NO: 54.

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 shown in SEQ ID NOs: 36, 37, 38, 18, 19, and 20. In certain embodiments, the amino acid sequence contains 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 compared to the exemplary sequence shown in SEQ ID NOS: 36, 37, 38, 18, 19 and 20.

Thus, embodiments of the invention include an antigen-binding protein 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 at least 99% identical to an amino acid sequence selected from the group of sequences shown in SEQ ID NOs: 36, 37, 38, 18, 19 and 20.

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 shown in SEQ ID NOs: 33, 34, 35, 15, 16, and 17. In certain embodiments, the amino acid sequence contains 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 compared to the exemplary sequence shown in SEQ ID NOS: 33, 34, 35, 15, 16 and 17.

Thus, embodiments of the invention include an antigen-binding protein 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 shown in SEQ ID NOs: 33, 34, 35, 15, 16 and 17.

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 shown in SEQ ID NOs: 30, 31, 32, 12, 13, and 14. In certain embodiments, the amino acid sequence contains 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 compared to the exemplary sequence shown in SEQ ID NOS: 30, 31, 32, 12, 13 and 14.

Thus, embodiments of the invention include an antigen-binding protein 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 at least 99% identical to an amino acid sequence selected from the group of sequences shown in SEQ ID NOs: 30, 31, 32, 12, 13 and 14.

The antigen binding proteins of the invention comprise conventional antibody scaffolds, including human and monoclonal antibodies, bispecific antibodies, diabodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as antibody mimetics), chimeric antibodies, fusion antibodies (sometimes referred to herein as antibody conjugates) and fragments of each, respectively. The above CDRs, including various combinations

CDRs may be grafted into any of the following scaffolds.

As used herein, the term antibody refers to various forms of monomeric or multimeric proteins containing one or more polypeptide chains that specifically bind to an antigen, as comprehensively defined herein. In certain embodiments of the invention, antibodies are obtained using recombinant DNA technology. In additional embodiments of the invention, antibodies are obtained by enzymatic or chemical cleavage of naturally occurring antibodies. In another aspect, the antibody is selected from the group consisting of a) a human antibody; b) a humanized antibody; c) a chimeric antibody; d) a monoclonal antibody; e) a polyclonal antibody; f) a recombinant antibody; g) an antigen-binding fragment; h) single chain antibody; i) diabody; j) triabody, k) tetrabody, l) Fab fragment; m) F(ab')2 fragment, n) IgA antibody, o) IgD antibody, p) IgE antibody, q) IgG1 antibody, r) IgG2 antibody, s) IgG3 antibody, t) IgG4 antibody, and u) IgM antibody.

The variable region or domain contains at least three heavy or light chain CDRs embedded in a framework (frame designations are FR1, FR2, FR3, and FR4). Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, MD. Traditional structural units of antibodies, as a rule, contain a tetramer. Each tetramer typically consists of two identical pairs of polypeptide chains, with each pair containing one light and one heavy chain. The amino terminal portion of each chain contains a variable region of about 100 to 110 or more 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, which determines the isotype of the antibody - IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgM1 IgM2. Embodiments of the invention include all such classes and subclasses of antibodies that contain the variable domain or CDRs of antigen-binding proteins as described herein.

Some naturally occurring antibodies, such as those found in camels and llamas, are dimers of two heavy chains and do not contain light chains. Included in the present invention are dimeric antibodies consisting of two heavy chains, or fragments thereof, that are capable of binding to OSMR.

Heavy and light chain variable regions generally share the same general structure with respect to conserved framework regions (FRs) connected by three hypervariable regions, ie. complementarity determining regions or CDRs. CDRs are primarily responsible for antigen recognition and binding. The CDRs of the two strands of each pair are aligned by framework regions, allowing binding to a specific epitope. In the N-terminal to C-terminal direction, both the light and heavy chains contain the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 domains. The assignment of amino acids to each of the domains is carried out in accordance with the definitions of Kabat.

The CDRs form the main contact sites for antigen binding. The light chain CDR3, and in particular the heavy chain CDR3, may form the most important determinants of antigen binding within the light and heavy chain variable regions. In some antibodies, the heavy chain CDR3 forms the main interface between antigen and antibody. In vitro selection schemes that vary only the CDR3 can be used to vary the binding properties of an antibody to determine which residues are relevant for antigen binding.

Antibodies of natural origin, as a rule, contain a signal sequence that directs the antibody into the cellular pathway for protein secretion and which is usually not present in the mature antibody. A polynucleotide encoding an antibody of the invention may encode a naturally occurring signal sequence or a heterologous signal sequence, as described below.

In one embodiment of the invention, the antigen-binding protein is an antibody containing one to six exemplary CDRs described herein. The antibodies of the invention may be of any type, including IgM, IgG (including IgG1, IgG2, IgG3, IgG4), IgD, IgA or IgE antibodies. In a particular embodiment, the antigen binding protein is an IgG type antibody, eg an IgG1 antibody.

In some embodiments, such as those in which the antigen binding protein is an antibody with complete heavy and light chains, all CDRs are from the same species, such as a human. Alternatively, for example, in embodiments of the invention in which the antigen binding protein contains less than six CDRs from the above sequences, additional CDRs may be from a different species or may be a CDR,

- 14 041171 other than those illustrated in the exemplary sequences. For example, HCDR3 and LCDR3 regions derived from the respective sequences defined herein can be used in conjunction with HCDR1, HCDR2, LCDR1, and LCDR2, optionally selected from other species or other human antibody sequences, or combinations thereof. For example, the CDRs of the invention can replace the CDR regions of corresponding commercial chimeric or humanized antibodies.

In particular embodiments of the invention, scaffold components of antigen-binding proteins are used that are human components. However, in some embodiments of the invention, the frame components may be a combination obtained from different species. Therefore, if the antigen binding protein is an antibody, such antibody may be a chimeric antibody and/or a humanized antibody. In general, both chimeric antibodies and humanized antibodies refer to antibodies containing regions derived from more than one species. For example, chimeric antibodies typically comprise mouse (or in some cases rat) variable region(s) and human constant region(s).

Humanized antibodies generally refer to non-human antibodies in which the variable domain framework regions have been replaced with sequences found in human antibodies. In the general case of a humanized antibody, all of the 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. CDRs, some or all of which are encoded by nucleic acids of non-human origin, are grafted into the folded beta layer of a human antibody variable region framework to produce an antibody whose specificity is determined by the grafted CDRs. The production 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., 2004, Biotechnol. Prog. 20:639-654). In exemplary embodiments of the invention described herein, certain CDRs are human and thus both humanized and chimeric antibodies in this context contain some non-human CDRs; for example, humanized antibodies can be made to contain HCDR3 and LCDR3 regions from one or more other CDR regions belonging to a different species.

In one embodiment, the OSMR antigen-binding protein is a multispecific antibody, namely a bispecific antibody, also referred to as a diabody. These are antibodies that bind to two or more different antigens or different epitopes of the same antigen. In certain embodiments, the bispecific antibody binds OSMR and a human effector cell (eg, T-cell) antigen. Such antibodies are useful for targeting an effector cell response against OSMR expressing cells, such as an OSMR expressing tumor cell. In preferred embodiments, the human effector cell antigen is CD3, US Pat. No. 7,235,641. Methods for producing bispecific antibodies are known in the art. One such method involves constructing the Fc portion of the heavy chains to provide ridges and valleys that promote the formation of heavy chain heterodimers when co-expressed in a cell. Another method also involves constructing the Fc portion of the heavy chain, but uses electrostatic interaction to stimulate the formation of heterodimers, while inhibiting the formation of heavy chain heterodimers in the case of co-expression in the cell, WO 09/089004, which is fully incorporated into this text by reference.

In one embodiment, the OSMR antigen-binding protein is a minibody. Mini-bodies are minimized antibody-like proteins containing scFv coupled to a CH3 domain (Hu et al., 1996, Cancer Res. 56:3055-3061).

In one embodiment, the OSMR antigen-binding protein is a domain antibody; see, for example, US Pat. No. 6,248,516. Domain antibodies (dAbs) are functional antibody binding domains corresponding to the variable regions of both the heavy (VH) and light (VL) chains of human antibodies. dABs have a molecular weight of approximately 13 kDa, or less than one tenth the size of a complete antibody. dABs are well expressed in a variety of host organisms, including bacterial, yeast, and mammalian cell systems. In addition, dAbs are very stable and remain active even after exposure to harsh conditions such as lyophilization or thermal denaturation. See, for example, US Pat. Nos. 6,291,158; 6582915; 6593081; 6172197; USA No. 2004/0110941; European patent 0368684; US patents No. 6696245, WO 04/058821, WO 04/003019 and WO 03/002609.

In one embodiment, the OSMR antigen-binding protein is an antibody fragment, i. a fragment of any of the antibodies listed herein that retains binding specificity for OSMR. In various embodiments, the antigen-binding proteins comprise, without limitation, F(ab), F(ab'), F(ab') ₂ , Fv fragments, or single chain Fv fragments. As used herein, an antibody comprises at least a polypeptide that can specifically bind to OSMR and contains all or part of a light or heavy chain variable region, such as one or more CDRs.

Additional examples of OSMR-binding antibody fragments include, but are not limited to, (i) a Fab fragment consisting of V _L , VH, C _L and C _H 1 domains, (ii) an Fd fragment consisting of VH and C _H 1 domains, (iii) an Fv fragment consisting of the V _L and VH domains of a single antibody; (iv) dAb fragment (Ward et al., 1989, Nature, 341:544-546), which consists of a single variable region, (v) isolated CDR regions, (vi) F(ab')2 fragments, divalent fragment , containing two linked Fab fragments, (vii) single-stranded Fv molecules (scFv) in which the VH domain and the V _L domain are linked by a peptide linker that allows association of the two domains 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) single chain Fv bispecific dimers (PCT/US92/09965) and (ix) diabody or triabody, multivalent or multispecific fragments constructed by gene fusion (Tomlinson et. al., 2000, Methods Enzymol. 326:461-479; WO 94/13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6444-6448). Antibody fragments may be modified. For example, molecules can be stabilized by incorporating 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.

In one embodiment, the OSMR antigen-binding protein is a fully human antibody. In this embodiment, as indicated above, the specific structures comprise complete heavy and light chains containing CDR regions. In further embodiments, one or more CDRs of the invention are used along with other CDRs, framework regions, J and D regions, constant regions, etc. derived from other human antibodies. For example, the CDRs of the invention may replace the CDRs of any number of human antibodies, in particular the corresponding commercial antibodies.

The formation of single chain antibodies can occur by linking fragments of the heavy and light chain variable domains (Fv regions) through an amino acid bridge (short peptide linker), which leads to the formation of a single polypeptide chain. Such single chain Fvs (scFvs) were generated by fusing the DNA encoding the peptide linker with the DNA encoding the two variable domain polypeptides (V _L and V _H ). The resulting polypeptides may self-conjugate or they may form multimers (eg, dimers, trimers, or tetramers) depending on the length of the flexible linker between the two variable domains (Kortt et al., 1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol Eng 18:95-108). By combining polypeptides containing different V _L and V H, multimeric scFvs can be obtained that bind to different epitopes (Kriangkum et al., 2001, Biomol. Eng. 18:31-40). Methods developed to produce single chain antibodies include those described in US Pat. 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 derived from antibodies provided herein, including but not limited to scFv, containing combinations of Ab1 LCv/Ab1 HCv variable domains (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), as well as combinations thereof, are included in the present invention. Typical single chain antibodies contain the following combinations of variable domains: SEQ ID NO: 27/SEQ ID NO: 53; and SEQ ID NO: 28/SEQ ID NO: 54.

In one embodiment, the OSMR antigen-binding protein is an antibody fusion protein (sometimes referred to as an antibody conjugate). The conjugation partner can be either protein or non-protein; the latter option is obtained using the functional groups of the antigen-binding protein and conjugation partner. In certain embodiments, the antibody is conjugated to a non-protein chemical (drug) to form an antibody-drug conjugate.

In one embodiment, the OSMR antigen-binding protein is an antibody analog, sometimes referred to as a synthetic antibody. For example, a large number of works use both alternative protein scaffolds and artificial scaffolds with grafted CDRs. Such scaffolds include, but are not limited to, mutations introduced to stabilize the three-dimensional structure of the binding protein, as well as fully synthetic scaffolds consisting of, for example, biocompatible polymers. See, 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. In addition, peptidic antibody mimetics (PAMs) can be used, as well as compounds based on antibody mimetics that use fibronectin components as scaffolds.

As used herein, a protein refers to at least two covalently linked amino acids, and the term includes proteins, polypeptides, oligopeptides, and peptides. In some embodiments of the invention, two or more covalently linked amino acids are connected by a peptide bond. The protein may be composed of naturally occurring amino acids and contain peptide bonds, for example when the protein is produced recombinantly using expression systems and host cells as described below. Alternatively, the protein may contain synthetic amino acids (eg, homophenylalanine, citrulline, ornithine, and norleucine) or peptidomimetic structures, ie. peptide or protein analogs such as peptoids (see, Simon et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:9367, incorporated herein by reference), which may be resistant to proteases or other physiological conditions and/or storage conditions. In particular, such synthetic amino acids may be incorporated during in vitro synthesis of the antigen-binding protein using conventional methods well known in the art. In addition, any combination of peptidomimetics, synthetic and natural residues/structures can be used. The term amino acid also includes amino acid residues such as proline and hydroxyproline. The amino acid R-group or side chain can be in both (L)- and ^-configuration. In a specific embodiment of the invention, the amino acids are in the (L)- or ^-configuration.

In certain aspects, the invention provides recombinant antigen-binding proteins that bind OSMR and, in some embodiments, recombinant human OSMR or a portion thereof. In this context, a recombinant protein is a protein obtained using recombinant technologies using any methods and methods known in the art, i. by expressing the recombinant nucleic acid as described herein. Methods and methods for obtaining recombinant proteins are well known in the art. Embodiments of the invention include recombinant antigen-binding proteins that bind wild-type OSMR and variants thereof.

Consisting predominantly of means that the amino acid sequence may differ by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% of the sequence shown in SEQ ID NO:, but still retain biological activity as described herein.

In some embodiments, the antigen-binding proteins of the invention are isolated proteins or substantially purified proteins. The isolated protein contains at least some of the material with which it is normally associated in its natural state, for example such material may comprise at least about 5% or at least about 50% by weight of the total protein in a given sample. It should be understood that the isolated protein may comprise from 5 to 99.9% by weight of the total protein content, depending on the circumstances. For example, a protein can be produced at a significantly higher concentration through the use of an inducible promoter or a highly expressed promoter, resulting in the production of a protein at an increased concentration level. This definition includes the production of antigen-binding protein in a large number of organisms and/or host cells, which are known in the art.

In the case of amino acid sequences, sequence identity and/or similarity is determined using standard methods known in the art, including, but not limited to, the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, Identical Serial Needleman-Wunsch Alignment Algorithm, 1970, J. Mol. Biol. 48:443, Pearson and Lipman's similarity search method, 1988, Proc. Nat. Acad. sci. U.S.A. 85:2444, computerized versions of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence alignment program described in Devereux et al. , 1984, Nucl. Acid Res. 12:387-395, preferably using default or discretionary options. Preferably, percent identity is calculated using FastDB based on the following parameters: mismatch penalty - 1; gap penalty -1; gap size penalty - 0.33; and a compound penalty of 30, Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, p. 127-149 (1988), Alan R. Liss, Inc.

An example of a suitable algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive pairwise alignment. You can also build a tree showing the relationship groups used to create the alignment. PILEUP uses a simplified progressive alignment method by Feng and Doolittle, 1987, J. Mol. Evol. 35:351-360; this method is similar to that described by Higgis and Sharp, 1989, CABIOS 5:151-153. Applicable PILEUP parameters include a default gap weight of 3.00, a default gap length of 0.10, and gaps with weighted ends.

Another example of a suitable algorithm is the BLAST algorithm 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. U.S.A. 90:5873-5787. In particular, a suitable BLAST program is the WU-BLAST-2 program, which was obtained in accordance with Altschul et al., 1996, Methods in Enzymology, 266:460-480. WU-BLAST-2 uses several search parameters, most of which are set by default. The adjustable parameters are set to the following values: overlap length = 1, overlap fraction = 0.125, threshold word length (T) = II. The parameters HSP S and HSP S2 are dynamic values and are determined by the program itself depending on the composition of a particular sequence and the composition of a particular database, which is used to search for a sequence of interest; however, these values can be adjusted to increase the sensitivity.

An additional suitable algorithm is the gapped BLAST described in Altschul et al., 1993, Nucl. Acids Res. 25:3389-3402. Gap BLAST uses the BLOSUM-62 substitution weighting matrix; the threshold parameter T is set to 9; the double match method is used to initiate extensions without gaps, the cost for a gap length equal to k is 10+k; X _u is set to 16 and Xg is set to 40 in the database search step and 67 in the output step of the algorithms. Gap alignment is initiated at a weight corresponding to about 22 bits.

In general, the amino acid homology, similarity or identity between individual variant CDRs and the sequence described herein is at least 80%, and more often preferably has an increasing degree of homology or identity of at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and almost 100%. Likewise, percent (%) nucleotide sequence identity with a nucleotide sequence of binding proteins defined herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical to nucleotide residues of an antigen binding protein coding sequence. The specific method uses the BLASTN module WU-BLAST-2 with default parameters and overlap length and overlap ratio set to 1 and 0.125, respectively.

In general, the homology, similarity, or nucleotide sequence identity between the nucleotide sequences encoding individual variant CDRs and the nucleotide sequences recited herein is at least 80, and more often preferably has an increasing degree of homology or identity of at least 80, 81, 82 , 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and almost 100%.

Thus, a variant CDR is a region with a certain homology, similarity or identity to the main CDR according to the invention, which has a biological function of, without limitation, at least 80, 81, 82, 83, 84, 85, 86, 87, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the specificity and/or activity of the main CDR.

While the site or site for introducing an amino acid sequence variation is predetermined, a mutation per se need not be predetermined. For example, to optimize the introduction of a mutation at a given site, random mutagenesis at the target codon or site can be performed and CDR variants of the antigen binding protein can be examined for the optimal combination of desired activity. Methods for introducing substitutional mutations at predetermined regions of DNA having a known sequence are well known, such as M13 primer mutagenesis and PCR mutagenesis. Mutants are screened using antigen-binding protein activity assays such as OSMR binding.

Amino acid substitutions typically include one residue; insertions are typically on the order of about one to about twenty amino acid residues, although significantly larger insertions are possible. The range of deletions is from about one to about twenty amino acid residues, although in some cases there may be significantly more deletions.

Substitutions, deletions, insertions, or any combination thereof can be used to produce the final derivative or variant. In general, these changes are carried out for a small number of amino acids in order to minimize changes in the molecule, in particular the immunogenicity and specificity of the antigen binding protein. In this case, under certain circumstances, large changes are possible. Conservative substitutions are carried out in the General case in accordance with the following scheme, given in the form of table. 3.

- 18 041171

Table 3

Original remainder Typical substitutions

Ala Ser Arg Lys Asn Gin, His asp Glu Cys Ser gin Asn Glu asp gly Pro His Asn, Gin He Leu, Vai Leu lie, Vai Lys Arg, Gin, Glu Met Leu, lie Phe Met, Leu, Lyr Ser Thr Thr Ser trp Tyr Tyr Trp, Phe Vai He, Leu

When choosing substitutions that are less conservative than those given in table. 3, significant changes in function and immunological identity occur. For example, substitutions can be made that most strongly affect: the structure of the polypeptide backbone in the area of change, for example, the structure of the alpha helix or beta folding; the charge or hydrophobicity of the molecule at the target site; or side chain volume. Substitutions for which the greatest changes in polypeptide properties are generally expected include those in which (a) a hydrophilic residue, eg seryl or threonyl, is replaced by a hydrophobic residue, eg leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) the cysteine or proline is replaced by any other residue; (c) a residue having an electropositive side chain, such as lysyl, arginyl or histidyl, is replaced by an electronegative residue, such as glutamyl or aspartyl; or (d) a residue having a bulky side chain, eg phenylalanine, is replaced by a residue having no side chain, eg glycine.

The variants generally exhibit qualitatively identical biological activity and elicit an identical immune response as the naturally occurring analogue, although variants that modify the characteristics of the antigen binding protein can be selected as desired. Alternatively, a variant can be engineered to alter the biological activity of the antigen binding protein. For example, as discussed herein, glycosylation sites can be altered or removed.

Other anti-OSMR antibody derivatives within the scope of this invention include covalent or aggregative conjugates of anti-OSMR antibodies or fragments thereof with other proteins or polypeptides, such as those obtained by recombinant expression of fusion proteins and containing heterologous polypeptides fused to the N-terminus or C -end of the anti-OSMR antibody polypeptide. For example, the conjugated peptide may be a heterologous signal (or leader) polypeptide, such as a yeast alpha factor leader peptide or a peptide such as an epitope tag. Anti-OSMR antibody fusion proteins may contain peptides added to facilitate purification or detection of an anti-OSMR antibody (eg, poly-His). An anti-OSMR antibody polypeptide can also be coupled to a FLAG peptide as described in Hopp et al., Bio/Technology, 6:1204, 1988 and US Pat. No. 5,011,912. mAb), which enables rapid analysis and facilitates purification of the expressed recombinant protein. Reagents used to generate fusion proteins in which the FLAG peptide is fused to the desired polypeptide are commercially available (Sigma, St. Louis, MO).

In one embodiment of the invention, the oligomer is obtained using polypeptides derived from immunoglobulins. The production of fusion proteins containing certain heterologous polypeptides fused to different portions of antibody-derived polypeptides (including the Fc domain) have been 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, in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11.

- 19 041171

One embodiment of the present invention relates to a dimer containing two fusion proteins obtained by fusion of the OSMR binding fragment of an anti-OSMR antibody to the Fc region of the antibody. A dimer can be obtained, for example, by inserting a gene fusion product encoding a fusion protein into an appropriate expression vector, expressing the gene fusion product in host cells transformed with the recombinant expression vector, and allowing the fusion protein to assemble in an antibody-like manner, in which Fc components are formed interchain disulfide bonds to form a dimer.

As used herein, the term Fc polypeptide includes native forms and muteins of polypeptides derived from the Fc region of an antibody. Also included are truncated forms of such polypeptides containing a hinge region that promotes dimerization. Fc-containing fusion proteins (and the oligomers formed therefrom) have the advantage of being easier to purify by affinity chromatography on protein A and protein G columns.

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. Another suitable Fc polypeptide is the Fc mutein described in US Pat. No. 5,457,035 and in Baum et al., 1994, EMBO J. 13:3992-4001. The amino acid sequence of this mutein is identical to the native Fc sequence shown in WO 93/10151, except that amino acid 19 was changed from Leu to Ala, amino acid 20 was changed from Leu to Glu, and amino acid 22 was changed from Gly to Ala. The mutein shows reduced affinity for Fc receptors.

In other embodiments, the variable portion of the heavy and/or light chain of an anti-OSMR antibody may be replaced by the variable portion of the heavy and/or light chain of the antibody.

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

Covalent modifications of antigen-binding proteins are within the scope of this invention and are generally, but not always, carried out after translation. For example, certain types of covalent modifications to an antigen-binding protein are introduced into a molecule by reacting certain amino acid residues of the antigen-binding protein with an organic derivatizing agent that is capable of reacting with selected side chains or N- or C-terminal residues.

Cystenyl residues are most commonly reacted with α-haloacetates (and the corresponding amines) such as chloroacetic acid or chloroacetamide to give carboxymethyl or carboxyamidomethyl derivatives. Cystenyl residues are also derivatized by reacting with bromtrifluoroacetone, a-bromo-b-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl-2-pyridyl disulfide, p-chloromercury benzoate, 2- chlormercury-4-nitrophenol or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reacting with diethyl pyrocarbonate at pH 5.5-7.0, since this agent is relatively specific for the histidyl side chain. Parabromophenacyl bromide is also applicable; the reaction is preferably carried out in a 0.1 M solution of sodium cacodylate at pH 6.0.

Lysinyl residues and amino-terminal residues are reacted with succinic anhydride or anhydrides of other carboxylic acids. Derivatization with these agents can lead to charge reversal of lysinyl residues. Other suitable reagents for derivatizing alpha-amino residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione and glyoxylate in a reaction catalyzed by transaminase.

Arginyl residues are modified by reacting with one or more conventional reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin.

- 20 041171

Derivatization of arginine residues requires that the reaction be carried out in an alkaline environment due to the high pK _a of the guanidine functional group. In addition, such reagents can react with lysine groups, as well as with the arginine epsilon-amino group.

It is also possible to carry out certain modifications of tyrosyl residues, of particular interest are the cases of introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. The most commonly used are N-acetylimidizole and tetranitromethane to form O-acetyl tyrosyl and 3-nitro derivatives, respectively. Tyrosyl residues are iodinated using ¹²⁵ I or ¹³¹ I to obtain labeled proteins for use in radioimmunoassay, whereby the method described above using chloramine T is suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'-N=C=N--R'), where R and R' are optionally different alkyl groups such as 1-cyclohexyl-3-( 2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4azonia-4,4-dimethylphenyl)carbodiimide. In addition, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reacting with ammonium ions.

Derivatization with bifunctional agents is used to cross-link antigen-binding proteins to a water-insoluble support matrix or support surface for use in a wide variety of methods. Commonly used cross-linking agents include, for example, 1,1-bis-(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters, such as '-dithiobis-(succinimidyl propionate), and difunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate allow the preparation of photoactivated intermediates that are capable of crosslinking in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and reactive substrates are used to immobilize proteins, as described in US Pat.

The glutaminyl and asparaginyl residues are often deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Any form of these residues is within the scope of this invention.

Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of α-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 1983, p 79-86), acetylation of the N-terminal amine and amidation of any C-terminal carboxyl group.

Another type of covalent modification of an antigen binding protein that is within the scope of this invention involves altering the glycosylation profile of the protein. As is known in the art, glycosylation profiles can depend both on the protein sequence (eg, the presence or absence of specific glycosylation amino acid residues discussed below) and on the host cell or organism in which the protein is produced. Specific expression systems are discussed below.

Glycosylation of polypeptides is typically N-linked or O-linked. N-linkage refers to the attachment of a 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 recognition sequences for enzymatic attachment of a carbohydrate component to the side chain of asparagine. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the addition 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 may also be used.

The addition of glycosylation sites to an antigen binding protein is traditionally accomplished by changing the amino acid sequence to include one or more of the tripeptide sequences described above (in the case of N-linked glycosylation sites). The change can also be made by adding or replacing one or more serine or threonine residues in the original sequence (in the case of O-linked glycosylation sites). To facilitate the task, the amino acid sequence of the antigen binding protein is preferably altered by changes at the DNA level, in particular by introducing a mutation at preselected bases in the DNA encoding the target polypeptide, so that codons are generated that will be translated into the required amino acids.

Other ways to increase the amount of carbohydrate components of the antigen-binding protein are enzymatic or chemical conjugation of protein glycosides. The advantage of these methods is that they do not require the production of protein in the host cell, which is characterized by the possibility of glycosylation in the case of N - and O-linked glycosylation. Depending on the applied

- 21 041171 conjugation schemes sugar (a) can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as cysteine groups, (d) free hydroxyl groups, such as serine groups, threonine or hydroxyproline, (e) aromatic residues such as phenylalanine, tyrosine or tryptophan residues, or (f) glutamine amide group. These methods are described in WO 87/05330, published September 11, 1987, and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., p. 259-306.

Removal of the carbohydrate components present in the original antigen-binding protein can be carried out chemically or enzymatically. Chemical deglycosylation requires exposing the protein to a trifluoromethanesulfonic acid compound or equivalent. This treatment results in removal of most or all of the sugars except the binding sugars (N-acetylglucosamine or N-acetylgalactosamine) while leaving the polypeptide itself intact. Chemical deglycosylation is described in Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and in Edge et al., 1981, Anal. Biochem. 118:131. Enzymatic cleavage of the carbohydrate components of polypeptides can be accomplished using various endo- and exoglycosidases as described in Thotakura et al., 1987, Meth. Enzymol. 138:350. Prevention of glycosylation at potential glycosylation sites can be achieved through the use of a tunicamycin compound as described in Duskin et al., 1982, J. Biol. Chem. 257:3105. Tunikamycin blocks the formation of protein-M-glycoside bonds.

Another type of covalent modification of an antigen-binding protein includes coupling the antigen-binding protein with various non-protein polymers, including, but not limited to, various polyols such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, by methods described in US Pat. Nos. 4,640,835; 4496689; 4301144; 4670417; 4,791,192 or 4,179,337. Also, as is known in the art, amino acid substitutions can be made at various positions on the antigen-binding protein to facilitate the addition of polymers such as PEG.

In some embodiments of the invention, the covalent modification of antigen-binding proteins according to the invention includes the addition of one or more labels.

The term labeling group refers to any detectable label. Examples of suitable labeling groups include, but are not limited to, the following components: radioisotopes or radionuclides (e.g. ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ⁿ¹ In, ¹²⁵ I, ¹³¹ I), fluorescent groups (e.g., FITC, rhodamine, lanthanide complex phosphors), enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups, or predetermined polypeptide epitopes recognized by the secondary reporter (e.g., paired sequences leucine zippers, secondary antibody binding sites, metal-binding domains, epitope tags). In some embodiments, the tag group is coupled to the antigen-binding protein via spacers of varying length to reduce potential steric mismatch. Various protein labeling methods are known in the art and can be used to carry out the present invention.

In general, labels are divided into different classes depending on the method of detection: a) isotopic labels, which may be radioactive or heavy isotopes; b) magnetic marks (eg magnetic particles); c) redox-active components; d) optical dyes; enzyme groups (eg horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); e) biotinylated groups; and f) predefined polypeptide epitopes recognized by the secondary reporter (eg, paired leucine zipper sequences, secondary antibody binding sites, metal binding domains, epitope tags). In some embodiments, the tag group is coupled to the antigen-binding protein via spacers of varying length to reduce potential steric mismatch. Various protein labeling methods are known in the art and can be used to carry out the present invention.

Specific labels include optical dyes, including but not limited to chromophores, luminophores and fluorophores, the latter being specific in many cases. Fluorophores can be either low molecular weight fluorophores or protein fluorophores.

By fluorescent meta is meant any molecule that can be identified by its characteristic fluorescent properties. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methylcoumarins, pyrene, malachite 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 (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa 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, Cy7 ( Amersham Life Science, Pittsburgh, PA). Suitable optical dyes, including fluorophores, are described in the Molecular Probes Handbook (Richard P. Haugland), incorporated herein by reference.

- 22 041171

Suitable protein fluorescent labels also include, but are not limited to, green fluorescent protein, including GFP Renilla, Ptilosarcus or Aequorea species (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 85:26032607) and Renilla (WO 92/15673, WO 95/07463, WO 98/14605, WO 98/26277, WO 99/49019 , US Pat. Nos. 5,292,658; 5,418,155; 5,683,888; 5,741,668; All of the above references are incorporated herein by reference.

The exemplary antigen-binding proteins described herein have properties associated with the unique OSMR epitope bound by the antigen-binding protein. The term epitope refers to the amino acids of a target molecule that are in contact with an antigen-binding protein, such as an antibody, when the antigen-binding protein binds to the target molecule. An epitope may be continuous or discontinuous (e.g. (i) in a single-stranded polypeptide, these are amino acid residues that are not continuous with each other in the polypeptide sequence, but which, in the case of a target molecule, are bound by an antigen-binding protein, or (ii) in a multimeric receptor containing two or more separate components, such as OSMR receptor A and gp130 or OSMR and IL-31, these are amino acid residues present in one or more of the individual components that are bound by an antigen binding protein). Epitope determinants may include reactive surface groups of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics and/or specific charge characteristics. In general, antigen-binding proteins specific for a particular target molecule preferentially recognize an epitope on the target molecule in a complex mixture of proteins and/or macromolecules.

Methods for characterizing epitope binding by an antigen-binding protein are well known in the art, including, but not limited to, sorting (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. The B-cell Epitope of the Monoclonal Anti-Factor VIII Antibody ESH8 Characterized by Peptide Array Analysis, 2008, Thromb. Haemost. 99, 634-7), mutagenesis techniques such as chimeric generation (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 (Lim et al. A diversity of antibody epitopes can induce signaling through the erythropoietin receptor, Biochemistry (2010), 49, 3797-3804), hydrogen-deuterium exchange methods (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 methods (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, J. Mol. Biol (2009), 394, 905-21). These methods differ in the level of detail they provide for the amino acids that make up the epitope. Example 4 describes a typical method for sorting epitopes.

The antigen-binding proteins of the present invention include those containing an epitope overlapping with the exemplary antigen-binding protein described herein, such as Ab1, Ab2 or Ab3. In certain embodiments of the invention, the antigen-binding protein contains an epitope that is identical to typical antigen-binding proteins. In other embodiments, the antigen-binding protein binds only a subset of the same amino acids as a typical antigen-binding protein.

In certain embodiments, the antigen-binding protein to OSMR contains an identical or overlapping epitope with Ab1, Ab2 or Ab3 and contains a) a light chain variable domain having at least 90% identity, at least 95% identity, or identical to the amino acid sequence shown 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 identical to the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11; or c) a light chain variable domain according to a) and a heavy chain variable domain according to b).

In certain embodiments, the antigen-binding protein to OSMR contains an identical or overlapping epitope with Ab1, Ab2 or Ab3 and contains a light chain variable domain having at least 90%, at least 95% or identical amino acid sequence.

- 23 041171 of the value shown in SEQ ID NO: 27, and a heavy chain variable domain having at least 90%, at least 95% or identical to the amino acid sequence shown in SEQ ID NO: 9; contains a light chain variable domain having at least 90%, at least 95% or identical to the amino acid sequence shown in SEQ ID NO: 28, and a heavy chain variable domain having at least 90%, at least 95% or identical to the amino acid sequence shown in SEQ ID NO: 10; and contains a light chain variable domain having at least 90%, at least 95% or identical to the amino acid sequence shown in SEQ ID NO: 29, and a heavy chain variable domain having at least 90%, at least 95% or identical to the amino acid sequence shown in SEQ ID NO: 11.

In certain embodiments, the antigen-binding protein to OSMR contains an identical or overlapping epitope with Ab1, Ab2 or Ab3 and contains a) a light chain variable domain containing no more than ten or no more than five amino acid additions, deletions or substitutions compared to the amino acid sequence shown in SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29; b) a heavy chain variable domain containing no more than ten or no more than five amino acid additions, deletions or substitutions compared to the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11; or c) a light chain variable domain according to a) and a heavy chain variable domain according to b).

In certain embodiments, the antigen-binding protein to OSMR contains an identical or overlapping epitope with Ab1, Ab2 or Ab3 and contains a light chain variable domain containing no more than ten or no more than five amino acid additions, deletions or substitutions compared to the amino acid sequence shown in SEQ ID NO: 27, and a heavy chain variable domain containing no more than ten or no more than five amino acid additions, deletions or substitutions compared to the amino acid sequence shown in SEQ ID NO: 9; contains a light chain variable domain containing no more than ten or no more than five amino acid additions, deletions or substitutions compared to the amino acid sequence shown in SEQ ID NO: 28, and a heavy chain variable domain containing no more than ten or no more than five amino acid additions , deletions or substitutions compared to the amino acid sequence shown in SEQ ID NO: 10; and contains a light chain variable domain containing no more than ten or no more than five amino acid additions, deletions or substitutions compared to the amino acid sequence shown in SEQ ID NO: 29, a heavy chain variable domain containing no more than ten or no more than five amino acid additions , deletions or substitutions compared to the amino acid sequence shown 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 position corresponding to position 73 of SEQ ID NO: 9. The amino acid sequence shown in SEQ ID NO: 53 is 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 a position corresponding to position 73 of SEQ ID NO: 10. The amino acid sequence shown in SEQ ID NO: 54 is is an example of a heavy chain variable domain variant of SEQ ID NO: 10.

In certain embodiments, the OSMR antigen-binding protein contains an identical or overlapping epitope with Ab1, Ab2, or Ab3 and contains a light chain variable domain comprising:

a) an LCDR1 containing no more than three amino acid additions, deletions or substitutions compared to the LCDR1 sequence shown in SEQ ID NO: 30;

An LCDR2 containing no more than three amino acid additions, deletions or substitutions compared to the LCDR2 sequence shown in SEQ ID NO: 33; And

An LCDR3 containing no more than three amino acid additions, deletions or substitutions compared to the LCDR3 sequence shown in SEQ ID NO: 36;

b) LCDR1 containing no more than three amino acid additions, deletions or substitutions compared to the LCDR1 sequence shown in SEQ ID NO: 31;

LCDR2 containing no more than three amino acid additions, deletions or substitutions compared to the LCDR2 sequence shown in SEQ ID NO: 34; And

LCDR3 containing no more than three amino acid additions, deletions or substitutions compared to the LCDR3 sequence shown in SEQ ID NO: 37; or

c) an LCDR1 containing no more than three amino acid additions, deletions or substitutions compared to the LCDR1 sequence shown in SEQ ID NO: 32;

LCDR2 containing no more than three amino acid additions, deletions or substitutions compared to the LCDR2 sequence shown in SEQ ID NO: 35; And

LCDR3 containing no more than three amino acid additions, deletions or substitutions compared to the LCDR3 sequence shown in SEQ ID NO: 38; And

- 24 041171 heavy chain variable domain containing:

d) HCDR1 containing no more than three amino acid additions, deletions or substitutions compared to the HCDR1 sequence shown in SEQ ID NO: 12;

HCDR2 containing no more than three amino acid additions, deletions or substitutions compared to the HCDR2 sequence shown in SEQ ID NO: 15; And

HC DR3 containing no more than three amino acid additions, deletions or substitutions compared to the HCDR3 sequence shown in SEQ ID NO: 18;

e) HCDR1 containing no more than three amino acid additions, deletions or substitutions compared to the HCDR1 sequence shown in SEQ ID NO: 13;

HCDR2 containing no more than three amino acid additions, deletions or substitutions compared to the HCDR2 sequence shown in SEQ ID NO: 16; And

HCDR3 containing no more than three amino acid additions, deletions or substitutions compared to the HCDR3 sequence shown in SEQ ID NO: 19; or

f) HCDR1 containing no more than three amino acid additions, deletions or substitutions compared to the HCDR1 sequence shown in SEQ ID NO: 14;

HCDR2 containing no more than three amino acid additions, deletions or substitutions compared to the HCDR2 sequence shown in SEQ ID NO: 17; And

HCDR3 containing no more than three amino acid additions, deletions or substitutions compared to the HCDR3 sequence shown in SEQ ID NO: 20.

Preferred OSMR antigen-binding proteins described immediately above include those comprising a light chain variable domain according to a) and a heavy chain variable domain according to d); those containing the light chain variable domain according to b) and the heavy chain variable domain according to e); and those containing a light chain variable domain according to c) and a heavy chain variable domain according to f).

OSMR antigen-binding proteins comprising a light chain variable domain according to a) and a heavy chain variable domain according to d) may optionally contain an amino acid other than asparagine (e.g., aspartic acid) at the position corresponding to position 73 in SEQ ID NO: 9. In such embodiments, the heavy chain variable domain optionally comprises the amino acid sequence shown in SEQ ID NO: 53.

OSMR antigen-binding proteins comprising a light chain variable domain according to b) and a heavy chain variable domain according to e) may 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 shown in SEQ ID NO: 54.

Antigen-binding proteins that contain an identical epitope or an overlapping epitope often show cross-competition for antigen binding. Thus, in certain embodiments of the invention, the antigen-binding protein of the invention cross-competes with Ab1, Ab2, or Ab3. Cross-competing or cross-competing means that antigen-binding proteins compete for the same epitope or binding site on a target. The presence of such competition can be determined by assay methods in which a standard antigen-binding protein (eg, an antibody, or an antigen-binding portion thereof) prevents or inhibits specific binding of the antigen-binding protein of interest, and vice versa. To determine whether a molecule of interest competes with a reference molecule for binding, numerous kinds of competitive binding assays can be used. Examples of assay methods that can be used include solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (ELISA), competition sandwich assay (see, e.g., Stahli et al. (1983), Methods in Enzymology, 9: 242-253), direct ELISA with biotin and avidin (see e.g. Kirkland et al., (1986), J. Immunol. labeled, 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). A typical method for determining cross-competition is described in Example 5. Typically, if the competing antigen-binding protein is present in excess, it will inhibit the binding of the standard antigen-binding protein to the total 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 antigen-binding proteins for OSMR.

Included in the invention are nucleic acids or isolated nucleic acids encoding OSMR antigen-binding proteins, including antibodies as defined herein. Preferred nucleic acids include those that encode the exemplary light and heavy chains described herein.

- 25 041171

An exemplary nucleic acid encoding Ab1 LC is a nucleic acid containing the sequence shown in SEQ ID NO: 21.

An exemplary nucleic acid encoding Ab2 LC is a nucleic acid containing the sequence shown in SEQ ID NO: 22.

An exemplary nucleic acid encoding Ab3 LC is a nucleic acid containing the sequence shown in SEQ ID NO: 23.

An exemplary nucleic acid encoding Ab1 HC is a nucleic acid containing the sequence shown in SEQ ID NO: 3.

An exemplary nucleic acid encoding Ab2 HC is a nucleic acid containing the sequence shown in SEQ ID NO: 4.

An exemplary nucleic acid encoding Ab3 HC is a nucleic acid containing the sequence shown in SEQ ID NO: 5.

An exemplary nucleic acid encoding the Ab1 HC variant is a nucleic acid containing the sequence shown in SEQ ID NO: 47.

An exemplary nucleic acid encoding the Ab2 HC variant is a nucleic acid containing the sequence shown in SEQ ID NO: 48.

An exemplary nucleic acid encoding the Ab3 HC variant is a nucleic acid containing the sequence shown in SEQ ID NO: 49.

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

Aspects of the invention include a large number of embodiments, including, but not limited to, the following exemplary embodiments.

An isolated nucleic acid containing a polynucleotide, said polynucleotide encoding one or more polypeptides containing an amino acid sequence selected from the group consisting of:

(A) 1) a light chain variable domain sequence at least 90% identical to the light chain variable domain sequence set forth in SEQ ID NOs: 27-29;

2) a heavy chain variable domain sequence at least 90% identical to the heavy chain variable domain sequence set forth in SEQ ID NOs: 9-11;

3) a light chain variable domain according to (1) and a heavy chain variable domain according to (2); and (B) a light chain variable domain containing CDR1, CDR2, CDR3 and/or a heavy chain variable domain containing CDR1, CDR2, CDR3 that are identical or differ by no more than three amino acid additions, substitutions and/or deletions in each CDR from the following sequences:

1) Light chain CDR1 (SEQ ID NO: 30), CDR2 (SEQ ID NO: 33), CDR3 (SEQ ID NO: 36) or CDR1 (SEQ ID NO: 12), CDR2 (SEQ ID NO: 15), CDR3 (SEQ ID NO: 18) Ab1 heavy chain;

2) Light chain CDR1 (SEQ ID NO: 31), CDR2 (SEQ ID NO: 34), CDR3 (SEQ ID NO: 37) or CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 16), CDR3 (SEQ ID NO: 19) Ab2 heavy chain;

3) Light chain CDR1 (SEQ ID NO: 32), CDR2 (SEQ ID NO: 35), CDR3 (SEQ ID NO: 38) or CDR1 (SEQ ID NO: 14), CDR2 (SEQ ID NO: 17), CDR3 (SEQ ID NO: 20) Ab3 heavy chain.

In some embodiments, the nucleic acid encodes a polypeptide that contains the amino acid sequence shown in SEQ ID NO: 53 or SEQ ID NO: 54.

In some embodiments, the nucleic acid encodes a polypeptide that contains the amino acid sequence shown in SEQ ID NO: 50, SEQ ID NO: 51, or SEQ ID NO: 52.

In preferred embodiments, the polypeptide encoded by the nucleic acid or isolated nucleic acid is a component of an antigen-binding protein that binds OSMR.

Nucleotide sequences corresponding to the amino acid sequences described herein for use as probes or primers for nucleic acid isolation, or as query sequences for database searches, can be obtained by reverse translation from amino acid sequences or by determining regions of amino acid identity with polypeptides. for which the DNA coding sequence has been determined. The well-known polymerase chain reaction (PCR) procedure can be used to isolate and amplify the DNA sequence encoding antigen-binding proteins for OSMR or the desired combinations of polypeptide fragments of antigen-binding proteins for OSMR. Oligonucleotides that define the desired ends of the combination of DNA fragments are used as 5' and 3' primers. The oligonucleotides may further contain restriction endonuclease recognition sites to facilitate insertion of the amplified combination of DNA fragments into the expression vector. PCR methods are described in Saiki et al., Science, 239:487 (1988); Recombinant DNA Methodology, Wu et al., eds., Academic Press, Inc., San

- 26 041171

Diego (1989), p. 189-196; and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., Academic

Press, Inc. (1990).

Nucleic acid molecules according to the invention include DNA and RNA in both single-stranded and double-stranded form, as well as the corresponding complementary sequences. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, PCR amplified DNA, and combinations thereof. Nucleic acid molecules according to the invention include full-length genes or cDNA molecules, as well as combinations of their fragments. The nucleic acid molecules of the invention are preferably derived from human sources, but the invention also includes those derived from non-human species.

In some embodiments, the nucleic acids of the invention are isolated nucleic acids. An isolated nucleic acid is a nucleic acid that has been separated from adjacent genetic sequences present in the genome of the organism from which the nucleic acid was isolated, in the case of nucleic acids isolated from sources of natural origin. In the case of nucleic acids synthesized enzymatically from a template or chemically, such as, for example, PCR products, cDNA molecules or oligonucleotides, it is to be understood that the nucleic acids thus obtained are isolated nucleic acids. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a single fragment or as a component of a larger nucleotide construct. In one preferred embodiment of the invention, the nucleic acids are substantially free of contaminating endogenous material. Preferably, the nucleic acid molecule is derived from DNA or RNA isolated at least once in a substantially pure form and in an amount and concentration that allows detection, manipulation and recovery of its constituent nucleotide sequences using standard biochemical methods (such as those that described in Sambrook et al., Molecular Cloning: A Laboratory Manual, ^2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)). Such sequences are preferably obtained and/or designed in the form of an open reading frame that is not interrupted by internal untranslated sequences or introns that are commonly found in eukaryotic genes. Non-translated DNA sequences may be present 5' or 3' from the open reading frame, where their presence does not interfere with manipulation or expression of the coding region.

The present invention also includes nucleic acids or isolated nucleic acids that hybridize under moderate stringency, and more preferably under high stringency conditions, to nucleic acids encoding OSMR antigen-binding proteins as described herein. The main parameters that influence the choice of hybridization conditions and guidance on selecting suitable conditions are given 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), they can easily be determined by those skilled in the art based on, for example, length and/or composition of DNA bases. One way to obtain moderate stringency conditions involves the use of a prewash solution containing 5xSSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), a hybridization buffer of about 50% formamide, 6xSSC and a hybridization temperature of about 55° C (or other similar hybridization solutions, such as a solution containing about 50% formamide, with a hybridization temperature of about 42°C), and washing conditions corresponding to about 60°C in 0.5xSSC, 0.1% SDS. In general, high stringency conditions are defined as the hybridization conditions above, but washing is carried out at approximately 68°C, 0.2xSSC, 0.1% SDS. SSPE (1xSSPE is 0.15M NaCl, 10mM NalkPO: and 1.25mM EDTA, pH 7.4) can be replaced with SSC (1xSSC is 0.15M NaCl and 15mM sodium citrate) in the hybridization and wash buffers; washing is carried out for 15 minutes after hybridization is completed. It should be understood that the wash temperature and wash salt concentration can be adjusted as necessary to obtain the desired degree of stringency by applying the basic principles that govern hybridization reactions and coil stability, as is 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 length of the hybrid is considered to be the length of the hybridizing nucleic acid. In the behavior of hybridization of nucleic acids with known sequences, the length of the hybrid can be determined by aligning the nucleic acid sequences and determining the region or regions of optimal sequence complementarity. The hybridization temperature for hybrids expected to be less than 50 bp in length should be 5-10° C. less than the melting point (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs long: Tm (°C)=2(base no. A+T)+4(base no. G+C). For hybrids longer than 18 base pairs: Tm (°C)=81.5+16.6(log ₁₀

- 27 041171

[Na ⁺ ])+0.41(% G+C)-(600/N), where N is the number of bases in the hybrid, and [Na ⁺ ] is the concentration of sodium ions in the hybridization buffer ([Na ⁺ ] for 1xSSC=0.165 M). Preferably, each hybridizing nucleic acid is at least 15 nucleotides in length (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 nucleic acid length acid of the invention with 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%, smaller m here 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%) with the nucleic acid of the invention to which it hybridizes, wherein sequence identity is determined by comparing the sequences of hybridizing nucleic acids when aligned in such a way as to maximize overlap and identity and minimize sequence gaps as described in more detail above.

Variants of the invention are typically generated by site-directed nucleotide mutagenesis in the DNA encoding the antigen binding protein using cassettes or PCR mutagenesis or other methods known in the art to obtain the DNA encoding the variant and then expressing the recombinant DNA in cell culture, as described in this document. However, fragments of antigen-binding proteins containing variant CDRs having up to about 100-150 residues can be obtained by in vitro synthesis using conventional methods. In general, variants exhibit qualitatively the same biological activity as the naturally occurring analogue, eg binding to OSMR, although variants having modified characteristics may be selected, as described in more detail below.

Those skilled in the art will recognize that, due to the degeneracy of the genetic code, a very large number of nucleic acids can be obtained that all encode the CDRs (as well as the heavy and light chain or other antigen binding protein components) of the present invention. Thus, once a particular amino acid sequence has been determined, any number of different nucleic acids can be generated by those skilled in the art by simply modifying the sequence of one or more codons so that the amino acid sequence of the encoded protein does not change.

The present invention also provides expression systems and constructs in the form of plasmids, expression vectors, transcription or expression cassettes that contain at least one of the polynucleotides described above. In addition, the invention provides host cells containing such expression systems or constructs.

As a rule, used in the case of any host cell, expression vectors contain sequences for maintaining the plasmid and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively termed flanking sequences, in some embodiments, typically comprise 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 containing a donor and a splicing acceptor site, a sequence encoding a leader sequence for secreting a polypeptide, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element. Each of these sequences is discussed below.

Optionally, the vector may contain a label encoding sequence, i. e. an oligonucleotide molecule located at the 5'- and 3'-end of the sequence encoding the antigen-binding protein to OSMR; the oligonucleotide sequence encodes polyHis (such as hexaHis) or another label such as FLAG, HA (influenza hemagglutinin) or myc for which there are commercially available antibodies. This label is typically fused to the polypeptide after expression of the polypeptide and may serve as a means to affinity purify or detect the antigen binding protein for OSMR in the host cell. Affinity purification can be carried out, for example, using column chromatography, using antibodies against the specified label as an affinity matrix. Optionally, the label can subsequently be removed from the purified OSMR antigen-binding protein by various methods, such as the use of peptidases for cleavage.

Flanking sequences can be homologous (i.e. derived from the same species and/or from the same strain as the host cell), heterologous (i.e. derived from the species and/or from

- 28 041171 strain other than the host cell), hybrid (ie a combination of sequences from more than one source), synthetic or native. Accordingly, the source of the flanking sequence can be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequence is functional in the host cell and can be activated by the host cell mechanism.

The flanking sequences used in the vectors of this invention can be obtained by any of several methods well known in the art. In general, the flanking sequences used herein have been previously identified by mapping and/or restriction endonuclease digestion and can therefore be isolated from a suitable tissue source using the appropriate restriction endonucleases. In some cases, the complete nucleotide sequence of the flanking sequence may be known. In this case, the flanking sequence can be synthesized using the methods described herein for the synthesis or cloning of nucleic acids.

Whether all or only a portion of the flanking sequence is known, it can be obtained by using polymerase chain reaction (PCR) and/or by screening the genomic library with a suitable probe such as an oligonucleotide and/or flanking sequence fragment derived from that the same or from another species. If the flanking sequence is known, it is possible to isolate a DNA fragment containing the flanking sequence from a larger stretch of DNA, which may contain, for example, a coding sequence or even another gene or genes. Isolation can be accomplished by restriction endonuclease digestion to obtain the appropriate DNA fragment, followed by isolation by agarose gel purification, Qiagen® column chromatography (Chatsworth, CA), or other methods known to one of skill in the art. The choice of suitable enzymes for this purpose is obvious to a person skilled in the art.

The origin of replication is typically part of a commercially available prokaryotic expression vector and facilitates amplification of the vector in the host cell. If the selected vector does not contain a site corresponding to the origin of replication, it can be chemically synthesized based on the known sequence and ligated into the vector. For example, the origin of replication from plasmid pBR322 (New England Biolabs, Beverly, MA) is suitable for most gram-negative bacteria, and different viral origins (for example, SV40, polyomas, adenovirus, vesicular stomatitis virus (VVS) or papillomaviruses such as HPV or papilloma virus KPC) are suitable for cloning vectors in mammalian cells. In general, the origin of replication component is not necessary for mammalian expression vectors (eg, the SV40 point is often used only because it also contains an early viral promoter).

A transcription termination sequence is typically located at the 3' end of the coding region of a polypeptide and serves to terminate transcription. Typically, the transcription termination sequence in prokaryotic cells is a rich G-C fragment followed by a poly-T sequence. Although the sequence can be easily cloned from a library or even purchased commercially, it is also not difficult to synthesize using nucleic acid synthesis methods such as those described herein.

The selectable marker gene encodes a protein necessary for the survival and growth of a host cell growing in a selective culture medium. Exemplary selectable marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, eg ampicillin, tetracycline, or kanamycin in the case of prokaryotic host cells; (b) supplement the cell's auxotrophic deficiency; or (c) supply essential nutrients not available from a complex or defined environment. Specific selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. It is also advantageous to use the neomycin resistance gene for selection in both prokaryotic and eukaryotic host cells.

Other selectable genes can be used to amplify the gene to be expressed. Amplification is a process in which there is a tandem reiteration of genes necessary for the production of a protein necessary for cell growth or survival within the chromosomes of successive generations of recombinant cells. Examples of suitable mammalian cell selectable markers include dihydrofolate reductase (DHFR) genes and promoterless thymidine kinase genes. Mammalian cell transformants are placed under selection pressure conditions in which only transformants are adapted to survive due to the presence of a selectable gene in the vector. Selection pressure is carried out by culturing the transformed cells under conditions in which the concentration of the selection agent in the medium gradually increases, resulting in the amplification of both the gene to be selected and the DNA encoding another gene, such as an antigen-binding protein antibody that binds to the polypeptide.

- 29 041171

OSMR. As a result, an increased amount of a polypeptide, such as antigen-binding protein for OSMR, is synthesized from the amplified DNA.

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

In some instances where glycosylation is required in the eukaryotic host cell expression system, different pre or pro sequences can be manipulated to improve glycosylation or yield. For example, changes can be made to the peptidase cleavage site of a particular signal peptide, or prosequences can be added that can also affect glycosylation. The final protein product may contain at position -1 (relative to the first amino acid of the mature protein) one or more additional amino acids intrinsic to expression that have not been completely removed. For example, the final protein product may contain one or two amino acid residues located at the peptidase cleavage site attached to the amino terminus. Alternatively, the use of certain enzyme cleavage sites may result in a slightly truncated form of the desired polypeptide if the enzyme cleavage occurs at such a region within the mature polypeptide.

Expression and cloning vectors of the invention typically contain a promoter that is recognized by the host organism and is operably linked to a molecule encoding an OSMR antigen binding protein. Promoters are non-transcribed sequences located upstream (ie, in the 5' direction) of the start codon of a structural gene (generally ranging from about 100 to 1000 bp) that regulate transcription of the structural gene. Traditionally, promoters have been classified into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from their DNA in response to certain changes in culture conditions, such as the presence or absence of nutrients, or changes in temperature. On the other hand, constitutive promoters constantly transcribe the gene with which they are functionally linked, i.e. poorly regulate or do not regulate gene expression. A large number of promoters are well known and recognized by a variety of potential host cells. A suitable promoter is operably linked to a DNA encoding a heavy chain or a light chain containing an OSMR antigen binding protein of the invention by removing the promoter from the original DNA by restriction enzyme digestion and inserting the desired promoter sequence into the vector.

Promoters suitable for use in yeast hosts are also well known in the art. Yeast enhancers are preferably used with yeast promoters. Promoters suitable for use in mammalian host cells are well known and include, but are not limited to, derived from the genomes of viruses such as polyoma virus, fowl pox virus, adenovirus (such as adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and most preferably simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, for example, heat shock promoters or the actin promoter.

Additional promoters that may be of interest include, but are not limited to, the SV40 early promoter (Benoist et al., 1981, Nature, 290:304-310); CMV promoter (Thomsen et al., 1984, Proc. Natl. Acad. U.S.A. 81:659-663); a promoter contained in the 3' long terminal repeat of the Rous sarcoma virus (Yamamoto et al., 1980, Cell, 22:787-797); herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1444-1445); the promoter and regulatory sequences of the metallothionein gene (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. U.S.A. 75:3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25). Also of interest are the following transcriptional regulatory regions of animal origin that exhibit tissue specificity and are used in transgenic animals: elastase I gene regulatory region, which is active in pancreatic acinar cells (Swift et al., 1984, Cell, 38:639646; Ornitz et al. ., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology, 7:425-515); the regulatory region of the insulin gene, which is active in pancreatic beta cells (Hanahan, 1985, Nature, 315:115-122); an immunoglobulin gene regulatory region that 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, Mol. Cell Biol 7:1436-1444); the mouse mammary tumor virus regulatory region, which is active in testis, mammary, lymphoid and mast cells (Leder et al., 1986, Cell, 45:485-495); regulatory region of the albumin gene, which is active in

- 30 041171 liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); an alpha-fetoprotein gene regulatory region that 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 regulatory region, which is active in the liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); the regulatory region of the beta-globin gene, which is active in myeloid cells (Mogram et al., 1985, Nature, 315:338-340; Kollias et al., 1986, Cell, 46:89-94); the regulatory region of the myelin basic protein gene, which is active in brain oligodendrocyte cells (Readhead et al., 1987, Cell, 48:703-712); myosin light chain-2 gene regulatory region, which is active in skeletal muscle (Sani, 1985, Nature, 314:283-286); a regulatory region of the gonadotropic releasing hormone gene that is active in the hypothalamus (Mason et al., 1986, Science, 234:1372-1378).

An enhancer sequence can be inserted into a vector to increase the transcription of a DNA encoding a light chain or a heavy chain containing an OSMR antigen-binding protein of the invention by higher eukaryotes. Enhancers are cis-acting DNA elements, typically 10-300 bp in length, that act on a promoter to increase transcription. Enhancers are relatively orientationally and positionally independent and can be found in both the 5' and 3' direction of the transcription unit. Several enhancer sequences are known that are available from mammalian genes (eg, globin, elastase, albumin, alpha-fetoprotein, and insulin). In this case, as a rule, enhancers obtained from viruses are used. The SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, and adenovirus enhancers are known in the art and are exemplary enhancer elements for the activation of eukaryotic promoters. Although the enhancer can be located in the vector in both the 5' and 3' direction relative to the coding sequence, as a rule, it is located in the 5' direction from the promoter. The expression vector may include a sequence encoding an appropriate native or heterologous signal sequence (leader sequence or signal peptide) to stimulate extracellular secretion of antibodies. The choice of signal or leader peptide depends on the type of host cells in which the antibody is to be produced, and the native signal sequence can be replaced with a heterologous signal sequence. Examples of signal peptides functional in mammalian host cells include the following peptides: the signal sequence of interleukin-7 (IL-7), described in US patent No. 4965195; the signal sequence of the interleukin 2 receptor described in Cosman et al., 1984, Nature, 312:768; an interleukin-4 receptor signal peptide as described in European Patent No. 0367566; the signal peptide of the type I interleukin-1 receptor described in US patent No. 4968607; type II interleukin-1 receptor signal peptide described in European Patent No. 0460846.

The vector may contain one or more elements that facilitate expression when the vector is integrated into the genome of the host cell. Examples include the EASE element (Aldrich et al. 2003, Biotechnol. Prog. 19:1433-38) and the matrix attachment site (MAR). MARs mediate the structural organization of chromatin and may protect the integrated vector from a positional effect. Thus, MARs are extremely useful when the vector is used to generate stable transfectants. A large number of natural and synthetic MAR-containing nucleic acids are known in the art, for example, US patents No. 6239328; 7326567; 6177612; 6388066; 6245974; 7259010; 6037525; 7422874;7129062.

Expression vectors according to the invention can be designed based on the original vector, for example, a commercially available vector. Such vectors may or may not contain all of the necessary flanking sequences. If one or more of the flanking sequences described herein are not present in the vector, they can be obtained separately and ligated into the vector. Methods for obtaining each of the flanking sequences are well known to the person skilled in the art.

Once the vector has been constructed and a nucleic acid molecule encoding a light chain, a heavy chain, or a light chain and a heavy chain containing an OSMR antigen-binding sequence has been inserted into the appropriate region of the vector, the complete vector can be introduced into a suitable host cell for amplification and /or expression of the polypeptide. Transfer of an expression vector for an antigen-binding protein to OSMR into a host cell of choice can be accomplished by well-known methods, including transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-dextran-mediated transfection, or other known methods. The method chosen depends in part on the host cell used. These methods and other suitable methods are well known to the person skilled in the art and are described, for example, in Sambrook et al., 2001, supra.

When cultured under suitable conditions, the host cell synthesizes antigen-binding protein to OSMR, which can subsequently be removed from the culture medium (if secretion from the host cell occurs into the medium) or directly from the host cell producing it (if secretion does not occur). The selection of an appropriate host cell depends on various factors such as desired levels of expression, desired or required polypeptide modifications for activity.

- 31 041171 (such as glycosylation or phosphorylation), and ease of folding into a biologically active molecule. The host cell may be eukaryotic or prokaryotic.

Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC) and any known in the art cell lines used in expression systems that can be used to obtain recombinant polypeptides according to the invention. In general, host cells are transformed with a recombinant expression vector that contains 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 E. coli or bacilli. Higher eukaryotic cells include insect cells and resistant cell lines of mammalian origin. Examples of suitable mammalian cell lines include the monkey kidney line COS-7 (ATCC CRL 1651) (Gluzman et al., 1981, Cell, 23:175), L cells, 293 cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or their derivatives such as Veggie CHO and related cell lines that grow in serum-free media (Rasmussen et al., 1998, Cytotechnology, 28:31), HeLa cells, BHK cell lines (ATCC CRL 10) and a CVI/EBNA cell line derived from the African green monkey kidney line CVI (ATCC CCL 70) as described in McMahan et al., 1991, EMBO J. 10:2821, human embryonic kidney cells such as 293, 293 EBNA or MSR 293, A431 human epidermal cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro primary tissue culture, primary explants, HL-60, U937, HaK or Jurkat cells. Optionally, mammalian cell lines such as HepG2/3B, KB, NIH 3T3, or S49, for example, can be used to express the polypeptide if the polypeptide is to be used in different signal transduction assays or reporter assays. Alternatively, the polypeptide can be produced in lower eukaryotes such as yeast or prokaryotes such as bacteria. Suitable yeasts include strains of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces, 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 polypeptides are produced in yeast or bacteria, it may be necessary to modify such a polypeptide, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain a functional polypeptide. Such covalent attachments can be carried out using known chemical or enzymatic methods. The polypeptide can also be obtained by operably linking the nucleic acid or isolated nucleic acid of the invention with suitable regulatory sequences in one or more insect expression vectors and using an insect expression system. Materials and methods for preparing baculovirus/insect cell expression systems are available commercially in kit form, for example, from Invitrogen, San Diego, CA, U.S.A. (MaxBac® kit), and such methods are well known in the art and are 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 using RNA derived from the nucleic acid constructs described herein can also be used to produce polypeptides. Suitable cloning and expression vectors for use in bacterial, fungal, yeast and mammalian host cells are described in Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985). A host cell that contains a nucleic acid or an isolated nucleic acid according to the invention, preferably operably linked to at least one expression regulation sequence, is a recombinant host cell.

In certain embodiments of the invention, the selection of cell lines can be carried out by determining which cell lines have high levels of expression and consistently produce antigen-binding proteins with OSMR binding properties. In another embodiment of the invention, a cell line can be selected from a B cell line of differentiation that does not produce its own antibodies, but has the ability to produce and secrete heterologous antibodies.

Cell-depleting antigen-binding proteins to OSMR

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 certain embodiments of the invention antigen-binding protein to OSMR binds OSMR and targets the depletion of cells expressing OSMR. In various aspects, the antigen-binding protein to OSMR binds OSMR and inhibits the binding of OSM and/or IL-31 and targets the depletion of OSMR cells.

Cell-depleting OSMR antigen-binding proteins are extremely useful in the treatment of diseases or disorders associated with OSMR overexpression, eg, autoimmune diseases, inflammatory diseases, diseases or disorders associated with extracellular matrix accumulation or remodeling, or tumors characterized by OSMR expression. Methods for targeting cells with antigen-binding proteins, such as antibodies, are well known in the art. Exemplary embodiments of the invention are discussed below.

Antibody drug conjugates.

Embodiments of the invention include antibody drug conjugates (ADCs). In general, an ADC contains an antibody conjugated to a chemotherapeutic agent, such as a cytotoxic agent, a cytostatic agent, a toxin, or a radioactive agent. A linker molecule can be used to conjugate the drug to the antibody. A large number of linkers and drugs useful in ADC technology are known in the art and can be used in embodiments of the present invention. (See US 20090028856; US 2009/0274713; US 2007/0031402; Wo 2005/084390; Wo 2009/099728; US 5208020; 5416064; 5475092; 5585499; 6436931; 6372738; and 6340701, which are all included in this document by link ).

Linkers.

In certain embodiments of the invention, the ADC contains a linker consisting of one or more linker components. Exemplary linker components include 6-maleimidocaproyl, maleimidopropanoyl, valine-citrulline, alanine-phenylalanine, p-aminobenzyloxycarbonyl, and those derived from conjugation with linker components including, but not limited to, N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimudyl-4-(Nmaleimidomethyl)cyclohexane-1 carboxylate (SMCC, also referred to herein as MCC), and N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB).

Linkers can be cleavable linkers or non-cleavable linkers (Ducry and Stump, Bioconjugate Chem. 2010, 21, 5-13; incorporated herein by reference in its entirety). Cleavable linkers are designed to be released from the drug upon exposure to certain environmental factors, such as absorption into the target cell. Cleavable linkers include acid labile linkers, protease sensitive linkers, photolabile linkers, dimethyl linkers or disulfide-containing linkers. Non-cleavable linkers remain covalently linked to at least one amino acid of the antibody and drug after uptake and degradation in the target cell. An exemplary non-cleavable linker is MCC.

Medications.

In certain embodiments of the invention, the antibody is conjugated to a chemotherapeutic agent. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbokwon, meturedopa and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecin (including the synthetic analog of topotecan); bryostatin; callistatin; CC-1065 (including its synthetic analogues adocelesin, carcelesin and bicelesin); cryptophycins (in particular, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues of KW-2189 and CBI-TMI); eleuterobine; pancratistatin; sarcodictin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphasine, holofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembihin, phenesterin, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as enediyne antibiotics (for example, calicheamicin, in particular calicheamicin gamma 1 and calicheamicin theta I, see, for example, Angew Chem. Intl. Ed. Engl. 33:183-186 (1994); dynemycin, including dynemycin A; esperamycin, as well as neocarzinostatin chromophore and related chromoprotein enediine antibiotic chromophores); aclacinomysins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, carabicin, kaminomycin, carcinophyllin; chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5oxo-E-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, nitomycins, pelivomycins, olivomycophenolic acid , potfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, cynostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocytabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; adrenal cortex inhibitors such as aminoglutethimide, mitotane, trilostane; folic acid fortifier such as frolinic acid; aceglatone; aldophosphamide gilko- 33 041171 zid; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraksat; defofamine; demecolcin; diaziquon; elfomitin; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pyrarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofuran; spirohemania; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (particularly toxin T-2, verracurin A, roridin A, and anguidin); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; hacytosine; arabinosyl (Ara-C); cyclophosphamide; thiotepa; taxoids, eg paclitaxel (TAXOL®, Bristol Myers Squibb Oncology, Princeton, NJ) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbin; novantron; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine and pharmaceutically acceptable salts, acids or derivatives of any of the above.

Also included in this definition are antihormonal agents that act to regulate or inhibit the action of hormones on tumors, such as antiestrogen, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; miRNA and pharmaceutically acceptable salts, acids or derivatives of any of the above drugs. Other chemotherapeutic agents that may be used in the present invention are disclosed in US Patent Publication No. 20080171040 or US Patent Publication No. 20080305044, each of which is incorporated herein by reference in its entirety.

It is contemplated that an antibody may be conjugated to two or more different chemotherapeutic agents, or a pharmaceutical composition may contain a mixture of antibodies, wherein the components of the antibodies are identical except that they are conjugated to different chemotherapeutic agents. Such embodiments of the invention may be useful when targeting multiple biological pathways of the target cell.

In preferred embodiments, the ADC comprises an antibody conjugated to one or more maytansinoid molecules, which are mitotic inhibitors that act to inhibit tubulin polymerization. Maytansinoids, including various modifications, are described in US Pat. Nos. 3,896,111; 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. The maytansinoid components of a drug product can be isolated from natural sources, obtained by recombinant technology, or obtained synthetically. Exemplary maytansinoids include C-19-dechlor (US Pat. No. 4,256,746), C-20-hydroxy (or C-20-demethyl) ± C-19-dechlor (US Pat. Nos. 4,307,016 and 4,361,650), C-20-demethoxy (or C-20-acyloxy (-OCOR), ± dechlor (US Pat. No. 4,294,757), C-9-SH (US Pat. No. 4,424,219), C-14-alkoxymethyl (demethoxy/CH2OR) (US Pat. No. 4,331,598), C- 14-hydroxymethyl or acyloxymethyl (CH2OH or _CH2OAc ) (US Pat. No. 4,450,254), C-15-hydroxy/acyloxy (US Pat. No. 4,364,866), C-15-methoxy (US Pat. Nos. 4,313,946 and 4,315,929), C-18 -N-demethyl (US Pat. Nos. 4,362,663 and 4,322,348) and 4,5-deoxy (US Pat. No. 4,371,533).

Depending on the required type of connection, different positions of the maytansinoid compounds can be used as a connecting position. For example, the C-3 position containing a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with a hydroxyl group, and the C-20 position containing a hydroxyl group are suitable for ester bond formation (US Pat. Nos. 5,208,020, RE39151 and 6,913,748; US Patent Applications Nos. 20060167245 and 20070037972 and WO 2009/099728).

Preferred maytansinoids include those known in the art as DM1, DM3 and DM4 (US Patent Applications Nos. 2009/030924 and 2005/0276812, incorporated herein by reference).

ADCs containing maytansinoids, methods for preparing such ADCs, and their therapeutic uses are disclosed in US Pat. Linkers that can be used to make maytansinoid ADCs are known in the art (US Patent No. 5,208,020 and US Patent Applications Nos. 2005/016993 and 2009/0274713; all incorporated herein by reference). Maytansinoid ADCs containing the SMCC linker can be prepared as described in US Patent Application No. 2005/0276812.

Antibodies with enhanced effector function.

One of the functions of the Fc portion of an antibody is to interact with the immune system when the antibody binds to its target. This is called the effector function. The interaction leads to antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC). ADCC and ADCP are mediated through Fc binding to Fc receptors on the surface of immune system cells. CSC is mediated through the binding of Fc to complement proteins such as C1q.

IgG subclasses differ in their ability to mediate effector functions. For example, IgG1 is far superior to IgG2 and IgG4 in mediating ADCC and CDC. Thus, in embodiments in which an OSMR expressing cell is targeted for destruction, an anti-OSMR IgG1 antibody is preferred.

The effector function of an antibody can be increased or decreased by introducing one or more Fc mutations. Embodiments of the invention include antigen-binding proteins, such as antibodies containing an Fc engineered to enhance effector function (U.S. Patent 7,317,091 and Strohl, Curr. Opin. Biotech., 20:685-691, 2009; incorporated herein by reference in its entirety ). Exemplary IgG1 Fc molecules with enhanced effector function include (based on the Kabat numbering scheme) those containing the following substitutions:

3239D/I332E

S239D/A33CS/I332E

Ξ239D/A33CL/I332E

3298A/D333A/K334A

P247I/A339D

P247I/A339Q

E280H/K29CS

D280H/K29CS/S298D

D280H/K29CS/S298V

F243L/R292P/Y300L

F243L/R292P/Y300L/P396L

F243L/R292P/Y300L/V305I/RZ96L

G236A/S239D/I332E

K326A/E333A

KZ26W/E333S

K290Ε/Ξ298G/T299A

K290Ν/Ξ298G/T299A

K290E/S298G/T299A/K326E

K290N/S298G/T299A/K326E

Additional embodiments of the invention include antigen-binding proteins, for example, antibodies containing Fc, designed to reduce effector function. Exemplary Fc molecules with reduced effector function include (based on the Kabat numbering scheme) those containing the following substitutions:

N2 97A (IgGl)

L234A/L235A (IgGl)

V234A/G237A (IgG2)

L235A/G237A/E318A (IgG4)

H268Q/V309L/A330S/A33IS (IgG2)

C220S/C2263/O229S/P238S (IgGl)

C2 26S/C2293/E233P/L234V/L235A (IgGl)

L234F/L235E/P33 IS (IgGl)

S267E/L328F (IgGl)

Another way to increase the effector function of IgG Fc-containing proteins is to reduce Fc fucosylation. Removal of core fucose from Fc-linked 2-anthenary complex-type oligosaccharides significantly increased ADCC effector function without altering antigen binding or CSC effector function. There are several ways to reduce or eliminate the fucosylation of Fc-containing molecules, such as antibodies. These include recombinant expression in several mammalian cell lines, including the FUT8 gene knockout cell line, the CHO Lec13 variant line, the YB2/0 rat hybridoma cell line, a small interfering RNA cell line, specifically against the FUT8 gene, and a cell line co-expressing c-1,4-K-acetylglucosaminyltransferase III Golgi α-mannosidase II. Alternatively, the Fc-containing molecule can be expressed in a non-mammalian cell, such as a plant cell, a yeast cell, or a prokaryotic cell, such as E. coli.

Thus, in certain embodiments of the invention, the composition contains an antibody, for example, Ab1, Ab2 or Ab3, characterized by reduced fucosylation or lack of fucosylation.

pharmaceutical compositions.

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

Preferably, the formulation materials are non-toxic to recipients at the dosages and concentrations used. In specific embodiments, the invention provides pharmaceutical compositions comprising a therapeutically effective amount of an antigen-binding protein to OSMR, such as an OSMR-binding antibody.

In certain embodiments of the invention, the pharmaceutical composition may contain materials to modify, maintain or maintain, for example, pH, osmolarity, viscosity, clarity, color, isotonicity, aroma, sterility, stability, dissolution or release rate, adsorption or permeability of the composition. In such embodiments, suitable materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, proline, or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrosulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrins); proteins (such as serum albumin, gelatin, or immunoglobulins); dyes, flavors and diluents; emulsifying agents; 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; surfactants or wetting agents (such as pluronics, PEGs, sorbitol esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, triloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol, sorbitol); means of delivery; diluents; excipients and/or pharmaceutical adjuvants. See REMINGTON'S PHARMACEUTICAL SCIENCES, ^18th Edition, (A. Genrmo, ed.), 1990, Mack Publishing Company.

In certain embodiments of the invention, the optimal pharmaceutical composition is determined by a person skilled in the art depending on, for example, the intended route of administration, the delivery format and the desired dosage. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In certain embodiments of the invention, such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the antigen-binding proteins of the invention. In certain embodiments of the invention, the main filler or carrier in the pharmaceutical composition may be aqueous or non-aqueous. For example, a suitable vehicle or carrier may be water for injection, physiological saline, or artificial cerebrospinal fluid, optionally with the addition of other materials conventionally used in parenteral compositions. Additional typical vehicles are neutral buffered saline or saline mixed with serum albumin. In specific embodiments of the invention, the pharmaceutical composition contains a Tris buffer with a pH of about 7.08.5 or an acetate buffer with a pH of about 4.0-5.5 and may additionally contain sorbitol or a suitable substitute. In certain embodiments, antigen-binding protein compositions for OSMR can be prepared for storage by mixing the selected composition, having the desired degree of purity, with optional agents (REMINGTON'S PHARMACEUTICAL SCIENCES, supra) in the form of a lyophilized tablet or aqueous solution. Additionally, in certain embodiments, the OSMR antigen-binding protein product can be formulated as a lyophilisate using appropriate excipients such as sucrose.

Pharmaceutical compositions according to the invention may be intended for parenteral delivery. Alternatively, the compositions may be designed to be delivered via

- 36 041171 digestive tract, for example oral. The preparation of such pharmaceutically acceptable compositions is within the art. The components of the formulation are preferably present at concentrations that are acceptable given the site of administration. Buffers are used in certain embodiments of the invention to maintain the composition at a physiological pH or slightly lower pH, typically within the range of about 5 to about 8.

If parenteral administration is contemplated, the therapeutic compositions for use in the present invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired OSMR antigen-binding protein in a pharmaceutically acceptable vehicle. The most suitable vehicle for parenteral injection is distilled water with antigen-binding protein to OSMR dissolved in it in the form of a sterile, properly stored isotonic solution. In certain embodiments of the invention, the preparation may include the association of the desired molecule with an agent, such as injectable microspheres, biodegradable particles, polymeric compounds (such as polylactic acid or polyglycolic acid), granules or liposomes, which can provide controlled or delayed release of the product, which can be delivered via depot injection. In certain embodiments of the invention, you can also use hyaluronic acid, which has the effect of prolonging the time spent in the circulatory system. In certain embodiments of the invention, implantable drug delivery devices may be used to introduce the desired antigen-binding protein.

Pharmaceutical compositions according to the invention can be prepared for inhalation. In such embodiments, OSMR antigen-binding proteins are preferably formulated as a dry, inhalable powder. In particular embodiments of the invention, OSMR antigen-binding protein inhalation solutions can be prepared with an aerosol delivery propellant. In certain embodiments of the invention, the solutions may be sprayable. Inhalation administration and methods for preparing appropriate formulations are further described in International Patent Application No. PCT/US94/001875, which is incorporated by reference and describes the inhalation delivery of chemically modified proteins.

It is also contemplated that the formulations may be administered orally. OSMR antigen-binding proteins administered by this route can be formulated with or without the carriers conventionally used to formulate solid dosage forms such as tablets and capsules. In certain embodiments of the invention, the capsule can be prepared in such a way that the active part of the composition is released at the point of the gastrointestinal tract with maximum bioavailability and minimum presystemic degradation. Additional agents may be included to facilitate absorption of the antigen binding protein to the OSMR. It is also fashionable to use diluents, flavors, low melting waxes, vegetable oils, lubricants, suspending agents, disintegrating agents and binders.

The existence of additional pharmaceutical compositions is apparent to those skilled in the art, including formulations containing OSMR antigen-binding proteins in delayed or controlled delivery formulations. Also known to those skilled in the art are methods for preparing a wide variety of other sustained or controlled delivery vehicles, such as liposome carriers, biodegradable microparticles or porous granules, and depot injections. See, for example, International Patent Application No. PCT/US93/00829, which is incorporated by reference and describes the controlled release of polymeric microparticles for the delivery of pharmaceutical compositions. Sustained release formulations may contain semi-permeable polymeric matrices in the form of molded articles such as films or microcapsules. Sustained release matrices may contain polyesters, hydrogels, polylactides (as disclosed in US Pat. No. 3,773,919 and European Patent Application No. EP 058481, which are incorporated by reference), copolymers of L-glutamic acid, and gamma ethyl-E-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 No. EP 133988). Also, sustained release compositions may contain liposomes, which can be prepared by any of several methods known in the art. See, for example, Eppstein et al., 1985, Proc. Natl. Acad. sci. U.S.A. 82:3688-3692; European Patent Applications No. EP 036676; EP 088046 and EP 143949 incorporated by reference.

Pharmaceutical compositions used for in vivo administration are generally in the form of sterile preparations. Sterilization can be accomplished by filtration through sterile filtration membranes. When the composition is lyophilized, sterilization using this method can be carried out both before and after lyophilization and reconstitution. Compositions for parenteral administration may 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 pierced by a hypodermic injection needle.

- 37 041171

Aspects of the invention include self-buffering antigen-binding protein formulations to

OSMRs that can be used as pharmaceutical compositions as described in International Patent Application WO 06138181A2 (PCT/US2006/022599), which is incorporated herein by reference in its entirety.

As discussed above, certain embodiments of the invention provide antigen-binding protein compositions for OSMR, in particular pharmaceutical compositions of antigen-binding proteins for OSMR, which contain, in addition to the antigen-binding protein for OSMR, one or more excipients, such as those illustratively described in this section. and elsewhere in this document. Auxiliaries can be used within the scope of this invention for numerous purposes such as adjusting the physical, chemical or biological properties of formulations, such as adjusting viscosity, and/or in the methods of the invention to improve the performance and/or to stabilize such formulations and ways to avoid degradation and spoilage due to, for example, stresses that occur during production, transport, storage, pre-formulation, administration, etc.

A large number of guidelines for the stabilization of proteins and formulation materials, as well as methods used for this purpose, are available, for example, 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, Carpenter and Manning, eds. Pharmaceutical Biotechnology. 13:61-84 (2002) and Randolph et al., Surfactant-protein interactions, Pharm. Biotechnol. 13:159-75 (2002), which are incorporated herein by reference in their entirety, in particular those parts relating to excipients and related methods for self-buffering protein formulations according to the present invention, in particular protein pharmaceutical products and methods for use in veterinary and/or human medicine.

In accordance with certain embodiments of the invention, salts can be used, for example, to adjust the ionic strength and/or isotonicity of the composition and/or improve the solubility and/or physical stability of the protein or other ingredient of the composition in accordance with the invention.

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 the charged and polar groups of the protein, as well as reducing the strength of their electrostatic interaction, attraction and repulsion. The ions can also stabilize the denatured state of the protein, in particular by binding to denatured peptide bonds (-CONH) of the protein. In addition, ionic interaction with charged and polar groups in a protein can also reduce intermolecular electrostatic interaction and thereby prevent or reduce protein aggregation and insolubility.

The types of ions differ significantly in the effect they have on proteins. A large number of methods have been developed for categorizing ions and their effect on proteins, which can be used in formulating pharmaceutical compositions in accordance with the invention. One example is the Hofmeister series, in which ionic and polar non-ionic solutes are ranked according to their effect on the conformational stability of proteins in solution. Stabilizing soluble components are called cosmotropic. Destabilizing solute components are called chaotropic. Cosmotropes are typically used at high concentrations (eg, >1 mol/L ammonium sulfate) to precipitate proteins from solution (salting out). Chaotropes are usually used to denature and/or dissolve proteins (salting-in). The relative effectiveness of ions in salting out and salting out determines their position in the Hofmeister series.

OSMR antigen-binding protein formulations can use free amino acids as bulking agents, stabilizers, and antioxidants, among other standard uses, in various embodiments. To stabilize the proteins in the composition, you can use lysine, proline, serine and alanine. Glycine is used during lyophilization to ensure the proper structure and properties of the tablet. Arginine can be used to inhibit protein aggregation in both liquid and lyophilized formulations. Methionine is used as an antioxidant.

Polyols include sugars such as mannitol, sucrose, and sorbitol, as well as polyhydric alcohols such as, for example, glycerol and propylene glycol, and for the purposes of discussion herein, polyethylene glycol (PEG) and related substances. The polyols are cosmotropic. They are used as stabilizing agents in both liquid and lyophilized formulations to protect proteins from physical and chemical degradation processes. Also, polyols are used to adjust the tonicity of the compositions.

Among the polyols useful in selected embodiments of the invention is mannitol, commonly used to provide structural stability to the tablet in lyophilized

- 38 041171 compositions. It provides structural stability to the tablet. It is usually used in conjunction with a lyoprotectant such as sucrose. Sorbitol and sucrose are among the preferred agents for adjusting tonicity and as stabilizers to protect against freeze-thaw stresses during transport or preparation of large volumes of the drug during the manufacturing process. A decrease in sugars (which contain free aldehyde or ketone groups), such as glucose and lactose, can glycate surface lysine and arginine residues. Therefore, in general, they are not among the preferred polyols for use in accordance with the invention. In addition, sugars which are reactive species such as sucrose, which hydrolyze to fructose and glucose under acidic conditions and thus carry a risk of glycation, are also not among the preferred polyols of the invention. PEG is used to stabilize proteins and as a cryoprotectant, so in this respect it can be used in this invention.

Embodiments of OSMR antigen-binding protein formulations further include surfactants. Protein molecules can be susceptible to adsorption to surfaces and to denaturation and subsequent aggregation at air-liquid, solid-liquid and liquid-liquid interfaces. These effects are generally inversely proportional to protein concentration. These detrimental interactions are generally inversely related to protein concentration and are typically exacerbated by physical shaking such as occurs during product handling and handling.

Surfactants are traditionally used to prevent, minimize or reduce surface adsorption. Surfactants useful in this invention include polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan polyethoxylates, and poloxamer 188.

Also, surfactants are traditionally used to regulate the conformational stability of a protein. The use of surfactants for this purpose is protein specific, as any given surfactant will generally stabilize some proteins and destabilize others.

Polysorbates are susceptible to oxidative degradation and often contain sufficient peroxides to cause oxidation of the side chains of protein residues, especially methionine. Therefore, polysorbates should be used with caution and, if used, at the lowest effective concentration. In this regard, polysorbates illustrate the general rule that excipients should be used at the lowest effective concentration.

Embodiments of OSMR antigen-binding protein formulations further include one or more antioxidants. Harmful oxidation of proteins in pharmaceutical formulations can be prevented to some extent by maintaining appropriate external oxygen levels and temperature and by avoiding exposure to light. Auxiliary antioxidants can also be used to prevent oxidative degradation of proteins. Antioxidants useful in this regard include reducing agents, oxygen/free radical scavengers, and chelating agents. Antioxidants for use in therapeutic protein formulations according to the invention are preferably water soluble and retain their activity throughout the shelf life of the product. In this regard, the preferred antioxidant according to the invention is EDTA.

Antioxidants can damage protein. For example, reducing agents, such as, in particular, glutathione, can disrupt intramolecular disulfide bonds. Therefore, the antioxidants for use in the invention are selected such that, among other things, they eliminate or significantly reduce the possibility of them damaging the proteins in the formulations.

The compositions according to the invention may contain metal ions which are protein cofactors and are necessary for the formation of protein coordination complexes, for example zinc which is necessary for the formation of certain insulin suspensions. Also, metal ions can inhibit some of the processes that lead to the destruction of proteins. At the same time, metal ions also catalyze physical and chemical processes that lead to the destruction of proteins.

Magnesium ions (10-120 mM) can be used to inhibit the isomerization of aspartic acid to isoaspartic acid. Ions Ca ⁺² (up to 100 mm) can increase the stability of human deoxyribonuclease. However, Mg ⁺² , Mn ⁺² and Zn ⁺² can destabilize rhDNase. Similarly, Ca ⁺² and Sr ⁺² can stabilize VIII, but it can be destabilized by Mg ⁺² , Mn ⁺² and Zn ⁺² , Cu ⁺² and Fe ⁺² , and its aggregation can be enhanced by Al ⁺³ ions.

Embodiments of OSMR antigen-binding protein formulations further include one or more preservatives. Preservatives are needed when developing multi-dose parenteral formulations that provide more than one set from the same container. Their main function is to inhibit the growth of bacteria and ensure the sterility of the product during the shelf life or shelf life of the medicinal product. Traditionally used preservatives include benzyl alcohol, phenol and m-cresol. Although preservatives have a long history of use with small molecule parenterals, it can be difficult to develop protein formulations that contain preservatives. Preservatives almost always have desta

- 39 041171 bilizing effect (leading to aggregation) on proteins, and this has become a major factor in limiting their use in multi-dose protein formulations. To date, most protein drugs are prepared for single use only. However, where multi-dose formulations are possible, they have the advantage of patient convenience and increased competitiveness. A good example is that of human growth hormone (hGH), in which case the development of preservative-containing formulations has led to the commercialization of a more convenient reusable pen. At least four such devices containing preservative formulations of hGH are available on the market today. Norditropin (liquid, Novo Nordisk), Nutropin AQ (liquid, Genentech) and Genotropin (lyophilized, dual chamber cartridge, Pharmacia & Upjohn) contain phenol, while Somatrope (Eli Lilly) is formulated with m-cresol.

During the formulation and development of dosage forms containing preservatives, several aspects must be considered. It is necessary to optimize the effective concentration of the preservative in the medicinal product. This requires testing a given preservative in a dosage form in a concentration range that provides antibacterial efficacy and does not adversely affect protein stability.

As might be expected, the development of liquid formulations containing preservatives is more difficult than the development of lyophilized formulations. Freeze-dried products can be lyophilized without the use of preservatives and reconstituted in preservative-containing diluent at the time of use. This reduces the amount of time that the preservative is in contact with the protein, greatly minimizing the risk of instability. In the case of liquid formulations, preservative effectiveness and stability must be maintained throughout the shelf life of the product (about 18 to 24 months). It is important to note that the effectiveness of the preservative should be manifested in the final composition containing the active drug and all auxiliary components.

In general, OSMR antigen-binding protein formulations are designed for specific routes and routes of administration, for specific dosages of administration and frequency of administration, for specific treatments for specific diseases, with different ranges of bioavailability and tolerability. Thus, formulations can be designed in accordance with the 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 intra-arterial injection, intramuscular injection, and subcutaneous injection.

Once a pharmaceutical composition has been prepared, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated or lyophilized powder. Such formulations can be stored either in ready-to-use form or in a form (eg, lyophilized) that must be reconstituted prior to administration. The invention also provides kits for obtaining a single dose for administration. Kits according to the invention may contain a first container containing a dry protein and a second container containing an aqueous composition. In certain embodiments, kits are provided that contain single and multi-chamber pre-filled syringes (eg, liquid syringes and lyophilisate syringes).

The therapeutically effective amount of a pharmaceutical composition containing an OSMR antigen-binding protein to be used depends, for example, on the therapeutic context and goals. It will be appreciated by one of ordinary skill in the art that appropriate dosage levels for treatment will vary depending in part on the molecule being delivered, the indication for which the antigen-binding protein is applied to OSMR, the route of administration, and size (body weight, body surface area, and organ size). ) and/or condition (age and general health) of the patient. In certain embodiments of the invention, the attending physician may titrate the dosage and modify the route of administration to provide the optimal therapeutic effect. A typical dosage may be in the range of about 0.1 μg/kg to about 30 mg/kg or more, depending on the above factors. In particular embodiments, the dosage may be in the range of about 0.1 µg/kg to about 20 mg/kg, optionally 10 µg/kg to about 10 mg/kg, or 100 µg/kg to about 5 mg/kg.

A therapeutically effective amount of antigen-binding protein to OSMR preferably results in a reduction in the severity of the symptoms of the disease, an increase in the frequency or duration of asymptomatic periods, or the prevention of deterioration or incapacitation due to disease involvement.

Pharmaceutical compositions can be administered using a medical device. Examples of medical devices for the administration of pharmaceutical compositions are described in US patents No. 4475196;4439196;4447224; 4447233;4486194; 4487603; 4596556;4790824;4941880;5064413;5312335; 5312335; 5383851 and 5399163, incorporated herein by reference.

Methods for diagnosing or treating an OSMR-related disease or disorder.

- 40 041171

Antigen binding proteins to OSMR according to the invention are extremely useful for detecting OSMR in a biological sample. In certain embodiments of the invention, a biological sample obtained from a patient is brought into contact with an antigen-binding protein for OSMR. The binding of antigen-binding protein to OSMR to OSMR is then detected to determine the presence or relative amount of OSMR in the sample. Such methods can be used in diagnosing or identifying patients amenable to treatment with antigen-binding protein to OSMR.

In certain embodiments, the OSMR antigen-binding protein of the invention is used to diagnose, detect, or treat an autoimmune disease, an inflammatory disease, or a disease associated with extracellular matrix accumulation or remodeling.

In the treatment of these diseases, an OSMR antigen-binding protein may be targeted to OSMR-expressing cells of the immune system for destruction and/or may block the interaction of OSMR with OSM and/or IL-31.

Diseases and disorders that are associated with OSMR-mediated signaling are exceptionally susceptible to treatment with one or more OSMR antigen-binding proteins disclosed herein. Such disorders include, but are not limited to, inflammation, pain, pruritus, pruritus nodosum, dermatitis, asthma, autoimmune disease, paraneoplastic autoimmune diseases, cartilage inflammation, fibrosis (including but not limited to pulmonary fibrosis and skin fibrosis), fibrosing diseases, chronic obstructive pulmonary disease (COPD), interstitial pneumonitis, abnormal collagen accumulation, systemic cutaneous amyloidosis, primary cutaneous amyloidosis, Behcet's disease, nasal polyposis, liver cirrhosis, cartilage destruction, bone destruction, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic-onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome ropathy), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, pauciarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic-onset rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, SEA (seronegative enthesopathy and arthropathy syndrome), dermatomyositis, psoriatic arthritis, scleroderma, scleroderma-associated interstitial lung disease, vasculitis, myelitis, poliomyelitis, dermatomyelitis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, scleroderma, sclerosis, primary biliary ductal sclerosing cholangitis, Sjögren's syndrome, psoriasis, patchy psoriasis, guttate psoriasis, fold 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, rhinosinusitis with polyps, eosinophilic esophagitis, eosinophilic bronchitis, Guillain-Barré disease, diabetes mellitus type I, thyroiditis (eg, Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, GVHD, transplant rejection, kidney damage, cardiovascular disease, infection, sepsis, HIV infection, trauma, allograft nephropathy, IgA nephropathy, diabetic nephropathy , diabetic retinopathy, macular degeneration, biliary atresia, congestive heart failure, atherosclerosis, restenosis, radiation fibrosis, chemotherapy-induced fibrosis, burns, surgical trauma, glomerulosclerosis, and the like.

In preferred embodiments, the autoimmune disease, inflammatory disease, or extracellular matrix accumulation or remodeling disease is fibrosis, cartilage destruction, arthritis, rheumatoid arthritis, scleroderma, scleroderma-associated interstitial lung disease, idiopathic pulmonary fibrosis, cirrhosis, psoriasis , atopic dermatitis, systemic cutaneous amyloidosis, primary cutaneous amyloidosis, inflammation, pruritic inflammation, nodular pruritus and pain.

In certain embodiments, the OSMR antigen-binding protein is used to diagnose, detect, or treat a cancer or oncogenic disorder. In the treatment of a cancer or oncogenic disorder, an OSMR antigen-binding protein may be targeted to OSMR-expressing cells for destruction and/or may block the interaction of OSMR with OSM and/or IL31, thereby reducing OSMR-mediated signaling. It is hypothesized that OSMR antigen-binding proteins that block OSM and/or IL-31 mediated signaling may be useful in improving survival among cancer patients. Cancer or oncogenic disorders that can be diagnosed, detected, or treated with OSMR antigen-binding protein include, but are not limited to, broadly defined solid tumors, lung cancer, ovarian cancer, breast cancer, prostate cancer, endometrial cancer, kidney cancer , esophageal cancer, pancreatic cancer,

- 41 041171 squamous cell carcinoma, uveal melanoma, cervical cancer, colorectal cancer, bladder cancer, brain cancer, pancreatic cancer, head cancer, neck cancer, liver cancer, leukemia, lymphoma and Hodgkin's disease, multiple myeloma, melanoma, gastric cancer, astrocytoma, gastric cancer and lung adenocarcinoma.

Antigen binding proteins can be used to suppress tumor growth, progression and/or the appearance of metastases. Such suppression can be tracked in various ways. For example, suppression may result in both a reduction in tumor size and/or a reduction in metabolic activity in the tumor. Both of these parameters are determined, for example, using an MRI or PET scan. Suppression can also be monitored by biopsy to determine the level of necrosis, tumor cell death, and the level of blood vessels in the tumor. The degree of spread of metastases can be determined by known methods.

In the context of this document, the use of any or all examples or comparative expressions (such as) is intended to provide a better understanding of embodiments of the invention and does not limit the scope of the invention unless otherwise indicated. No wording in the text of the description should be taken as defining any element not listed in the claims as essential to the practice of the invention.

Examples

The following examples, both practical and forward-looking, are provided for the purpose of illustrating certain embodiments and features of the present invention and do not limit its scope.

Example 1 Generation of Anti-OSMR Antibodies Using the XENOMOUSE® Platform

Fully human antibodies directed against human OSMR were generated using XENOMOUSE® technology (as described in US Pat. Nos. 6,114,598; 6,162,963; 6,833,268; 7,049,426; Nature 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 Kellermann et al. , Current Opinion in Biotechnology, 13:593-7, 2002).

To obtain antibodies to OSMR, two different strains of XENOMOUSE® animals, i. XMG2-KL and XMG4-KL mice were immunized with human soluble OSMR-Fc proteins (obtained by Amgen, Seattle, WA). An appropriate amount of immunogen (i.e., 10 μg/mouse soluble human OSMR-Fc protein) was used to prime XENOMOUSE® animals according to the methods disclosed in US Patent Application No. 08/759620 published December 3, 1996, and International Patent Application Nos. WO 98/24893, published June 11, 1998, and WO 00/76310, published December 21, 2000, the disclosure of which is incorporated herein by reference. After the primary immunization, subsequent booster immunizations with the immunogen (5 μg/mouse soluble human OSMR-Fc protein) were carried out according to the schedule and for the time necessary to induce a suitable titer of anti-OSMR antibodies in mice.

Serum samples were collected approximately four weeks after the first injection and specific titers were determined by ELISA. The protocol used to titrate XENOMOUSE® animals was as follows: Costar 3368 binding media plates were coated with neutravidin @ 8 µg/ml (50 µl/well) and incubated at 4° C. in 1xPBS/0.05% azide overnight. The plates were washed using 3 wash cycles of TiterTek reverse osmosis demineralized water. The plates were blocked with 250 μl 1xPBS/1% milk and incubated for at least 30 minutes at RT. The block was washed using 3 cycles of TiterTek reverse osmosis demineralized water washes. Biotinylated huOSMR-FNFH (obtained by Amgen, Seattle, WA) was then captured at 2 mg/ml in 1xPBS/1% milk/10 mM Ca ²⁺ (assay diluent), 50 μl/well and incubated for 1 hour at RT . Washing was then carried out using 3 cycles of TiterTek reverse osmosis demineralized water washes. In the case of primary antibodies, serum samples were titrated 1:3 in duplicate from 1:100. This was done in assay diluent, 50 µl/well, and incubated for 1 hour at RT. Washing was then carried out using 3 cycles of TiterTek reverse osmosis demineralized water washes. The secondary antibody was goat anti-human IgG Fc HRP @, 400 ng/ml in assay diluent at 50 μl/well. It was incubated for 1 h at RT. Washing was then carried out using 3 cycles of TiterTek reverse osmosis demineralized water washes and dried with paper towels. For the substrate, a one-step TMB solution (Neogen, Lexington, Kentucky) (50 μl/well) was used and the substrate was allowed to form for 30 min at RT.

Animals showing appropriate titers were identified. Five XMG2KL animals were identified with a specific IgG immune response to OSMR. The spleen and drainage lymph nodes were excised from these animals and combined to obtain a hybridoma. Tissue samples from five XMG4KL animals with specific immune responses were collected in a similar manner and prepared for a separate fusion screening study. Enriched B cells from immune animals were fused with P3x63Ag8.653 non-secretory myeloma cells (American Type Culture Collection CRL-1580; Kearney et al., J. Immunol. 123:1548-50, 1979) to obtain hybridomas using standard methods (Kohler et al., Nature, 256, 495-7, 1975).

The hybridomas were then plated at high density (multiple different hybridoma clones per well) in 96-well tissue culture plates and grown for four weeks. Hybridoma line supernatants were tested for binding to full-length human and cynomolgus OSMR expressed by transiently transfected 293T cells using a fluorometric microvolume assay (FMAT) (Applied Biosystems, Foster City, CA). Briefly, 40 µl of a mixture of 3,000 OSMR-transfected 293T cells and 15,000 parenteral 293T cells was mixed in 384-well FMAT plates with 15 µl of hybridoma supernatant and 10 µl of anti-human light chain (h-kappa/h-lambda) labeled Alexa647 (Invitrogen, Carlsbad, CA). ) secondary antibody (1.0 µg/ml at final concentration). The plates were then incubated for 3 hours at room temperature and fluorescence was read using an FMAT reader. These studies identified 885 hybridoma lines that bind to both human and cynomolgus monkey OSMR.

Example 2 Human OSMR blocking assay.

The ability of anti-OSMR antibodies to block signaling through human OSMR was determined using a two-step assay with human oncostatin M (OSM) or human interleukin 31 (IL-31) as ligand. Together, this assay was used to determine if antibodies could inhibit OSMR signaling induced by OSM and/or IL-31 binding.

In the first study, antibodies were evaluated for their ability to block OSM signaling via OSMR. Stimulation of primary normal human lung fibroblasts by OSM induces phosphorylation of STAT3 and its subsequent translocation to the nucleus. Cells were seeded at 3000 cells/well in 384-well Costar plates and allowed to settle overnight. Cells were pretreated for 20 min with antibody supernatant and then stimulated with 80 pM human OSM for 30 min.

Cells were then washed 3X in PBS, fixed in 3.5% formaldehyde, washed (3X in PBS), and permeabilized with 0.5% Triton X-100. Cells were then stained with anti-phosphoSTAT3 antibody for 1 hour, washed and stained with AlexaFluor conjugated antibody (all from Cellomics HitKit). The plates were coated and read using the ArrayScan device using Cellomics proprietary algorithm to generate a nuclear intensity value and a cytoplasmic intensity value. The results were presented as the difference between these two values and further normalized to control data including maximally stimulated cells and media-treated cells (POC).

In a second study, antibodies were evaluated for their ability to inhibit IL-31 proliferative signal via OSMR in a stable cell line overexpressing 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 is only able to proliferate in response to human IL-31 and therefore can be used specifically to assess the blocking ability of anti-OSMR antibodies. BaF3 cells were seeded in 96-well plates at a density of 20,000 cells/well. Antibodies and ligand (huIL-31, Peprotech) were added to the wells to reach a final volume of 100 μl, and the plates were incubated for 72 h in a humid chamber at 5% CO ₂ , 37C. 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 (570-600 nm) plate reader at various time points after the addition of Alamar Blue.

The results of the two analyzes are presented below in table. 4. More than 3,000 hybridoma supernatants were tested for blocking power in these two assays; The 200 most potent blockers were further tested in a 4-point titration, after which 14 were selected for recombinant protein production and further testing. The IC ₅₀ values for the three generic antibodies (antibodies 1-3) are shown for both assays. Some antibodies inhibited OSM-induced STAT3 translocation to a greater extent than they inhibited IL-31-induced proliferation, and vice versa. All three antibodies were effective inhibitors of OSM- and IL-31-mediated signaling.

Table 4

IC50 AI АЪ2 ALZ ΟΞΜ 157 pM 252 pM 1.35 pM IL-31 35.2 pM 27.6 pM 780 pM

Example 3 Java Macaque OSMR Blocking Assay.

The ability of anti-OSMR antibodies to block cynomolgus monkey OSMR signaling was investigated using a two-step assay with human OSM or human IL-31 as ligand.

In the first study, antibodies were evaluated for their ability to block signaling

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OSM through cynomolgus monkey OSMR using a primary kidney epithelial cell line. Stimulation of these cynomolgus monkey (cyno) OSM cells induces phosphorylation of STAT3 and its subsequent translocation to the nucleus. Cells were seeded at 3000 cells/well in 384-well Costar plates and allowed to settle overnight. Cells were pretreated for 20 min with antibody supernatant and then stimulated with 80 pM cynomolgus monkey OSM for 30 min. Cells were then washed 3X in PBS, fixed in 3.5% formaldehyde, washed (3X in PBS), and permeabilized with 0.5% Triton X-100. Cells were then stained with anti-phospho-STAT3 antibody for one hour, washed and stained with AlexaFluor conjugated antibody (all from Cellomics' HitKit). The plates were coated and read using the ArrayScan device using Cellomics proprietary algorithm to generate a nuclear intensity value and a cytoplasmic intensity value. The results were presented as the difference between these two values and further normalized to control data including maximally stimulated cells and media-treated cells (POC).

In a second study, the antibodies were evaluated for their ability to inhibit IL-31 proliferative signal via cynomolgus OSMR in a stable cell line overexpressing IL-31RA4 and cynomolgus OSMR. As in example 2, BaF3 cells were seeded in 96-well plates at a density of 20,000 cells/well. Antibodies and ligand (huIL-31 cynomolgus monkey, internal, i.e. from Amgen, Seattle, WA) were added to the wells to reach a final volume of 100 μl, and the plates were incubated for 72 hours in a humid chamber at 5% CO ₂ . 37°C. 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 (570-600 nm) plate reader at various time points after the addition of Alamar Blue.

The results of the two analyzes are presented in table. 5 with IC ₅₀ values for both analysis steps. The results confirm that antibodies 1, 2 and 3 are effective inhibitors of OSM and IL-31 mediated signaling.

Table 5

IC50 Abi AL2 ALZ OSM 1.26 Nm 518 pM 1.24 nM IL-31 225 pM 29.3 pM 6, 8 7 nM

Example 4 Sorting of anti-OSMR antibody epitopes.

Antibody competition studies were performed in order to characterize anti-OSMR antibody epitopes in xeno mice. Antibodies that compete with each other can be considered to bind to the same site on the target. In these experiments, anti-OSMR antibodies or irrelevant antibodies were captured on streptavidin-coated Luminex beads precoupled with a capture antibody (biotinylated monovalent mouse anti-human IgG Fc antibody). The OSMR antigen or buffer (containing no antigen) was added to the wells, after which a probe antibody was added to each well and detection was performed with a PE-labeled monovalent mouse anti-human IgG Fc antibody. For each well, the average fluorescence intensity was measured. For a full description, see Jia et al., J. Immunol. Methods, 288:91-8, 2004. Detection of fluorescence in a given well indicated that the probe antibody was able to bind to OSMR even in the presence of another anti-OSMR antibody, demonstrating that they bind to different epitopes. At least three groups were found, as shown in Table 1. 6.

Table 6

Example 5 Determination of the affinity of anti-OSMR antibodies.

The affinity of anti-OSMR antibodies was determined. Kinetic rate constants were determined to investigate the interaction of antibodies 1-3 (Ab 1-3) with human OSMR.

Biosensor analysis was performed at 25°C in HBS-EP+ (1X) buffer system (10 mM HEPES, pH 7.4, 150 mM NaCl, 3.0 mM EDTA, 0.05% surfactant P20) using a Biacore 3000 optical biosensor, equipped with CM5 sensor chip. Prior to injection, all reagents were stored at 8°C. Goat anti-human IgG (Jackson ImmunoResearch, no. 109-005-098) was immobilized (~3000 KE) on the sensor chip by standard amine coupling with flow cells 1 and 2 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 (0.25 to 0.5 μg/ml) in running buffer. Antibodies were injected (15 µl) through flow cell 2 at a flow rate of 10 µl/min. Approximately 50 KE antibodies were captured. The surfaces were allowed time to stabilize (90 s) and then each concentration (150, 50.0, 16.7, 5.56, 1.85 and

0.617) hOSMR was run through flow cells 1 and 2 at a flow rate of 50 μl/min to observe association (5 min) and dissociation (5 min). The samples were examined in duplicate in random order.

Empty analyte-free buffer (0 nM hOSMR) was injected before, between and after sample injections. Antibodies were injected (15 µl) through flow cell 2 at a flow rate of 10 µl/min. Approximately 50 CU antibodies were captured. The surface was allowed time to stabilize (90 s) and then each concentration (150 nM) hOSMR was run through flow cells 1 and 2 at a flow rate of 50 μl/min to observe association (5 min) and dissociation (1-2 h). The samples were examined in triplicate.

Empty, analyte-free buffer (0 nM hOSMR) was injected before and after sample injections. The surface was repaired at a flow rate of 50 μl/min with two injections of 10 mM glycine (pH 1.5, 50 μl). This was followed by an injection of empty buffer (15 s).

Data was analyzed using Scrubber 2.0 software as follows. Data for flow cell 2 was subtracted from data for flow cell 1 (blank standard). Then, standard subtracted data (2-1) were subtracted (double standardized) from closest data for 0 nM concentration. After double standardization, long term dissociation data were fitted with a 1:1 binding model to determine the dissociation rate constant (cd). This dissociation rate constant was used as a fixed parameter to fit short term dissociation data after double standardization with a 1:1 binding model to determine the association rate constant (k _a ) and the equilibrium dissociation constant (Kd).

Under experimental conditions, the behavior of the reagents was normal and the data (see Table 7) fit well with the 1:1 binding model.

Table 7

Antibody Antigen k* (1/Ms) < P/s) ίπΜ) huOSM? 9.95 xu ⁵ 221 AL2 huOSMR 5.5CxlCP 1, 91 x10 ^x 32.7 ALZ huOSMR 9, 47x10 ⁴ 1, C2 V10 1 1080

Example 6 Anti-OSMR Antibodies.

Fully human antibodies directed against human OSMR were generated using the XENOMOUSE® technology described in Example 1 above. Antibodies 1, 2 and 3 were all shown to be effective inhibitors of OSM and/or IL-31 mediated signaling. The sequences of antibodies 1, 2, and 3 (i.e., Abi, AL2, and AL3) were determined and are shown in Table 1. 8.

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Table 8

Description 3EQ ID no: Subsequence LY - Heavy chain nucleotide 3 caggtgcagctggcgcagtccggggctgaggtgaagaa gcctggggcctcagtgaaggcctcctgcaaggcttctg gaLacaccLLGdcccigLLaLgaLaLcaa.GLgggLgeqa caggccactggacaggggctcgagtggatgggatggat gaaccc LaaLag LggLaacacagacLaLgcacagaagL tccagggcagagtcaccatgaccaggaacatttccaca agcacggcctacaetgagctgagcagGctgagatctga ggacacggccgttcattactgtgcgagagatatggtgg etgegaataeggaotactacttctactacggtatggac gtctggggccaagggaccacggtcac.cgtctcctcagc tagcaccaagggcccatcggtcttccccctggcgccct gctccaggagoacctccgagagcacagc.ggccc.tgggc tgcctggtcaaggactacttccccgaaccggtgacggt gtcgtggaactcaggcgctccgaccagcggcgtgcaca ccLLcceagcLgLccLacagcccLcaggacLcLacLcc ctcagcagcgtggcgaccgtgccctccagcaacttegg cacccagacctacacctgcaacgtagatcacaagccca gcaacaccaaggtggacaagacagttgagcgcaaatgt tgtqtcgagtqcccaccqtgcccaqcaccacctqtqqc aggaccgtcagtcc.tcctct r.ccccccaaaacccaagg acacccteatgatetcccggacccctgaggtcacgtgc gtggtggtggacgcgagccacgaagaccGcgaggtGca gttcaactggtacgtggacggcgtggaggtgcataatg ccaagacaaagccacgggaggagcagttc.aacagcacg ttccgtgtggtcagcgtcctcaccgttgtgcaccagga cLggcLgaacggca aggagLacaagLgcaaggL^L^Ld acaaaggcctcccagcccccatcgagaaaaccatctcc aaaaccaaagggcagccccgagaaccacagg LgLacac cctgcccccatcccgggaggagatgaccaagaaccagg tcagcctgacctgcctggtcaaaggcttctaccccagc gacatcgccgtggagtgggagagcaatgggcagccgga gaacaactacaaqaccacaccteccatgctqqactccq

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acggcTcctTcttcctctacagoaagctcaccgtggac aagagcagguggcagcaggggaacgtcttctcatgcTc cgtgacgcacgaggctgcacaaccactacacgcaga agagcctctccctgtctccgggtaaa Άο2 - Heavy chain nucleotide 4 caggtgcagctggcgcagtctggggctgaggtgaagaa gcctggggcctcagtgaaggtctcctgcaaggcttcTg gatacacctccaccagttatgaaatcaactgggtgcga caggccactggacaagggcttgagtggatgggatggat gaacccLaacagLgg L LacacagguLaLgcacagaaqL tccagggcagagtcaccatgaccaggaacacctccaca agcacaqcccacaTqgaaatgaqcaqcctgagatctqa ggacacggccgtguattactgtgcgagagatatagtgg cqgcgaatacggaqtactacttctattatggtatggac gcctggggccaagggaccacggtcaccgtctcctcagc tagcaccaagggcccatcggtcttccccctggcgccct gctccaggagcacctccgagagcacagcggccctgggc tgcctggtcaaggactacttccccgaaccggtgacggt gTcgtggaactcaggcgctctgaccagcggcgtgcaca ccttcccagctgtcotacagtcctcaggactctactcc c7.cagcagcgtgg7.gaccgtgcccts-:cagcaacttcgg cacccagacctacacctgcaacgtagatcacaagccca gcaacaccaaggtggacaagacagttgagcgcaaatgt tgtgtcgagcgcccaccgtgcccagcaccacctgtggc aggaeegLuag LuтLeeLeiLccccccaaaacccaagg acaccctcacgatctcccggacccctgaggtcacgtgc gcggtggtggacgcgagccacgaagaccccgaggtcca gctcaactggtacgtggacggcgtggaggtgcataacg ccaaqacaaagccacgqgaggaqcaqttcaacagcacg t r.ccgr.gtggtcagcgtcctcaccgttgtgcaccagga clggcugaacggcaa ggagtacaagtgcaaggtctcca acaaaggccccccagcccccatcgagaaaaccatctcc aaaaccaaagggcagccccgagaaccacaggtgtacac

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gaacaactacaagaccacacctcccatgctggacnccg acggctccttcttcctctacagcaagctcaccgtggac aagagcaggtggcagcaggggaacgtcttcccatgctc eg Lga LgcaLgaggcLc Lgcacaaccac Lacacgcaga agagcctctccctgtcTccgggTaaa AL3 - Heavy chain nucleotide cagg LLcaLcLgg LgcagLcLggagc LgaggLgaagaa gcctggggcctcagtgaaggtczcctgcaaggctnctg gttocacctttoccagctatggcatcagctgggtgcgo caggcccctggacaagggcttgagtggatgggatggct caqcacttacaqtqqtaacacaaactatqcacaqaaqc tccagggcagagtcaccatgaccacagacacatccacg ageacagcctacatqgagctgagqagoctgagatetqa cgacacggccgtgtat cactgtgcgagagggaacr.t et actactacggtatggacgtctggggcoaggggaccacg gtcaccgtctcctcagctagcaccaagggcccatcggt cttccccctggcgcccqgctccaggagcacctccgaga gcacagcggcccLgggcLgccLggLcaaggacLacL Lc cccgaaccggtgacggtgtcgtggaactcaggcgctct gaccagcggcgtgcacaccttcccagctgtcctacagt cctcaggactctactcostcageagcgtggtgaccgtg ccctccagcaacttcggcacccagacctacacctgcaa cgtagatcacaagcccaqcaacaccaaqqtqqacaaga cagttgagcgcaaatgztgtgtcgagtgcccaccgtgc ccagcaccacctgtggcaggaccgtcagtcttcccctt ccccccaaaacccaaggacaccctcatgatctcccgga cccctgaggtcacgtgcgtggtggtggacgcgagccac gaagaccccgaggtccagLLcaaclggLacgtggacgg cgtggaggtgcataatgccaagacaaagccacgggagg agcaq LLcaacagcacgLLccgtgLgg Leageg LccLc accgttgtgcaccaggactggccgaacggcaaggagta caagtqcaaqqtctc caacaaaqqcctcccaqcccca tcgagaaaaccatctccaaaaccaaagggcacccccga qaaccacaqqtgtacacc.ctgcccccatcccqgqaqqa gatgaccaagaaccaggtcagcctgacctgcct.ggtca aaggcttctaccccagcgacatcgccgtggagtgggag

- 48 041171 agcaatgggcagccggagaacaaclacaagaccacacc tcccatgctggactccgacggctccttcttcctctaca goaagctcaccgoggacaagagcaggtggcagcagggg aacgtcttc-caAgczccgtgatgcatgaggctctgca cauccuclaGucgcagaagugGcLGLGCcLg LcLccgg gtaa

Abi - Heavy chain protein b QVQLVQSGAEVKKPGA5VKVSCKASGYTFGSYDINWVR QATGQGLEWMGWMNPNSGNTDYAQKFQGRVTMTRNTSI S TAYIELS SLRSEDTAVYYCARDMVAANTDYY FYYGMD VWGQGTTVTVSSASTKGPSVFPLAFCSRSTSESTAALG CLVKDYFPEPVTVSWNSGALTSC-VHTFPAVLGSSGLYS LSGWTVPSGNFGTQTYTCETJbHKPSNTWDKTVERKC CVECPPCPAPPVAGPSVFLFPPKPKDTLMTSRTPEVTC VVVJVSHI^DPI^VQFNWYVDGVKVHNAKTKPREEQFXS·]' FRWSVLTVVLQDWLNGKEYKCKVSNKGLPAPTEKTIS KTKGQPRZPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DTAVEWESNGQPENNYKTTPPMT.DSPGSFFT.YSKTYVD KSRWQQGWFSCSV2-1HEALHNHYTQKSLSLSPGK Ab2 - Protein heavy chain 7 QVQLVQSGAEVKKPGA3VKVSGKASGYTFTSYEINWVR QATGQGLEWMGWMNPNSGYTGYAQKFQGRVTMTRNGSI STAYMEMSSLRSEDTAVYYCARDIVAANTDYYFYYGMD VWGQGrmWSSASTKGPSVh'PJ.APCSRSYSESTAAl >G CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPSSNFGTGTYTCWDHKFSNTKVDKTVERKC CVECPPCPAPPVAGPSVKA'PPKPKirri M : SRTPEVTC WVDVSYEDPTVQFNWYVDCA^VHNAKTYPREEaFXST FRWSVLTWHQDL ⁷ LNGKEYKCKVSNKGLPA?TEK ^m IS KTKGQPREPQVYTLPFPREDMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLGVD KSRWQQGWFSCS ⁷ 7MHEALHNHYTQKSLSLS?GK AL3 - Heavy chain protein 6 QVHn^QSGAEVKKPGASVKVSCAASGYTFTSYGISWVR QAPGQGLEWMGWLSTYSGNTNYAQKLQGRVTMTTETST STAYMELRSLRSDDTAVYYCARC-NFYYYGMDVWGQGTT VTVSSASTKGPSVFPLAFCSRSTSESTAALGCLVKDYF PEPVVTVSWNSGALTSCVHTFPAVLQSSGLYfjLSSV’v’TV

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P5SNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPC PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVQFNWYVDGVEVHNARTKPREEQFNSTFRWSVL TWHQDWLNGREYKCKVSNXGLPAPIEKTISKPKGQPR Е PQVYTLPPSREEMT KXQVS L T C LVKG FY P S D PAVE ИE SNGQPENNYKTTPPMEDSDGS hTLYSKI .TVDKSRWQQG NVFSCSWHEALHNHYGQKSLSLSPGK Ahl Heavy chain variable region 9 QVQLVQSGAEVKKPGASVKVSCKASGYTFFSYDINWVR QATGQGDEWMGWMNPNSGNTDYAQKFQGRVTMTRN1SI STAY 1 EbSSlRSKDTAV YYCARDMVAANTDYYKYYGMh WGQGTTVTVSS #2 - Variable region of severe chains 10 QVQLVQSGAEVKKPGASVKVSCXASGYTFTSYEIMWVR QATGQGLEWHGWMNPNSGYTGYAQKFQGRVTMTRNTSI 5 TAYMEMSSbRSEDTAVYYCARD I VAANTDYY FYYGM1) WGQGTTVTVSS ALZ - Variable region of severe chains eleven QVHLVQSGAEVKKPGASVKVSCRASGYTFESYGISWVR QAPGQGLEWMGWLSTYSGNTNYAQKLQGRVTMTTDTST STAYMEbRSGRSDOTAV YYCARGN?'Y YYGMDVWGQGT'l' VTVSS Ahl - heavy chain GDR1 12 SYD] N AL2 - heavy chain CDR1 13 SYEIN ALZ - CDR1 heavy chain 14 SYGIS AI - CDR2 heavy chain 15 WMNPNSGNTDYAQKFQG AL2 - CDR2 heavy value 16 WMGWMNPNSGYTGYAQKFQG ALZ - CDR2 heavy target 17 WLSTYSGNTNYAQKLQG Abi - C1ZhZ heavy chain 10 3#AAN TD Y Y FY YGMDV L2 - GDR3 heavy chain 19 DIVAAN T DY Y FY YGMDV

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Ab3 - heavy chain CDR3 AI - Easy Target Nucleotide 20 21 GNFYYYGMDV cagtctgtgctgactcagccacccrcagcatctgggac ccccqggcaqaqqqtcaccatctcrtqttctgqaaqca gctccaacgccggaagtaatactgtaagctggtaccaa cagctcccaggaacggcccccaaactcctcatctatac taataatcggcggccctccggggtccctgaccgattct ctggctcxaagtctggcacctcagcctccctggccatc agtgggctccagectgaggatgaggctgattatttctg tgcagcgttagatgacagtctgaatggtgtggtattcg gcggagggaccaaaccgaccg-.cc-.aggccaaccgaaa gcggegcccr-cggtcactctgrtcccgccctcctct ga ggagettcaagccaacaaggccacactggtgtgtctca LaagLgacLccLacccgggagccgtgacaglggccLgg aaggcagatagcagccccgtcaaggcgggagtggagac caccacaccctccaaacaaagcaacaacaagtacgcgg ccaqcaqctatctgaqcctgacqcctqaqcagtqgaaq tcccacagaagceacagczgccaggtcacgcatgaagg gagcaccgLggagaagacagLggccccLacagaaLgL L ca Le2 - Light chain nucleotide 22 cagtctgtgctgactcagccacccrcagcgtctgggac сииоьддсададдд LcaccaLcLct Lg LLclggaagca actccaacar.cggaagtaatactgrcaactggtaccac cagctcccaggaacggcccccaaactcctcatctataa tattaataagcggccctcaggggtccctgaccgattet etggetccaagtctggctcctcagcctccctggccatc agtgggctccagoctgaggatgaggctgattattactg СЬсааоаЬдддатдасадссЪддатддЪдЪддНаНЪсд gcggagggaccaagccgacc.gccccaggccaaccgaaa дсддсдсссдeggteactetgrtcccgccctcctctда ggagcttcaagccaacaaggccacactggtgtgtctca taagtgacttctacccgggagccgtgacagtggcctgg aaggcagatagcagccccgtcaaggcgggagtggagac caccacaccctccaaacaaagcaacaacaagtacgcgg спад cage La Lc tgagcGtgac.gccLgagсад Lggaag

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tcccacagaagctacagctgccaggtcacgcatgaagg gagcacog Lggugaagaoag LggcocoLucagaa Lg LL ca Άό3 - Light chain nucleotide 2 3 gaaattgtgrtgacgcagrctccaggcaccetgtcttt gtctccaggggaaagagccaccctctcctgcagggcca gLeacugLgt Lag garden cage Lac. Ltagее LggLaecag cagaaacctggccaggctcccaggctoctcatctttgg tgcttccagcagggccacrggcatcccagacaggttca gtggcagtgggtctgggacagactccactctcaecatc aqcaqactqgagcctgaagatettgeagtgtattactg tcageagtaeggragetcgccrccgatcacctteggee aagggacacgacrggagartaaacgtacggtggetgea ccatctgterteatertcccgccaretgatgageagtt gaaatctggaacrgcctcrgttgtgtgcctgctgaata acLLcLaLcecagagaggceaaagtacagLggaagg Lg gataacgccctccaatcgggtaacTcccaggagagtgt cacagagcaggacagcaaggacagcacctacagcctca qcaQcaccctgucgctgaqcaaagcaqucLacqagaaa cacaaagtcracgcccgcgaagtcacccatcagggcct gagctcgcccgtcacaaagagcttcaacaggggagagt gt Ahl - Light chain protein 24 QSVLTQPPSASGTPGQRVTISCSGSSSNVGSNTVSWYQ QLPGTAPZKLLIYTNNRRPSGVPDRGGGSKSGTSASLAI SGLQSEPZADYFCAALDDSLNGWYGGGTKLTVLGQPK МРБУТЪБРРЗЗЕБЬОАИКАТБУББТЗЕГУРСАУТУАК KADSSPV:<AGVK ^r ]igTSKQSNN:<YAASSY IS IG'PEQWK SERSYSCQVTHEGSTVEKTVAPTECS AL2 - Light chain protein 25 Q SVLIQPP SAS GT PGQRVTIS CS GSNSNIGSNTЖYH QLPGTAPKLLIYNINKRPSGVPDRTSGSKSGSSASLAI SGI .QSbJT-'Al iYYGSTWDLiSI ₁ IpGVV? GGGTK].TV],GQPK AAPSVTLFPPSSETLQANKATLVCLISEFYPGAVTVAW KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ^ SERSYSCaVTREGSTVEKTVAFTECS AL3 - Light chain protein 26 B 1 Vl.TQSPGTIiS GSPGKRATI.SCRASQSVSSSYI.AWY^ QKPGQAPRLLIFGASSRATGIPDRFSGSGSGTDFTTLTI

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5RLEPEDFAVYYCQQYGSSPPIТFGQGTRLEIKRTVAA PSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVCWKV DNALQSGNSQESVIEQDSKDSTYSLSSTLTLSKAPYEK IIKVYACEVTIIQGLSSPVTKSFNRGEC AI Variable region of the patch chain 27 QSVLTQPPSASGTPGQRVTISCSGSSSNVGSNTVSWYQ QLPGTAPRLLIYTNNRRPSGVPDRFSGSRSGTSASLAI SGLQSEL^ADYFCAALDDSLNGVVFGGGTKLTVLG No. 2 Variable region of the leg chain 29 QSVLTQPPSASGTPGQRVTISCSGSNSNIGSNTVNWYH QLPGTAPKLLIYNINKRPSGVPDRFSGSKSGSSASLAI SGLQSEDEADYYCSTWDDSLDGWFGGGTKLTVLG AL3 Variable region of the fly chain 29 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIFGASSPATGIPDRFSGSGSGTDFTTLTI SRLEPEDFAVYYCQQYGSSPPITFGQGTRLEIKR AI - CDR1 light chain thirty SGSSSNVGSNTVS AL2 - CDR1 light chain and 31 SGSNSMIG3NWN #3 - CDR1 light chain and 32 RASQSVSS5YPA AI - CDR2 light chain 33 TNNRRPS ALZ - GPR2 light chain 34 NINKRP3 A03-CDR2 light chains 39 CASSRAT AI - CDR3 light chain 36 AALDDSLNGW AI - CDR3 light chain 3 7 S Th cDG ld GW ALZ - CDR3 easy price 38 QQYGSPPIT

Example 7 Modified anti-OSMR antibodies.

Received modified versions of AL, AL2 and AL3. In the case of all three modified forms of antibodies, the lysine at the C-terminus of the heavy chain was removed. In the case of AL and AL2, the glycosylation site at position 73 was removed by replacing asparagine 73 with aspartic acid. These variants were named Abl-N73D and Ab2-N73D. The sequence of the modified antibodies are given in table. 9 (modified nucleotides and amino acids are underlined).

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Table 9

Description SEQ ID no: Subsequence Abi version 2 Heavy chain nucleotide (SZB/Sterminal lysine removed! 47 caggtgcagctggtgcagtctggggctgaggtgaagaa gccLggggccLcagLgaaggLcLccLgcaaggcgLcLg дадасассttcaccagttatgatatcaactgggcgcga caggccactggacaggggcttgagtggaBgggagggat gaaccctaataqcqgtaacacaqactatqcacaqaaqt Ёссадддсададдcaccatgaccagggacatttccat a agcacggcctacattgagctgagcagccдgagaдctga ggacacggccg LgLaL Lac-Lg Lgcgagaga Lalggigg ctgcgaacacggattactacttctactacggtacggac gtctggggocaagggaccacggtcaccgr.ctccncagc tagcaccaagggcccatcggtcttccccctggcgccct gccccaggagcacctccgagagcacagcggccccgggc tgcctggccaaggactacttccccgaaccggtgacggt gtcgtgqaactcagqcgctctgaccagcqgcgtqcaca ccctcccagctgccctacagtcctcaggactctactcc ctcagcagcgtggtgac:cgtgccctccagc.aacctcgg cacccagacctacacctgcaacgtagatcacaagccca gcaacaGcaagggggacaagacagttgagcgcaaatgt tqr.qtcqagtqcccaccgtqcccaqcaccacctgtqqc aggaccgicagtcttcctcttccccccaaaacccaagg acacccLca LguDcLcucggaccccLgaggLcacg Lgc gtggtggnggacgtgagccacgaagaccccgaggtcca gt:7.caar:Tggtacgtggacggcgtggaggtgcanaacg ccaagacaaagccacgggaqgaqcagttcaacaqcacq

- 54 041171

ttccgtgtggtcagcgtcctcaccgttgtgcaccagga ctggctgaacggcaaggagtacaagtgcaaggtctcca acaaagqcctcccagcccccatcqagaaaacca'tctcc aaaaccaaagggcagccccgagaaccacaggtgoacac ccLGCGCCsaLeeegggaggagaLgaccaagaaccagg tcagcctgacctgcctggtcaaaggcttctaccccagc gacatcgccgtggagtgggagagcaatgggcagccgga gaacaaceacasqaccacacctcccatqctggactccq aeggetocttetc octetacagcaagetcaccgoggac aagagcaggLggcagcaggggaacgLcL^cLcaogcoc cgtgatgcatgaggctctgcacaaccaceacacgcaga agagcctctcccogtctccgggt AL2 version 2 - Heavy chain nucleotide (N73D/C-terminal lysine deleted) 4S caggtgcagctggtgcagtctggggctgaggtgaagaa gcctggggcctcagtgaaggtctcctgcaaggcctcog gatacaocttcaccagttatgaaatcaactgggcgcga caggccactggacaagggcttgagtggaogggaeggat gaaccctaacagcggttacacaggctatgcacagaagt tccagggcagagccaccatgaccaggg;acacctccat a agcacagcctacatggaaatgagcagcctgagatctga ggaeacggccgtgtattactgtgcgagagatatagtgg ctgcgaaoacggattactacttctattatggtaoggao gtctggqgccaaqqgaccacggtcaccqtctcctcaqc tagcaccaagggcccatcggtcttccccctggcgccct gcoccaggagcaccLccgagagcacagcggcccogggc tgcctggocaaggactacttccccgaaccggtgacggt gtcgtggaactcaggcgctctgaccagcggcgtgcaca ccttcGcaqctgtcctacaqtcctcaggactctactcc ctcagcagcgtggtgaccgtgccctccagcaacotcgg cacccagacctacacctgcaacgLagaLcacaagccca gcaacaccaaggoggacaagacagttgagcgcaaatgt tgtgtcgagtgcccaccgtgcccagcaccacctgtggc aggaccgocagtcttcctcttccccccaaaacccaaqq acaccctcatgaoctcccggacccctgaggtcacgtgc g LggLggoggacgLgagccacgaagaccccgaggLc.ca gtccaacoggtacgtggacggcgtggaggtgcao.aaog

- 55 041171

ccaagacaaagccacgggaggagcagttcaacagcacg LLccg LgLggLcagcgLccLcaccg L Lg Lgcaccagga ctggotgaacggcaaggagtacaagtgcaaggtctcca acaaaggccccccagcccccaccgagaaaaccatctcc aaaaccaaagggcagccccgagaaccacaggtgtacac cctgcccccatcccgggaggaqatgaccaagaaccagg tcagGctgacctgcceggecaaaggcttctaccccagc gacatcgccgtggag ^- .gggagagcaatgggcagccgga gaacaactacaagaccacaccecccatgctggactccg acgqctcctecteccrctacaqcaaqctcaccqtgqac aagagcaggcggcagcaggggaacgtcttctcatgctc cgtgatgcacgaggccctgcacaaccactacacgcaga agagcctctcccogtctccgggt Ap3 - Heavy chain nucleotide (C-terminal lysine removed) 4 9 caqgttcatctqgtqcagTctqgagctqaqqtqaaqaa gcctggggcctcagtgaaggtctcctgcaaggcttctg gttacacctr.taccagctatggtar.c.agctgggtgcga caggcccctggacaagggcttgagcggatgggatggct cagcacttacagoggcaacacaaactatgcacagaagc tccagggcagagrcaccargaccacagacacatccacg agcacagccracatggagctgaggagcctgagatctga cgacaoqgccgtgtaTtactqTgcqaqaqqqaacttct actactacggtacggacgcctggggccaggggaccacg g t caccgt ст. rest. cage tagcaccaagggcc cat cggt cttccccctggcgccctgctccaggagcacctccgaga дсасадсддссстдддсРдсстддтсааддасЬасЪЪс cccgauccggLgacggLgLcg LggaacLcuggcgcLcL gaccagcggcgtgcacaccttcccagctgtcctacagt cctcaggaccctactcccccagcagcgtggtgaccgtg ccctccagcaacotcggcacccagacctacacctgcaa cgLacatcucaagcccagcddJdcutiaggLggacaaga cagttgagcgcaaatgttgtgccgagtgcccaccgtgc ccagcaccacctgtggcaggaccgccagtcttcctctt coccccaaaacccaaggacaccctcatgatctcccgga cccctqagqtcacqtgcqtqqtqqtqgacqtgaqccac gaagaccccgaggtccagctcaaccggtacgtggacgg

-56041171

AL2 sersya 2 Her.pok heavy chain (X7 3D/Terminal life removed) 50 cgtggaggtgcaYaaTgccaagacaaagccacgggagg адсад L Lcaacagcacg LscogLg sgg Lcagcg Lee Lc accgttgtgcaccaggaccggctgaacggcaaggagta caagtgcaaggtctccaacaaaggcctcccagccccca tcgagaaaaccaxctccaaaaccaaagggcagccccga даассасаддЕд7.асассс5дссссса5сссдддадда gatgaccaagaaccaggtcagcctgacctgcctggtca aagget tctaccccagcgacar.cgccgtggagtgggag agcaat gggcagccggagaacaaccacaagaccacacc tcccatgctggactccgacggctccttcttcctctaca gcaagcLcaccg gggacaagagcaggLggcagcagggg aacgtcttcccaLgccccgtgatgcatgaggctctgca caaccactacacgcagaagagcctctccctgtctccgg gt QVQLVQSGAEVKKPGASVKVSCZAASGYTFTSYDINWVR. QATiGQGLZWMGWMNPNSGNTDYAOKFQGAVTMTRrZSI S TAYIELS SLRS EDTAVYYCARDMVAANTDYYFYYGMD WGQGTTVTVSSASTKGPS\ ^Z FPLAPCSRSTSESTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS5NFGTQTYTCPWDHKPSNT37ZDK?VERKC CVHCPPCPAPPVAGPSVFOb'PPKPKD'ri я: SRYPEVTC W/DVSHE DPEVQFNWYVDGVEVHNAKTXPREEQ FXST FRWSVLIWHQDWLNCЖE¥KC:<ASNKGLPA?ΞEKTIS KTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVD ksrwqqgnvescs\™ealhnhytq:<slsls?g AL2 version 2 Heavy chain protein IX73D/Ctrgdshalyi lyshn deleted) 51 QVQLVQSGAEVKKPGASVKVSCKASGYTFYSYEINWVR QATGQGLE^Gi^NPNSGYTGYAQKFQGRVTMYRCYSI S TAYMEMS SLRS EDTAVYYCARDIVAANTDYYFYYGMD VWGQGTTVTVSSASTKGPSVFPLAPCSRSDSESTAALG CLVKDYFPEPVTVSWNSGALTSGVHIFPAVLQSSGLYS LSSvA ^T TVPSSNFGIQTYTCWDHKPSNT?GZDKEVERKC CVECPPCPAPPVAGPSVFLFPFKPKDTLMISREFEVTC FRWS\ ⁷ LTWHQDiiLNGKEYKCKVSNKGLPA?2EK ^m IS

- 57 041171

KTX0RBBRAUUTRR5KEBMTKYaUZTSBUKSGUR5 OIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVD KSRWQQGWFSCS^HEALHNHYTQKSLSLSPG AL3 Persia 2 Heavy chain protein (Terminal lysine removed) 52 QVHLVQSCAEVKKPGASVKVSCKASGYTFTSYGISWVR QAPGQGLEWMGWLETYSGNTNYAQKLQGRVTMTTETGT STAYMELRSLRSDDTAVYYCARC-NFYYYGMDVWGQGTT VTVSSASTKGPSVFPGAPCSRSTSES'rAALGCLVKDYb' FEPVTVSWNSGALTSGVHTFPAVLQ3SGLYSLSSVVTV rSSNFGTQTYTCNVDHKPSNTKVDKIVERKCCVECPPC FAPHVAGPEVHi- ΡΡΚΡΚΙΙΊΊιΜ 1 S RT PEV7CWVEVSH EDPEVQFNWYVDGVEVHNAXTKPREEQFNSTFRWPVL TWHQDWLNGKEYKCKVSNKGLPAFIEKTISKTKGQPR EPQVYTT.PPSRF.EMTKXQVST.TCT.VXGFYPSrTAVEWE SNGQPENNYKTTPPMLDSDGSFFLYSKLTVDK3RWQQG NVF S C S VMHEAL HNHYTQKSLSLSPG AI version 2 Heavy chain variable region (X73B/Sterminal lysine deleted) 53 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVR QATGQGLEWMGWMNPNEGNTDYAQKFQGRVTMTRDISI STAYIELSSLRSEDTAVYYCARDMVAANTDYYFYYGMD WGQGTTVTVSP AL2 version 2 Heavy chain variable region (X73B/Sterminal lysine deleted) 54 QVQLVQSGAEVKKPGASVKVSCXASGYTFTSYEINWVR QATGQGLEWMGWMNPNSGYTGYAQKFQGRVTMTRPTSI STAYMEMSSLRSEDTAVYYCARDIVAANTDYYFYYGME VWGQGTTVTVS3

ELISA experiments were performed in different formats (capture ELISA in case of less avidity format; sandwich ELISA in case of liquid phase format and direct ELISA in case of solid phase format with avidity) using antibodies containing variable regions Ab1 or Ab2 (or N73D- variant Ab1 or Ab2) with different Fc regions.

Ab1 and Ab2 contain the C _H 1, CH2, CH3 domains of IgG2 of human origin. As used herein, the terms Ab1 and Ab1 IgG2 WT refer to the same antibody. Similarly, the terms Ab21 and Ab2 IgG2 WT refer to the same antibody.

Antibodies defined as IgG4P agly/IgG1 contain the variable regions of Ab1 or Ab2 (or the N73D variant of Ab1 or Ab2) fused to the CH1 domain of human IgG4, a human IgG4 hinge domain with a Ser to Pro substitution mutation (at position 228), to to reduce shuffling, the CH2 domain of human IgG4 with a mutation involving the change of Asn to Gln (at position 297) to remove the N-linked glycosylation site, and the CH3 domain of human IgG1. The IgG4P agly/IgG1 framework region is described in US Patent Application No. US 2012/0100140.

The ELISA results indicate that the removal of glycosylation sites by replacing N73D does not affect the binding of the modified antibodies to OSMR, see table. 10.

_________________________________________________ Table 10

Antibody Capture (ΞG53) hM Sandwich 'Έθ50) nM Direct (EC50) nM Abi IgG2 WT 10.2 0.561 0.164 Abi Ν73Γ IgG2 4, 35 0.359 0.0728 Ab. N 7 31J ]qG4P agly/lgGl 2.36 0.05 0.0626 Ab2 IgG2 WT 3, 63 0.366 0.162 Ab2 N73E IgG2 5, 49 0.34 3 0.179 Ab2 N73D IgG4P agly/lgGl 1, 61 0.054 0.0558

The binding study was performed using the BIAcore method. Antibodies containing Ab1 or Ab2 variable regions (or N73D variant Ab1 or Ab2) with different Fc regions 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 by replacing N73D improved the binding affinity. In the case of Ab1, the substitution improved the rate of K _acc , while in the case of

Ab2, it improved the rate K _diss , see Table. eleven.

Table 11

The stability of the Fab fragments was determined by assessing the thermal unfolding of the antibodies. The high melting point of the Fab fragments is directly related to the increased stability. Removal of glycosylation sites on Ab2 by replacing N73D did not affect the thermal stability of the Fab fragments and had a negligible effect on Ab1 as assessed by differential scanning fluorometry experiments, see table. 12.

The ability of modified anti-OSMR antibodies to block signaling through human OSMR was evaluated. The modified antibodies were evaluated for their ability to inhibit the proliferation of the hu-IL31R/OSMR/gp130 BaF cell line in the presence of IL31, OSM, or IL31 and OSM. The results are presented in table. 13 and 14 with the IC ₅₀ values given for each antibody. The results confirm that the modified versions of Ab1 and Ab2 are effective inhibitors of OSM- and IL-31-mediated signaling.

________________________________________________ Table 13

Antibody IL31 ; ic50) OSM (IC50j IL31/OSM (1258) AL2 0.3828 0.3528 '1.9 Abi IgG2 WT 0.6691 0.5298 4, 304 Abi N7 3D IgG2 0.7565 0.5226 3.7 02 Abi Ν73Ό TqG4P agly/TgGl 0.4672 0.5657 3, 380

Table 14

Antibody IL31 (ICbC) OSM (ICbO) IL31/OSM iIC-0i Ab 2 0.4426 0.4019 1/8 Abz 1 gG2 WT 0.367Ί 0.475V 2,049 Ab2 N7 3D IgG2 0.2131 0.1859 1,474 Ab2 N73D IgG4F aqly/IqGl 0.2838 0.2401 1.276

The invention has been described in terms of specific embodiments that include, or are intended to include, certain schemes for practicing the invention. For specialists in the art, it is obvious that there are various modifications and variations of the described invention, which can be carried out without going beyond the scope and essence of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the claimed invention should not be limited to these specific embodiments. Indeed, various modifications of the described embodiments of the invention that are obvious to those skilled in the art are included within the scope of the following claims.

Claims (21)

CLAIM 1. An antibody to the oncostatin M receptor (OSMR) containing the light chain variable domain containing the complementarity determining region of the light chain 1 (LCDR1) shown in SEQ ID NO: 31; complementarity determining region of the light chain 2 (LCDR2), shown in SEQ ID NO: 34; and the light chain complementarity determining region 3 (LCDR3) shown in SEQ ID NO: 37; and a heavy chain variable domain comprising the heavy chain complementarity determining region 1 (HCDR1) set forth in SEQ ID NO: 13; complementarity determining region of the heavy chain 2 (HCDR2), shown in SEQ ID NO: 16; and the heavy chain complementarity determining region 3 (HCDR3) shown in SEQ ID NO: 19. 2. Anti-OSMR antibody according to claim 1, wherein the light chain variable domain has the sequence shown in SEQ ID NO: 28. 3. An anti-OSMR antibody according to claim 1, wherein the heavy chain variable domain has the sequence shown in SEQ ID NO: 54. 4. An anti-OSMR antibody according to claim 1, wherein the light chain variable domain has the sequence shown in SEQ ID NO: 28 and the heavy chain variable domain has the sequence shown in SEQ ID NO: 54. 5. An anti-OSMR antibody according to claim 1, wherein the light chain of said antibody contains the sequence shown in SEQ ID NO: 25. 6. An anti-OSMR antibody according to claim 1, wherein said antibody is a monoclonal antibody. 7. An anti-OSMR antibody according to claim 1, wherein said antibody is a human antibody. 8. An anti-OSMR antibody according to claim 1, wherein said antibody inhibits the binding of human oncostatin M (OSM) or human IL-31 to human OSMR. 9. The anti-OSMR antibody of claim 1, wherein said antibody reduces human OSM mediated or human IL-31 mediated OSMR signaling in human OSMR expressing cells. 10. Anti-OSMR antibody according to claim 4, wherein said antibody is a monoclonal antibody. 11. An anti-OSMR antibody according to claim 10, wherein said antibody is a human antibody. 12. An anti-OSMR antibody according to claim 4, wherein the light chain of said antibody contains the amino acid sequence shown in SEQ ID NO: 25. 13. An anti-OSMR antibody according to claim 4, wherein said antibody inhibits the binding of human OSM or human IL-31 to human OSMR. 14. The anti-OSMR antibody of claim 4, wherein said antibody reduces human OSM mediated or human IL-31 mediated OSMR signaling in human OSMR expressing cells. 15. A pharmaceutical composition for reducing OSMR mediated signaling, comprising a therapeutically effective amount of an anti-OSMR antibody according to any one of the preceding claims and a carrier. 16. The pharmaceutical composition according to claim 15, wherein said carrier is aqueous. 17. The pharmaceutical composition of claim 15, wherein said carrier is non-aqueous. 18. The pharmaceutical composition of claim 15, wherein said pharmaceutical composition is a solution. 19. A pharmaceutical composition according to claim 15, wherein said pharmaceutical composition is lyophilized. 20. The use of an anti-OSMR antibody according to any one of claims 1 to 14 in the treatment of pruritus in a patient in need thereof. 21. The use of a pharmaceutical composition according to any one of claims 15 to 19 in the treatment of pruritus in a patient in need thereof.