Antibodies against canid and feline oncostatin M receptor beta, and their use

Abstract

The present invention provides an isolated antibody, or antigen-binding portion thereof, that specifically binds to canine or feline oncostatin M receptor beta (OSMR-β), or both, wherein the antibody antagonizes IL-31-mediated signaling, OSM-mediated signaling, or both, in canine and / or feline cells.

Description

[Technical Field]

[0001] This invention belongs to the field of immunotherapy. More specifically, this invention relates to the oncostatin M (OSM) receptor, and its modifiers, such as monoclonal antibodies that bind to the canine and / or feline oncostatin M receptor beta subunit (OSMR-β). This invention also relates to the diagnosis and / or treatment of diseases in canines and felines associated with OSM and IL-31 using anti-OSMR-β antibodies. [Background technology]

[0002] Cytokines comprise a large population of small proteins that play crucial roles in the development and regulation of immune responses. Certain cytokines are associated with the initiation and persistence of pathological pain behavior, including nerve and skin injury. Interleukin-31 (IL-31), a more recently discovered cytokine, is associated with the induction of chronic skin inflammation (Dillon et al. (2004) Nat. Immunol. 5, 752-760). Human and mouse data show high expression of IL-31 associated with pruritus, alopecia, skin lesions, and severe inflammatory skin disorders including atopic dermatitis (AD), as well as other controlled allergic diseases such as asthma (Dillon et al. (2004) (see above), Neis et al. (2006) J. Allergy Clin. Immunol. 118, 930-937, Rabenhorst et al. (2014) Curr. Allergy Asthma Rep. 14, 423, Cornelissen et al. (2012) Eur. J. Cell Biol. 91, 552-566, Takaoka et al. (2006) Exp. Dermatol. 15, 161-167, Sonkoly et al. (2006) J. Allergy Clin. Immunol. 117, 411-417, Lewis et al. al. (2017) J. Eur. Acad. Dermatology Venereol. 31, 142-150).

[0003] Experimental animal models of human AD have reported a strong correlation between itchiness-related scratching behavior and IL-31 mRNA expression in NC / Nga mice (Takaoka et al. (see above)). Compared to healthy controls, elevated serum IL-31 levels were found in adult patients with AD (Raap et al. (2008) J. Allergy Clin. Immunol. 122, 421-423) and in pediatric patients with AD flares and remission (Ezzat et al. (2011) J. Eur. Acad. Dermatology Venereol. 25, 334-339).

[0004] Furthermore, these data suggest that IL-31 represents an important target for the development of treatments for such inflammatory skin diseases in humans. Antagonist anti-IL-31 monoclonal antibodies (mAbs) are currently under development for human health (Pantazi et al. (2017) Nat. Rev. Drug Discov. 17, 237-238, Nemoto et al. (2016) Br. J. Dermatol. 174, 296-304). In addition, ant-IL-31 mAbs have already been developed for animal health (US Patent No. 8,790,651B2 against Bammert et al., Michels et al. (2016) Vet. Dermatol. 27, 478-e129). For example, the anti-hIL-31RA mAb CIM331 binds to IL-31RA, inhibits IL-31 signaling, and reduces severe itching (Nemoto et al. (2016) (above)). In veterinary medicine, lokivetomab, a "canine-derived" anti-IL-31 mAb, has shown efficacy in clinical trials for canine pruritus and is currently approved as a treatment for Alzheimer's disease in dogs (Michels et al. (2016) (above)).

[0005] IL-31 is a member of the IL-6 cytokine superfamily, preferentially produced by T helper type 2 cells (Dillon et al. (2004) (see above)). Mature human IL-31 (hIL-31) has a predicted topology of four antiparallel helices (Le Saux et al. (2010) J. Biol. Chem. 285, 3470-3477) and is composed of 141 amino acids (Dillon et al. (2004) (see above)). The IL-31 signaling pathway is thought to be mediated via the gp130-like type 1 cytokine receptor (IL-31RA, also known as GPL) and the oncostatin M receptor (OSMR) (Dillon et al. (above), Le Saux et al. (2010) (above), Diveu et al. (2004) Eur. Cytokine Netw. 15, 291-302, Zhang et al. (2008) Cytokine Growth Factor Rev. 19, 347-356). Both receptors belong to the type 1 cytokine receptor family, sharing a common cytokine-binding domain (CBD) formed by two fibronectin type III-like domains (Diveu et al. (2003) J. Biol. Chem. 278, 49850-49859).

[0006] Previous studies have provided immunoprecipitation evidence that human IL-31RA (hIL-31RA) directly binds to hIL-31. In these same studies, immunoprecipitation results failed to detect direct binding of human OSMR (hOSMR) to hIL-31 (Le Saux et al. (2010) Molecular dissection of human interleukin-31-mediated signal transduction through site-directed mutagenesis. J. Biol. Chem. 285, 3470-3477, Diveu et al. (2004) (above)). However, when hIL-31RA and hOSMR are combined, there is a significant increase in binding, suggesting that hIL-31 first binds to hIL-31RA, at which point hOSMR is recruited to form a ternary complex (Le Saux et al. (2010) (above), Diveu et al. (2004) (above)). In this model, the ternary complex activates numerous downstream signaling pathways (Dillon et al. (2004) (see above), Le Saux et al. (2010) (see above), Diveu et al. (2004) (see above), Dambacher et al. (2007) Gut 56, 1257-1265, Dreuw et al. (2004) J. Biol. Chem. 279, 36112-36120).

[0007] Based on the structure of the IL-6 / IL-6α-receptor / gp130 complex (Boulanger et al. (2003) Science. 300, 2101-2104), the IL-6 cytokine superfamily is thought to interact with its receptor via three distinct contact binding sites (sites I, II, and III). Le Saux et al., using computational analysis and sparse alanine scanning, depicted sites II and III only as binding sites important for the interaction between hIL-31 and its receptor (Le Saux et al. (2010) (above)). In particular, Glu44, Glu106, and His110 were identified as important residues for binding site II, while Lys134 was identified within binding site III.

[0008] The applicant initiated the study to gain insights into the interaction between feline IL-31 (fIL-31) and its feline receptors fOSMR and fIL-31RA, and to map conformational epitopes for anti-fIL-31 mAbs, referred to as mAb #1 or 15H05 (Medina-Cucurella AV et al, Feline Interleukin-31 Shares Overlapping Epitopes with the Oncostatin M Receptor and IL-31RA. Biochemistry. 2020 Jun 16;59(23):2171-2181), WO2019 / 177697A2 (Zoetis Services LLC). In contrast to previous studies performed on human homologs that showed OSMR could not interact with IL-31 in the absence of IL-31RA, the applicant discovered, by multiple biophysical methods, that fOSMR directly binds to fIL-31 and partially interferes with fIL-31RA binding. The applicant identified potential fIL-31 binding sites for fOSMR, fIL31-RA, and anti-fIL-31 mAbs using a predictive structural model combined with fine epitope mapping (Medina-Cucurella (2019) Methods Mol. Biol. 1764, 101-121), yeast surface display (Chao et al. (2006) Nat. Protoc. 1, 755-768), Nicking mutagenesis (Wrenbeck et al. (2016) Nat. Methods 13, 928-930), and deep sequencing (Araya and Fowler (2011) Trends Biotechnol. 29, 435-442). The constructed binding sites coincided with sites previously discovered by Le Saux et al. (above) and indicated additional overlapping sites between the two receptors, which the applicant calls “shared sites.” Specifically, the binding sites for IL31-RA included positions E20 and K82 of fIL-31, while the binding sites for OSMR included the "PADNFERK" motif (P103-K110) and positions G39 and K100, which were consistent with previous studies on human homologs.However, the applicant's results also revealed a novel overlapping site between the two feline receptors, consisting of locations R69, R72, P73, D76, D81, and E97, which had not been previously reported in humans. The conformational epitopes of anti-feline IL-31 mAbs that inhibit both OSMR and IL-31RA were also mapped to this shared site. Together, the applicant's results indicate that fIL-31 binds independently to IL-31RA and OSMR via a partially shared epitope. These results suggest that the mechanism of IL-31 signaling in companion animals such as cats may differ from that in humans. This, in turn, implies that effective therapeutic strategies for antagonizing IL-31-mediated signaling in companion animals may differ from therapeutic strategies for antagonizing IL-31-mediated signaling in higher organisms.

[0009] It would be desirable to provide a therapeutic monoclonal antibody that binds to the canid and / or feline oncostatin M receptor beta subunit (OSMR-β) and antagonistizes OSM-mediated and / or IL-31-mediated signaling in canids and / or felines. Such an antibody would preferably be useful in the diagnosis and / or treatment of diseases in canids and felines associated with OSM and IL-31, including severe inflammatory skin disorders such as allergic dermatitis and atopic dermatitis, as well as fibrotic conditions such as dermatofibrosis or renal fibrosis.

[0010] Cell models explain the role of OSM in regulating human keratinocyte function by influencing the expression of genes encoding proteins involved in support structure / metabolism, inflammation / innate immunity, and tissue remodeling. (Boniface K et al; Oncostatin M secreted by skin infiltrating T lymphocytes is a potent keratinocyte activator involved in skin inflammation. J Immunol. 2007 Apr 1;178(7):4615-22). These researchers provide compelling evidence for the role of OSM-mediated OSMR activation in skin inflammation resulting from keratinocyte activation in cell models using reconstructed primary epidermal keratinocytes. OSM-mediated OSMR activation in these keratinocyte cultures upregulates gene expression patterns consistent with psoriasis and atopic diseases in the skin. These data were supported by increased expression of both OSM and OSMR in patients with inflammatory skin disorders (psoriasis or atopic dermatitis (AD)). Targeting OSMR-β with a blocking monoclonal antibody would block the function of both IL-31 and OSM cytokines. Blocking IL-31 function is expected to achieve antipruritic and anti-inflammatory properties similar to lokivetomab, but further anti-inflammatory and anti-fibrotic activity exceeding that of lokivetomab may be achieved due to OSM inhibition. [Overview of the project]

[0011] The present invention provides an isolated antibody or antigen-binding moiety that specifically binds to canid or feline oncostatin M receptor beta (OSMR-β), or both, wherein the antibody or antigen-binding moiety antagonistizes IL-31-mediated signaling or OSM-mediated signaling, or both, in canid and / or feline cells. In one embodiment, the antibody or antigen-binding moiety antagonistizes both IL-31-mediated signaling and OSM-mediated signaling in canid and / or feline cells. In one embodiment, the IL-31-mediated signaling is pSTAT signaling (e.g., pSTAT3 signaling, but not limited to). In another embodiment, the OSM-mediated signaling is pSTAT signaling (e.g., pSTAT3 signaling, but not limited to).

[0012] In one embodiment, the antibody of the present invention, or its antigen-binding portion, 1) 02D09: Variable heavy chain (VH)-CDR1 (DYGMH) of SEQ ID NO: 1, VH-CDR2 (YISSGSRAVFFADTVKG) of SEQ ID NO: 2, VH-CDR3 (DRYDGRGFAY) of SEQ ID NO: 3, Variable light chain (VL)-CDR1 (RASQSISNNLH) of SEQ ID NO: 4, VL-CDR2 (YASQSIS) of SEQ ID NO: 5, and VL-CDR3 (QQSNSWPLT) of SEQ ID NO: 6, 2) 09E09: VH-CDR1 (SYAMS) of sequence number 7, VH-CDR2 (YISSGGDYIYYADTVKG) of sequence number 8, VH-CDR3 (DPITGTFAY) of sequence number 9, VL-CDR1 (RASQDINNYLN) of sequence number 10, VL-CDR2 (YTSTLHS) of sequence number 11, and VL-CDR3 (QQGNTLPWT) of sequence number 12, 3) 10F07: VH-CDR1 (SYAMS) of SEQ ID NO: 13, VH-CDR2 (YISSGGDYFYYADTVKG) of SEQ ID NO: 14, VH-CDR3 (DPITGTFAY) of SEQ ID NO: 15, VL-CDR1 (RASQDITNYLN) of SEQ ID NO: 16, VL-CDR2 (YTSTLHS) of SEQ ID NO: 17, and VL-CDR3 (QQGHMLPWT) of SEQ ID NO: 18, 4) 14C04: VH-CDR1 (NYWMN) of SEQ ID NO: 19, VH-CDR2 (QIYPGHVNTNYNGNFKD) of SEQ ID NO: 20, VH-CDR3 (SADNSGFVLFAY) of SEQ ID NO: 21, VL-CDR1 (RASKSVSTSGYSYLH) of SEQ ID NO: 22, VL-CDR2 (LASNLES) of SEQ ID NO: 23, and VL-CDR3 (QHSRELPLT) of SEQ ID NO: 24, 5) 19F07: VH-CDR1 (DYYMA) of SEQ ID NO: 25, VH-CDR2 (NINYDGSSTYYLDSLKS) of SEQ ID NO: 26, VH-CDR3 (GLTWDFDV) of SEQ ID NO: 27, VL-CDR1 (KASQDVDTAVA) of SEQ ID NO: 28, VL-CDR2 (LASTRHT) of SEQ ID NO: 29, and VL-CDR3 (QQYSRFPLT) of SEQ ID NO: 30, or 6) It includes a combination of CDR sequences selected from the complementarity-determining region (CDR) variants of 1, 2, 3, 4, or 5.

[0013] In some embodiments of the CDR variants of the parental antibody, the amino acid residues located at the mutation positions shown in Table A of the Examples section in the parental antibodies 02D09, 09E09, 10F07, and 19F07 are conserved in the CDR variants of their respective parental antibodies.

[0014] In other embodiments, the CDR variants of the 19F07 parental antibody are provided in Table B of the Examples section. Specifically, Table B provides a general description for each CDR of the 19F07 antibody and shows the allowed amino acid substitutions for each CDR of 19F07.

[0015] In another embodiment, the antibody according to the present invention (a) is a variable heavy chain, Sequence ID 31(MU_02D09_VH)EVQLVESGGGLVKPGGSLTLSCAASGFTFSDYGMHWLRQAPEKGLEWVAYISSGSRAVFFADTVKGRFTISRDNAKNTLFLQMTSLRSDDTAMYYCARDRYDGRGFAYWGQGTLVTVSA, Sequence ID 35(MU_09E09_VH)DVKLVESGEGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVAYISSGGDYIYYADTVKGRFTISRDNARNTLYLQMSSLKSEDTAMYYCTRDPITGTFAYWGQGTLVTVSA, Sequence ID 39(MU_10F07_VH)DVKLVESGEGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVTYISSGGDYFYYADTVKGRFTISRDNARNTLYLQMSSLKSEDTAMYYCTRDPITGTFAYWGQGTLVTVSA, Sequence ID 43 (MU_14C04_VH) EVQLQESGAELVKPGASVKISKASGYAFSNYWMNWMKQRPGKGLEWIGQIYPGHVNTNYNGNFKDKATLTADK SSSTAYMQLSSLTSEDSAVYFCARSADNSGFVLFAYWGQGTLVTVS, Sequence ID 47 (MU_19F07_VH) EVKLVESEGGLVQPGSSMKLSCTASGFTFSDYYMAWVRQVPEKGLEWVANINYDGSSTYYLDSLKSRFIISRDNAKNILYLQMSSLKSEDTATYYCARGLTWDFDVWGTGTTVTVSS, Sequence ID 51(FEL_02D09_VH1)DVQLVESGGDLVKPGGSLRLTCVASGFTYSDYGMHWVRQAPGKGLQWVAYISSGSRAVFFADTVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCVRDRYDGRGFAYWGQGTLVTVSS, Sequence ID 53(FEL_02D09_VH2)DVQLVESGGDLVKPGGSLRLTCVASGFTFSDYGMHWVRQAPGKGLQWVAYISSGSRAVFFADTVKGRFTISRDNAKNTLYLQMNGLRTEDTATYYCARDRYDGRGFAYWGQGTLVTVSS, Sequence ID 59(CAN_09E09_VH1)EVQLVESGGDLVKPGGSLRLSCVASGFTFSSYAMSWVRQAPGKGLQWVAYISSGGDYIYYADTVKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCVRDPITGTFAYWGQGTLVTVSS, Sequence ID 61(CAN_09E09_VH2)EVQLVESGGDLVKPAGSLTLSCLASGFTFSSYAMSWVRQTPEKGLQWVAYISSGGDYIYYADTVKGRFTISRDNAKNTLYLQMNSLRDEDTAVYYCARDPITGTFAYWGQGTLVTVSS, Sequence ID 67(FEL_09E09_VH1)DVQLVESGGDLVKPGGSLRLTCVASGFTYSSYAMSWVRQAPGKGLQWVAYISSGGDYIYYADTVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCVRDPITGTFAYWGQGTLVTVSS, Sequence ID 69(FEL_09E09_VH2)DVQLVESGGNLVKPGGSLRLTCVASGFTFSSYAMSWVRQAPGKGLQWVAYISSGGDYIYYADTVKGRFTISKDNAKNTLYLQMNSLKTEDTATYYCARDPITGTFAYWGQGTLVTVSS, Sequence ID 75(CAN_10F07_VH1)EVQLVESGGDLVKPGGSLRLSCVASGFTFSSYAMSWVRQAPGKGLQWVAYISSGGDYFYYADTVKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCVRDPITGTFAYWGQGTLVTVSS, Sequence ID 79(CAN_10F07_VH2)EVQLVESGGDLVKPAGSLTLSCLASGFTFSSYAMSWVRQTPEKGLQWVAYISSGGDYFYYADTVKGRFTISRDNAKNTLYLQMNSLRDEDTAVYYCARDPITGTFAYWGQGTLVTVSS, Sequence ID 83(FEL_10F07_VH1)DVQLVESGGDLVKPGGSLRLTCVASGFTYSSYAMSWVRQAPGKGLQWVAYISSGGDYFYYADTVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCVRDPITGTFAYWGQGTLVTVSS, Sequence ID 87(FEL_10F07_VH2)DVQLVESGGDLVKPGGSLRLTCVASGFTFSSYAMSWVRQAPGKGLQWVAYISSGGDYFYYADTVKGRFTISRDDAKNTLYLQMSSLKTEDTATYYCTGDPITGTFAYWGQGTLVTVSS, Sequence ID 91(CAN_19F07_VH1)EVQLVESGGDLVKPGGSLRLSCVASGFTFSDYYMAWVRQAPGKGLQWVANINYDGSSTYYLDSLKSRFTISRDNAKNTLYLQMNSLRAEDTAMYYCVRGLTWDFDVWGQGTLVTVSS, Sequence ID 95(CAN_19F07_VH2)EVQLVESGGDLVKPAGSLTLSCLASGFTFSDYYMAWVRQTPEKGLQWVANINYDGSSTYYLDSLKSRFTISRDNAKNTLYLQMNSLRDEDTAVYYCARGLTWDFDVWGQGTLVTVSS, Sequence ID 99(FEL_19F07_VH1)DVQLVESGGDLVKPGGSLRLTCVASGFTYSDYYMAWVRQAPGKGLQWVANINYDGSSTYYLDSLKSRFTISRDNAKNTLYLQMNSLKTEDTATYYCVRGLTWDFDVWGQGTLVTVSS, Sequence ID 103(FEL_19F07_VH2)DVQLVESGGNLVKPGGSLRLTCVASGFTFSDYYMAWVRQAPGKGLQWVANINYDGSSTYYLDSLKSRFTISRDNAKNTLYLQMNSLKTEDTATYYCARGLTWDFDVWGQGTLVTVSS, Sequence ID 127. (CAN_14C04_VH1) EVQLVESGGDLVKPGGSLRLSCVASGFTFSNYWMNWVRQAPGKGLQWVAQIYPGHVNTNYNGNFKDRFTISRDNARNTVYLQMNSLRAEDTAVYYCARSADNSGFVLFAYWGQGTLVTVSS, or Sequence ID 129. (CAN_14C04_VH2) Variable heavy chains including EVQLVESGGDLVKPGGSLRLSCVASGFTFSNYWMNWVRQSPGKGLQWVAQIYPGHVNTNYNGNFKDRFTISRDNAKNTLYLQMNSLRAEDTAVYFCARSADNSGFVLFAYWGQGTLVTVSS, and (b) Variable light chain, Sequence ID 33(MU_02D09_VL)DIVLTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQTSHESPRLLITYASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPLTFGAGTKLELK, Sequence ID 37(MU_09E09_VL)DLQMTQTTSSLSASLGDRVTISCRASQDINNYLNWYQQKPDGTVKLLIYYTSTLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIK, Sequence ID 41(MU_10F07_VL)DIQMTQTTSSLSASLGDRVTISCRASQDITNYLNWYQQKPDGTVKLLIYYTSTLHSGVPSRFSGSGSGTDFSLTISNLEQEDIATYFCQQGHMLPWTFGGGTKLEIK, Sequence ID 45(MU_14C04_VL)DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIFLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLELK, Sequence ID 49(MU_19F07_VL)DIVMTQSHKFMSPSVGDRVSITCKASQDVDTAVAWYQQKPGQSPKLLIYLASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSRFPLTFGAGTKLELK, Sequence ID 55(FEL_02D09_VL1)EIQMTQSPSSLSASPGDRVTITCRASQSISNNLHWYQQKPGKVPKLLIYYASQSISGVPSRFSGSGSGTDFTLTISSLEPEDAATYYCQQSNSWPLTFGQGT, Sequence ID 57(FEL_02D09_VL2)DIVMTQTPLSLSVTPGESASISCRASQSISNNLHWYLQKSGQSPRRLIYYASQSISGVPDRFSGSGSGTDFTLRISRVEADDVGVYYCQQSNSWPLTFGQGT, Sequence ID 63(CAN_09E09_VL1)EIVMTQSPASLSLSQEEKVTITCRASQDINNYLNWYQQKPGQAPKLLIYYTSTLHSGVPSRFSGSGSGTDFSFTISSLEPEDVAVYYCQQGNTLPWTFGQGT, Sequence ID 65(CAN_09E09_VL2)DIVLTQPTSVSGSLGQRVTISCRASQDINNYLNWYQQLPGKAPKLLVYYTSTLHSGVPDRFSGSNSGSSATLTITGLQAEDEADYYCQQGNTLPWTFGQGT, Sequence ID 71(FEL_09E09_VL1)EIQMTQSPSSLSASPGDRVTITCRASQDINNYLNWYQQKPGKVPKLLIYYTSTLHSGVPSRFSGSGSGTDFTLTISSLEPEDAATYYCQQGNTLPWTFGQGT, Sequence ID 73(FEL_09E09_VL2)DITMTQSPGSLAGSPGQQVTMNCRASQDINNYLNWYQQKPGQHPKLLIYYTSTLHSGVPDRFSGSGSGTDFTLTISNLQAEDVASYYCQQGNTLPWTFGQGT, Sequence ID 77(CAN_10F07_VL1)EIVMTQSPASLSLSQEEKVTITCRASQDITNYLNWYQQKPGQAPKLLIYYTSTLHSGVPSRFSGSGSGTDFSFTISSLEPEDVAVYYCQQGHMLPWTFGQGT, Sequence ID 81(CAN_10F07_VL2)DIVLTQPTSVSGSLGQRVTISCRASQDITNYLNWYQQLPGKAPKLLVYYTSTLHSGVPDRFSGSNSGSSATLTITGLQAEDEADYYCQQGHMLPWTFGQGT, Sequence ID 85(FEL_10F07_VL1)EIQMTQSPSSLSASPGDRVTITCRASQDITNYLNWYQQKPGKVPKLLIYYTSTLHSGVPSRFSGSGSGTDFTLTISSLEPEDAATYYCQQGHMLPWTFGQGT, Sequence ID 89(FEL_10F07_VL2)DITMTQSPGSLAGSPGQQVTMNCRASQDITNYLNWYQQKPGQHPKLLIYYTSTLHSGVPDRFSGSGSGTDFTLTISNLQAEDVASYYCQQGHMLPWTFGQGT, Sequence ID 93(CAN_19F07_VL1)EIVMTQSPASLSLSQEEKVTITCKASQDVDTAVAWYQQKPGQAPKLLIYLASTRHTGVPSRFSGSGSGTDFSFTISSLEPEDVAVYYCQQYSRFPLTFGQGT, Sequence ID 97(CAN_19F07_VL2)DIVMTQTPLSLSVSPGETASISCKASQDVDTAVAWFRQKPGQSPQRLIYLASTRHTGVPDRFSGSGSGTDFTLRISRVEADDTGVYYCQQYSRFPLTFGQGT, Sequence ID 101(FEL_19F07_VL1)EIQMTQSPSSLSASPGDRVTITCKASQDVDTAVAWYQQKPGKVPKLLIYLASTRHTGVPSRFSGSGSGTDFTLTISSLEPEDAATYYCQQYSRFPLTFGQGT, Sequence ID 105(FEL_19F07_VL2)DITMTQSPGSLAGSPGQQVTMNCKASQDVDTAVAWYQQKPGQHPKLLIYLASTRHTGVPDRFSGSGSGTDFTLTISNLQAEDVASYYCQQYSRFPLTFGQGT, Sequence ID 131. (CAN_14C04_VL1) EIVMTQSPASLSLSQEEKVTITCRASKSVSTSGYSYLHWYQQKPGQAPKLLIYLASNLESGVPSRFSGSGSGTDFSFTISSLEPEDVAVYYCQHSRELPLTFGQGT, or Sequence ID 133. (CAN_14C04_VL2) It includes at least one of a variable heavy chain and a variable light chain, such as a variable light chain containing DIVMTQTPLSLSVSPGETASISCRASKSVSTSGYSYLHWYLQKPGQSPQLLIYLASNLESGVSKRFSGSGSGTDFTLRISRVEADDTGIYYCQHSRELPLTFGQGT.

[0016] In one embodiment, the antibody is a chimeric antibody. In another embodiment, the antibody is canine or feline.

[0017] In one embodiment, the antibody inhibits or neutralizes IL-31-mediated or OSM-mediated pruritic or allergic conditions in dogs or cats. In one embodiment, the IL-31-mediated or OSM-mediated pruritic conditions are selected from atopic dermatitis, eczema, psoriasis, scleroderma, and pruritus. In another embodiment, the IL-31-mediated or OSM-mediated allergic conditions are selected from allergic dermatitis, summer eczema, urticaria, heaves, inflammatory airway disease, recurrent airway obstruction, airway hypersensitivity, chronic obstructive pulmonary disease, and inflammatory processes resulting from autoimmunity.

[0018] In one embodiment, the antibody inhibits IL-31-mediated or OSM-mediated fibrous or inflammatory disorders. In one embodiment, the IL-31-mediated or OSM-mediated fibrous disorders are selected from renal fibrosis, pulmonary fibrosis, and cutaneous fibrosis. In another embodiment, the IL-31-mediated or OSM-mediated inflammatory disorders are selected from autoimmune inflammatory processes, inflammation of the skin or joints in animals suffering from osteoarthritis, immune-mediated polyarthritis, chronic bronchitis, allergic asthma, atopic dermatitis, allergic dermatitis, suppurative traumatic dermatitis, atherosclerosis, and cardiovascular disease.

[0019] In further embodiments, the antibody reduces IL-31-mediated or OSM-mediated inflammatory pain. In one embodiment, the IL-31-mediated or OSM-mediated inflammatory pain is osteoarthritis pain.

[0020] The present invention also provides a veterinary composition containing a therapeutically effective amount of the antibody described above.

[0021] The present invention also provides a method for treating an IL-31-mediated or OSM-mediated disorder in a subject, comprising administering an antibody according to the present invention to the subject. In one embodiment of this method, the IL-31-mediated or OSM-mediated disorder is selected from pruritic conditions, allergic conditions, fibrotic disorders, inflammatory disorders, or inflammatory pain.

[0022] In another embodiment of the method of treatment, the IL-31-mediated or OSM-mediated pruritic condition is selected from the group consisting of atopic dermatitis, eczema, psoriasis, scleroderma, and pruritus. In yet another embodiment of the method, the IL-31-mediated or OSM-mediated allergic condition is selected from allergic dermatitis, summer eczema, urticaria, respiratory fatigue, inflammatory airway disease, recurrent airway obstruction, airway hypersensitivity, chronic obstructive pulmonary disease, and inflammatory processes resulting from autoimmunity.

[0023] In a further embodiment of the method of treatment, the IL-31-mediated or OSM-mediated fibrosis is selected from renal fibrosis, pulmonary fibrosis, or cutaneous fibrosis. In another embodiment of the method, the IL-31-mediated or OSM-mediated inflammatory disorder is selected from autoimmune inflammatory processes, inflammation of the skin or joints in animals suffering from osteoarthritis, immune-mediated polyarthritis, chronic bronchitis, allergic asthma, atopic dermatitis, allergic dermatitis, suppurative traumatic dermatitis, atherosclerosis, or cardiovascular disease.

[0024] In a further embodiment of the treatment method, IL-31-mediated or OSM-mediated inflammatory pain is osteoarthritis pain.

[0025] In one embodiment of the treatment method, the subject is a dog or a cat.

[0026] The present invention further provides a method for inhibiting IL-31 and / or OSM activity in dogs or cats, comprising administering the aforementioned antibody to the dogs or cats.

[0027] A method for detecting OSMR beta in a sample is also provided. This method includes incubating a sample containing OSMR beta in the presence of the aforementioned antibody and detecting the antibody that binds to OSMR beta in the sample. In one embodiment of this method, the antibody includes a label. In one embodiment, the detection method further includes quantifying OSMR beta in the sample.

[0028] The present invention also provides host cells and nucleic acids that can be used to produce the antibody or its antigen-binding moiety. In one embodiment, the present invention provides a host cell comprising the following combination of complementarity-determining region (CDR) sequences: 1) 02D09: Variable heavy chain (VH)-CDR1 (DYGMH) of SEQ ID NO: 1, VH-CDR2 (YISSGSRAVFFADTVKG) of SEQ ID NO: 2, VH-CDR3 (DRYDGRGFAY) of SEQ ID NO: 3, Variable light chain (VL)-CDR1 (RASQSISNNLH) of SEQ ID NO: 4, VL-CDR2 (YASQSIS) of SEQ ID NO: 5, and VL-CDR3 (QQSNSWPLT) of SEQ ID NO: 6, 2) 09E09: VH-CDR1 (SYAMS) of sequence number 7, VH-CDR2 (YISSGGDYIYYADTVKG) of sequence number 8, VH-CDR3 (DPITGTFAY) of sequence number 9, VL-CDR1 (RASQDINNYLN) of sequence number 10, VL-CDR2 (YTSTLHS) of sequence number 11, and VL-CDR3 (QQGNTLPWT) of sequence number 12, 3) 10F07: VH-CDR1 (SYAMS) with sequence number 13, VH-CDR2 (YISSGGDYFYYADTVKG) with sequence number 14, VH-CDR3 (DPITGTFAY) with sequence number 15, VL-CDR1 (RASQDITNYLN) with sequence number 16, VL-CDR2 (YTSTLHS) with sequence number 17, and VL-CDR3 (QQGHMLPWT) with sequence number 18, 4) 14C04: VH-CDR1 (NYWMN) of sequence number 19, VH-CDR2 (QIYPGHVNTNYNGNFKD) of sequence number 20, VH-CDR3 (SADNSGFVLFAY) of sequence number 21, VL-CDR1 (RASKSVSTSGYSYLH) of sequence number 22, VL-CDR2 (LASNLES) of sequence number 23, and VL-CDR3 (QHSRELPLT) of sequence number 24, 5) 19F07: VH-CDR1 (DYYMA) with sequence number 25, VH-CDR2 (NINYDGSSTYYLDSLKS) with sequence number 26, VH-CDR3 (GLTWDFDV) with sequence number 27, VL-CDR1 (KASQDVDTAVA) with sequence number 28, VL-CDR2 (LASTRHT) with sequence number 29, and VL-CDR3 (QQYSRFPLT) with sequence number 30, or 6) A host cell is provided that produces an isolated antibody or its antigen-binding moiety containing at least one of the CDR variants 1, 2, 3, 4, or 5.

[0029] The present invention also relates to isolated nucleic acids, comprising the following combinations of variable heavy chain complementarity-determining region (CDR) sequences: 1) 02D09: Variable heavy chain (VH)-CDR1 (DYGMH) of SEQ ID NO: 1, VH-CDR2 (YISSGSRAVFFADTVKG) of SEQ ID NO: 2, and VH-CDR3 (DRYDGRGFAY) of SEQ ID NO: 3, 2) 09E09: VH-CDR1 (SYAMS) with sequence number 7, VH-CDR2 (YISSGGDYIYYADTVKG) with sequence number 8, and VH-CDR3 (DPITGTFAY) with sequence number 9, 3) 10F07: VH-CDR1 (SYAMS) with sequence number 13, VH-CDR2 (YISSGGDYFYYADTVKG) with sequence number 14, and VH-CDR3 (DPITGTFAY) with sequence number 15, 4) 14C04: VH-CDR1 (NYWMN) of sequence number 19, VH-CDR2 (QIYPGHVNTNYNGNFKD) of sequence number 20, and VH-CDR3 (SADNSGFVLFAY) of sequence number 21, 5) 19F07: VH-CDR1 (DYYMA) of sequence number 25, VH-CDR2 (NINYDGSSTYYLDSLKS) of sequence number 26, and VH-CDR3 (GLTWDFDV) of sequence number 27, or 6) Provide an isolated nucleic acid comprising a nucleic acid sequence encoding at least one of the CDR variants 1, 2, 3, 4, or 5.

[0030] In one embodiment, the above-mentioned nucleic acid is a combination of the following variable light chain CDR sequences: 1) 02D09: Variable light chain (VL)-CDR1 (RASQSISNNLH) of SEQ ID NO: 4, VL-CDR2 (YASQSIS) of SEQ ID NO: 5, and VL-CDR3 (QQSNSWPLT) of SEQ ID NO: 6 2) 09E09: VL-CDR1 (RASQDINNYLN) of sequence number 10, VL-CDR2 (YTSTLHS) of sequence number 11, and VL-CDR3 (QQGNTLPWT) of sequence number 12, 3) 10F07: VL-CDR1 (RASQDITNYLN) with sequence number 16, VL-CDR2 (YTSTLHS) with sequence number 17, and VL-CDR3 (QQGHMLPWT) with sequence number 18, 4) 14C04: VL-CDR1 (RASKSVSTSGYSYLH) of sequence number 22, VL-CDR2 (LASNLES) of sequence number 23, and VL-CDR3 (QHSRELPLT) of sequence number 24, 5) 19F07: VL-CDR1 (KASQDVDTAVA) with sequence number 28, VL-CDR2 (LASTRHT) with sequence number 29, and VL-CDR3 (QQYSRFPLT) with sequence number 30, or 6) Further comprising a nucleic acid sequence encoding at least one of the CDR variants of 1, 2, 3, 4, or 5.

[0031] In another embodiment, the nucleic acid sequence according to the present invention is a combination of the following variable light chain CDR sequences: 1) 02D09: Variable light chain (VL)-CDR1 (RASQSISNNLH) of SEQ ID NO: 4, VL-CDR2 (YASQSIS) of SEQ ID NO: 5, and VL-CDR3 (QQSNSWPLT) of SEQ ID NO: 6 2) 09E09: VL-CDR1 (RASQDINNYLN) of sequence number 10, VL-CDR2 (YTSTLHS) of sequence number 11, and VL-CDR3 (QQGNTLPWT) of sequence number 12, 3) 10F07: VL-CDR1 (RASQDITNYLN) with sequence number 16, VL-CDR2 (YTSTLHS) with sequence number 17, and VL-CDR3 (QQGHMLPWT) with sequence number 18, 4) 14C04: VL-CDR1 (RASKSVSTSGYSYLH) of sequence number 22, VL-CDR2 (LASNLES) of sequence number 23, and VL-CDR3 (QHSRELPLT) of sequence number 24, 5) 19F07: VL-CDR1 (KASQDVDTAVA) with sequence number 28, VL-CDR2 (LASTRHT) with sequence number 29, and VL-CDR3 (QQYSRFPLT) with sequence number 30, or 6) Code at least one of the CDR variants 1, 2, 3, 4, or 5.

[0032] The present invention further provides a vector containing the nucleic acid described above.

[0033] Furthermore, the present invention provides a method for producing antibodies. This method comprises culturing the aforementioned host cells under conditions that induce antibody production, and isolating the antibodies from the host cells or the culture medium of the host cells. [Brief explanation of the drawing]

[0034] [Figure 1A] a) Canid DH82 cells and b) Feline Fcwf4 cells are shown in graphs illustrating the pSTAT3 dose response produced by treatment with canid and feline OSM. [Figure 1B] a) Canid DH82 cells and b) Feline Fcwf4 cells are shown in graphs illustrating the pSTAT3 dose response produced by treatment with canid and feline OSM. [Figure 2] This flowchart shows the steps used to select the anti-OSMR antibody described in this application. [Figure 3-1] This is an alignment of the variable heavy chain sequence and variable light chain sequence of the anti-OSMR antibody described herein. [Figure 3-2] Continuation of Figure 3-1. [Figure 4-1] This matrix shows the RMSD values ​​of the CDR after pairwise comparison of the structures of homology models generated for the five anti-OSMR antibodies described herein. [Figure 4-2] Continuation of Figure 4-1. [Figure 4-3] Continuation of Figure 4-2. [Figure 5]Figure 5A) This homology model shows a dimer of canine OSM bound to a dimer of canine OSMR protein. The black circles highlight the binding interface between OSM and OSMR. Figure 5B) This shows an enlarged region of the binding interface, highlighting amino acids known to be important in human OSM:OSMR interaction. [Figure 6] This specification shows the logical location where OSM binds to OSMR, as well as the epitope-binding sites of the antibodies described herein, based on the sequence diversity among human, canid, and feline OSMR proteins. [Figure 7] This paper also shows the quantification of OSMR mRNA in feline skin using an RNAscope ISH probe. The control tissue was derived from a normal feline skin biopsy, while the diseased tissue was derived from a feline skin biopsy with allergic skin disease. [Figure 8] A single representative image from IHC staining of OSMR protein from control (non-allergic) skin tissue is shown for diseased (allergic) skin tissue. [Figure 9] Figure 9A) Shows proliferation of canine synovial cells in response to dose-increasing canine OSM protein over a 24-hour period. Figure 9B) Shows inhibition of OSM-induced canine synovial cell proliferation using anti-OSMR mouse antibody 10F07 (described herein) compared with a control antibody (also incubated for 24 hours). [Figure 10] Figure 10A) Shows the induction of canine MCP-1 protein in canine synovial cells in response to dose-increasing canine OSM protein over a 24-hour period. Figure 10B) Shows the inhibition of OSM-induced canine synovial cell MCP-1 production using anti-OSMR mouse antibody 10F07 (as described herein) compared with a control antibody (also incubated for 24 hours). [Figure 11] This study demonstrates the induction of canine MCP-1 protein in primary canine renal fibrosis in response to dose-increasing canine OSM protein over a 72-hour period. [Figure 12]Figure 12A) Shows pre- and post-treatment pruritus scores in a canine model of IL-31-induced pruritus, comparing anti-OSMR mouse:canine chimeras 19F07(T01) and 10F07(T02), both administered subcutaneously to 6 animals per group at a dose of 12.0 mg / kg on day 0. Figure 12B) Shows summary statistics of post-challenge scores by treatment group, and paired t-tests with mean and 90% confidence intervals. [Modes for carrying out the invention]

[0035] A brief explanation of arrays Sequence ID 1 is a variable heavy chain CDR1 referred to herein as 02D09-VH-CDR1, Sequence ID 2 is a variable heavy chain CDR2 referred to herein as 02D09-VH-CDR2, Sequence ID 3 is a variable heavy chain CDR3 referred to herein as 02D09-VH-CDR3, Sequence ID 4 is a variable light chain CDR1 referred to herein as 02D09-VL-CDR1, Sequence ID 5 is a variable light chain CDR2 referred to herein as 02D09-VL-CDR2, Sequence ID 6 is a variable light chain CDR3 referred to herein as 02D09-VL-CDR3, Sequence ID 7 is a variable heavy chain CDR1 referred to herein as 09E09-VH-CDR1, Sequence ID 8 is a variable heavy chain CDR2 referred to herein as 09E09-VH-CDR2, Sequence ID 9 is a variable heavy chain CDR3 referred to herein as 09E09-VH-CDR3, Sequence ID 10 is a variable light chain CDR1 referred to herein as 09E09-VL-CDR1, Sequence ID 11 is a variable light chain CDR2 referred to herein as 09E09-VL-CDR2, Sequence ID 12 is a variable light chain CDR3 referred to herein as 09E09-VL-CDR3, Sequence ID 13 is a variable heavy chain CDR1 referred to herein as 10F07-VH-CDR1, Sequence ID 14 is a variable heavy chain CDR2 referred to herein as 10F07-VH-CDR2, Sequence ID 15 is a variable heavy chain CDR3 referred to herein as 10F07-VH-CDR3, Sequence ID 16 is a variable light chain CDR1 referred to herein as 10F07-VL-CDR1, Sequence ID 17 is a variable light chain CDR2 referred to herein as 10F07-VL-CDR2, Sequence ID 18 is a variable light chain CDR3 referred to herein as 10F07-VL-CDR3, Sequence ID 19 is a variable heavy chain CDR1 referred to herein as 14C04-VH-CDR1, Sequence ID 20 is a variable heavy chain CDR2 referred to herein as 14C04-VH-CDR2, Sequence ID 21 is a variable heavy chain CDR3 referred to herein as 14C04-VH-CDR3, Sequence ID 22 is a variable light chain CDR1 referred to herein as 14C04-VL-CDR1, Sequence ID 23 is a variable light chain CDR2 referred to herein as 14C04-VL-CDR2, Sequence ID 24 is a variable light chain CDR3 referred to herein as 14C04-VL-CDR3, Sequence ID 25 is a variable heavy chain CDR1 referred to herein as 19F07-VH-CDR1, Sequence ID 26 is a variable heavy chain CDR2 referred to herein as 19F07-VH-CDR2, Sequence ID 27 is a variable heavy chain CDR3 referred to herein as 19F07-VH-CDR3, Sequence ID 28 is a variable light chain CDR1 referred to herein as 19F07-VL-CDR1, Sequence ID 29 is a variable light chain CDR2 referred to herein as 19F07-VL-CDR2, Sequence ID 30 is a variable light chain CDR3 referred to herein as 19F07-VL-CDR3, Sequence ID 31 is a variable heavy chain sequence referred to herein as MU_02D09_VH, Sequence ID 32 is a nucleotide sequence that encodes a variable heavy chain sequence referred to herein as MU_02D09_VH, Sequence ID 33 is a variable light chain sequence referred to herein as MU_02D09_VL, Sequence ID 34 is a nucleotide sequence that encodes a variable light chain sequence referred to herein as MU_02D09_VL, Sequence ID 35 is a variable heavy chain sequence referred to herein as MU_09E09_VH, Sequence ID 36 is a nucleotide sequence that encodes a variable heavy chain sequence referred to herein as MU_09E09_VH, Sequence ID 37 is a variable light chain sequence referred to herein as MU_09E09_VL, Sequence ID 38 is a nucleotide sequence that encodes a variable light chain sequence referred to herein as MU_09E09_VL, Sequence ID 39 is a variable heavy chain sequence referred to herein as MU_10F07_VH, Sequence ID 40 is a nucleotide sequence that encodes a variable heavy chain sequence referred to herein as MU_10F07_VH, Sequence ID 41 is a variable light chain sequence referred to herein as MU_10F07_VL, Sequence ID 42 is a nucleotide sequence that encodes a variable light chain sequence referred to herein as MU_10F07_VL, Sequence ID 43 is a variable heavy chain sequence referred to herein as MU_14C04_VH, Sequence ID 44 is a nucleotide sequence that encodes a variable heavy chain sequence referred to herein as MU_14C04_VH, Sequence ID 45 is a variable light chain sequence referred to herein as MU_14C04_VL, Sequence ID 46 is a nucleotide sequence that encodes a variable light chain sequence referred to herein as MU_14C04_VL, Sequence ID 47 is a variable heavy chain sequence referred to herein as MU_19F07_VH, Sequence ID 48 is a nucleotide sequence that encodes a variable heavy chain sequence referred to herein as MU_19F07_VH, Sequence ID 49 is a variable light chain sequence referred to herein as MU_19F07_VL, Sequence ID 50 is a nucleotide sequence that encodes a variable light chain sequence referred to herein as MU_19F07_VL, Sequence ID 51 is a variable heavy chain sequence referred to herein as FEL_02D09_VH1, Sequence ID 52 is a nucleotide sequence that encodes a variable heavy chain sequence referred to herein as FEL_02D09_VH1, Sequence ID 53 is a variable heavy chain sequence referred to herein as FEL_02D09_VH2, Sequence ID 54 is a nucleotide sequence that encodes a variable heavy chain sequence referred to herein as FEL_02D09_VH2, Sequence ID 55 is a variable light chain sequence referred to herein as FEL_02D09_VL1, Sequence ID 56 is a nucleotide sequence that encodes a variable light chain sequence referred to herein as FEL_02D09_VL1, Sequence ID 57 is a variable light chain sequence referred to herein as FEL_02D09_VL2, Sequence ID 58 is a nucleotide sequence that encodes a variable light chain sequence referred to herein as FEL_02D09_VL2, Sequence ID 59 is a variable heavy chain sequence referred to herein as CAN_09E09_VH1, Sequence ID 60 is a nucleotide sequence that encodes a variable heavy chain sequence referred to herein as CAN_09E09_VH1, Sequence ID 61 is a variable heavy chain sequence referred to herein as CAN_09E09_VH2, Sequence ID 62 is a nucleotide sequence that encodes a variable heavy chain sequence referred to herein as CAN_09E09_VH2, Sequence ID 63 is a variable light chain sequence referred to herein as CAN_09E09_VL1, Sequence ID 64 is a nucleotide sequence that encodes a variable light chain sequence referred to herein as CAN_09E09_VL1, Sequence ID 65 is a variable light chain sequence referred to herein as CAN_09E09_VL2, Sequence ID 66 is a nucleotide sequence that encodes a variable light chain sequence referred to herein as CAN_09E09_VL2, Sequence ID 67 is a variable heavy chain sequence referred to herein as FEL_09E09_VH1, Sequence ID 68 is a nucleotide sequence that encodes a variable heavy chain sequence referred to herein as FEL_09E09_VH1, Sequence ID 69 is a variable heavy chain sequence referred to herein as FEL_09E09_VH2, Sequence ID 70 is a nucleotide sequence that encodes a variable heavy chain sequence referred to herein as FEL_09E09_VH2, Sequence ID 71 is a variable light chain sequence referred to herein as FEL_09E09_VL1, Sequence ID 72 is a nucleotide sequence that encodes a variable light chain sequence referred to herein as FEL_09E09_VL1, Sequence ID 73 is a variable light chain sequence referred to herein as FEL_09E09_VL2, Sequence ID 74 is a nucleotide sequence that encodes a variable light chain sequence referred to herein as FEL_09E09_VL2, Sequence ID 75 is a variable heavy chain sequence referred to herein as CAN_10F07_VH1, Sequence ID 76 is a nucleotide sequence that encodes a variable heavy chain sequence referred to herein as CAN_10F07_VH1, Sequence ID 77 is a variable light chain sequence referred to herein as CAN_10F07_VL1, Sequence ID 78 is a nucleotide sequence that encodes a variable light chain sequence referred to herein as CAN_10F07_VL1, Sequence ID 79 is a variable heavy chain sequence referred to herein as CAN_10F07_VH2, Sequence ID 80 is a nucleotide sequence that encodes a variable heavy chain sequence referred to herein as CAN_10F07_VH2, Sequence ID 81 is a variable light chain sequence referred to herein as CAN_10F07_VL2, Sequence ID 82 is a nucleotide sequence that encodes a variable light chain sequence referred to herein as CAN_10F07_VL2, Sequence ID 83 is a variable heavy chain sequence referred to herein as FEL_10F07_VH1, Sequence ID 84 is a nucleotide sequence that encodes a variable heavy chain sequence referred to herein as FEL_10F07_VH1, Sequence ID 85 is a variable light chain sequence referred to herein as FEL_10F07_VL1, Sequence ID 86 is a nucleotide sequence that encodes a variable light chain sequence referred to herein as FEL_10F07_VL1, Sequence ID 87 is a variable heavy chain sequence referred to herein as FEL_10F07_VH2, Sequence ID 88 is a nucleotide sequence that encodes a variable heavy chain sequence referred to herein as FEL_10F07_VH2, Sequence ID 89 is a variable light chain sequence referred to herein as FEL_10F07_VL2, Sequence ID 90 is a nucleotide sequence that encodes a variable light chain sequence referred to herein as FEL_10F07_VL2, Sequence ID 91 is a variable heavy chain sequence referred to herein as CAN_19F07_VH1, Sequence ID 92 is a nucleotide sequence that encodes a variable heavy chain sequence referred to herein as CAN_19F07_VH1, Sequence ID 93 is a variable light chain sequence referred to herein as CAN_19F07_VL1, Sequence ID 94 is a nucleotide sequence that encodes a variable light chain sequence referred to herein as CAN_19F07_VL1, Sequence ID 95 is a variable heavy chain sequence referred to herein as CAN_19F07_VH2, Sequence ID 96 is a nucleotide sequence that encodes a variable heavy chain sequence referred to herein as CAN_19F07_VH2, Sequence ID 97 is a variable light chain sequence referred to herein as CAN_19F07_VL2, Sequence ID 98 is a nucleotide sequence that encodes a variable light chain sequence referred to herein as CAN_19F07_VL2, Sequence ID 99 is a variable heavy chain sequence referred to herein as FEL_19F07_VH1, Sequence ID 100 is a nucleotide sequence that encodes a variable heavy chain sequence referred to herein as FEL_19F07_VH1, Sequence ID 101 is a variable light chain sequence referred to herein as FEL_19F07_VL1, Sequence ID 102 is a nucleotide sequence that encodes a variable light chain sequence referred to herein as FEL_19F07_VL1, Sequence ID 103 is a variable heavy chain sequence referred to herein as FEL_19F07_VH2, Sequence ID 104 is a nucleotide sequence that encodes a variable heavy chain sequence referred to herein as FEL_19F07_VH2, Sequence ID 105 is a variable light chain sequence referred to herein as FEL_19F07_VL2, Sequence ID 106 is a nucleotide sequence that encodes a variable light chain sequence referred to herein as FEL_19F07_VL2, Sequence ID 107 is the amino acid sequence of a canine OSMR-Fc fusion protein, denoted herein as Canine_OSMR_hIgG1_Fc. Sequence ID 108 is a nucleotide sequence encoding a canine OSMR-Fc fusion protein, which is referred to herein as Canine_OSMR_hIgG1_Fc. Sequence ID 109 is an amino acid sequence corresponding to the canine OSM referred to herein as Canine_OSM, Sequence ID 110 is the amino acid sequence corresponding to the feline OSM-Fc fusion protein referred to herein as Feline_OSM_hIgG1_Fc, Sequence ID 111 is a nucleotide sequence corresponding to the feline OSM-Fc fusion protein referred to herein as Feline_OSM_hIgG1_Fc, Sequence ID 112 is the amino acid sequence of the feline OSMR-Fc fusion protein, denoted herein as Feline_OSMR_hIgG1_Fc. Sequence ID 113 is a nucleotide sequence encoding a feline OSMR-Fc fusion protein, which is referred to herein as Feline_OSMR_hIgG1_Fc. Sequence ID 114 is the amino acid sequence for the constant region of the heavy chain of a canine animal, referred to herein as Canine_HC_65_1. Sequence ID 115 is a nucleotide sequence that encodes a constant region of the heavy chain of a canine animal, referred to herein as Canine_HC_65_1. Sequence ID 116 is the amino acid sequence for the constant region of the light chain of a canine animal, referred to herein as Canine_LC_Kappa. Sequence ID 117 is a nucleotide sequence that encodes a constant region of the canine light chain, referred to herein as Canine_LC_Kappa. Sequence ID 118 is the amino acid sequence for the constant region of the heavy chain of a feline animal, referred to herein as Feline_HC_AlleleA_1. Sequence ID 119 is a nucleotide sequence that encodes the constant region of the heavy chain of a feline animal, referred to herein as Feline_HC_AlleleA_1. Sequence ID 120 is the amino acid sequence for the constant region of the light chain of a feline animal, referred to herein as Feline_LC_Kappa_G_minus. Sequence ID 121 is a nucleotide sequence that encodes the amino acid sequence for the constant region of the light chain of a feline animal, referred to herein as Feline_LC_Kappa_G_minus. Sequence ID 122 is the amino acid sequence for the human OSMR protein, referred to herein as Human_OSMR. Sequence ID 123 is the amino acid sequence for the canine IL31 protein, referred to herein as Canine_IL31. Sequence ID 124 is a nucleotide sequence encoding the canine IL31 protein, referred to herein as Canine_IL31. Sequence ID 125 is the amino acid sequence for the feline IL31 protein, referred to herein as Feline_IL31. Sequence ID 126 is a nucleotide sequence encoding the feline IL31 protein, referred to herein as Feline_IL31. Sequence ID 127 is a variable heavy chain sequence referred to herein as CAN_14C04_VH1, Sequence ID 128 is a nucleotide sequence that encodes a variable heavy chain sequence referred to herein as CAN_14C04_VH1, Sequence ID 129 is a variable heavy chain sequence referred to herein as CAN_14C04_VH2, Sequence ID 130 is a nucleotide sequence that encodes a variable heavy chain sequence referred to herein as CAN_14C04_VH2, Sequence ID 131 is a variable heavy chain sequence referred to herein as CAN_14C04_VL1, Sequence ID 132 is a nucleotide sequence that encodes a variable heavy chain sequence referred to herein as CAN_14C04_VL2, Sequence ID 133 is a variable heavy chain sequence referred to herein as CAN_14C04_VL1, Sequence ID 134 is a nucleotide sequence that encodes a variable heavy chain sequence referred to herein as CAN_14C04_VL2, Sequence ID 135 is the amino acid sequence of the human LIFR protein, which is referred to herein as Human_LIFR. Sequence ID 136 is the amino acid sequence of the human LIF protein, denoted herein as Human_LIF.

[0036] definition Before describing the present invention in detail, we define some terms used in the context of the present invention. In addition to these terms, other terms will be defined elsewhere in this specification as necessary. Unless otherwise expressly defined herein, technical terms used herein have the meanings recognized in their respective art.

[0037] As used herein and in the claims, singular forms such as “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. For example, a reference to “antibody” includes multiple such antibodies.

[0038] As used herein, the term “comprising” is intended to mean that a composition or method includes the listed elements but does not exclude other elements.

[0039] As used herein, an epitope refers to an antigenic determinant recognized by the CDR of an antibody. In other words, an epitope refers to any part of a molecule that is recognized by and can be bound to by an antibody. Unless otherwise indicated, as used herein, the term “epitope” refers to the region of OSMR beta to which an anti-OSMR beta agent reacts.

[0040] An "antigen" is a molecule or a part of a molecule that can be bound by an antibody, recognized by an antibody, and bound by an antibody (the corresponding antibody-binding region may be called a paratope). Generally, an epitope consists of a classification of the chemically active surface of a molecule, such as an amino acid or sugar side chain, and has specific three-dimensional structural and specific charge characteristics.

[0041] In the context of antibody binding, the term "specifically" refers to the high avidity and / or high affinity binding of an antibody to a specific antigen, i.e., a polypeptide or epitope. In many embodiments, the specific antigen is an antigen (or fragment or partial fragment of an antigen) used by antibody-producing cells to immunize an isolated animal host. Antibodies that specifically bind to an antigen are stronger than the binding of the same antibody to other antigens. Antibodies that specifically bind to a polypeptide may be able to bind to other polypeptides at a weak but detectable level (e.g., less than 10% of the binding shown to the polypeptide of interest). Such weak binding, or background binding, can be easily distinguished from the binding of the specific antibody to the polypeptide of interest, for example, by the use of an appropriate control. Generally, specific antibodies are 10 -7 M or less, for example, 10 -8 M or less (for example, 10 -9 M or less, 10 -10 The following 10 -11 The following 10 -12 The following, or 10 -13 The following K D It binds to the antigen with binding affinity.

[0042] As used herein, the term “antibody” refers to an intact immunoglobulin having two light chains and two heavy chains. Thus, a single isolated antibody or fragment may be a polyclonal antibody, a monoclonal antibody, a synthetic antibody, a recombinant antibody, a chimeric antibody, a heterochimeric antibody, a canine-derived antibody, or a feline-derived antibody. The term “antibody” preferably refers to monoclonal antibodies and their fragments, as well as their immunological conjugate equivalents, that can bind to OSMR beta protein and its fragments. Both terms are used to refer to a homogeneous molecule or a mixture such as a serum product composed of multiple different molecular entities.

[0043] "Natural antibodies" and "natural immunoglobulins" are generally heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intra-chain disulfide cross-links. Each heavy chain has a variable domain (V H ) at one end, followed by a number of constant domains. Each light chain has a variable domain (V L ) at one end and a constant domain at its other end, and the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Certain amino acid residues are thought to form the interface between the light chain variable domain and the heavy chain variable domain.

[0044] The term "antibody fragment" refers to something less than the intact antibody structure, including, but not limited to, isolated single antibody chains, Fv constructs, Fab constructs, Fc constructs, light chain variable or complementarity determining region (CDR) sequences, etc.

[0045] The term "variable" region includes framework and CDR (also known as hypervariable), referring to the fact that certain parts of the variable domain differ extensively in sequence between antibodies, and are used in the binding and specificity of each particular antibody to that particular antigen. However, variability is not evenly distributed throughout the variable domain of an antibody. Variability is concentrated in three segments called hypervariable regions in both the light chain and heavy chain variable domains. The more highly conserved parts of the variable domain are called framework regions (FRs). The natural heavy chain and light chain variable domains each contain multiple FRs, primarily employing β-sheet structures, connected by three hypervariable regions that form loops connecting α-sheet structures, and in some cases form parts of them. The hypervariable regions within each chain are held together in close proximity by the fiber-retaining domain (FR), and together with the hypervariable regions from other chains, they contribute to the formation of the antibody's antigen-binding site (see Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), pages 647-669). The constant domain does not directly participate in antibody-antigen binding, but exhibits various effector functions, such as the involvement of antibodies in antibody-dependent cytotoxicity.

[0046] As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody involved in antigen binding. The hypervariable region includes amino acid residues from the “complementarity-determining region” or “CDR” (Kabat, et al. (1991), above) and / or those residues from the “hypervariable loop” (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “Framework” or “FR” residues are variable domain residues other than those of the hypervariable region as defined herein.

[0047] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments, each having a single antigen-binding site, and the remaining "Fc" fragment, a name reflecting its ability to readily crystallize. Pepsin treatment yields an F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking antigens.

[0048] "Fv" is the smallest antibody fragment containing complete antigen recognition and antigen-binding sites. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain in a close, non-covalent association. The three hypervariable regions of each variable domain interact to form V H -V L It is in this configuration that the antigen-binding site on the surface of the dimer is defined. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of Fv containing only the three antigen-specific hypervariable regions) can recognize and bind to the antigen, albeit with lower affinity than the entire binding site.

[0049] The Fab fragment also contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment by the addition of several residues at the carboxyl terminus of the heavy chain CH1 domain, which contains one or more cysteines from the antibody hinge region. Fab'-SH is a notation used herein for Fab' in which the cysteine ​​residue of the constant domain has a free thiol group. The F(ab')2 antibody fragment was originally produced as a pair of Fab' fragments having hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0050] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two distinct types called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0051] Depending on the amino acid sequence of the constant domains of their heavy chains, immunoglobulins can be assigned to different classes. Currently, there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2 (defined by mouse and human notation). The heavy chain constant domains corresponding to different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in multiple species. The incidence of individual isotypes and functional activity associated with these constant domains is species-specific and must be defined experimentally.

[0052] As defined herein, a “monoclonal antibody” is an antibody produced by a single clone of a cell (specifically, a single clone of a hybridoma cell), and is therefore a single, pure, homogeneous antibody. All monoclonal antibodies produced from the same clone are identical and have the same antigen specificity. The term “monoclonal” refers to a single clone of a cell, a single cell, and its offspring.

[0053] The monoclonal antibodies of the present invention specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species. Fragments of such antibodies are also included, insofar as they exhibit the desired biological activity. Typically, a chimeric antibody is an antibody in which the light chain and heavy chain genes are constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species. For example, a variable segment of a gene from a mouse monoclonal antibody can be linked to a canid constant segment. In this embodiment, the antigen-binding site is derived from mouse, but on the other hand, F C The part is a canid.

[0054] The "canine-modified" form of a non-canine (e.g., mouse) antibody is a genetically modified antibody that contains a minimal amount of sequences derived from non-canine immunoglobulins. A canine-modified antibody is a canine immunoglobulin sequence (recipient antibody) in which the hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-canine species (donor antibody), such as mouse, that possess the desired specificity, affinity, and capabilities. In some cases, framework region (FR) residues of the canine immunoglobulin sequence are replaced by corresponding non-canine residues. Furthermore, canine-modified antibodies may contain residues not found in the recipient or donor antibody. These modifications are made to further improve antibody performance. Generally, a canine-modified antibody contains at least one, typically two, variable domains substantially all of them, with all or substantially all of the hypervariable region corresponding to that of the non-canine immunoglobulin sequence, and all or substantially all of the FRs being from the canine immunoglobulin sequence. Canine-derived antibodies may also optionally include immunoglobulin constant regions (Fc), typically the entire or at least a portion of the constant region (Fc) of a canine immunoglobulin. In one embodiment, a mouse CDR is grafted onto a canine framework.

[0055] The "felineized" form of a non-feline (e.g., mouse) antibody is a genetically modified antibody that contains a minimal amount of sequences derived from non-feline immunoglobulins. A felineized antibody is a feline immunoglobulin sequence (recipient antibody) in which the hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-feline species (donor antibody), such as mouse, that possess the desired specificity, affinity, and capabilities. In some cases, framework region (FR) residues of the feline immunoglobulin sequence are replaced by corresponding non-feline residues. Furthermore, felineized antibodies may contain residues not found in the recipient or donor antibody. These modifications are made to further improve antibody performance. Generally, a felineized antibody contains substantially all of at least one, typically two, variable domains, with all or substantially all of the hypervariable region corresponding to that of the non-feline immunoglobulin sequence, and all or substantially all of the FRs being from the feline immunoglobulin sequence. Feline-derived antibodies may also optionally include the constant region (Fc) of an immunoglobulin, typically the entire constant region (Fc) of a feline immunoglobulin, or at least a portion thereof.

[0056] As defined herein, the term "heterochimeric" refers to an antibody in which one of its antibody chains (heavy or light) is canidized, while the other is chimeric. In one embodiment, a canidized variable heavy chain (all of the CDRs are mouse and all of the FRs are canid) pairs with a chimeric variable light chain (all of the CDRs are mouse and all of the FRs are mouse). In this embodiment, both the variable heavy chain and the variable light chain are fused to a canid constant region.

[0057] A "variant" anti-OSMR beta antibody, as used herein, refers to a molecule whose amino acid sequence differs from that of the "parent" anti-OSMR beta antibody due to the addition, deletion, and / or substitution of one or more amino acid residues in the parent antibody sequence, and which retains at least one desired activity of the parent anti-OSMR beta antibody. Desired activities may include the ability to specifically bind to an antigen, the ability to reduce, inhibit, or neutralize OSM activity and / or IL-31 activity in animals, and the ability to inhibit OSM-mediated and / or IL-31-mediated pSTAT signaling (STAT phosphorylation) in cell-based assays. In one embodiment, the variant comprises one or more amino acid substitutions in one or more hypervariable (CDR) and / or framework regions of the parent antibody. For example, the variant may comprise at least one, e.g., about 1 to about 15, preferably about 2 to about 5, or about 2 to about 10 substitutions in one or more hypervariable (CDR) and / or framework regions of the parent antibody. In one embodiment, a variant may contain at least approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions in one or more CDR regions of the parent antibody. In another embodiment, a variant may contain up to approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions in one or more CDR regions of the parent antibody. It should be understood that, with respect to a variant antibody, the number of amino acid substitutions in any given CDR of the parent antibody may differ from the number of substitutions in other CDRs of the parent antibody. In one embodiment, each variant may independently have variable heavy chain CDR1, variable heavy chain CDR2, and variable heavy chain CDR3 amino acid sequences that have at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% amino acid sequence identity with the variable heavy chain CDR1, variable heavy chain CDR2, and variable heavy chain CDR3 amino acid sequences of the parent antibody.In another embodiment, the variant may have variable light chain CDR1, variable light chain CDR2, and variable light chain CDR3 amino acid sequences, each independently having at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% amino acid sequence identity with the variable light chain CDR1, variable light chain CDR2, and variable light chain CDR3 amino acid sequences of the parent antibody. Typically, the variant may have an amino acid sequence having at least 50% amino acid sequence identity, more preferably at least 65%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity with the heavy chain or light chain variable domain sequence of the parent antibody. Sequence identity or homology is defined herein as the percentage of amino acid residues in the candidate sequence that are identical to the parent antibody residues after the sequences have been aligned and, if necessary, gaps have been introduced to achieve the maximum sequence identity percentage. N-terminal, C-terminal, or internal extensions, deletions, or insertions into the antibody sequence should not be interpreted as affecting sequence identity or homology. The variant retains the ability to bind to OSMR beta and preferably has a desired activity superior to that of the parent antibody. For example, the variant may have stronger binding affinity, an enhanced ability to reduce, inhibit, or neutralize OSM activity and / or IL-31 activity in animals, and / or an enhanced ability to inhibit OSM-mediated and / or IL-31-mediated pSTAT signaling in cell-based assays.

[0058] In this specification, a “variant” nucleic acid refers to a molecule whose sequence differs from that of the “parent” nucleic acid. Diversity in polynucleotide sequences can arise from mutations such as the deletion, substitution, or addition of one or more nucleotides. Each of these changes may occur once or more times in a given sequence, either individually or in combination.

[0059] The “parent” antibody of the present invention is encoded by an amino acid sequence used for variant preparation. Preferably, the parent antibody has a canine framework region and, if present, a canine antibody constant region. For example, the parent antibody may be a canine-derived antibody or a canine antibody. Another example is that the parent antibody may be a feline-derived antibody or a feline antibody. Yet another example is that the parent antibody is a mouse monoclonal antibody.

[0060] The term “isolated” means that the material (e.g., antibody or nucleic acid) is separated and / or recovered from components of its natural environment. Contaminating components of its natural environment are those that would interfere with the diagnostic or therapeutic use of the material and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. With respect to nucleic acids, isolated nucleic acids may include those separated from the 5'→3' sequence that it normally associates in chromosomes. In preferred embodiments, the material is purified to over 95% by weight, most preferably over 99% by weight. Since at least one component of the material’s natural environment is absent, the isolated material contains the material in situ within recombinant cells. However, typically, isolated material will be prepared by at least one purification step.

[0061] As used herein, the term “label” refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody or nucleic acid. The label may be detectable itself (e.g., radioisotope labeling or fluorescent labeling), or, in the case of enzymatic labeling, may catalyze a chemical change in a detectable substrate compound or composition.

[0062] The terms “nucleic acid,” “polynucleotide,” and “nucleic acid molecule” may be used interchangeably herein and refer to a set of nucleotide bases (also called “nucleotides”) in DNA and RNA. Nucleic acids may include deoxyribonucleotides, ribonucleotides, and / or analogues thereof. The term “nucleic acid” includes, for example, single-stranded and double-stranded molecules. Nucleic acids may be, for example, genes or gene fragments, exons, introns, DNA molecules (e.g., cDNA), RNA molecules (e.g., mRNA), recombinant nucleic acids, plasmids, and other vectors, primers, and probes. Both 5'→3' (sense) and 3'→5' (antisense) polynucleotides are included.

[0063] "Subject" or "patient" refers to an animal requiring treatment that may be affected by the molecules of the present invention. Examples of animals that can be treated according to the present invention include vertebrates, with mammals such as canids, felines, and equines being particularly preferred examples.

[0064] The “therapeutic effective dose” (or “effective dose”) refers to the amount of the active ingredient, such as the drug according to the present invention, that is sufficient to produce a beneficial or desired result when administered to a subject or patient. The effective dose may be administered in one or more doses, applications, or dosages. The therapeutic effective dose of the composition according to the present invention can be readily determined by those skilled in the art. In the context of the present invention, the “therapeutic effective dose” is the amount that produces an objectively measurable change in one or more parameters related to the treatment of a pruritic or allergic condition, including clinical improvement of symptoms. Naturally, the therapeutic effective dose will vary depending on the specific subject and condition being treated, the subject’s weight and age, the severity of the disease condition, the specific composition selected, the drug regimen to be followed, the timing of administration, the mode of administration, etc., all of which can be readily determined by those skilled in the art.

[0065] As used herein, the term “therapeutic” encompasses the entire spectrum of treatment for a disease or disorder. The “therapeutic” agents of the present invention may act in a prophylactic or preventive manner (genomic pharmacology), including by incorporating procedures designed to target animals that can be identified as being at risk, or in a manner that is essentially improvement or cure, or by slowing the rate or degree of progression of at least one symptom of the disease or disorder being treated.

[0066] The terms "treatment" and "treating" refer to both therapeutic measures and preventive or protective measures. Animals in need of treatment include animals already suffering from a disorder and animals for which the disorder should be prevented. The terms "treatment" or "treating" of a disease or disorder include prevention or protection against the disease or disorder (i.e., not causing the cause of clinical symptoms), inhibition of the disease or disorder (i.e., preventing or suppressing the onset of clinical symptoms, and / or mitigation of the disease or disorder (i.e., causing regression of clinical symptoms)). As can be understood, it is not always possible to distinguish between "preventing" and "suppressing" a disease or disorder, as the final inducing event or event may be unknown or latent. Therefore, the term "prophylaxis" will be understood to constitute a type of "treatment" that encompasses both "preventing" and "suppressing." Thus, the term "treatment" includes "prevention."

[0067] The term “allergic condition” is defined herein as a disorder or disease caused by the interaction between the immune system and a foreign substance in the body. This foreign substance is called an “allergen.” Common allergens include pollen, dust, mold, dust mite proteins, and airborne allergens such as saliva injected from insect bites. Examples of allergic conditions include, but are not limited to, allergic dermatitis, summer eczema, urticaria, shortness of breath, inflammatory airway disease, recurrent airway obstruction, airway hypersensitivity, chronic obstructive pulmonary disease, and inflammatory processes resulting from autoimmunity, such as irritable bowel syndrome (IBS).

[0068] The term “pruritus” is defined herein as a disorder or condition characterized by an intense itch that compels the person to rub or scratch the skin in order to obtain pain relief. Examples of pruritus include, but are not limited to, atopic dermatitis, eczema, psoriasis, scleroderma, and pruritus.

[0069] The term "fibrotic disorder" is defined herein as a disease or disorder characterized by fibrosis, which is the accumulation of extracellular matrix components in an organ or tissue that alters its structure and disrupts its normal function. Fibrosis can occur in almost any organ or tissue and is associated with a wide variety of diseases. Examples of types of fibrotic disorders include, but are not limited to, renal fibrosis, pulmonary fibrosis, and cutaneous fibrosis.

[0070] The term "inflammatory disorders" encompasses a vast number of disorders and conditions characterized by inflammation. Examples include, but are not limited to, inflammatory processes resulting from autoimmunity, inflammation of the skin or joints in animals with osteoarthritis, immune-mediated polyarthritis, chronic bronchitis, allergic asthma, atopic dermatitis, allergic dermatitis, suppurative traumatic dermatitis, atherosclerosis, and cardiovascular diseases.

[0071] As used herein, the term “inflammatory pain” refers to a spontaneous hypersensitivity to pain arising in response to tissue damage and inflammation (e.g., postoperative pain, trauma, arthritis). In one non-limiting example, inflammatory pain is pain associated with osteoarthritis.

[0072] As used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms include their offspring, which are any and all subsequent generations. It is understood that all offspring may not be identical due to planned or accidental mutations. In the context of the expression of heterologous nucleic acid sequences, “host cell” refers to a prokaryotic or eukaryotic cell (e.g., bacterial cells, yeast cells, mammalian cells, and insect cells), whether located in vitro or in vivo. For example, a host cell may be located in a transgenic animal. A host cell may include any transformable organism that can be used as a recipient for a vector and is capable of replicating the vector and / or expressing the heterologous nucleic acid encoded by the vector.

[0073] "Composition" is intended to mean a combination of an active agent, which may be inert (e.g., labeled) or active (e.g., an adjuvant), and another compound or composition.

[0074] As defined herein, pharmaceutically acceptable carriers suitable for use in the present invention are well known to those skilled in the art. Such carriers include, but are not limited to, water, physiological saline, buffered physiological saline, phosphate buffer, alcohol / aqueous solution, emulsion, or suspension. Other conventionally used diluents, adjuvants, and excipients may be added according to conventional techniques. Such carriers include ethanol, polyols, and suitable mixtures thereof, vegetable oils, and injectable organic esters. Buffers and pH adjusters may also be used. Buffers include, but are not limited to, salts prepared from organic acids or bases. Typical buffers include, but are not limited to, organic acid salts, e.g., salts of citrate (e.g., citrate), ascorbic acid, gluconic acid, histidine-HCl, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid salts, Tris, trimethamine hydrochloride, or phosphate buffers. Parenteral carriers may include sodium chloride solution, ringer's dextrose, dextrose, trehalose, sucrose, and sodium chloride, lactated Ringer's solution, or fixative oil. Intravenous carriers may include fluids and nutritional supplements, electrolyte supplements (such as those based on ringer's dextrose), etc. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents (e.g., EDTA), and inert gases, may also be provided in the pharmaceutically acceptable carrier. The present invention is not limited by the choice of carrier. The preparation of these pharmaceutically acceptable compositions from the above-mentioned components having appropriate pH isotonicity, stability, and other conventional characteristics is within the realm of the art. For example, please refer to texts such as Remington: The Science and Practice of Pharmacy, 20th ed, Lippincott Williams & Wilkins, publ., 2000, and The Handbook of Pharmaceutical Excipients, 4th sup.th edit, eds. RCRowe et al, APhA Publications, 2003.

[0075] The term “conservative amino acid substitution” refers to any amino acid substitution for a given amino acid residue, where the substituted residue is chemically very similar to that of the given residue, and therefore does not result in a substantial decrease in polypeptide function (e.g., enzyme activity). Conservative amino acid substitutions are commonly known in the art, and examples of them are described, for example, in U.S. Patents 6,790,639, 6,774,107, 6,194,167, or 5,350,576. In preferred embodiments, a conservative amino acid substitution is any that occurs within one of the following six groups: ●1. Small aliphatic substantially nonpolar residues: Ala, Gly, Pro, Ser, and Thr, ●2. Large aliphatic nonpolar residues: Ile, Leu, and Val, Met, ●3. Polar negatively charged residues and their amides: Asp and Glu, ●4. Amides of negatively charged polar residues: Asn and Gln, His, ●5. Polarly charged positive residues: Arg and Lys, His, and ●6. Large aromatic residues: Trp, Tyr, and Phe. In preferred embodiments, a conserved amino acid substitution is one of the following listed as native residue (conserved substitution) pairs: Ala(Ser);Arg(Lys);Asn(Gln;His);Asp(Glu);Gln(Asn);Glu(Asp);Gly(Pro);His(Asn;Gln);Ile(Leu;Val);Leu(Ile;Val);Lys(Arg;Gln;Glu);Met(Leu;Ile);Phe(Met;Leu;Tyr);Ser(Thr);Thr(Ser);Trp(Tyr);Tyr(Trp;Phe), and Val(Ile;Leu).

[0076] Just as polypeptides can contain conserved amino acid substitutions, their polynucleotides can contain conserved codon substitutions. A codon substitution is considered conserved if, when expressed, it produces the aforementioned conserved amino acid substitutions. Degenerate codon substitutions that do not result in amino acid substitutions are also useful in the polynucleotides according to the present invention. Therefore, for example, a polynucleotide encoding a selected polypeptide useful in one embodiment of the present invention can be mutated by degenerate codon substitutions to approximate the codon usage frequency shown by the expressing host cell transformed with it, or to improve its expression in other ways.

[0077] Detailed description of the invention It should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein, and that these themselves may change. The technical terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is defined solely by the claims.

[0078] Unless otherwise defined, the scientific and technical terms used in relation to the antibodies described herein shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise required by the context, singular terms shall include plural forms and plural terms shall include singular forms. In general, the terminology and techniques used in relation to cell and tissue culture, molecular biology, and the chemistry and hybridization of proteins and oligonucleotides or polynucleotides described herein are well known and commonly used in the art.

[0079] Standard techniques are used for recombinant DNA and oligonucleotide synthesis, and tissue culture and transfection (e.g., electroporation, lipofection). Enzyme reactions and purification techniques are carried out according to the manufacturer's specifications, as commonly achieved in the art, or as described herein. The aforementioned techniques and procedures are generally carried out according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., MOLECULAR CLONING: LAB. MANUAL (3rd ed., Cold Spring Harbor Lab. Press, Cold Spring Harbor, NY, 2001), and Ausubel et al., Current Protocols in Molecular Biology (New York: Greene Publishing Association / Wiley Interscience), 1993. The terminology and laboratory procedures and techniques used in relation to analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques are used in chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, as well as in the treatment of patients.

[0080] Except in the operating examples, or unless otherwise indicated, all figures representing the quantities of components or reaction conditions used herein should be understood to be modified in all cases by the term "approximately".

[0081] All specified patents and other publications are expressly incorporated herein by reference for the purpose of describing and disclosing methodologies described in such publications that may be used in connection with the present invention. These publications are provided only for disclosures prior to the filing date of this application.

[0082] This invention provides recombinant monoclonal antibodies and peptides, as well as their use in clinical and scientific procedures, including diagnostic procedures. With the advent of molecular biology methods and recombinant techniques, it is possible to produce antibodies and antibody-like molecules by recombinant means, and thereby generate gene sequences encoding specific amino acid sequences found in the polypeptide structure of the antibody. Such antibodies can be produced by either cloning the gene sequence encoding the polypeptide chain of the antibody, or by directly synthesizing the polypeptide chain and assembling the synthesized chain to form an active tetramer (H2L2) structure having affinity for a specific epitope and antigenic determinant. This has made it possible to immediately produce antibodies having sequences characterized by neutralizing antibodies from different species and sources.

[0083] Regardless of how they are recombinantly constructed or synthesized—whether from an antibody source, or using transgenic animals (laboratory-sized or commercially available large cell cultures), transgenic plants, or by direct chemical synthesis without the use of living organisms at any stage of the process—all antibodies have a similar three-dimensional structure overall. This structure is often provided as H2L2, referring to the fact that antibodies generally contain two light-chain (L) amino acids and two heavy-chain (H) amino acids. Both chains have regions capable of interacting with structurally complementary antigen targets. The target-interacting region is referred to as the "variable" or "V" region and is characterized by differences in the amino acid sequence from antibodies of different antigen specificities. The variable region of either the H or L chain contains an amino acid sequence capable of specifically binding to the antigen target.

[0084] As used herein, the term “antigen-binding region” refers to a portion of an antibody molecule that contains amino acid residues that interact with an antigen and confer its specificity and affinity to the antigen to the antibody. The antibody-binding region includes “framework” amino acid residues necessary to maintain the proper conformation of the antigen-binding residues.

[0085] Within the variable regions of the H or L chains that provide the antigen-binding domain, there are smaller sequences called "hypervariable" regions, which are the reason for the extreme variability between antibodies of different specificities. Such hypervariable regions are also called "complementarity-determining regions" or "CDR regions." These CDR regions are responsible for the fundamental specificity of antibodies to specific antigenic determinant structures.

[0086] The CDR represents a discontinuous range of amino acids within the variable region. However, regardless of the species, the positions where these important amino acid sequences are located within the variable heavy and light chain regions have been found to be similar within the amino acid sequence of the variable chain. The variable heavy and light chains of all antibodies each have three CDR regions, each discontinuous with the others.

[0087] In all mammalian species, antibody peptides contain both a constant (i.e., highly conserved) region and a variable region, the latter of which includes a CDR and a so-called "framework region" consisting of amino acid sequences that are within the variable region of the heavy or light chain but outside the CDR.

[0088] Antigen determinants recognized by the CDR region of an antibody are also called "epitopes." In other words, an epitope refers to one or more parts of any molecule that are recognized by an antibody (the corresponding antibody-binding region may be called a paratope) and can be bound by the antibody.

[0089] An "antigen" is a molecule or a part of a molecule that can be bound by an antibody and can induce an animal to produce an antibody that can bind to the epitope of that antigen. An antigen may have one or more epitopes. The specific reaction mentioned above means that the antigen reacts with its corresponding antibody in a highly selective manner and does not react with a large number of other antibodies that may be induced by other antigens.

[0090] The term "antibody" includes both intact immunoglobulin molecules and parts, fragments, peptides and their derivatives such as Fab, Fab', F(ab')2, Fv, Fse, CDR regions, paratopes, etc., or any part or peptide sequence of an antibody that can bind to an antigen or epitope. An antibody is said to be "capable of binding" to a molecule if it reacts specifically with the molecule, thereby enabling the molecule to bind to the antibody.

[0091] Antibodies also include, but are not limited to, chimeric antibodies, heterochimeric antibodies, canine-derived antibodies, or feline-derived antibodies, as well as fragments, parts, regions, peptides, or derivatives thereof, provided by any known technique such as enzymatic cleavage, peptide synthesis, or recombinant technology. Such antibodies of the present invention are capable of specifically binding to at least one of canine OSMR beta or feline OSMR beta. The antibody fragments or parts lack the Fc fragment of the intact antibody, may be cleared more rapidly from circulation, and may exhibit less nonspecific tissue binding than the intact antibody. Examples of antibody fragments can be produced from the intact antibody by proteolytic cleavage using methods well known in the art, for example, by enzymatic cleavage such as papain (to produce a Fab fragment) or pepsin (to produce an F(ab')2 fragment). See, for example, Wahl et al., 24 J. Nucl. Med. 316-25 (1983). Some antibodies can be produced by any of the methods described above, or by expressing a portion of the recombinant molecule. For example, the CDR region of a recombinant antibody may be isolated and subcloned into a suitable expression vector. See, for example, U.S. Patent No. 6,680,053.

[0092] Clones 02D09, 09E09, 10F07, 14C04, and 19F07 nucleotide and amino acid sequences In some embodiments, the present invention provides novel monoclonal antibodies that specifically bind to at least one of canine OSMR beta or feline OSMR beta. In one embodiment, the monoclonal antibody of the present invention binds to canine OSMR beta or feline OSMR beta and prevents or inhibits the activity of an IL-31 coreceptor complex comprising IL-31 receptor A (IL-31Ra) and oncostatin M-specific receptor (OsmR or OSMR beta). The monoclonal antibodies of the present invention are identified herein by "02D09", "09E09", "10F07", "14C04", and "19F07", referring to the number assigned to their hybridoma clones. In this specification, “02D09,” “09E09,” “10F07,” “14C04,” and “19F07” also refer to paratopes or CDRs that are part of monoclonal antibodies that specifically bind to the OSMR beta-epitopes identified as 02D09, 09E09, 10F07, 14C04, and 19F07, respectively, due to their ability to bind to the 02D09, 09E09, 10F07, 14C04, and 19F07 antibodies. Several recombinants, chimeras, heterochimers, canine and / or feline forms of 02D09, 09E09, 10F07, 14C04, and 19F07 described herein may be referred to by the same names.

[0093] In one embodiment, the antibody of the present invention, or its antigen-binding portion, 1) 02D09: Variable heavy chain (VH)-CDR1 (DYGMH) of SEQ ID NO: 1, VH-CDR2 (YISSGSRAVFFADTVKG) of SEQ ID NO: 2, VH-CDR3 (DRYDGRGFAY) of SEQ ID NO: 3, Variable light chain (VL)-CDR1 (RASQSISNNLH) of SEQ ID NO: 4, VL-CDR2 (YASQSIS) of SEQ ID NO: 5, and VL-CDR3 (QQSNSWPLT) of SEQ ID NO: 6, 2) 09E09: VH-CDR1 (SYAMS) of sequence number 7, VH-CDR2 (YISSGGDYIYYADTVKG) of sequence number 8, VH-CDR3 (DPITGTFAY) of sequence number 9, VL-CDR1 (RASQDINNYLN) of sequence number 10, VL-CDR2 (YTSTLHS) of sequence number 11, and VL-CDR3 (QQGNTLPWT) of sequence number 12, 3) 10F07: VH-CDR1 (SYAMS) with sequence number 13, VH-CDR2 (YISSGGDYFYYADTVKG) with sequence number 14, VH-CDR3 (DPITGTFAY) with sequence number 15, VL-CDR1 (RASQDITNYLN) with sequence number 16, VL-CDR2 (YTSTLHS) with sequence number 17, and VL-CDR3 (QQGHMLPWT) with sequence number 18, 4) 14C04: VH-CDR1 (NYWMN) of sequence number 19, VH-CDR2 (QIYPGHVNTNYNGNFKD) of sequence number 20, VH-CDR3 (SADNSGFVLFAY) of sequence number 21, VL-CDR1 (RASKSVSTSGYSYLH) of sequence number 22, VL-CDR2 (LASNLES) of sequence number 23, and VL-CDR3 (QHSRELPLT) of sequence number 24, 5) 19F07: VH-CDR1 (DYYMA) with sequence number 25, VH-CDR2 (NINYDGSSTYYLDSLKS) with sequence number 26, VH-CDR3 (GLTWDFDV) with sequence number 27, VL-CDR1 (KASQDVDTAVA) with sequence number 28, VL-CDR2 (LASTRHT) with sequence number 29, and VL-CDR3 (QQYSRFPLT) with sequence number 30, or 6) Includes a combination of CDR sequences selected from the complementarity-determining region (CDR) variants 1, 2, 3, 4, or 5. In some embodiments, the amino acid residues located at the mutation sites shown in Table A of the Examples section for parent antibodies 02D09, 09E09, 10F07, and 19F07 are preserved in the CDR variants of the respective parent antibodies. For example, based on the information in Table A and the sequence listing, in some embodiments, the underlined amino acid residues shown below are preserved in the CDR variants of parent antibodies 02D09, 09E09, 10F07, and 19F07. 02D09:

number

number

number

number

[0094] Based on the results described in Example 19, alanine substitution mutations at the underlined positions adversely affected antibody binding to the OSMR target. By inference, the ununderlined residues are substituted in the CDR variant because alanine mutations at those positions did not adversely affect antibody binding to the OSMR target.

[0095] Furthermore, the information in Table B of the Examples section supports the fact that, in some embodiments, the CDR variant of the 19F07 parent antibody may have the acceptable substitutions specified in the sequence definitions in that table, which are reproduced below. [Table a-1]

[0096] In another embodiment, the antibody according to the present invention is (a) Variable heavy chain, Sequence ID 31(MU_02D09_VH)EVQLVESGGGLVKPGGSLTLSCAASGFTFSDYGMHWLRQAPEKGLEWVAYISSGSRAVFFADTVKGRFTISRDNAKNTLFLQMTSLRSDDTAMYYCARDRYDGRGFAYWGQGTLVTVSA, Sequence ID 35(MU_09E09_VH)DVKLVESGEGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVAYISSGGDYIYYADTVKGRFTISRDNARNTLYLQMSSLKSEDTAMYYCTRDPITGTFAYWGQGTLVTVSA, Sequence ID 39(MU_10F07_VH)DVKLVESGEGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVTYISSGGDYFYYADTVKGRFTISRDNARNTLYLQMSSLKSEDTAMYYCTRDPITGTFAYWGQGTLVTVSA, Sequence ID 43 (MU_14C04_VH) EVQLQESGAELVKPGASVKISKASGYAFSNYWMNWMKQRPGKGLEWIGQIYPGHVNTNYNGNFKDKATLTADK SSSTAYMQLSSLTSEDSAVYFCARSADNSGFVLFAYWGQGTLVTVS, Sequence ID 47 (MU_19F07_VH) EVKLVESEGGLVQPGSSMKLSCTASGFTFSDYYMAWVRQVPEKGLEWVANINYDGSSTYYLDSLKSRFIISRDNAKNILYLQMSSLKSEDTATYYCARGLTWDFDVWGTGTTVTVSS, Sequence ID 51(FEL_02D09_VH1)DVQLVESGGDLVKPGGSLRLTCVASGFTYSDYGMHWVRQAPGKGLQWVAYISSGSRAVFFADTVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCVRDRYDGRGFAYWGQGTLVTVSS, Sequence ID 53(FEL_02D09_VH2)DVQLVESGGDLVKPGGSLRLTCVASGFTFSDYGMHWVRQAPGKGLQWVAYISSGSRAVFFADTVKGRFTISRDNAKNTLYLQMNGLRTEDTATYYCARDRYDGRGFAYWGQGTLVTVSS, Sequence ID 59(CAN_09E09_VH1)EVQLVESGGDLVKPGGSLRLSCVASGFTFSSYAMSWVRQAPGKGLQWVAYISSGGDYIYYADTVKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCVRDPITGTFAYWGQGTLVTVSS, Sequence ID 61(CAN_09E09_VH2)EVQLVESGGDLVKPAGSLTLSCLASGFTFSSYAMSWVRQTPEKGLQWVAYISSGGDYIYYADTVKGRFTISRDNAKNTLYLQMNSLRDEDTAVYYCARDPITGTFAYWGQGTLVTVSS, Sequence ID 67(FEL_09E09_VH1)DVQLVESGGDLVKPGGSLRLTCVASGFTYSSYAMSWVRQAPGKGLQWVAYISSGGDYIYYADTVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCVRDPITGTFAYWGQGTLVTVSS, Sequence ID 69(FEL_09E09_VH2)DVQLVESGGNLVKPGGSLRLTCVASGFTFSSYAMSWVRQAPGKGLQWVAYISSGGDYIYYADTVKGRFTISKDNAKNTLYLQMNSLKTEDTATYYCARDPITGTFAYWGQGTLVTVSS, Sequence ID 75(CAN_10F07_VH1)EVQLVESGGDLVKPGGSLRLSCVASGFTFSSYAMSWVRQAPGKGLQWVAYISSGGDYFYYADTVKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCVRDPITGTFAYWGQGTLVTVSS, Sequence ID 79(CAN_10F07_VH2)EVQLVESGGDLVKPAGSLTLSCLASGFTFSSYAMSWVRQTPEKGLQWVAYISSGGDYFYYADTVKGRFTISRDNAKNTLYLQMNSLRDEDTAVYYCARDPITGTFAYWGQGTLVTVSS, Sequence ID 83(FEL_10F07_VH1)DVQLVESGGDLVKPGGSLRLTCVASGFTYSSYAMSWVRQAPGKGLQWVAYISSGGDYFYYADTVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCVRDPITGTFAYWGQGTLVTVSS, Sequence ID 87(FEL_10F07_VH2)DVQLVESGGDLVKPGGSLRLTCVASGFTFSSYAMSWVRQAPGKGLQWVAYISSGGDYFYYADTVKGRFTISRDDAKNTLYLQMSSLKTEDTATYYCTGDPITGTFAYWGQGTLVTVSS, Sequence ID 91(CAN_19F07_VH1)EVQLVESGGDLVKPGGSLRLSCVASGFTFSDYYMAWVRQAPGKGLQWVANINYDGSSTYYLDSLKSRFTISRDNAKNTLYLQMNSLRAEDTAMYYCVRGLTWDFDVWGQGTLVTVSS, Sequence ID 95(CAN_19F07_VH2)EVQLVESGGDLVKPAGSLTLSCLASGFTFSDYYMAWVRQTPEKGLQWVANINYDGSSTYYLDSLKSRFTISRDNAKNTLYLQMNSLRDEDTAVYYCARGLTWDFDVWGQGTLVTVSS, Sequence ID 99(FEL_19F07_VH1)DVQLVESGGDLVKPGGSLRLTCVASGFTYSDYYMAWVRQAPGKGLQWVANINYDGSSTYYLDSLKSRFTISRDNAKNTLYLQMNSLKTEDTATYYCVRGLTWDFDVWGQGTLVTVSS, Sequence ID 103(FEL_19F07_VH2)DVQLVESGGNLVKPGGSLRLTCVASGFTFSDYYMAWVRQAPGKGLQWVANINYDGSSTYYLDSLKSRFTISRDNAKNTLYLQMNSLKTEDTATYYCARGLTWDFDVWGQGTLVTVSS, Sequence ID 127. (CAN_14C04_VH1) EVQLVESGGDLVKPGGSLRLSCVASGFTFSNYWMNWVRQAPGKGLQWVAQIYPGHVNTNYNGNFKDRFTISRDNARNTVYLQMNSLRAEDTAVYYCARSADNSGFVLFAYWGQGTLVTVSS, or Sequence ID 129. (CAN_14C04_VH2) Variable heavy chains including EVQLVESGGDLVKPGGSLRLSCVASGFTFSNYWMNWVRQSPGKGLQWVAQIYPGHVNTNYNGNFKDRFTISRDNAKNTLYLQMNSLRAEDTAVYFCARSADNSGFVLFAYWGQGTLVTVSS, and (b) Variable light chain, Sequence ID 33(MU_02D09_VL)DIVLTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQTSHESPRLLITYASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPLTFGAGTKLELK, Sequence ID 37(MU_09E09_VL)DLQMTQTTSSLSASLGDRVTISCRASQDINNYLNWYQQKPDGTVKLLIYYTSTLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIK, Sequence ID 41(MU_10F07_VL)DIQMTQTTSSLSASLGDRVTISCRASQDITNYLNWYQQKPDGTVKLLIYYTSTLHSGVPSRFSGSGSGTDFSLTISNLEQEDIATYFCQQGHMLPWTFGGGTKLEIK, Sequence ID 45(MU_14C04_VL)DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIFLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLELK, Sequence ID 49(MU_19F07_VL)DIVMTQSHKFMSPSVGDRVSITCKASQDVDTAVAWYQQKPGQSPKLLIYLASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSRFPLTFGAGTKLELK, Sequence ID 55(FEL_02D09_VL1)EIQMTQSPSSLSASPGDRVTITCRASQSISNNLHWYQQKPGKVPKLLIYYASQSISGVPSRFSGSGSGTDFTLTISSLEPEDAATYYCQQSNSWPLTFGQGT, Sequence ID 57(FEL_02D09_VL2)DIVMTQTPLSLSVTPGESASISCRASQSISNNLHWYLQKSGQSPRRLIYYASQSISGVPDRFSGSGSGTDFTLRISRVEADDVGVYYCQQSNSWPLTFGQGT, Sequence ID 63(CAN_09E09_VL1)EIVMTQSPASLSLSQEEKVTITCRASQDINNYLNWYQQKPGQAPKLLIYYTSTLHSGVPSRFSGSGSGTDFSFTISSLEPEDVAVYYCQQGNTLPWTFGQGT, Sequence ID 65(CAN_09E09_VL2)DIVLTQPTSVSGSLGQRVTISCRASQDINNYLNWYQQLPGKAPKLLVYYTSTLHSGVPDRFSGSNSGSSATLTITGLQAEDEADYYCQQGNTLPWTFGQGT, Sequence ID 71(FEL_09E09_VL1)EIQMTQSPSSLSASPGDRVTITCRASQDINNYLNWYQQKPGKVPKLLIYYTSTLHSGVPSRFSGSGSGTDFTLTISSLEPEDAATYYCQQGNTLPWTFGQGT, Sequence ID 73(FEL_09E09_VL2)DITMTQSPGSLAGSPGQQVTMNCRASQDINNYLNWYQQKPGQHPKLLIYYTSTLHSGVPDRFSGSGSGTDFTLTISNLQAEDVASYYCQQGNTLPWTFGQGT, Sequence ID 77(CAN_10F07_VL1)EIVMTQSPASLSLSQEEKVTITCRASQDITNYLNWYQQKPGQAPKLLIYYTSTLHSGVPSRFSGSGSGTDFSFTISSLEPEDVAVYYCQQGHMLPWTFGQGT, Sequence ID 81(CAN_10F07_VL2)DIVLTQPTSVSGSLGQRVTISCRASQDITNYLNWYQQLPGKAPKLLVYYTSTLHSGVPDRFSGSNSGSSATLTITGLQAEDEADYYCQQGHMLPWTFGQGT, Sequence ID 85(FEL_10F07_VL1)EIQMTQSPSSLSASPGDRVTITCRASQDITNYLNWYQQKPGKVPKLLIYYTSTLHSGVPSRFSGSGSGTDFTLTISSLEPEDAATYYCQQGHMLPWTFGQGT, Sequence ID 89(FEL_10F07_VL2)DITMTQSPGSLAGSPGQQVTMNCRASQDITNYLNWYQQKPGQHPKLLIYYTSTLHSGVPDRFSGSGSGTDFTLTISNLQAEDVASYYCQQGHMLPWTFGQGT, Sequence ID 93(CAN_19F07_VL1)EIVMTQSPASLSLSQEEKVTITCKASQDVDTAVAWYQQKPGQAPKLLIYLASTRHTGVPSRFSGSGSGTDFSFTISSLEPEDVAVYYCQQYSRFPLTFGQGT, Sequence ID 97(CAN_19F07_VL2)DIVMTQTPLSLSVSPGETASISCKASQDVDTAVAWFRQKPGQSPQRLIYLASTRHTGVPDRFSGSGSGTDFTLRISRVEADDTGVYYCQQYSRFPLTFGQGT, Sequence ID 101(FEL_19F07_VL1)EIQMTQSPSSLSASPGDRVTITCKASQDVDTAVAWYQQKPGKVPKLLIYLASTRHTGVPSRFSGSGSGTDFTLTISSLEPEDAATYYCQQYSRFPLTFGQGT, Sequence ID 105(FEL_19F07_VL2)DITMTQSPGSLAGSPGQQVTMNCKASQDVDTAVAWYQQKPGQHPKLLIYLASTRHTGVPDRFSGSGSGTDFTLTISNLQAEDVASYYCQQYSRFPLTFGQGT, Sequence ID 131. (CAN_14C04_VL1) EIVMTQSPASLSLSQEEKVTITCRASKSVSTSGYSYLHWYQQKPGQAPKLLIYLASNLESGVPSRFSGSGSGTDFSFTISSLEPEDVAVYYCQHSRELPLTFGQGT, or Sequence ID 133. (CAN_14C04_VL2) It includes at least one of a variable heavy chain and a variable light chain, such as a variable light chain containing DIVMTQTPLSLSVSPGETASISCRASKSVSTSGYSYLHWYLQKPGQSPQLLIYLASNLESGVSKRFSGSGSGTDFTLRISRVEADDTGIYYCQHSRELPLTFGQGT.

[0097] In another embodiment, the present invention provides a host cell that produces the above-mentioned antibody.

[0098] The present invention also includes, to the extent of its scope, nucleotide sequences encoding the variable regions of the light and heavy chains of the anti-OSMR beta antibody of the present invention. The scope of the present invention also includes any nucleotide sequences encoding the amino acid sequences of the 02D09, 09E09, 10F07, 14C04, and 19F07 antibodies, or their antigen-binding moieties.

[0099] The present invention also relates to isolated nucleic acids, comprising the following combinations of variable heavy chain complementarity-determining region (CDR) sequences: 1) 02D09: Variable heavy chain (VH)-CDR1 (DYGMH) of SEQ ID NO: 1, VH-CDR2 (YISSGSRAVFFADTVKG) of SEQ ID NO: 2, and VH-CDR3 (DRYDGRGFAY) of SEQ ID NO: 3, 2) 09E09: VH-CDR1 (SYAMS) with sequence number 7, VH-CDR2 (YISSGGDYIYYADTVKG) with sequence number 8, and VH-CDR3 (DPITGTFAY) with sequence number 9, 3) 10F07: VH-CDR1 (SYAMS) with sequence number 13, VH-CDR2 (YISSGGDYFYYADTVKG) with sequence number 14, and VH-CDR3 (DPITGTFAY) with sequence number 15, 4) 14C04: VH-CDR1 (NYWMN) of sequence number 19, VH-CDR2 (QIYPGHVNTNYNGNFKD) of sequence number 20, and VH-CDR3 (SADNSGFVLFAY) of sequence number 21, 5) 19F07: VH-CDR1 (DYYMA) of sequence number 25, VH-CDR2 (NINYDGSSTYYLDSLKS) of sequence number 26, and VH-CDR3 (GLTWDFDV) of sequence number 27, or 6) Provide an isolated nucleic acid comprising a nucleic acid sequence encoding at least one of the CDR variants 1, 2, 3, 4, or 5.

[0100] In one embodiment, the above-mentioned nucleic acid is a combination of the following variable light chain CDR sequences: 1) 02D09: Variable light chain (VL)-CDR1 (RASQSISNNLH) of SEQ ID NO: 4, VL-CDR2 (YASQSIS) of SEQ ID NO: 5, and VL-CDR3 (QQSNSWPLT) of SEQ ID NO: 6 2) 09E09: VL-CDR1 (RASQDINNYLN) of sequence number 10, VL-CDR2 (YTSTLHS) of sequence number 11, and VL-CDR3 (QQGNTLPWT) of sequence number 12, 3) 10F07: VL-CDR1 (RASQDITNYLN) with sequence number 16, VL-CDR2 (YTSTLHS) with sequence number 17, and VL-CDR3 (QQGHMLPWT) with sequence number 18, 4) 14C04: VL-CDR1 (RASKSVSTSGYSYLH) of sequence number 22, VL-CDR2 (LASNLES) of sequence number 23, and VL-CDR3 (QHSRELPLT) of sequence number 24, 5) 19F07: VL-CDR1 (KASQDVDTAVA) with sequence number 28, VL-CDR2 (LASTRHT) with sequence number 29, and VL-CDR3 (QQYSRFPLT) with sequence number 30, or 6) Further comprising a nucleic acid sequence encoding at least one of the CDR variants of 1, 2, 3, 4, or 5.

[0101] In another embodiment, the nucleic acid sequence according to the present invention is a combination of the following variable light chain CDR sequences: 1) 02D09: Variable light chain (VL)-CDR1 (RASQSISNNLH) of SEQ ID NO: 4, VL-CDR2 (YASQSIS) of SEQ ID NO: 5, and VL-CDR3 (QQSNSWPLT) of SEQ ID NO: 6 2) 09E09: VL-CDR1 (RASQDINNYLN) of sequence number 10, VL-CDR2 (YTSTLHS) of sequence number 11, and VL-CDR3 (QQGNTLPWT) of sequence number 12, 3) 10F07: VL-CDR1 (RASQDITNYLN) with sequence number 16, VL-CDR2 (YTSTLHS) with sequence number 17, and VL-CDR3 (QQGHMLPWT) with sequence number 18, 4) 14C04: VL-CDR1 (RASKSVSTSGYSYLH) of sequence number 22, VL-CDR2 (LASNLES) of sequence number 23, and VL-CDR3 (QHSRELPLT) of sequence number 24, 5) 19F07: VL-CDR1 (KASQDVDTAVA) with sequence number 28, VL-CDR2 (LASTRHT) with sequence number 29, and VL-CDR3 (QQYSRFPLT) with sequence number 30, or 6) Code at least one of the CDR variants 1, 2, 3, 4, or 5.

[0102] The present invention further provides vectors containing the nucleic acids described above. A single expression vector may contain a nucleic acid sequence encoding a variable light chain CDR sequence and a nucleic acid sequence encoding a variable heavy chain CDR sequence. Alternatively, the nucleic acid sequence encoding the variable light chain CDR sequence may be contained in one vector, while the nucleic acid sequence encoding the variable heavy chain CDR sequence may be contained in separate vectors.

[0103] Because the genetic code is degenerate, more than one codon can be used to code for a particular amino acid. Using the genetic code, one or more different nucleotide sequences can be identified, each of which may be capable of coding for an amino acid. The actual probability that a particular oligonucleotide constitutes an actual XXX coding sequence can be estimated by considering unusual base pairing relationships and the frequency with which a particular codon is actually used (to code for a particular amino acid) in eukaryotic or prokaryotic cells expressing anti-OSMR beta antibodies or parts thereof. Such “codon usage rules” are disclosed by Lathe, et al., 183 J. Molec. Biol. 1-12 (1985). Using Lathe’s “codon usage rules,” it is possible to identify a single nucleotide sequence, or a set of nucleotide sequences, that contains the theoretically “most likely” nucleotide sequence capable of coding for an anti-OSMR beta sequence.

[0104] Furthermore, antibody coding regions for use in the present invention may also be provided by modifying existing antibody genes using standard molecular biology techniques that result in variants (agonists) of the antibody and antigen-binding portions described herein. Such variants include, but are not limited to, deletions, additions, and substitutions in the amino acid sequence of anti-OSMR beta antibodies or their antigen-binding portions.

[0105] For example, one class of substitutions is conserved amino acid substitutions. Such substitutions replace a given amino acid in an anti-OSMR beta antibody sequence with another amino acid that has similar characteristics. Typical examples of conserved substitutions include the substitution of aliphatic amino acids Ala, Val, Leu, and Ile with each other, the exchange of hydroxyl residues Ser and Thr, the exchange of acidic residues Asp and Glu, the substitution of amide residues Asn and Gln, the exchange of basic residues Lys and Arg, and the substitution of aromatic residues Phe and Tyr. Guidance on which amino acid changes are likely to be phenotypic silent can be found in Bowie et al., 247 Science 1306-10 (1990).

[0106] A variant or agonist anti-OSMR beta antibody or its antigen-binding moiety may be fully functional or may lack function in one or more activities. Fully functional variants typically contain only conserved mutations or mutations in non-essential residues or regions. Functional variants may also contain similar amino acid substitutions that do not alter function or alter it only slightly. Alternatively, such substitutions may have some beneficial or detrimental effects. Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or tip breaks, or substitutions, insertions, inversions, or deletions in essential residues or regions.

[0107] The amino acids essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis. Cunningham et al., 244 Science 1081-85 (1989). The latter procedure involves introducing a single alanine mutation into all residues in the molecule. The resulting mutant molecule is then tested for biological activity, such as epitope binding or in vitro ADCC activity. Sites crucial for ligand-receptor binding can also be determined by structural analysis, such as crystallography, nuclear magnetic resonance, or photoaffinity labeling. Smith et al., 224 J.Mol.Biol.899-904 (1992), de Vos et al., 255 Science 306-12 (1992).

[0108] Furthermore, polypeptides often contain amino acids other than the 20 "naturally occurring" amino acids. Moreover, many amino acids, including terminal amino acids, can be modified by natural processes such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent bonding of flavins, covalent bonding of heme moieties, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, covalent bonding of phosphotidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking, cystine formation, pyroglutamate formation, formylation, gammacarboxylation, glycosylation, GPI anchor formation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolysis, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation, and other transfer-RNA mediated additions of amino acids to proteins, as well as ubiquitination.

[0109] Such modifications are well known to those skilled in the art and are described in detail in the scientific literature. Several particularly common modifications, such as glycosylation, lipid attachment, sulfated, gamma-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues, are described in the most basic texts, for example, *Proteins--Structure and Molecular Properties* (2nd ed., TECreighton, WH Freeman & Co., NY, 1993). Numerous detailed reviews on this subject are available, including *Wold, Posttranslational Covalent Modification of Proteins*, 1-12 (Johnson, ed., Academic Press, NY, 1983), *Seifter et al. 182 Meth. Enzymol. 626-46* (1990), and *Rattan et al. 663 Ann. NY Acad. Sci. 48-62* (1992).

[0110] Therefore, the antibodies and their antigen-binding moieties of the present invention also include derivatives or analogs in which the substituted amino acid residues are not encoded by the gene code.

[0111] Similarly, additions and substitutions in the amino acid sequence, as well as the mutations and modifications described above, may be equally applicable to the amino acid sequence of the OSM beta antigen and / or its epitope, and are therefore encompassed by the present invention. As mentioned above, the gene encoding the monoclonal antibody according to the present invention is specifically effective for the recognition of OSM beta.

[0112] antibody derivative The scope of this invention includes antibody derivatives. “Derivatives” of antibodies typically contain additional chemical portions that are not part of the protein. Covalent modifications of proteins are included within the scope of this invention. Such modifications can be introduced into a molecule by reacting a targeted amino acid residue of an antibody with an organic derivatizing agent capable of reacting with a selected side chain or terminal residue. For example, derivatization using bifunctional agents well known in the art is useful for crosslinking antibodies or fragments to a water-insoluble support matrix or other macromolecule carrier.

[0113] The derivatives also include radiolabeled monoclonal antibodies that are labeled. For example, radioactive iodine ( 125 I, 131 I), carbon ( 14 C), sulfur ( 35 S), Indium ( 111 In), tritium ( 3 H) and the like; conjugates of monoclonal antibodies with biotin or avidin, enzymes such as horseradish peroxidase, alkaline phosphatase, beta-D-galactosidase, glucose oxidase, glucoamylase, carboxylic acid anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, or glucose-6-phosphate dehydrogenase; and further, conjugates of monoclonal antibodies with bioluminescent agents (such as luciferase), chemiluminescent agents (such as acridine ester), or fluorescent agents (such as phycovir protein).

[0114] Another derivative bifunctional antibody of the present invention is a bispecific antibody produced by combining portions of two separate antibodies that recognize two different antigenic groups. This can be achieved by crosslinking or recombinant techniques. In addition, the portion can be attached to the antibody or a portion thereof to increase the in vivo half-life (for example, by extending the time to clearance from the bloodstream). Such a technique is, for example, the attachment of a PEG portion (also known as pegylation), which is well known in the art. See U.S. Patent Application Publication No. 2003 / 0031671.

[0115] Recombinant expression of antibodies In some embodiments, the nucleic acid encoding the subject monoclonal antibody is directly introduced into host cells, and the cells are incubated under conditions sufficient to induce the expression of the encoded antibody. After the nucleic acid of the subject is introduced into the cells, the cells are incubated for a period of about 1 to 24 hours, typically at 37°C and possibly under selective conditions, to allow antibody expression. In one embodiment, the antibody is secreted into the supernatant of the culture medium in which the cells are growing.

[0116] Conventionally, monoclonal antibodies have been produced as innate molecules in mouse hybridoma strains. In addition to that technique, the present invention provides recombinant DNA expression of monoclonal antibodies. This enables the production of canine and feline-derived antibodies in selected host species, as well as the generation of spectra for antibody derivatives and fusion proteins.

[0117] The present invention's at least one anti-OSMR beta antibody, or a nucleic acid sequence encoding its antigen-binding region, can be recombined with vector DNA according to conventional techniques including blunt or protruding ends for ligation, restriction enzyme digestion to provide suitable ends, proper packing of sticky ends, alkaline phosphatase treatment to avoid undesirable binding, and ligation with a suitable ligase. Techniques for such operations are disclosed, for example, by Maniatis et al., MOLECULAR CLONING, LAB. MANUAL, (Cold Spring Harbor Lab. Press, NY, 1982 and 1989), and Ausubel et al. 1993 (above), and can be used to construct a monoclonal antibody molecule or a nucleic acid sequence encoding its antigen-binding region.

[0118] Nucleic acid molecules such as DNA contain nucleotide sequences that provide transcriptional and translational regulatory information, and if such sequences are "operably linked" to nucleotide sequences that encode polypeptides, then the polypeptide is said to be "expressible." An operable linkage is a linkage in which the regulatory DNA sequence and the DNA sequence to be expressed are connected in a manner that enables gene expression as an anti-OSMR beta polypeptide or antibody portion in a recoverable amount. The precise properties of the regulatory region required for gene expression can vary from organism to organism, as is well known in similar technologies. See, for example, Sambrook et al., 2001 (above) and Ausubel et al., 1993 (above).

[0119] Accordingly, the present invention encompasses the expression of anti-OSMR beta antibodies or their antigen-binding moieties in either prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including bacteria, yeast, insects, fungi, birds, and mammalian cells, in vivo or in situ, or in host cells of mammalian, insect, bird, or yeast origin. Mammalian cells or tissues may be of human, primate, hamster, rabbit, rodent, cattle, pig, sheep, horse, goat, dog, or cat origin, but any other mammalian cells may be used.

[0120] In one embodiment, the nucleotide sequence to be introduced would be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors can be used for this purpose. See, for example, Ausubel et al., 1993 (above). Important factors in the selection of a particular plasmid or viral vector include the ease with which recipient cells containing the vector can be recognized and selected from recipient cells that do not contain the vector; the desired number of copies of the vector in a particular host; and whether it is desirable that the vector can be "shuttle" between host cells of different species.

[0121] Examples of known prokaryotic vectors in the art include plasmids that can replicate in E. coli (e.g., pBR322, ColE1, pSC101, pACYC184, pi.VX, etc.). Such plasmids are disclosed, for example, by Maniatis et al., 1989 (above) and Ausubel et al., 1993 (above). Examples of Bacillus plasmids include pC194, pC221, and pT127. Such plasmids are disclosed by Gryczan in THE MOLEC.BIO.OF THE BACILLI 307-329 (Academic Press, NY, 1982). Suitable Streptomyces plasmids include pIJ101 (Kendall et al., 169 J.Bacteriol. 4177-83 (1987)) and Streptomyces bacteriophages, such as phi.C31 (Chater et al., in SIXTH INT'L SYMPOSIUM ON ACTINOMYCETALES BIO. 45-54 (Akademiai Kaido, Budapest, Hungary 1986)). Pseudomonas plasmids are outlined in John et al, 8 Rev.Infect.Dis. 693-704 (1986), Izaki, 33 Jpn.J.Bacteriol. 729-42 (1978), and Ausubel et al., 1993 (above).

[0122] Alternatively, useful gene expression elements for expressing anti-OSMR beta antibodies encoding cDNA or their antigen-binding moieties include, but are not limited to, (a) viral transcription promoters and their enhancer elements, e.g., the early SV40 promoter (Okayama et al., 3 Mol. Cell. Biol. 280 (1983)), Rous sarcoma virus LTR (Gorman et al., 79 Proc. Natl. Acad. Sci., USA 6777 (1982)), and Moloney mouse leukemia virus LTR (Grosschedl et al., 41 Cell 885 (1985)), (b) splice regions and polyadenylation sites, e.g., those derived from the late SV40 region (Okayarea et al., 1983), and (c) polyadenylation sites, e.g., those in SV40 (Okayama et al., 1983).

[0123] The immunoglobulin cDNA gene can be expressed using the SV40 early promoter and its enhancer, the mouse immunoglobulin H chain promoter enhancer, the SV40 late region mRNA splicing, the rabbit S-globin intervention sequence, the immunoglobulin, the rabbit S-globin polyadenylation site, and the SV40 polyadenylation element as expression elements, as described by Weidle et al., 51Gene 21 (1987).

[0124] In immunoglobulin genes composed of some cDNA and some genomic DNA (Whittle et al., 1 Protein Engine. 499 (1987)), the transcription promoter may be human cytomegalovirus, the promoter enhancer may be cytomegalovirus and mouse / human immunoglobulin, and the mRNA splicing and polyadenylation regions may be native chromosomal immunoglobulin sequences.

[0125] In one embodiment, in the expression of a cDNA gene in rodent cells, the transcription promoter is a viral LTR sequence, the transcription promoter enhancer is either or both a mouse immunoglobulin heavy chain enhancer and a viral LTR enhancer, the splice region contains an intron of more than 31 bp, and the polyadenylation and transcription termination regions are derived from the native chromosome sequence corresponding to the synthesized immunoglobulin chain. In other embodiments, a cDNA sequence encoding another protein is combined with the expression elements listed above to achieve protein expression in mammalian cells.

[0126] Each fusion gene can be assembled into an expression vector or inserted into an expression vector. Recipient cells capable of expressing the chimeric immunoglobulin chain gene product are then transfected individually with a gene encoding an anti-OSMR beta immunoglobulin chain or a chimeric H or chimeric L chain, or co-transfected with both the chimeric H and chimeric L chain genes. The transfected recipient cells are cultured under conditions that enable the expression of the incorporated genes, and the expressed immunoglobulin chain or intact antibody or fragment is recovered from the culture.

[0127] In one embodiment, a fusion gene encoding anti-OSMR beta-immunoglobulin H and L chains, or chimeric H and L chains, or a portion thereof, is assembled in a separate expression vector and then used to co-transfect recipient cells. Alternatively, the fusion gene encoding the chimeric H and L chains may be assembled on the same expression vector.

[0128] For transfection of expression vectors and generation of chimeric antibodies, the recipient cell line may be myeloma cells. Myeloma cells can synthesize, assemble, and secrete immunoglobulins encoded by transfected immunoglobulin genes and possess a mechanism for immunoglobulin glycosylation. Myeloma cells can grow in culture or in the peritoneal cavity of mice, where secreted immunoglobulins can be obtained from ascites fluid. Other suitable recipient cells include lymphocytes such as human or non-human B lymphocytes, human or non-human hybridoma cells, or interspecies heterohybridoma cells.

[0129] Expression vectors containing the chimeric, canine or feline antibody constructs, or anti-OSMR beta-immunoglobulin gene constructs of the present invention can be introduced into suitable host cells by any of a variety of suitable means, including biochemical means such as transformation, transfection, conjugation, protoplast fusion, and calcium phosphate precipitation, as well as polycation application such as diethylaminoethyl (DEAE) dextran, and mechanical means such as electroporation, direct microinjection, and particle guns. Johnston et al., 240 Science 1538 (1988).

[0130] Yeast can offer a substantial advantage over bacteria in the production of immunoglobulin H and L chains. Yeast performs post-translational peptide modifications, including glycosylation. Several recombinant DNA strategies currently exist that utilize strong promoter sequences and high-copy-number plasmids that can be used to generate desired proteins in yeast. Yeast recognizes the leader sequence of a cloned mammalian gene product and secretes a peptide (i.e., a pre-peptide) that carries the leader sequence. Hitzman et al., 11th Int'l Conference on Yeast, Genetics & Molec. Biol. (Montpelier, France, 1982).

[0131] Yeast gene expression systems can be evaluated in the usual manner for the production, secretion, and stability levels of anti-OSMR beta antibody sequences, antibodies, and assembled mice, as well as chimeric, heterochimeric, canine or feline-derived antibodies, and their fragments and regions. One of a series of yeast gene expression systems incorporating promoters and termination elements from actively expressed genes encoding glycolytic enzymes, which are produced in large quantities when yeast is grown in glucose-rich media, may be utilized. Known glycolytic genes can also provide highly efficient transcriptional regulatory signals. For example, promoter and terminator signals from the phosphoglycerate kinase (PGK) gene may be utilized. Several approaches can be selected to evaluate the optimal expression plasmid for the expression of cloned immunoglobulin cDNA in yeast. See Vol. II DNA Cloning, 45-66, (Glover, ed., IRL Press, Oxford, UK 1985).

[0132] Bacterial strains may also be used as hosts for the production of antibody molecules or peptides described in this invention. Plasmid vectors containing replicons and control sequences derived from species compatible with the host cells are used in conjunction with these bacterial hosts. The vectors carry replication sites and specific genes capable of providing phenotypic selection in transformed cells. Several approaches can be taken to evaluate expression plasmids for the production of mouse, chimeric, heterochimeric, canine or feline-derived antibodies, fragments and regions, or antibody chains encoded by cloned immunoglobulin cDNA in bacteria (see Glover, 1985 (above), Ausubel, 1993 (above), Sambrook, 2001 (above), Colligan et al., eds., Current Protocols in Immunology, John Wiley & Sons, NY, NY (1994-2001), Colligan et al., eds., Current Protocols in Protein Science, John Wiley & Sons, NY, NY (1997-2001)).

[0133] Host mammalian cells can be grown in vitro or in vivo. Mammalian cells provide post-translational modifications to immunoglobulin protein molecules, including leader peptide removal, H and L chain folding and assembly, antibody molecule glycosylation, and secretion of functional antibody proteins.

[0134] In addition to the lymphoid cells mentioned above, mammalian cells that may be useful as hosts for antibody protein production include fibroblast-derived cells such as Vero (ATCC CRL81) or CHO-K1 (ATCC CRL61) cells.

[0135] Many vector systems are available for the expression of cloned anti-OSMR beta H and L chain genes in mammalian cells (see Glover, 1985 (above)). Different approaches may be followed to obtain a complete H2L2 antibody. It is possible to co-express the H and L chains in the same cells and achieve intracellular association and ligation of the H and L chains to the complete tetrameric H2L2 antibody and / or anti-OSMR beta fragment (e.g., its antigen-binding portion). Co-expression can be performed using either the same or different plasmids in the same host. The genes for both the H and L chains and / or the anti-OSMR beta fragment may be placed in the same plasmid, which can then be transfected into cells to directly select cells expressing both chains. Alternatively, cells can be first transfected with a plasmid encoding one chain, e.g., the L chain, and then the resulting cell line can be transfected with an H chain plasmid containing a second selectable marker. Cell lines producing anti-OSMR beta amino acid sequences and / or H2L2 molecules via either pathway were transfected with plasmids encoding further copies of the peptide, H, L, or H and L chains, combined with additional selectable markers, to generate cell lines with enhanced properties, such as increased production of assembled H2L2 antibody molecules or enhanced stability of the transfected cell lines.

[0136] Stable expression can be used for the long-term high-yield production of recombinant antibodies. For example, cell lines that stably express antibody molecules can be engineered. Rather than using expression vectors containing viral replications, host cells may be transformed with immunoglobulin expression cassettes and selectable markers. Following the introduction of exogenous DNA, engineered cells may be grown in concentrated medium for 1-2 days, then switched to selective medium. Selectable markers in recombinant plasmids confer resistance to selection, allowing cells to stably incorporate the plasmid into their chromosomes and grow, thereby forming a focus that can be cloned and expanded into cell lines. Such engineered cell lines may be particularly useful in screening and evaluating compounds / components that directly or indirectly interact with antibody molecules.

[0137] Once the antibody of the present invention is generated, it can be purified by any method known in the art for the purification of immunoglobulin molecules, for example, chromatography (e.g., ion exchange, affinity, particularly affinity for a specific antigen after protein A, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for the purification of proteins. In many embodiments, the antibody is secreted from cells into a culture medium and then collected from the culture medium.

[0138] Pharmaceutical uses The anti-OSMR beta antibody or its antigen-binding moiety of the present invention can be used to treat various conditions in companion animals, such as dogs and cats. These conditions include pruritus, allergic conditions, inflammatory conditions, fibrous conditions, and inflammation-related pain. Specific but non-limiting examples of these types of conditions are disclosed herein. It should be understood that in some cases, a particular disorder may be considered to fall into more than one category. For example, a particular allergic condition may also be considered an inflammatory condition. More specifically, the present invention further provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and an antibody or peptide according to the present invention as an active ingredient. The antibody may be a chimeric, heterochimeric, canine, or feline antibody according to the present invention. Intact immunoglobulins or their conjugated fragments (e.g., Fab) are also conceivable. The antibodies and pharmaceutical compositions of the present invention are useful for parenteral administration, such as subcutaneous, intramuscular, or intravenous administration.

[0139] The anti-OSMR beta antibody and / or its antigen-binding moiety of the present invention may be administered either as an individual therapeutic agent or in combination with other therapeutic agents. They may be administered alone, but generally, they are administered with a pharmaceutical carrier selected based on a chosen route of administration and standard pharmaceutical practice.

[0140] The antibodies disclosed herein may be administered orally or to the airway surface by any suitable means, including parenteral injection (such as intraperitoneal, subcutaneous, or intramuscular injection), or by topical administration of the antibodies (typically performed in a pharmaceutical formulation). Topical administration to the airway surface may be performed by intranasal administration (e.g., by using a dropper, cotton swab, or inhaler). Topical administration of antibodies to the airway surface may also be performed by inhalation, such as by creating breathable particles of a pharmaceutical formulation (including both solid and liquid particles) containing the antibody as an aerosol suspension, and then having the subject inhale the breathable particles. Methods and apparatus for administering breathable particles of pharmaceutical formulations are well known, and any conventional techniques may be used. Oral administration may be, for example, in the form of an ingestible liquid or solid formulation.

[0141] In some preferred embodiments, the antibody is administered by parenteral injection. For parenteral administration, the anti-OSMR beta antibody or its antigen-binding moiety may be formulated as a solution, suspension, emulsion, or lyophilized powder associated with a pharmaceutically acceptable parenteral vehicle. For example, the vehicle may be a solution of the antibody or its cocktail dissolved in an acceptable carrier such as an aqueous carrier, such as water, saline, Ringer's solution, dextrose solution, trehalose or sucrose solution, or 5% serum albumin, 0.4% saline, 0.3% glycine, etc. Liposomes such as non-volatile oils and non-aqueous vehicles may also be used. These solutions are sterile and generally free of particulate matter. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffers, and toxicity modifiers, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The antibody concentration in these formulations may vary widely, for example, from less than about 0.5% by weight, typically from at least about 1% by weight to a maximum of 15% or 20% by weight, and will be selected mainly based on fluid volume, viscosity, etc., according to the specific dosage mode selected. The vehicle or lyophilized powder may contain additives to maintain isotonicity (e.g., sodium chloride, mannitol) and additives to maintain chemical stability (e.g., buffers and preservatives). The formulations are sterilized by commonly used techniques.

[0142] Practical methods for preparing parenterally administrative compositions are known or obvious to those skilled in the art and are described in detail, for example, in REMINGTON'S PHARMA.SCI. (15th ed., Mack Pub.Co., Easton, Pa., 1980).

[0143] The antibodies of the present invention can be lyophilized for storage and reconstituted in a suitable carrier before use. This technique has been shown to be effective for conventional immunoglobulins. Any suitable lyophilization and reconstitution technique may be used. Those skilled in the art will understand that lyophilization and reconstitution may result in varying degrees of antibody activity loss, which may need to be compensated for by adjusting the usage level.

[0144] Compositions containing these antibodies or cocktails thereof may be administered for the prevention of recurrence and / or therapeutic treatment of pre-existing diseases. Suitable pharmaceutical carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in the art.

[0145] In therapeutic applications, the composition is administered to a subject already suffering from the disease in an amount sufficient to cure, or at least partially halt or alleviate, the disease and its complications. The amount sufficient to achieve this is defined as the "therapeutic effective dose" or "therapeutic effective amount." The amount effective for this use depends on the severity of the disease and the general state of the subject's own immune system, but is generally in the range of about 0.1 mg to about 10 mg of antibody per kg of body weight, preferably about 0.3 mg to about 5 mg of antibody per kg of body weight. Given the minimization of heterogeneous substances and the lower probability of "foreign substance" rejection achieved by the canine and feline antibodies of the present invention, it may be possible to administer a considerably excessive amount of these antibodies.

[0146] The dosage administered naturally varies depending on known factors, such as the pharmacodynamic characteristics of a particular drug and its method and route of administration; the recipient's age, health, and weight; the nature and severity of symptoms, the type of concurrent treatment, the frequency of treatment, and the desired effect.

[0147] As a non-limiting example, the treatment of IL-31-related or OSM-related conditions in dogs or cats may be provided as a bi-weekly or monthly dose of the anti-OSMR beta antibody of the present invention, within the dosage range described above.

[0148] Examples of antibodies for therapeutic use in canids or felines include high-affinity (which are also high-avidity) antibodies with potent in vivo anti-OSMR beta activity according to the present invention, as well as fragments, regions, and derivatives thereof.

[0149] The composition may be administered as a single or multiple doses at dose levels and in patterns selected by the treating veterinarian. In any case, the pharmaceutical formulation should provide a sufficient amount of the antibody of the present invention to effectively treat the subject.

[0150] Diagnostic use The present invention also provides the above-mentioned anti-OSMR beta antibody and its antigen-binding moiety for use in a diagnostic method for detecting OSMR beta in companion animals that are known to have or suspected to have OSM and / or IL-31-mediated conditions, such as pruritus, allergic conditions, inflammatory conditions, fibrous conditions, or inflammation-related pain.

[0151] The anti-OSMR beta antibody and its antigen-binding moiety of the present invention are useful for immunoassays to detect or quantify OSMR beta or anti-OSMR beta antibodies in a sample. An immunoassay for OSMR beta typically comprises incubating a clinical or biological sample in the presence of the detectably labeled high-affinity (or high-avidity) anti-OSMR beta antibody of the present invention, which can selectively bind to OSMR beta, and detecting the bound labeled antibody in the sample. Various clinical assay procedures are well known in the art. For example, see IMMUNOASSAYS FOR THE 80'S (edited by Voller et al., Univ. Park, 1981). Such samples include tissue biopsies, blood, serum, and fecal samples, or liquids collected from animal subjects and subjected to ELISA analysis as described below.

[0152] In some embodiments, the binding of an antigen to an antibody is detected without the use of a solid support. For example, the binding of an antigen to an antibody may be detected in liquid form.

[0153] In other embodiments, the anti-OSMR beta antibody may be immobilized on, for example, nitrocellulose, or another solid support capable of immobilizing cells, cell particles, or soluble proteins. The support can then be washed with a suitable buffer and treated with a detectably labeled OSMR beta-specific antibody. The solid support can then be washed again with the buffer to remove any unbound labeled antibody. The amount of bound label on the solid support can then be detected by known method steps.

[0154] "Solid support" or "carrier" refers to any support capable of binding to a peptide, antigen, or antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, polyvinylidene fluoride (PVDF), dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The properties of the carrier may be partially soluble or insoluble for the purposes of this invention. The support material may have substantially any possible structural configuration, as long as the coupled molecule can bind to an OSMR beta or anti-OSMR beta antibody. Therefore, the configuration of the support may be spherical, as in beads, or cylindrical, as on the inner surface of a test tube, or a rod-shaped outer surface. Alternatively, the surface may be flat, such as a sheet, culture dish, or test strip. For example, the support may include polystyrene beads. Those skilled in the art know of, or can confirm by routine experiments, many other suitable carriers for binding to antibodies, peptides, or antigens.

[0155] A well-known method step can determine the binding activity of a given lot of anti-OSMR beta antibody. Those skilled in the art can determine the operational and optimal assay conditions through routine experiments.

[0156] The detection of OSMR beta-specific antibodies can be achieved by linking them to an enzyme for use in an enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA). The linked enzyme reacts with the exposed substrate to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorescence quantification, or visual means. Enzymes that can be used to detectably label OSMR beta-specific antibodies according to the present invention include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

[0157] By radiolabeling OSMR beta-specific antibodies, it is possible to detect OSMR beta through the use of radioimmunoassay (RIA). See Work et al., LAB. TECHNIQUES & BIOCHEM. 1N MOLEC. Bio. (No. Holland Pub. Co., NY, 1978). Radioisotopes can be detected by means such as the use of a gamma counter or scintillation counter, or by autoradiography. Isotopes particularly useful for the purposes of this invention include: 3 H, 125 I, 131 I, 35 S, 14 C, and 125 I is one example.

[0158] It is also possible to label the OSMR beta-specific antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of an appropriate wavelength, then its presence can be detected due to the fluorescence. The most commonly used fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine.

[0159] The OSMR beta-specific antibody can also 125 be delectably labeled using a fluorescent-emitting metal such as Eu, or another of the lanthanide series. These metals can be connected to the OSMR beta-specific antibody using a metal chelate group such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). [[ID=?]] [[ID=?]]

[0160] [[ID=?]] The OSMR beta-specific antibody can also be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescently labeled antibody is then determined by detecting the presence of the luminescence that occurs during the course of the chemical reaction. Examples of useful chemiluminescent labeling compounds are luminol, isoluminol, seromatic acridinium ester, imidazole, acridinium salts, and oxalate esters. [[ID=?]] [[ID=?]]

[0161] [[ID=?]] Similarly, bioluminescent compounds can be used to label the OSMR beta-specific antibody, part, fragment, polypeptide, or derivative of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems where a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of the bioluminescent protein is determined by detecting the presence of the luminescence. Important bioluminescent compounds for the purpose of labeling are luciferin, luciferase, and aequorin. [[ID=?]] [[ID=?]]

[0162] [[ID=?]] Detection of OSMR beta-specific antibodies, moieties, fragments, polypeptides, or derivatives can be achieved, for example, by a scintillation counter if the detectable label is a radioactive gamma emitter, or by a fluorometer if the label is a fluorescent material. In the case of enzymatic labeling, detection can be achieved by a colorimetric method using the substrate against the enzyme. Detection can also be achieved by a visual comparison of the degree of enzymatic reaction of the substrate compared to a similarly prepared standard.

[0163] For the purposes of the present invention, OSMR beta detected by the above assay can be present in a biological sample. Any sample containing OSMR beta can be used. For example, the sample may be a biological fluid such as blood, serum, lymph, urine, feces, inflammatory exudate, cerebrospinal fluid, amniotic fluid, tissue extract, or homogenate. The present invention is not limited to assays using only these samples, however, in light of this specification, those skilled in the art can determine suitable conditions that enable the use of other samples.

[0164] In-situ detection can be achieved by taking a histological specimen from an animal subject and providing such specimen with the combination of labeled antibodies of the present invention. The antibody (or portion thereof) can be provided by applying or overlaying the labeled antibody (or portion thereof) onto the biological specimen. Through the use of such a procedure, it is possible to determine not only the presence of OSMR beta but also the distribution of OSMR beta in the tissue examined. Using the present invention, those skilled in the art will readily recognize that any of the various histological methods (e.g., staining procedures) can be modified to achieve such in-situ detection.

[0165] The antibodies, fragments, or derivatives of the present invention can be adapted for use in immunoassays also known as "two-site" or "sandwich" assays. In a typical immunoassay, a certain amount of unlabeled antibody (or antibody fragment) is bound to a solid support that is insoluble in the liquid being tested, and a certain amount of detectably labeled soluble antibody is added to enable the detection and / or quantification of a ternary complex formed between the solid antibody, the antigen, and the labeled antibody.

[0166] Antibodies may be used to quantitatively or qualitatively detect OSMR beta in a sample, or to detect the presence of cells expressing OSMR beta. This can be achieved by immunofluorescence techniques (see below) using fluorescently labeled antibodies in combination with fluorescence microscopy, flow cytometry, or fluorescence measurement detection. For diagnostic purposes, the antibody may be labeled or unlabeled. Unlabeled antibodies can be used in combination with other labeled antibodies (second antibodies) that are reactive with the antibody, such as antibodies specific to the constant region of canine or feline immunoglobulins. Alternatively, the antibody may be directly labeled. A variety of labels may be used, such as radionuclides, fluorine, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, and ligands (especially haptens). Numerous types of immunoassays, such as those discussed above, are available and well known to those skilled in the art.

[0167] In one embodiment, the diagnostic method for detecting OSMR beta is a lateral flow immunoassay test. This is also known as an immunochromatographic assay, Rapid ImmunoMigration (RIM®), or strip test. A lateral flow immunoassay is essentially an immunoassay adapted to operate along a single axis to fit the form of a test strip. Many variations of this technique have been developed into commercial products, but they all operate according to the same basic principles. A typical test strip consists of the following components: (1) Sample pad - an absorbent pad to which the test sample is applied; (2) Conjugate or reagent pad - which contains antibodies specific to the target analyte conjugated into colored particles (usually colloidal gold particles or latex microparticles); (3) Reaction membrane - typically a hydrophobic nitrocellulose cellulose acetate membrane in which anti-target analyte antibodies are immobilized within lines crossing the membrane as capture zones or test lines (a control zone containing antibodies specific to the conjugate antibody may also be present); and (4) Core or waste container - a further absorbent pad designed to aspirate the sample across the reaction membrane by capillary action and collect it. The components of the strip are usually fixed to an inert backing material and may be in a simple dipstick form, or may be contained within a plastic casing having a sample port and reaction windows indicating the capture zone and the control zone.

[0168] There are two main types of lateral flow immunoassays used in microbiological testing: the double antibody sandwich assay and the competitive assay. In the double antibody sandwich assay, the sample moves from the sample pad through the conjugate pad, where any present target analytes bind to the conjugate. The sample then continues to move across the membrane until it reaches the capture zone, where the target / conjugate complex binds to the immobilized antibody, creating a visible line on the membrane. The sample then moves further along the strip until it reaches the control zone, where any excess conjugate binds, creating a second visible line on the membrane. This control line indicates that the sample moved across the membrane as intended. Two distinct lines on the membrane indicate a positive result. A single line in the control zone indicates a negative result. The competitive assay differs from the double antibody sandwich assay in that the conjugate pad contains antibodies already bound to the target analyte or its analogue. If the target analyte is present in the sample, it does not bind to the conjugate and remains unlabeled. As the sample moves along the membrane and reaches the capture zone, any excess unlabeled analyte binds to the immobilized antibody, blocking the capture of the conjugate, resulting in no visible line being produced. The unbound conjugate then binds to the antibody in the control zone, producing a visible control line. A single control line on the membrane indicates a positive result. Two visible lines, one in the capture zone and one in the control zone, indicate a negative result. However, if there is no excess unlabeled target analyte, a weak line may be produced in the capture zone, resulting in an inconclusive result. Several variations of the lateral flow technique exist. The capture zone on the membrane may contain immobilized antigen or enzyme instead of antibody, depending on the target analyte. Multiple capture zones can also be applied to create multiplex tests. For example, commercially available test strips have been developed that can detect both EHEC Shiga toxin ST1 and ST2 separately in the same sample.

[0169] Importantly, the antibodies of the present invention may be useful in diagnosing pruritic conditions, allergic conditions, inflammatory disorders, fibrotic disorders, inflammation-related pain, or combinations thereof in dogs or cats. More specifically, the antibodies of the present invention may identify overexpression of OSMR beta in companion animals. Thus, the antibodies of the present invention may provide an important immunohistochemical tool.

[0170] The antibodies of the present invention may be used in antibody arrays that are highly suitable for measuring gene expression profiles.

[0171] kit The scope of the present invention also includes kits for carrying out the subject matter. The kit comprises at least one of the following: the antibody of the present invention, the nucleic acid encoding it, or cells containing it. In one embodiment, the antibody of the present invention may be provided in a container, usually in a lyophilized form. The antibody, which may or may not be conjugated with a label or toxin, is typically included in the kit with buffers, stabilizers, biocides, inactive proteins such as Tris, phosphates, carbonates, etc., and serum albumin, etc. Generally, these materials are present in less than 5% by weight based on the amount of active antibody, and usually in a total amount of at least about 0.001% by weight again based on the antibody concentration. Often, it is desirable to include an inactive extender or excipient for diluting the active ingredient, and the excipient may be present in about 1% to 99% by weight of the total composition. If a second antibody that can bind to the primary antibody is used in the assay, it is usually present in a separate vial. The second antibody is typically conjugated with a label and formulated in a manner similar to the antibody formulations described above. The kit also typically includes a set of instructions for use.

[0172] In one embodiment, the kit according to the present invention is a test strip kit (lateral flow immunoassay kit) useful for detecting canine or feline OSMR beta protein in a sample. Such a test strip may typically include a sample pad to which the test sample is applied; a conjugate or reagent pad containing antibodies specific to canine or feline OSMR beta, with the antibodies conjugated into colored particles (usually colloidal gold particles); a reaction membrane on which anti-OSMR beta antibodies are immobilized within lines crossing the membrane as a capture zone or test line (a control zone containing antibodies specific to the conjugate antibody may also be present); and a further absorbent pad designed to aspirate the sample across the reaction membrane by capillary action and collect it. The test strip kit will generally also include instructions for use.

[0173] Cell-based assay for evaluating the functional activity of OSMR beta inhibitor candidates in canids or felines. The present invention also provides a cell-based assay for evaluating the functional activity of a candidate canine or feline OSMR beta inhibitor. The method comprises providing cells that endogenously express a receptor for canine or feline OSM and respond thereto; incubating canine or feline OSM in the presence of a composition comprising the candidate OSMR beta inhibitor or a vehicle control; treating the cells with the incubated canine or feline OSM; measuring the direct phosphorylation of proteins induced by the canine or feline OSM treatment; and determining whether the composition comprising the candidate canine or feline OSMR beta inhibitor inhibited canine or feline OSM-induced direct protein phosphorylation compared to a vehicle control.

[0174] In one embodiment, the measurement step includes measuring the direct phosphorylation of the STAT protein. In a particular embodiment, the STAT protein is STAT3.

[0175] In another embodiment of the cell-based assay, the cells are monocytes and / or macrophages. For example, in one embodiment, the cells are canine DH82 cells. DH82 cells are a macrophage-monocyte cell line derived from dogs with malignant histiocytosis.

[0176] In one embodiment, an increase in signaling occurs due to direct protein phosphorylation induced by canine or feline OSM treatment. In another embodiment, a decrease in signaling occurs due to inhibition of direct protein phosphorylation induced by a candidate canine or feline OSMR beta inhibitor.

[0177] In one embodiment, the canine or feline OSMR beta inhibitor candidate is a recombinant canine or feline anti-OSMR beta antibody. In a particular embodiment, the recombinant canine or feline OSMR beta antibody is contained in the supernatant of a hybridoma culture. In other embodiments, the canine or feline OSMR beta inhibitor candidate is a small molecule pharmacochemical compound.

[0178] In one embodiment, the canine or feline OSM is co-incubated with a candidate canine or feline OSM beta inhibitor before treating the cells with the OSM. In another embodiment, the cells are pre-incubated with gamma interferon for a period sufficient to increase OSM beta receptor expression before treatment with canine or feline OSM. In one embodiment, the cells are pre-incubated with canine gamma interferon. In some embodiments, after pre-incubation with canine gamma interferon, the cells are subsequently subjected to serum starvation before treatment with canine or feline OSM.

[0179] In some embodiments, a candidate canine or feline OSMR beta inhibitor that inhibits more than 50% of direct protein phosphorylation, compared to vehicle control, is selected for further purification and / or characterization. In one embodiment, the method is, for example, via a cell-based assay for any such candidate that inhibits more than 50% of direct protein phosphorylation, compared to vehicle control, to determine the IC 50 value of a candidate canine or feline OSMR beta inhibitor.

[0180] In one embodiment, inhibition of canine or feline OSM-induced direct protein phosphorylation observed in the assay correlates with inhibition of IL-31-mediated or OSM-mediated conditions in dogs or cats. Such disorders include, but are not limited to, the IL-31-mediated or OSM-mediated pruritic disorders, allergic disorders, inflammatory disorders, and fibrotic disorders described herein, as well as OSM-mediated inflammatory pain, such as osteoarthritis pain.

[0181] In a particular embodiment, the invention provides a cell-based assay for evaluating the functional activity of a candidate canine or feline IL-31 inhibitor, comprising providing canine DH82 cells that endogenously express a receptor for canine or feline OSM and respond thereto, incubating canine or feline OSM in the presence of a composition comprising a candidate OSMR beta inhibitor or a vehicle control, treating the DH82 cells with the incubated canine or feline OSM, measuring the biological activity in the DH82 cells induced by the canine or feline OSM treatment, and determining whether a composition comprising a candidate canine or feline OSMR beta inhibitor inhibits canine or feline OSM-induced biological activity compared to the vehicle control. This cell-based assay is hereinafter referred to as the DH82 assay.

[0182] In one embodiment of the DH82 assay, the biological activity is direct protein phosphorylation induced by canine or feline OSM treatment. In one embodiment of the DH82 assay, the protein is a STAT protein, e.g., STAT3. In another embodiment of the DH82 assay, an increase in signal occurs due to direct protein phosphorylation induced by canine or feline OSM treatment. In yet another embodiment of the DH82 assay, a decrease in signal occurs due to inhibition of direct protein phosphorylation induced by a candidate canine or feline OSMR beta inhibitor.

[0183] In further embodiments of the DH82 assay, the canine or feline OSMR beta inhibitor candidate is a recombinant canine or feline anti-OSMR beta antibody. Such recombinant canine or feline anti-OSMR beta antibody may be contained, for example, in the supernatant of a hybridoma culture. In other embodiments of the DH82 assay, the canine or feline OSMR beta inhibitor candidate is a small molecule pharmacologic compound.

[0184] In another embodiment of the DH82 assay, the canine or feline OSM is co-incubated with a candidate canine or feline OSMR beta inhibitor before treating the DH82 cells with the canine or feline OSM. In a further embodiment of the DH82 assay, the DH82 cells are pre-incubated with gamma interferon, e.g., canine gamma interferon, for a period sufficient to increase OSMR beta receptor expression before treating the canine or feline OSM. In one embodiment, the DH82 cells are then subjected to serum starvation before treating the canine or feline OSM.

[0185] In a specific embodiment of the DH82 assay, a canine or feline OSMR beta inhibitor candidate that inhibits more than 50% of direct protein phosphorylation compared to a vehicle control is selected for further purification and / or characterization. In one embodiment, for example, the method may include determining the IC50 value of the canine or feline OSMR beta inhibitor candidate via a DH82 cell-based assay.

[0186] In one embodiment of the DH82 assay, the inhibition of canid or feline OSM-induced direct protein phosphorylation observed in the assay is IL-31-mediated in dogs or cats. Alternatively, it correlates with the inhibition of OSM-mediated conditions. Such disorders include, but are not limited to, IL-31-mediated or OSM-mediated pruritic disorders, allergic disorders, inflammatory disorders, and fibrotic disorders as described herein, as well as OSM-mediated inflammatory pain, such as osteoarthritis pain. Description of claims at the time of international application [Section 1] An isolated antibody, or its antigen-binding moiety, that specifically binds to the canid or feline oncostatin M receptor beta (OSMR-β), or both, wherein the antibody, or its antigen-binding moiety, antagonistizes IL-31-mediated signaling or OSM-mediated signaling, or both, in canid and / or feline cells. [Section 2] The antibody according to claim 1, or the antigen-binding moiety thereof, wherein the antibody antagonistizes both IL-31-mediated signaling and OSM-mediated signaling in canid and / or feline cells. [Section 3] 1) 02D09: Variable heavy chain (VH)-CDR1 (DYGMH) of SEQ ID NO: 1, VH-CDR2 (YISSGSRAVFFADTVKG) of SEQ ID NO: 2, VH-CDR3 (DRYDGRGFAY) of SEQ ID NO: 3, Variable light chain (VL)-CDR1 (RASQSISNNLH) of SEQ ID NO: 4, VL-CDR2 (YASQSIS) of SEQ ID NO: 5, and VL-CDR3 (QQSNSWPLT) of SEQ ID NO: 6, 2) 09E09: VH-CDR1 (SYAMS) of sequence number 7, VH-CDR2 (YISSGGDYIYYADTVKG) of sequence number 8, VH-CDR3 (DPITGTFAY) of sequence number 9, VL-CDR1 (RASQDINNYLN) of sequence number 10, VL-CDR2 (YTSTLHS) of sequence number 11, and VL-CDR3 (QQGNTLPWT) of sequence number 12, 3) 10F07: VH-CDR1 (SYAMS) with sequence number 13, VH-CDR2 (YISSGGDYFYYADTVKG) with sequence number 14, VH-CDR3 (DPITGTFAY) with sequence number 15, VL-CDR1 (RASQDITNYLN) with sequence number 16, VL-CDR2 (YTSTLHS) with sequence number 17, and VL-CDR3 (QQGHMLPWT) with sequence number 18, 4) 14C04: VH-CDR1 (NYWMN) of sequence number 19, VH-CDR2 (QIYPGHVNTNYNGNFKD) of sequence number 20, VH-CDR3 (SADNSGFVLFAY) of sequence number 21, VL-CDR1 (RASKSVSTSGYSYLH) of sequence number 22, VL-CDR2 (LASNLES) of sequence number 23, and VL-CDR3 (QHSRELPLT) of sequence number 24, 5) 19F07: VH-CDR1 (DYYMA) with sequence number 25, VH-CDR2 (NINYDGSSTYYLDSLKS) with sequence number 26, VH-CDR3 (GLTWDFDV) with sequence number 27, VL-CDR1 (KASQDVDTAVA) with sequence number 28, VL-CDR2 (LASTRHT) with sequence number 29, and VL-CDR3 (QQYSRFPLT) with sequence number 30, or 6) Complementarity Determination Region (CDR) variants of 1, 2, 3, 4, or 5 An antibody according to claim 1 or 2, or its antigen-binding moiety, comprising a combination of CDR sequences selected from the group consisting of the following. [Section 4] (c) Variable heavy chain, Sequence ID 31(MU_02D09_VH)EVQLVESGGGLVKPGGSLTLSCAASGFTFSDYGMHWLRQAPEKGLEWVAYISSGSRAVFFADTVKGRFTISRDNAKNTLFLQMTSLRSDDTAMYYCARDRYDGRGFAYWGQGTLVTVSA, Sequence ID 35(MU_09E09_VH)DVKLVESGEGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVAYISSGGDYIYYADTVKGRFTISRDNARNTLYLQMSSLKSEDTAMYYCTRDPITGTFAYWGQGTLVTVSA, Sequence ID 39(MU_10F07_VH)DVKLVESGEGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVTYISSGGDYFYYADTVKGRFTISRDNARNTLYLQMSSLKSEDTAMYYCTRDPITGTFAYWGQGTLVTVSA, Sequence ID 43 (MU_14C04_VH) EVQLQESGAELVKPGASVKISKASGYAFSNYWMNWMKQRPGKGLEWIGQIYPGHVNTNYNGNFKDKATLTADK SSSTAYMQLSSLTSEDSAVYFCARSADNSGFVLFAYWGQGTLVTVS, Sequence ID 47 (MU_19F07_VH) EVKLVESEGGLVQPGSSMKLSCTASGFTFSDYYMAWVRQVPEKGLEWVANINYDGSSTYYLDSLKSRFIISRDNAKNILYLQMSSLKSEDTATYYCARGLTWDFDVWGTGTTVTVSS, Sequence ID 51(FEL_02D09_VH1)DVQLVESGGDLVKPGGSLRLTCVASGFTYSDYGMHWVRQAPGKGLQWVAYISSGSRAVFFADTVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCVRDRYDGRGFAYWGQGTLVTVSS, Sequence ID 53(FEL_02D09_VH2)DVQLVESGGDLVKPGGSLRLTCVASGFTFSDYGMHWVRQAPGKGLQWVAYISSGSRAVFFADTVKGRFTISRDNAKNTLYLQMNGLRTEDTATYYCARDRYDGRGFAYWGQGTLVTVSS, Sequence ID 59(CAN_09E09_VH1)EVQLVESGGDLVKPGGSLRLSCVASGFTFSSYAMSWVRQAPGKGLQWVAYISSGGDYIYYADTVKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCVRDPITGTFAYWGQGTLVTVSS, Sequence ID 61(CAN_09E09_VH2)EVQLVESGGDLVKPAGSLTLSCLASGFTFSSYAMSWVRQTPEKGLQWVAYISSGGDYIYYADTVKGRFTISRDNAKNTLYLQMNSLRDEDTAVYYCARDPITGTFAYWGQGTLVTVSS, Sequence ID 67(FEL_09E09_VH1)DVQLVESGGDLVKPGGSLRLTCVASGFTYSSYAMSWVRQAPGKGLQWVAYISSGGDYIYYADTVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCVRDPITGTFAYWGQGTLVTVSS, Sequence ID 69(FEL_09E09_VH2)DVQLVESGGNLVKPGGSLRLTCVASGFTFSSYAMSWVRQAPGKGLQWVAYISSGGDYIYYADTVKGRFTISKDNAKNTLYLQMNSLKTEDTATYYCARDPITGTFAYWGQGTLVTVSS, Sequence ID 75(CAN_10F07_VH1)EVQLVESGGDLVKPGGSLRLSCVASGFTFSSYAMSWVRQAPGKGLQWVAYISSGGDYFYYADTVKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCVRDPITGTFAYWGQGTLVTVSS, Sequence ID 79(CAN_10F07_VH2)EVQLVESGGDLVKPAGSLTLSCLASGFTFSSYAMSWVRQTPEKGLQWVAYISSGGDYFYYADTVKGRFTISRDNAKNTLYLQMNSLRDEDTAVYYCARDPITGTFAYWGQGTLVTVSS, Sequence ID 83(FEL_10F07_VH1)DVQLVESGGDLVKPGGSLRLTCVASGFTYSSYAMSWVRQAPGKGLQWVAYISSGGDYFYYADTVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCVRDPITGTFAYWGQGTLVTVSS, Sequence ID 87(FEL_10F07_VH2)DVQLVESGGDLVKPGGSLR LTCVASGFTFSSYAMSWVRQAPGKGLQWVAYISSGGDYFYYADTVKGRFTISRDDAKNTLYLQMSSLKTEDTATYYCTGDPITGTFAYWGQGTLVTVSS, Sequence ID 91(CAN_19F07_VH1)EVQLVESGGDLVKPGGSLRLSCVASGFTFSDYYMAWVRQAPGKGLQWVANINYDGSSTYYLDSLKSRFTISRDNAKNTLYLQMNSLRAEDTAMYYCVRGLTWDFDVWGQGTLVTVSS, Sequence ID 95(CAN_19F07_VH2)EVQLVESGGDLVKPAGSLTLSCLASGFTFSDYYMAWVRQTPEKGLQWVANINYDGSSTYYLDSLKSRFTISRDNAKNTLYLQMNSLRDEDTAVYYCARGLTWDFDVWGQGTLVTVSS, Sequence ID 99(FEL_19F07_VH1)DVQLVESGGDLVKPGGSLRLTCVASGFTYSDYYMAWVRQAPGKGLQWVANINYDGSSTYYLDSLKSRFTISRDNAKNTLYLQMNSLKTEDTATYYCVRGLTWDFDVWGQGTLVTVSS, Sequence ID 103(FEL_19F07_VH2)DVQLVESGGNLVKPGGSLRLTCVASGFTFSDYYMAWVRQAPGKGLQWVANINYDGSSTYYLDSLKSRFTISRDNAKNTLYLQMNSLKTEDTATYYCARGLTWDFDVWGQGTLVTVSS, Sequence ID 127. (CAN_14C04_VH1) EVQLVESGGDLVKPGGSLRLSCVASGFTFSNYWMNWVRQAPGKGLQWVAQIYPGHVNTNYNGNFKDRFTISRDNARNTVYLQMNSLRAEDTAVYYCARSADNSGFVLFAYWGQGTLVTVSS, or Sequence ID 129. (CAN_14C04_VH2) Variable heavy chains including EVQLVESGGDLVKPGGSLRLSCVASGFTFSNYWMNWVRQSPGKGLQWVAQIYPGHVNTNYNGNFKDRFTISRDNAKNTLYLQMNSLRAEDTAVYFCARSADNSGFVLFAYWGQGTLVTVSS, and (d) Variable light chain, Sequence ID 33(MU_02D09_VL)DIVLTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQTSHESPRLLITYASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPLTFGAGTKLELK, Sequence ID 37(MU_09E09_VL)DLQMTQTTSSLSASLGDRVTISCRASQDINNYLNWYQQKPDGTVKLLIYYTSTLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIK, Sequence ID 41(MU_10F07_VL)DIQMTQTTSSLSASLGDRVTISCRASQDITNYLNWYQQKPDGTVKLLIYYTSTLHSGVPSRFSGSGSGTDFSLTISNLEQEDIATYFCQQGHMLPWTFGGGTKLEIK, Sequence ID 45(MU_14C04_VL)DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIFLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLELK, Sequence ID 49(MU_19F07_VL)DIVMTQSHKFMSPSVGDRVSITCKASQDVDTAVAWYQQKPGQSPKLLIYLASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSRFPLTFGAGTKLELK, Sequence ID 55(FEL_02D09_VL1)EIQMTQSPSSLSASPGDRVTITCRASQSISNNLHWYQQKPGKVPKLLIYYASQSISGVPSRFSGSGSGTDFTLTISSLEPEDAATYYCQQSNSWPLTFGQGT, Sequence ID 57(FEL_02D09_VL2)DIVMTQTPLSLSVTPGESASISCRASQSISNNLHWYLQKSGQSPRRLIYYASQSISGVPDRFSGSGSGTDFTLRISRVEADDVGVYYCQQSNSWPLTFGQGT, Sequence ID 63(CAN_09E09_VL1)EIVMTQSPASLSLSQEEKVTITCRASQDINNYLNWYQQKPGQAPKLLIYYTSTLHSGVPSRFSGSGSGTDFSFTISSLEPEDVAVYYCQQGNTLPWTFGQGT, Sequence ID 65(CAN_09E09_VL2)DIVLTQPTSVSGSLGQRVTISCRASQDINNYLNWYQQLPGKAPKLLVYYTSTLHSGVPDRFSGSNSGSSATLTITGLQAEDEADYYCQQGNTLPWTFGQGT, Sequence ID 71(FEL_09E09_VL1)EIQMTQSPSSLSASPGDRVTITCRASQDINNYLNWYQQKPGKVPKLLIYYTSTLHSGVPSRFSGSGSGTDFTLTISSLEPEDAATYYCQQGNTLPWTFGQGT, Sequence ID 73(FEL_09E09_VL2)DITMTQSPGSLAGSPGQQVTMNCRASQDINNYLNWYQQKPGQHPKLLIYYTSTLHSGVPDRFSGSGSGTDFTLTISNLQAEDVASYYCQQGNTLPWTFGQGT, Sequence ID 77(CAN_10F07_VL1)EIVMTQSPASLSLSQEEKVTITCRASQDITNYLNWYQQKPGQAPKLLIYYTSTLHSGVPSRFSGSGSGTDFSFTISSLEPEDVAVYYCQQGHMLPWTFGQGT, Sequence ID 81(CAN_10F07_VL2)DIVLTQPTSVSGSLGQRVTISCRASQDITNYLNWYQQLPGKAPKLLVYYTSTLHSGVPDRFSGSNSGSSATLTITGLQAEDEADYYCQQGHMLPWTFGQGT, Sequence ID 85(FEL_10F07_VL1)EIQMTQSPSSLSASPGDRVTITCRASQDITNYLNWYQQKPGKVPKLLIYYTSTLHSGVPSRFSGSGSGTDFTLTISSLEPEDAATYYCQQGHMLPWTFGQGT, Sequence ID 89(FEL_10F07_VL2)DITMTQSPGSLAGSPGQQVTMNCRASQDITNYLNWYQQKPGQHPKLLIYYTSTLHSGVPDRFSGSGSGTDFTLTISNLQAEDVASYYCQQGHMLPWTFGQGT, Sequence ID 93(CAN_19F07_VL1)EIVMTQSPASLSLSQEEKVTITCKASQDVDTAVAWYQQKPGQAPKLLIYLASTRHTGVPSRFSGSGSGTDFSFTISSLEPEDVAVYYCQQYSRFPLTFGQGT, Sequence ID 97(CAN_19F07_VL2)DIVMTQTPLSLSVSPGETASISCKASQDVDTAVAWFRQKPGQSPQRLIYLASTRHTGVPDRFSGSGSGTDFTLRISRVEADDTGVYYCQQYSRFPLTFGQGT, Sequence ID 101 (FEL_19F07_VL1)EIQMTQSPSSLSASPGDRVTITCKASQDVDTAVAWYQQKPGKVPKLLIYLASTRHTGV PSRFSGSGSGTDFTLTISSLEPEDAATYYCQQYSRFPLTFGQGT, Sequence ID 105(FEL_19F07_VL2)DITMTQSPGSLAGSPGQQVTMNCKASQDVDTAVAWYQQKPGQHPKLLIYLASTRHTGVPDRFSGSGSGTDFTLTISNLQAEDVASYYCQQYSRFPLTFGQGT, Sequence ID 131. (CAN_14C04_VL1) EIVMTQSPASLSLSQEEKVTITCRASKSVSTSGYSYLHWYQQKPGQAPKLLIYLASNLESGVPSRFSGSGSGTDFSFTISSLEPEDVAVYYCQHSRELPLTFGQGT, or Sequence ID 133. (CAN_14C04_VL2) The antibody according to any one of claims 1 to 3, comprising at least one from the group consisting of variable light chains, including DIVMTQTPLSLSVSPGETASISCRASKSVSTSGYSYLHWYLQKPGQSPQLLIYLASNLESGVSKRFSGSGSGTDFTLRISRVEADDTGIYYCQHSRELPLTFGQGT. [Section 5] The antibody according to any one of claims 1 to 4, wherein the antibody is a chimeric antibody. [Section 6] The antibody according to any one of claims 1 to 4, wherein the antibody is canine-like or feline-like. [Section 7] The antibody according to any one of claims 1 to 4, wherein the antibody inhibits or neutralizes IL-31-mediated or OSM-mediated pruritic or allergic conditions in dogs or cats. [Section 8] The antibody according to claim 7, wherein the IL-31-mediated or OSM-mediated pruritic condition is selected from the group consisting of atopic dermatitis, eczema, psoriasis, scleroderma, and pruritus. [Section 9] The antibody according to claim 8, wherein the IL-31-mediated or OSM-mediated allergic condition is selected from the group consisting of allergic dermatitis, summer eczema, urticaria, respiratory fatigue, inflammatory airway disease, recurrent airway obstruction, airway hypersensitivity, chronic obstructive pulmonary disease, and inflammatory processes resulting from autoimmunity. [Section 10] The antibody according to any one of claims 1 to 4, wherein the antibody inhibits IL-31-mediated or OSM-mediated fibrous or inflammatory disorders. [Section 11] The antibody according to claim 10, wherein the IL-31-mediated or OSM-mediated fibrosis is selected from the group consisting of renal fibrosis, pulmonary fibrosis, and cutaneous fibrosis. [Section 12] The antibody according to claim 10, wherein the IL-31-mediated or OSM-mediated inflammatory disorder is selected from the group consisting of autoimmune inflammatory processes, inflammation of the skin or joints of animals suffering from osteoarthritis, immune-mediated polyarthritis, chronic bronchitis, allergic asthma, atopic dermatitis, allergic dermatitis, suppurative traumatic dermatitis, atherosclerosis, and cardiovascular disease. [Section 13] The antibody according to any one of claims 1 to 4, wherein the antibody reduces IL-31-mediated or OSM-mediated inflammatory pain. [Section 14] The antibody according to claim 13, wherein the IL-31-mediated or OSM-mediated inflammatory pain is osteoarthritis pain. [Section 15] A veterinary composition comprising a therapeutically effective amount of the antibody described in any one of claims 1 to 4. [Section 16] A method for treating an IL-31-mediated or OSM-mediated disorder in a subject, comprising administering an antibody according to any one of claims 1 to 4 to the subject. [Section 17] The method according to claim 16, wherein the IL-31-mediated or OSM-mediated disorder is selected from the group consisting of pruritic conditions, allergic conditions, fibrotic disorders, inflammatory disorders, and inflammatory pain. [Section 18] The method according to claim 17, wherein the IL-31-mediated or OSM-mediated pruritic condition is selected from the group consisting of atopic dermatitis, eczema, psoriasis, scleroderma, and pruritus. [Section 19] The method according to claim 17, wherein the IL-31-mediated or OSM-mediated allergic condition is selected from the group consisting of allergic dermatitis, summer eczema, urticaria, respiratory fatigue, inflammatory airway disease, recurrent airway obstruction, airway hypersensitivity, chronic obstructive pulmonary disease, and inflammatory processes resulting from autoimmunity. [Section 20] The method according to claim 17, wherein the IL-31-mediated or OSM-mediated fibrosis is selected from the group consisting of renal fibrosis, pulmonary fibrosis, and cutaneous fibrosis. [Section 21] The method according to claim 17, wherein the IL-31-mediated or OSM-mediated inflammatory disorder is selected from the group consisting of autoimmune inflammatory processes, inflammation of the skin or joints of animals suffering from osteoarthritis, immune-mediated polyarthritis, chronic bronchitis, allergic asthma, atopic dermatitis, allergic dermatitis, suppurative traumatic dermatitis, atherosclerosis, and cardiovascular disease. [Section 22] The method according to claim 17, wherein the IL-31-mediated or OSM-mediated inflammatory pain is osteoarthritis pain. [Section 23] The method according to any one of claims 16 to 22, wherein the subject is a dog or a cat. [Section 24] A method for inhibiting IL-31 and / or OSM activity in dogs or cats, comprising administering an antibody according to any one of claims 1 to 4 to the dogs or cats. [Section 25] A method for detecting OSMR beta in a sample, a) Incubating a sample containing OSMR beta in the presence of the antibody described in any one of claims 1 to 4, b) A method comprising detecting the antibody that binds to OSMR beta in the sample. [Section 26] The method according to claim 25, wherein the antibody includes a label. [Section 27] The method according to claim 25 or 26, further comprising quantifying the OSMR beta in the sample. [Section 28] Host cells with the following combinations of complementarity-determining region (CDR) sequences: 1) 02D09: Variable heavy chain (VH)-CDR1 (DYGMH) of SEQ ID NO: 1, VH-CDR2 (YISSGSRAVFFADTVKG) of SEQ ID NO: 2, VH-CDR3 (DRYDGRGFAY) of SEQ ID NO: 3, Variable light chain (VL)-CDR1 (RASQS) of SEQ ID NO: 4 ISNNLH), VL-CDR2 (YASQSIS) of sequence number 5, and VL-CDR3 (QQSNSWPLT) of sequence number 6, 2) 09E09: VH-CDR1 (SYAMS) of sequence number 7, VH-CDR2 (YISSGGDYIYYADTVKG) of sequence number 8, VH-CDR3 (DPITGTFAY) of sequence number 9, VL-CDR1 (RASQDINNYLN) of sequence number 10, VL-CDR2 (YTSTLHS) of sequence number 11, and VL-CDR3 (QQGNTLPWT) of sequence number 12, 3) 10F07: VH-CDR1 (SYAMS) with sequence number 13, VH-CDR2 (YISSGGDYFYYADTVKG) with sequence number 14, VH-CDR3 (DPITGTFAY) with sequence number 15, VL-CDR1 (RASQDITNYLN) with sequence number 16, VL-CDR2 (YTSTLHS) with sequence number 17, and VL-CDR3 (QQGHMLPWT) with sequence number 18, 4) 14C04: VH-CDR1 (NYWMN) of sequence number 19, VH-CDR2 (QIYPGHVNTNYNGNFKD) of sequence number 20, VH-CDR3 (SADNSGFVLFAY) of sequence number 21, VL-CDR1 (RASKSVSTSGYSYLH) of sequence number 22, VL-CDR2 (LASNLES) of sequence number 23, and VL-CDR3 (QHSRELPLT) of sequence number 24, 5) 19F07: VH-CDR1 (DYYMA) with sequence number 25, VH-CDR2 (NINYDGSSTYYLDSLKS) with sequence number 26, VH-CDR3 (GLTWDFDV) with sequence number 27, VL-CDR1 (KASQDVDTAVA) with sequence number 28, VL-CDR2 (LASTRHT) with sequence number 29, and VL-CDR3 (QQYSRFPLT) with sequence number 30, or 6) A host cell that produces an isolated antibody or its antigen-binding moiety, comprising at least one of the CDR variants 1, 2, 3, 4, or 5. [Section 29] Isolated nucleic acids, with the following combinations of complementarity-determining region (CDR) sequences: 1) 02D09: Variable heavy chain (VH)-CDR1 (DYGMH) of SEQ ID NO: 1, VH-CDR2 (YISSGSRAVFFADTVKG) of SEQ ID NO: 2, and VH-CDR3 (DRYDGRGFAY) of SEQ ID NO: 3, 2) 09E09: VH-CDR1 (SYAMS) with sequence number 7, VH-CDR2 (YISSGGDYIYYADTVKG) with sequence number 8, and VH-CDR3 (DPITGTFAY) with sequence number 9, 3) 10F07: VH-CDR1 (SYAMS) with sequence number 13, VH-CDR2 (YISSGGDYFYYADTVKG) with sequence number 14, and VH-CDR3 (DPITGTFAY) with sequence number 15, 4) 14C04: VH-CDR1 (NYWMN) of sequence number 19, VH-CDR2 (QIYPGHVNTNYNGNFKD) of sequence number 20, and VH-CDR3 (SADNSGFVLFAY) of sequence number 21, 5) 19F07: VH-CDR1 (DYYMA) of sequence number 25, VH-CDR2 (NINYDGSSTYYLDSLKS) of sequence number 26, and VH-CDR3 (GLTWDFDV) of sequence number 27, or 6) An isolated nucleic acid comprising a nucleic acid sequence encoding at least one of the CDR variants 1, 2, 3, 4, or 5. [Section 30] The following CDR sequence combinations: 1) 02D09: Variable light chain (VL)-CDR1 (RASQSISNNLH) of SEQ ID NO: 4, VL-CDR2 (YASQSIS) of SEQ ID NO: 5, and VL-CDR3 (QQSNSWPLT) of SEQ ID NO: 6 2) 09E09: VL-CDR1 (RASQDINNYLN) of sequence number 10, VL-CDR2 (YTSTLHS) of sequence number 11, and VL of sequence number 12 -CDR3(QQGNTLPWT), 3) 10F07: VL-CDR1 (RASQDITNYLN) with sequence number 16, VL-CDR2 (YTSTLHS) with sequence number 17, and VL-CDR3 (QQGHMLPWT) with sequence number 18, 4) 14C04: VL-CDR1 (RASKSVSTSGYSYLH) of sequence number 22, VL-CDR2 (LASNLES) of sequence number 23, and VL-CDR3 (QHSRELPLT) of sequence number 24, 5) 19F07: VL-CDR1 (KASQDVDTAVA) with sequence number 28, VL-CDR2 (LASTRHT) with sequence number 29, and VL-CDR3 (QQYSRFPLT) with sequence number 30, or 6) The nucleic acid according to claim 29, further comprising a nucleic acid sequence encoding at least one of the CDR variants 1, 2, 3, 4, or 5. [Section 31] A nucleic acid sequence, which is a combination of the following CDR sequences: 1) 02D09: Variable light chain (VL)-CDR1 (RASQSISNNLH) of SEQ ID NO: 4, VL-CDR2 (YASQSIS) of SEQ ID NO: 5, and VL-CDR3 (QQSNSWPLT) of SEQ ID NO: 6 2) 09E09: VL-CDR1 (RASQDINNYLN) of sequence number 10, VL-CDR2 (YTSTLHS) of sequence number 11, and VL-CDR3 (QQGNTLPWT) of sequence number 12, 3) 10F07: VL-CDR1 (RASQDITNYLN) with sequence number 16, VL-CDR2 (YTSTLHS) with sequence number 17, and VL-CDR3 (QQGHMLPWT) with sequence number 18, 4) 14C04: VL-CDR1 (RASKSVSTSGYSYLH) of sequence number 22, VL-CDR2 (LASNLES) of sequence number 23, and VL-CDR3 (QHSRELPLT) of sequence number 24, 5) 19F07: VL-CDR1 (KASQDVDTAVA) with sequence number 28, VL-CDR2 (LASTRHT) with sequence number 29, and VL-CDR3 (QQYSRFPLT) with sequence number 30, or 6) A nucleic acid sequence encoding at least one of the CDR variants 1, 2, 3, 4, or 5. [Section 32] A vector comprising the nucleic acid according to any one of claims 29, 30, or 31. [Section 33] A method for producing an antibody, comprising: culturing the host cells described in claim 28 under conditions that result in the production of the antibody; and isolating the antibody from the host cells or the culture medium of the host cells.

[0187] The present invention is further described here by the following non-limiting embodiments. [Examples]

[0188] Example 1. Generating recombinant proteins for use in this research Recombinant proteins were generated for the purpose of producing antibodies and evaluating the affinity and efficacy of antibody candidates. By homology with human homologs, cytokine-binding, Ig-like, and fibronectin III domains were identified for canid and feline OSMRs. Synthetic DNA constructs were designed for optimal expression of canid (SEQ ID NO: 107, Canine_OSMR_hIgG1_Fc) (its corresponding nucleotide sequence is (SEQ ID NO: 108, Canine_OSMR_hIgG1_Fc)) and feline (SEQ ID NO: 112, Feline_OSMR_hIgG1_Fc) (its corresponding nucleotide sequence is (SEQ ID NO: 113, Feline_OSMR_hIgG1_Fc)) OSMR proteins as human IgG1 Fc fusions. Furthermore, a synthetic DNA construct was designed for optimal expression of the feline OSM gene (SEQ ID NO: 110, Feline_OSM_hIgG1_Fc) (its corresponding nucleotide sequence is (SEQ ID NO: 111, Feline_OSM_hIgG1_Fc)). Canine OSM was purchased from Kingfisher Biotech, Inc. (Saint Paul, MN), and its protein sequence is (SEQ ID NO: 109, Canine_OSM). All synthetic cassettes were cloned into pcDNA3.1 using standard molecular biological methods and expressed in one of two mammalian suspension cell lines: Freestyle 293F (human fetal kidney) cells or EXPICHO-S (Chinese hamster ovary) cells.

[0189] The suspended cells were maintained in Freestyle 293 expression medium (Gibco) at a concentration of 0.15 to approximately 2.5 × 10⁶ cells / ml. On the day of transfection, the cells were diluted to 1.0 × 10⁶ cells / ml and transfected with a mixture of plasmid DNA and FectoPRO (Polyplus Transfection) reagent as described in the FectoPro Protocol, according to condition C. After approximately 24 hours, deviating from the FectoPro protocol, a diet consisting of 20% w / v Freestyle 293 medium diluted with Lipton was added to each culture. After 7 days of incubation, the cultures were harvested and clarified. For the EXPICHO-S suspension, the cells were maintained in EXPICHO expression medium (Gibco) at a concentration of 0.14 to 8.0 × 10⁶ cells / ml. The cells were diluted according to the ExpiCHO protocol user manual on day 1 and the day of transfection. Diluted cells were transfected according to the protocol using reagents supplied from the ExpiFectamine CHO Transfection Kit (Gibco) under Max Titer conditions. After 12–14 days of incubation, the cultures were harvested and clarified.

[0190] For the purification of hexahistidine-tagged proteins, a conditioned medium was prepared with 500 mM sodium chloride, 5 mM imidazole, and pH 7.4. This medium was then loaded onto IMAC resin (either Ni Sepharose Excel (GE Healthcare) or HisPur Cobalt (Thermo Scientific)) that had been pre-equilibrated with Buffer A (5 mM imidazole, 20 mM sodium phosphate, 500 mM sodium chloride, pH 7.4). After loading, the IMAC resin was thoroughly washed with Buffer A, and then eluted in the same buffer while increasing the imidazole concentration from 5 to 500 mM. The fractions were evaluated by SDS-PAGE. A pool was prepared, and the imidazole was dialyzed to the final buffer. Fc fusion proteins and antibodies were purified using Protein A chromatography. The acclimatization medium was loaded onto MabSelect Sure LX (GE Healthcare) or AmMag Protein A Magnetic beads (Genscript), which had been pre-equilibrated with PBS. After sample loading, the resin was washed with PBS, and then with 20 mM sodium acetate (pH 5.5). When magnetic beads were used, 0.05% tween-20 was added to the equilibration wash buffer. Using either method, the sample was eluted from the column with 20 mM acetic acid (pH 3.5). After elution, a pool was prepared and neutralized by adding 1 M sodium acetate to 4%. Depending on the available volume and intended use, the sample was sometimes replaced with a final buffer (e.g., PBS or other). The final protein concentration was measured by absorbance at 280 nm or by BCA protein assay.

[0191] The synthesis and characterization of the canid IL-31 protein (SEQ ID NO: 123, Canine_IL31) (whose corresponding nucleotide sequence is (SEQ ID NO: 124, Canine_IL31)) was previously described (U.S. Patent No. 8,790,651 to Bammert, et al.). The synthesis and characterization of the feline IL-31 protein (SEQ ID NO: 125, Feline_IL31) (whose corresponding nucleotide sequence is (SEQ ID NO: 126, Feline_IL31)) was previously described (U.S. Patent Application No. 2019 / 0284272 to Bammert, et al.).

[0192] Example 2. Identification of mouse monoclonal antibodies that recognize canid and / or feline OSMR Female AJ and CD1 mice were immunized using a mixture of recombinant canine and feline OSMR proteins, represented by (SEQ ID NO: 107, Canine_OSMR_hIgG1_Fc) (whose corresponding nucleotide sequence is (SEQ ID NO: 108, Canine_OSMR_hIgG1_Fc)) and (SEQ ID NO: 112, Feline_OSMR_hIgG1_Fc) (whose corresponding nucleotide sequence is (SEQ ID NO: 113, Feline_OSMR_hIgG1_Fc)), for the purpose of generating monoclonal antibodies. Mice were immunized using a 28-day rapid immunization protocol (RIMMS) including a series of low-dose immunizations administered over two weeks. Serum antibody titers from immunized mice were determined using an enzyme-linked immunosorbent assay (ELISA). Canine or feline OSMR (50 ng / well) was immobilized on polystyrene microplates and used as a capture antigen. A separate ELISA was performed to determine the antibody response to unrelated human IgG Fc fusion proteins. Before the assay, each plate was blocked with casein, and serum from immunized mice was diluted with phosphate-buffered saline containing 0.05% tween-20 (PBST). The presence of anti-OSMR (or anti-human IgG Fc) antibodies was detected by anti-mouse HRP-labeled secondary antibody. A chromogenic substrate (SureBlue Reserve TMB 1-Component Microwell Peroxidase Substrate, KPL, Inc., Gaithersburg, MD) was added, and the mixture was incubated at room temperature (RT) for 10 minutes, after which the reaction was stopped by adding 100 μL of 0.1N HCl. The absorbance of each well was determined by optical density (OD) at 450 nm.

[0193] Test blood samples were collected on day 20, and antiserum samples were evaluated to determine if they had reached a fusion-ready titer, defined as OD > 0.1 above background in a 1:31K serum dilution. In addition, anti-OSMR specificity was assessed using a human IgG absorption assay. Serum samples were spiked with pooled / purified human IgG before being applied to ELISA plates. Antibodies recognizing the "irrelevant" human IgG Fc component of the fusion protein were absorbed from the system. Antibodies that were OSMR-specific bound to OSMR on the ELISA plate. Post-absorption anti-OSMR signaling was an indicator of target-specific response. After anti-human IgG absorption, a single responsive CD-1 mouse with high specific titers against both canine and feline OSMR was selected. This mouse received a pre-fusion boost, and donor splenocytes were used for fusion on day 28.

[0194] Hybridoma supernatants were screened by ELISA for conditions that bind to canid and / or feline OSMR proteins but not to unrelated human IgG Fc. Candidate mouse anti-OSMR hybridomas that selectively bound to canid and / or feline OSMR were further subcloned to generate hybridomas that produce homogeneous antibodies for sequencing of various heavy and light chains. Cells producing antibodies with these desired characteristics were selected for sequencing analysis of RNA transcripts of variable heavy (VH) and variable light (VL) IgG chains.

[0195] Example 3. A method for determining the affinity of anti-OSMR antibodies to OSMR using surface plasmon resonance. The affinity of candidate mAbs to canid, feline, and human (SEQ ID NO: 122, Human_OSMR) (R&D Systems, Minneapolis, MN) OSMR was determined using surface plasmon resonance (SPR) on a Biacore system (Biacore Life Sciences (GE Healthcare), Uppsala, Sweden). To avoid affinity differences associated with differential surface preparation that may occur when immobilizing antibodies to surfaces, OSMRs from each species were directly conjugated to individual surfaces. Immobilization was achieved by amine coupling of 5 μg / mL OSMR using N-hydroxysuccinimide (NHS) / 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry. The tips were quenched with ethanolamine, and the affinity with all candidate mAbs bound to the immobilized OSMR was evaluated. All curves were fitted to a 1:1 model. 1 × 10⁻⁶ -11 An affinity constant (KD) less than M(1E-11M) is below the detection limit of the instrument. The results of the affinity measurement are described herein.

[0196] Example 4. A method for determining the efficacy of anti-OSMR antibodies evaluated by inhibition of canine and feline IL-31-induced pSTAT3 signaling in canine and feline macrophage cells. To identify candidate inhibitors, antibodies were evaluated for their ability to affect IL-31-mediated STAT3 phosphorylation in either canine or feline cell-based assays. STAT3 phosphorylation was determined in canine DH-82 (ATCC® CRL-10389®) or feline Fcwf-4 macrophage-like cells (ATCC CRL-2787). DH82 and Fcwf-4 cells were primed with 10 ng / mL canine interferon-gamma (R&D Systems, Minneapolis, MN) for 24 hours or 125 ng / mL feline interferon-gamma (R&D Systems, Minneapolis, MN) for 96 hours, respectively, to increase receptor expression. Both cell types were serum-starved for 2 hours prior to IL-31 and mAb treatment. All candidate mAbs were evaluated for their ability to inhibit either 1 μg / mL canine or 42 ng / mL feline IL-31-induced STAT3 phosphorylation using two independent methods. Assays were also performed to demonstrate cross-reactivity between canine and feline cytokines, as well as cross-functionality of antibodies in inhibiting signaling in both species. To ensure complex formation, a 1-hour co-incubation of mAbs and IL-31 cytokines was completed before cell stimulation. IL-31 cell stimulation was performed for 5 minutes. STAT3 phosphorylation was measured using the AlphaLISA SureFire ULTRA® technique (Perkin Elmer, Waltham, MA). When antibody concentration and purity were unknown, hybridoma supernatant was qualitatively measured for its ability to inhibit STAT3 phosphorylation after 1-hour co-incubation with 1 μg / mL canine or 42 ng / mL feline IL-31. The potency of individual monoclonal antibodies, defined by their ability to inhibit IL-31-mediated STAT3 phosphorylation in these assays, was considered a primary selection criterion for further advancement of the chosen antibodies. The term potency refers to the IC calculated from these assays. 50This value refers to the antibody concentration at which IL-31-induced signaling is reduced to half of its maximum value. The increased potency described herein is IC 50 It correlates with a decrease in value.

[0197] Example 5. A method for determining the efficacy of anti-OSMR antibodies evaluated by inhibition of canine and feline OSM-induced pSTAT3 signaling in canine and feline macrophage cells. To identify candidate antibodies with inhibitory activity, antibodies were evaluated for their ability to affect OSM-mediated STAT3 phosphorylation in either canine or feline cell-based assays. STAT3 phosphorylation was determined in canine DH-82 (ATCC CRL-10389®) or feline Fcwf-4 macrophage-like cells (ATCC CRL-2787). To assess the dynamic range of these assays, dose-response curves using canine and feline OSM were evaluated for both cell types. Cells were starved for 2 hours and then treated for 10 minutes using a 9-point semi-logarithmic curve of canine or feline OSM at concentrations of 1 μg / mL to 0.0001 μg / mL to induce STAT3 phosphorylation. The reaction was quenched with lysis buffer, and STAT3 phosphorylation was measured using the AlphaLISA SureFire ULTRA® technique (Perkin Elmer, Waltham, MA). EC 50 The value was determined as the concentration of OSM protein that induces 50% of the maximum signal. Figures 1A and 1B show the dose-response curves and EC2 for canine and feline OSM in canine DH-82 and feline Fcwf-4 cells, respectively. 50 Show the value.

[0198] To determine the potency of candidate antibodies by assaying their ability to inhibit OSM-mediated STAT3 phosphorylation, assays were performed in both canids and felines. Cells were serum-starved for 2 hours prior to OSM and mAb treatment. All candidate mAbs were evaluated for their ability to inhibit 0.02 μg / mL canine OSM-induced STAT3 phosphorylation using two independent methods. The assay was performed by incubating anti-OSM mAbs with serum-starved cells for 30 minutes for DH-82 cells or 20 minutes for Fcwf-4 cells. At this point, the supernatant was removed and a medium containing 0.02 μg / mL canine OSM was added over 10 minutes for cell stimulation. The reaction mixture was quenched with lysis buffer and STAT3 phosphorylation was measured. When antibody concentration and purity were unknown, hybridoma supernatant was incubated with cells for 30 or 20 minutes as described above, and then qualitatively measured for its ability to inhibit STAT3 phosphorylation after stimulation with canine OSM at 0.02 μg / mL. In these assays, the potency of individual monoclonal antibodies, defined by their ability to inhibit OSM-mediated STAT3 phosphorylation, was considered a primary selection criterion for further advancement of the selected antibodies. The term potency is defined as the IC calculated from these assays. 50 This value refers to the antibody concentration at which OSM-induced signaling decreases to half of its maximum value. The increase in potency described herein is IC 50 It correlates with a decrease in value.

[0199] Example 6. Selection of anti-OSMR monoclonal antibodies capable of neutralizing IL-31 and OSM-mediated STAT phosphorylation in canid and feline cells. Mouse anti-OSMR antibodies were selected based on the criteria outline in Figure 2. Mice were immunized with a combination of canine and feline OSMR proteins containing three cytokine-binding domains and a single fibronectin III domain fused to human IgG1 Fc to facilitate expression and purification. Antibodies produced from hybridomas were screened for binding to canine and feline OSMR by ELISA and counter-screened against unrelated human IgG. Hybridomas producing antibodies that bound to canine and / or feline OSMR but not to human IgG were selected for further analysis. The drained hybridoma supernatant was generated from these initial hits, and the antibodies were purified for further analysis. Antibodies from these nonclonal hybridomas were first assayed for binding to canine and feline OSMR using Biacore to confirm the results from the ELISA screening. These hybridoma candidates were further tested for their ability to inhibit IL-31 and OSM-mediated STAT phosphorylation in canine DH82 and feline Fcwf-4 cells. Hybridoma candidates that bound to canine and / or feline OSMR and exhibited some inhibitory activity in one of the IL-31 and OSM cell-based assays were selected for subcloning. Subcloning of hybridoma candidates was performed by limiting dilution of cells to confirm monoclonality. The supernatant of these cells producing monoclonal antibodies was confirmed by ELISA for binding to canine and / or feline OSMR proteins, and the cells producing these antibodies were used with isolated RNA for sequence analysis of variable weight and variable light IgG chains. Monoclonal antibodies purified from these cultures were also tested for binding to human OSMR. None of the antibody candidates described herein bound to human OSMR.

[0200] This hybridoma campaign initially generated five monoclonal antibodies, each possessing a unique combination of variable heavy and light chains. These mouse anti-OSMR antibodies include a variable heavy chain sequence (SEQ ID NO: 31, MU_02D09_VH) (whose corresponding nucleotide is (SEQ ID NO: 32, MU_02D09_VH)) and a variable light chain sequence (SEQ ID NO: 33, MU_02D09_VL) (whose corresponding nucleotide is (SEQ ID NO: 34, MU_02D09_VL)); and a variable heavy chain sequence (SEQ ID NO: 35, MU_09E09_VH) (whose corresponding nucleotide is (SEQ ID NO: 35, MU_09E09_VH)). The nucleotide is (SEQ ID NO: 36, MU_09E09_VH) and the variable light chain sequence (SEQ ID NO: 37, MU_09E09_VL) (the corresponding nucleotide is (SEQ ID NO: 38, MU_09E09_VL)); the variable heavy chain sequence (SEQ ID NO: 39, MU_10F07_VH) (the corresponding nucleotide is (SEQ ID NO: 40, MU_10F07_VH) and the variable light chain sequence (SEQ ID NO: 41, MU_10F07_VL) (the 10F07 having the corresponding nucleotide (SEQ ID NO: 42, MU_10F07_VL); 14C04 having the variable heavy chain sequence (SEQ ID NO: 43, MU_14C04_VH) (its corresponding nucleotide is (SEQ ID NO: 44, MU_14C04_VH) and the variable light chain sequence (SEQ ID NO: 45, MU_14C04_VL) (its corresponding nucleotide is (SEQ ID NO: 46, MU_14C04_VL)); and variable heavy chain sequence 19F07 has (SEQ ID NO: 47, MU_19F07_VH) (its corresponding nucleotide is (SEQ ID NO: 48, MU_19F07_VH)) and a variable light chain sequence (SEQ ID NO: 49, MU_19F07_VL) (its corresponding nucleotide is (SEQ ID NO: 50, MU_19F07_VL)). Figure 3 shows the clustalW alignment of these variable heavy and variable light chains, with the CDR highlighted in black squares. [Table 1] [Table 2]

[0201] Example 7. Chimeric antibody generation The antibody variable domain is involved in antigen binding. Grafting the complete variable domain to each constant region is expected to have little to no effect on the antibody's ability to bind to the OSMR immunogen. To simultaneously confirm the correct sequences of the heavy chain and light chain variable regions and to produce a homogeneous material, expression vectors were designed to produce recombinant chimeric antibodies in a mammalian expression system. The chimeric antibodies described herein consist of variable sequences (both CDRs and frameworks) from a host species antibody, grafted onto the respective heavy chain and light chain constant regions of a canid or feline IgG molecule (e.g., mouse variable:canid constant is referred to as mouse:canid chimera). Synthetic DNA sequences were constructed for the variable heavy chain (VH) and variable light chain (VL) sequences of selected antibodies.

[0202] For mouse canid chimeras, each mouse variable region was cloned into a mammalian expression plasmid containing either a canid IgG heavy (SEQ ID NO: 114, Canine_HC_65_1) (whose corresponding nucleotide sequence is (SEQ ID NO: 115, Canine_HC_65_1)) or a light chain (SEQ ID NO: 116, Canine_LC_Kappa) (whose corresponding nucleotide sequence is (SEQ ID NO: 117, Canine_LC_Kappa)) constant region. For mouse feline chimeras, each variable region was cloned into a mammalian expression plasmid containing either a feline IgG heavy (SEQ ID NO: 118, Feline_HC_AlleleA_1) (whose corresponding nucleotide sequence is (SEQ ID NO: 119, Feline_HC_AlleleA_1)) or a light chain (SEQ ID NO: 120, Feline_LC_Kappa_G_minus) (whose corresponding nucleotide sequence is (SEQ ID NO: 121, Feline_LC_Kappa_G_minus)) constant region. These antibodies are a mouse:canid 02D09 chimera having a variable heavy chain sequence (SEQ ID NO: 31, MU_02D09_VH) (whose corresponding nucleotide sequence is (SEQ ID NO: 32, MU_02D09_VH)) and a variable light chain sequence (SEQ ID NO: 33, MU_02D09_VL) (whose corresponding nucleotide sequence is (SEQ ID NO: 34, MU_02D09_VL)); and a variable heavy chain sequence (SEQ ID NO: 35, MU_09E09_VH) (whose corresponding nucleotide sequence is (SEQ ID NO: 36, MU_09E09_VH)) and a variable light chain sequence A mouse possessing (SEQ ID NO: 37, MU_09E09_VL) (whose corresponding nucleotide sequence is (SEQ ID NO: 38, MU_09E09_VL)): Canid 09E09 chimera; a mouse possessing a variable heavy chain sequence (SEQ ID NO: 39, MU_10F07_VH) (whose corresponding nucleotide sequence is (SEQ ID NO: 40, MU_10F07_VH)) and a variable light chain sequence (SEQ ID NO: 41, MU_10F07_VL) (whose corresponding nucleotide sequence is (SEQ ID NO: 42, MU_10F07_VL)): Canid 10F07 chimera;Mouse having a variable heavy chain sequence (SEQ ID NO: 43, MU_14C04_VH) (whose corresponding nucleotide sequence is (SEQ ID NO: 44, MU_14C04_VH)) and a variable light chain sequence (SEQ ID NO: 45, MU_10F07_VL) (whose corresponding nucleotide sequence is (SEQ ID NO: 46, MU_10F07_VL)): Canid 14C04 chimera; variable heavy chain sequence (SEQ ID NO: 47, MU_19F07_VH) (whose corresponding nucleotide sequence is (SEQ ID NO: 48, MU_19F07_VH)) and variable light chain sequence (sequence number Mice possessing sequence number 49, MU_19F07_VL) (whose corresponding nucleotide sequence is (sequence number 50, MU_19F07_VL)): Canid 19F07 chimera; Mice possessing a variable heavy chain sequence (sequence number 31, MU_02D09_VH) (whose corresponding nucleotide sequence is (sequence number 32, MU_02D09_VH)) and a variable light chain sequence (sequence number 33, MU_02D09_VL) (whose corresponding nucleotide sequence is (sequence number 34, MU_02D09_VL)): Felid 02D09 chimera; Mouse having a variable heavy chain sequence (SEQ ID NO: 35, MU_09E09_VH) (whose corresponding nucleotide sequence is (SEQ ID NO: 36, MU_09E09_VH)) and a variable light chain sequence (SEQ ID NO: 37, MU_09E09_VL) (whose corresponding nucleotide sequence is (SEQ ID NO: 38, MU_09E09_VL)): Feline 09E09 Chimera; Variable heavy chain sequence (SEQ ID NO: 39, MU_10F07_VH) (whose corresponding nucleotide sequence is (SEQ ID NO: 40, MU_10F07_VH)) and variable light chain sequence (sequence number Mice possessing sequence number 41, MU_10F07_VL) (whose corresponding nucleotide sequence is (sequence number 42, MU_10F07_VL)): feline 10F07 chimera; Mice possessing a variable heavy chain sequence (sequence number 43, MU_14C04_VH) (whose corresponding nucleotide sequence is (sequence number 44, MU_14C04_VH)) and a variable light chain sequence (sequence number 45, MU_14C04_VL) (whose corresponding nucleotide sequence is (sequence number 46, MU_14C04_VL)): feline 14C04 chimera;Furthermore, the mouse:Felid 19F07 chimera possesses a variable heavy chain sequence (SEQ ID NO: 47, MU_19F07_VH) (with its corresponding nucleotide sequence being (SEQ ID NO: 48, MU_19F07_VH)) and a variable light chain sequence (SEQ ID NO: 49, MU_19F07_VL) (with its corresponding nucleotide sequence being (SEQ ID NO: 50, MU_19F07_VL)). These sequences contain unique restriction endonuclease sites, Kozak consensus sequences, and N-terminal secretion leaders to facilitate the expression and secretion of chimeric recombinant antibodies from mammalian cell lines. Under the control of the CMV promoter, plasmids encoding each heavy and light chain were co-transfected into host cells as described herein and purified. [Table 3] [Table 4]

[0203] Example 8. Design and expression of speciation-typed anti-OSMR antibodies The formation of anti-drug antibodies (ADAs) can be associated with a loss of efficacy for any biological therapeutic protein, including monoclonal antibodies. A comprehensive review of the literature may find examples of immunogenic fully human mAbs and non-immunogenic chimeric mAbs, but it has been shown that speciation of monoclonal antibodies can reduce the tendency for mAbs to become immunogenic. Two methods of speciation are described herein. Canine speciation means grafting mouse CDRs onto a Canis species framework (e.g., Canis lupus familiaris or dog), and feline speciation means grafting mouse CDRs onto a Felis species framework (e.g., Felis catus or cat). Canine and feline speciation strategies were used to help mitigate the risks associated with ADA formation in the anti-OSMR monoclonal antibodies provided herein. These canine and feline speciation strategies were based on identifying the most appropriate canine or feline germline antibody sequences for CDR grafting. Following extensive analysis of all available germline sequences for both variable heavy and light chains, candidate germlines were selected based on their homology to mouse anti-OSMR mAbs, and CDRs from these mouse precursor mAbs were used to replace innate canine or feline CDRs. The objective was to maintain high affinity and cell-based activity using a canine or feline antibody framework to minimize potential immunogenicity in vivo.

[0204] Canine and feline mAbs are expressed, and their affinity for canine and feline OSMRs, as well as their efficacy in cell-based assays, are characterized. If a canine or feline antibody loses its ability to bind to a canine or feline OSMR, systematic dissociation is performed to identify 1) the chain responsible for loss of function, 2) the framework responsible for loss of function, and 3) the amino acid responsible for loss of function. The speciation antibodies described herein consist of variable sequences (both CDR and framework) expressed with the respective heavy and light chain constant regions of a canine or feline IgG molecule. Synthetic DNA sequences are constructed for the variable heavy chain (VH) and variable light chain (VL) sequences of the selected antibody. These sequences contain unique restriction endonuclease sites, Kozak consensus sequences, and N-terminal secretion leaders to facilitate the expression and secretion of recombinant antibodies from mammalian cell lines. For canine-derived antibodies, each mouse variable region is cloned into a mammalian expression plasmid containing either a canine IgG heavy (SEQ ID NO: 114, Canine_HC_65_1) (whose corresponding nucleotide sequence is (SEQ ID NO: 115, Canine_HC_65_1)) or a light chain (SEQ ID NO: 116, Canine_LC_Kappa) (whose corresponding nucleotide sequence is (SEQ ID NO: 117, Canine_LC_Kappa)) constant region. For feline-derived antibodies, each variable region is cloned into a mammalian expression plasmid containing either a feline IgG heavy (SEQ ID NO: 118, Feline_HC_AlleleA_1) (whose corresponding nucleotide sequence is (SEQ ID NO: 119, Feline_HC_AlleleA_1)) or a light chain (SEQ ID NO: 120, Feline_LC_Kappa_G_minus) (whose corresponding nucleotide sequence is (SEQ ID NO: 121, Feline_LC_Kappa_G_minus)) constant region. Under the control of the CMV promoter, plasmids encoding each heavy chain and light chain are co-transfected into host cells, expressed, and purified as described herein.

[0205] Felidae 02D09 1.1 is a variable heavy chain sequence (SEQ ID NO: 51, FEL_02D09_VH1) (its corresponding nucleotide sequence is (SEQ ID NO: 52, FEL_02D09_VH1)) and a variable light chain sequence (SEQ ID NO: 55, FEL_02D09_VL1) (its corresponding nucleotide sequence is (SEQ ID NO: 56, FEL_02D09_VL1)), and Felidae 02D09 2.1 is a variable heavy chain sequence (SEQ ID NO: 53, FEL_02D09_VH2) (its corresponding nucleotide sequence is (SEQ ID NO: 54, FEL_02D09_VH2)) and a variable light chain sequence (SEQ ID NO: 55, FEL_02D09_VL1) (its corresponding nucleotide sequence is (SEQ ID NO: 56, FEL_02D09_VL1)), and Felidae 02D09 1.2 is a variable heavy chain sequence (SEQ ID NO: 51, FEL_02D09_VH1) (its corresponding nucleotide sequence is (SEQ ID NO: 52, FEL_02D09_VH1)) and a variable light chain sequence (SEQ ID NO: 57, FEL_02D09_VL2) (its corresponding nucleotide sequence is (SEQ ID NO: 58, FEL_02D09_VL2)), and is a feline 02D09. 2.2 is a variable heavy chain sequence (SEQ ID NO: 53, FEL_02D09_VH2) (its corresponding nucleotide sequence is (SEQ ID NO: 54, FEL_02D09_VH2)) and a variable light chain sequence (SEQ ID NO: 57, FEL_02D09_VL2) (its corresponding nucleotide sequence is (SEQ ID NO: 58, FEL_02D09_VL2)).

[0206] Canid 09E09 1.1 has a variable heavy chain sequence (SEQ ID NO: 59, CAN_09E09_VH1) (its corresponding nucleotide sequence is (SEQ ID NO: 60, CAN_09E09_VH1)) and a variable light chain sequence (SEQ ID NO: 63, CAN_09E09_VL1) (its corresponding nucleotide sequence is (SEQ ID NO: 64, CAN_09E09_VL1)), and Canid 09E09 2.1 has a variable heavy chain sequence (SEQ ID NO: 61, CAN_09E09_VH2) (its corresponding nucleotide sequence is (SEQ ID NO: 62, CAN_09E09_VH2)) and a variable light chain sequence (SEQ ID NO: 63, CAN_09E09_VL1) (its corresponding nucleotide sequence is (SEQ ID NO: 64, CAN_09E09_VL1)), and Canid 09E09 1.2 is a variable heavy chain sequence (SEQ ID NO: 59, CAN_09E09_VH1) (its corresponding nucleotide sequence is (SEQ ID NO: 60, CAN_09E09_VH1)) and a variable light chain sequence (SEQ ID NO: 65, CAN_09E09_VL2) (its corresponding nucleotide sequence is (SEQ ID NO: 66, CAN_09E09_VL2)), and canids 09E09 2.2 is a variable heavy chain sequence (SEQ ID NO: 61, CAN_09E09_VH2) (its corresponding nucleotide sequence is (SEQ ID NO: 62, CAN_09E09_VH2)) and a variable light chain sequence (SEQ ID NO: 65, CAN_09E09_VL2) (its corresponding nucleotide sequence is (SEQ ID NO: 66, CAN_09E09_VL2)).Felidae 09E09 1.1 has a variable heavy chain sequence (sequence number 67, FEL_09E09_VH1) (its corresponding nucleotide sequence is (sequence number 68, FEL_09E09_VH1)) and a variable light chain sequence (sequence number 71, FEL_09E09_VL1) (its corresponding nucleotide sequence is (sequence number 72, FEL_09E09_VL1)), and Felidae 09E09 2.1 has a variable heavy chain sequence (sequence number 69, FEL_09E09_VH2) (its corresponding nucleotide sequence is (sequence number 70, FEL_09E09_VH2)) and a variable light chain sequence (sequence number 71, FEL_09E09_VL1) (its corresponding nucleotide sequence is (sequence number 72, FEL_09E09_VL1)), and Felidae 09E09 1.2 is a variable heavy chain sequence (sequence number 67, FEL_09E09_VH1) (its corresponding nucleotide sequence is (sequence number 68, FEL_09E09_VH1)) and a variable light chain sequence (sequence number 73, FEL_09E09_VL2) (its corresponding nucleotide sequence is (sequence number 74, FEL_09E09_VL2)), and is a feline 09E09 2.2 is a variable heavy chain sequence (sequence number 69, FEL_09E09_VH2) (its corresponding nucleotide sequence is (sequence number 70, FEL_09E09_VH2)) and a variable light chain sequence (sequence number 73, FEL_09E09_VL2) (its corresponding nucleotide sequence is (sequence number 74, FEL_09E09_VL2)).

[0207] Canid 10F07 1.1 is a variable heavy chain sequence (SEQ ID NO: 75, CAN_10F07_VH1) (its corresponding nucleotide sequence is (SEQ ID NO: 76, CAN_10F07_VH1)) and a variable light chain sequence (SEQ ID NO: 77, CAN_10F07_VL1) (its corresponding nucleotide sequence is (SEQ ID NO: 78, CAN_10F07_VL1)), and Canid 10F07 2.1 is a variable heavy chain sequence (SEQ ID NO: 79, CAN_10F07_VH2) (its corresponding nucleotide sequence is (SEQ ID NO: 80, CAN_10F07_VH2)) and a variable light chain sequence (SEQ ID NO: 77, CAN_10F07_VL1) (its corresponding nucleotide sequence is (SEQ ID NO: 78, CAN_10F07_VL1)), and Canid 10F07 1.2 is a variable heavy chain sequence (sequence number 75, CAN_10F07_VH1) (its corresponding nucleotide sequence is (sequence number 76, CAN_10F07_VH1)) and a variable light chain sequence (sequence number 81, CAN_10F07_VL2) (its corresponding nucleotide sequence is (sequence number 82, CAN_10F07_VL2)), and canid 10F07 2.2 is a variable heavy chain sequence (sequence number 79, CAN_10F07_VH2) (its corresponding nucleotide sequence is (sequence number 80, CAN_10F07_VH2)) and a variable light chain sequence (sequence number 81, CAN_10F07_VL2) (its corresponding nucleotide sequence is (sequence number 82, CAN_10F07_VL2)).Felidae 10F07 1.1 is a variable heavy chain sequence (sequence number 83, FEL_10F07_VH1) (its corresponding nucleotide sequence is (sequence number 84, FEL_10F07_VH1)) and a variable light chain sequence (sequence number 85, FEL_10F07_VL1) (its corresponding nucleotide sequence is (sequence number 86, FEL_10F07_VL1)), and Felidae 10F07 2.1 is a variable heavy chain sequence (sequence number 87, FEL_10F07_VH2) (its corresponding nucleotide sequence is (sequence number 88, FEL_10F07_VH2)) and a variable light chain sequence (sequence number 85, FEL_10F07_VL1) (its corresponding nucleotide sequence is (sequence number 86, FEL_10F07_VL1)), and Felidae 10F07 1.2 is a variable heavy chain sequence (sequence number 83, FEL_10F07_VH1) (its corresponding nucleotide sequence is (sequence number 84, FEL_10F07_VH1)) and a variable light chain sequence (sequence number 89, FEL_10F07_VL2) (its corresponding nucleotide sequence is (sequence number 90, FEL_10F07_VL2)), and is a feline 10F07. 2.2 is a variable heavy chain sequence (sequence number 87, FEL_10F07_VH2) (its corresponding nucleotide sequence is (sequence number 88, FEL_10F07_VH2)) and a variable light chain sequence (sequence number 89, FEL_10F07_VL2) (its corresponding nucleotide sequence is (sequence number 90, FEL_10F07_VL2)).

[0208] Canid 19F07 1.1 is a variable heavy chain sequence (SEQ ID NO: 91, CAN_19F07_VH1) (its corresponding nucleotide sequence is (SEQ ID NO: 92, CAN_19F07_VH1)) and a variable light chain sequence (SEQ ID NO: 93, CAN_19F07_VL1) (its corresponding nucleotide sequence is (SEQ ID NO: 94, CAN_19F07_VL1)), and Canid 19F07 2.1 is a variable heavy chain sequence (SEQ ID NO: 95, CAN_19F07_VH2) (its corresponding nucleotide sequence is (SEQ ID NO: 96, CAN_19F07_VH2)) and a variable light chain sequence (SEQ ID NO: 93, CAN_19F07_VL1) (its corresponding nucleotide sequence is (SEQ ID NO: 94, CAN_19F07_VL1)), and Canid 19F07 1.2 is a variable heavy chain sequence (sequence number 91, CAN_19F07_VH1) (its corresponding nucleotide sequence is (sequence number 92, CAN_19F07_VH1)) and a variable light chain sequence (sequence number 97, CAN_19F07_VL2) (its corresponding nucleotide sequence is (sequence number 98, CAN_19F07_VL2)), and canid 19F07 2.2 is a variable heavy chain sequence (sequence number 95, CAN_19F07_VH2) (its corresponding nucleotide sequence is (sequence number 96, CAN_19F07_VH2)) and a variable light chain sequence (sequence number 97, CAN_19F07_VL2) (its corresponding nucleotide sequence is (sequence number 98, CAN_19F07_VL2)).Felidae 19F07 1.1 is a variable heavy chain sequence (sequence number 99, FEL_19F07_VH1) (its corresponding nucleotide sequence is (sequence number 100, FEL_19F07_VH1)) and a variable light chain sequence (sequence number 101, FEL_19F07_VL1) (its corresponding nucleotide sequence is (sequence number 102, FEL_19F07_VL1)), and Felidae 19F07 2.1 is a variable heavy chain sequence (sequence number 103, FEL_19F07_VH2) (its corresponding nucleotide sequence is (sequence number 104, FEL_19F07_VH2)) and a variable light chain sequence (sequence number 101, FEL_19F07_VL1) (its corresponding nucleotide sequence is (sequence number 102, FEL_19F07_VL1)), and Felidae 19F07 1.2 is a variable heavy chain sequence (sequence number 99, FEL_19F07_VH1) (its corresponding nucleotide sequence is (sequence number 100, FEL_19F07_VH1)) and a variable light chain sequence (sequence number 105, FEL_19F07_VL2) (its corresponding nucleotide sequence is (sequence number 106, FEL_19F07_VL2)), and is a feline 19F07. 2.2 is a variable heavy chain sequence (sequence number 103, FEL_19F07_VH2) (its corresponding nucleotide sequence is (sequence number 104, FEL_19F07_VH2)) and a variable light chain sequence (sequence number 105, FEL_19F07_VL2) (its corresponding nucleotide sequence is (sequence number 106, FEL_19F07_VL2)).

[0209] Canid 14C04 1.1 has a variable heavy chain sequence (SEQ ID NO: 127, CAN_14C04_VH1) (its corresponding nucleotide sequence is (SEQ ID NO: 128, CAN_14C04_VH1)) and a variable light chain sequence (SEQ ID NO: 131, CAN_14C04_VL1) (its corresponding nucleotide sequence is (SEQ ID NO: 132, CAN_14C04_VL1)), and Canid 14C04 2.1 has a variable heavy chain sequence (SEQ ID NO: 129, CAN_14C04_VH2) (its corresponding nucleotide sequence is (SEQ ID NO: 130, CAN_14C04_VH2)) and a variable light chain sequence (SEQ ID NO: 131, CAN_14C04_VL1) (its corresponding nucleotide sequence is (SEQ ID NO: 132, CAN_14C04_VL1)), and Canid 14C04 1.2 is a variable heavy chain sequence (sequence number 127, CAN_14C04_VH1) (its corresponding nucleotide sequence is (sequence number 128, CAN_14C04_VH1)) and a variable light chain sequence (sequence number 133, CAN_14C04_VL2) (its corresponding nucleotide sequence is (sequence number 134, CAN_14C04_VL2)), and canids 14C04 2.2 is a variable heavy chain sequence (sequence number 129, CAN_14C04_VH2) (its corresponding nucleotide sequence is (sequence number 130, CAN_14C04_VH2)) and a variable light chain sequence (sequence number 133, CAN_14C04_VL2) (its corresponding nucleotide sequence is (sequence number 134, CAN_14C04_VL2)).

[0210] Example 9. Homology modeling of candidate anti-OSMR antibodies and comparison of structural variations between CDRs Generating homology models based on known protein structures is useful for understanding the three-dimensional structure of antibodies. Overlaying these protein models allows for direct comparison of similar and different regions of antibodies in three-dimensional space. These methods overcome the limitations of comparing proteins as linear amino acid sequence strings, which do not consider the collective representation of the physicochemical properties of each amino acid relative to its surroundings. The antibody models for the five mouse anti-OSMR antibodies described herein were generated using Molecular Operating Environment (MOE) software (Chemical Computing Group, Montreal, Canada), which is capable of creating such models as other available software. See, for example, Almagro et al.; Antibody Modeling Assessment; Proteins: Struct. Func. Bioinf. 79(2011) 3050-3066, of record.

[0211] To compare the structures of five different mouse anti-OSMR antibody models, mean squared deviation (RMSD) values ​​were determined from pairwise comparisons between each antibody CDR, which is considered most important for antigen binding. RMSD is a known criterion in the art used to determine the similarity of structures in three-dimensional space by comparing the coordinates of their alpha carbon atoms, which act as the scaffolding of the protein. When calculating RMSD, the MOE software generates a mean squared deviation representing the overall difference in the structures for the chosen region of interest, using a comparison of each alpha carbon with its respective aligned alpha carbon on other structures. This specification describes a comparison of only the CDR region between each antibody and the variable domain, not the entire region. When calculating RMSD values ​​between three antibody structures, sequence alignment is performed taking into account structural overlaps and regions where gaps are introduced to optimize alignment. In some situations, an overestimation of RMSD similarity (lower RMSD value) is obtained due to the failure to consider variations in loop length. This is the case when considering CDRH3 and CDRL1 from five different mouse anti-OSMR antibody candidates with different CDR lengths (Figure 3). RMSD of 2 angstroms or more represents significant separation in three-dimensional space.

[0212] Figure 4 shows six matrices using RMSDs obtained from pairwise comparisons of each CDR from the five mouse anti-OSMR antibodies described herein. Comparison of CDRH1 from these structural models shows that these antibodies have general structural similarities in this loop without having RMSD values ​​exceeding 2 angstroms. Note that mouse antibody 14C04, which has a variable heavy chain sequence (SEQ ID NO: 43, MU_14C04_VH) (whose corresponding nucleotide sequence is (SEQ ID NO: 44, MU_14C04_VH)) and a variable light chain sequence (SEQ ID NO: 45, MU_14C04_VL) (whose corresponding nucleotide sequence is (SEQ ID NO: 46, MU_14C04_VL)), has the most different CDRH1 structure (largest RMSD compared to the other antibodies) and is the only antibody that is specific only to canid OSMR. The differences in RMSD between the mouse anti-OSMR antibody 14C04 and other antibodies are more pronounced when comparing CDRH2 and RMSD values ​​greater than 2 for each comparison.

[0213] The RMSD results obtained from comparing the CDRH3 structures of five different mouse anti-OSMR antibodies highlight another important consideration regarding the structure-function relationship between the antibody and its target, OSMR. Mouse anti-OSMR antibody 14C04 had RMSD values ​​exceeding 2 angstroms in three of the four comparisons, and is structurally very different from the others in this CDR. As already mentioned, 14C04 is canid OSMR-specific (see Table 1 for affinity data). Other antibodies exhibit high structural differences from the other four in mouse anti-OSMR 02D09, which have a variable heavy chain sequence (SEQ ID NO: 31, MU_02D09_VH) (with its corresponding nucleotide sequence being (SEQ ID NO: 32, MU_02D09_VH)) and a variable light chain sequence (SEQ ID NO: 33, MU_02D09_VL) (with its corresponding nucleotide sequence being (SEQ ID NO: 34, MU_02D09_VL)). Mouse anti-OSMR 02D09 is interesting because it exhibits specificity for binding to feline OSMR (see Tables 1 and 3, Table 3 showing the higher affinity of the chimeric 02D09 antibody for feline OSMR (compared to canine OSMR) and the slower offrate, supporting its preferential specificity for the feline OSMR protein). To reiterate previously mentioned points, antibodies 02D09 and 14C04 also have longer CDRH3 loops, and these additional amino acids, while not considered in a direct structural comparison, significantly influence the different structures of these two CDRH3 loops when compared to the other three antibodies that bind to both canine and feline OSMR.

[0214] Continuing the comparison of light chain CDR structures, the CDRL1 matrix in Figure 4 shows that the RMSD comparison of the five anti-OSMR antibodies does not exceed a difference of 2 angstroms. Here again, the canid OSMR antibody 14C04 has a higher RMSD value, which indicates a tendency to distinguish the structure without considering the four additional amino acids in this CDR. These four additional amino acids in the CDR loop represent a significant structural difference compared to the other four antibodies, further supporting the intrinsic structural composition of the binding paratope for this antibody. Antibodies 02D09 and 14C04 have the largest structural variation in the CDRL2 loop, where the greatest significance (RMSD greater than 2 angstroms) exists in the comparison between them.

[0215] It is interesting to note that although the amino acid sequences of CDRL2 from antibodies 09E09 and 10F07 are identical, the RMSD comparison between them is not zero (the value is 0.31). An RMSD value of 0.31 indicates structures that are very similar to each other. The difference arises from the fact that antibodies 09E09 and 10F07 are not identical because they do not have the same variable light chain. Structural changes introduced in other regions of the protein chain can cause differences that occur distally and are reflected in subtle differences in RMSD values.

[0216] A comparison of the light chain CDRL3 again reveals the existence of significant structural differences between antibodies 02D09 and 09E09, and the tendency for these two species-specific anti-OSMR mAbs to exhibit large structural variations when compared reveals their bispecificity for canid and feline OSMR. While we do not wish to be constrained by a single specific hypothesis, the inventors present evidence supporting the relationship between the structure of the CDR loop of these anti-OSMR antibodies and their functionality in specific binding and blocking to canid and / or feline OSMR, which suggests that this binding inhibits the ability of IL-31 or OSM protein-induced pSTAT3 signaling.

[0217] Example 10. Homology modeling of OSM:OSMR receptors in canids and felines The OSMR receptor is characterized in the context of cytokines, which are bindings that result in cellular signaling responses. These cytokines (including OSMs) are members of the gp130 family of cytokines that induce multifaceted responses in the context of different cellular and tissue distributions, including cell differentiation, proliferation, hematopoiesis, immunity, and inflammatory responses (Richards CD.; The Enigmatic Cytokine Oncostatin M and Roles in Disease; ISRN Inflammation. 2013:512103 (2013)). The OSMR protein shares a similar structural architecture to LIFR in humans, forming a heterodimeric receptor complex with gp130 for signaling in response to both LIF and OSMs. This overlap in functional features allows for comparison of these receptor structures within and between species. While the protein structure of OSMR is not currently available, the crystal structure of leukemia inhibitory factor (LIF) in the LIF receptor (LIFR) (PDB ID: 2Q7N, 4 angstroms) is available (Huyton T et al.; An unusual cytokine:Ig-domain interaction revealed in the crystal structure of leukemia inhibitory factor (LIF) in complex with the LIF receptor. Proc Natl Acad Sci US A. 2007 Jul 31;104(31):12737-42). Although we do not wish to be constrained by a single hypothesis, the inventors present herein use this LIF:LIFR cocrystal structure as a template to present structural homology data to aid in understanding the interaction between IL-31, OSM, and OSMR. By combining this structural data with reports of functional mapping of amino acid residues involved in the interaction between human OSM and human OSMR, we can infer homologous regions in canid and feline OSMR, supporting our understanding of the epitopes to which inhibitory anti-OSMR antibodies bind.

[0218] Figure 5A shows a representative homology model generated using amino acids 28-329 of (SEQ ID NO: 107, Canine_OSMR_hIgG1_Fc) (whose corresponding nucleotide sequence is (SEQ ID NO: 108, Canine_OSMR_hIgG1_Fc) (the extracellular domain of feline OSMR)) on the template structure 2Q7N of human LIFR (SEQ ID NO: 135, Human_LIFR). A similar homology model was generated using amino acids 28-329 of (SEQ ID NO: 112, Feline_OSMR_hIgG1_Fc) (whose corresponding nucleotide sequence is (SEQ ID NO: 113, Feline_OSMR_hIgG1_Fc) (the extracellular domain of feline OSMR)) on the template structure 2Q7N of human LIFR (SEQ ID NO: 135, Human_LIFR). Figure 5 shows a homology model generated using a canine OSM (SEQ ID NO: 109, Canine_OSM) on the template structure 2Q7N of a human LIF (SEQ ID NO: 136, Human_LIF). A similar homology model was generated using amino acids 23-234 of a feline OSM (SEQ ID NO: 110, Feline_OSM_hIgG1_Fc) on the template structure 2Q7N human LIF (SEQ ID NO: 136, Human_LIF) (its corresponding nucleotide sequence is (SEQ ID NO: 111, Feline_OSM_hIgG1_Fc)).

[0219] Figure 5A shows the binding interface of a canine OSM homology model near the canine OSM protein, based on a LIF:LIFR cocrystal template. Figure 5B shows a magnified view of the region contained within the highlighted circle. This magnified view includes labeled amino acids identified from previous studies that define the relevant amino acids involved in human OSM:OSMR interaction (Adrian-Segarra JM et al.; The AB loop of Oncostatin M(OSM) determines species-specific signaling in humans and mice. J Biol Chem. 2018 Dec 28;293(52):20181-20199). While this publication used human proteins to evaluate the functionality of key amino acid residues involved in OSM binding to OSMR, it is important to note that the homologous amino acids labeled on this canine OSM:OSMR homology model are indeed located within this binding interface and therefore provide supporting evidence that inferences from human protein structures support canine and feline receptor systems.

[0220] Example 11. Determination of anti-OSMR binding regions for canid and feline OSMR based on homology to known OSM-binding domains and interspecies amino acid sequence variations. Figure 6 shows a diagram for logically defining the binding regions of five different mouse anti-OSMR antibodies against canids and / or felines. That is, due to the sequence diversity in the human OSMR protein, and the limited diversity between canid and feline OSMR, the number of amino acid residues defining the OSM (and possibly IL-31) binding sites on OSMR is narrow, resulting in only a very limited number of locations where these antibodies can specifically bind and induce the functional activity described herein. In Figure 6, the extracellular domain of human OSMR is represented by black-filled ovals and hatched rectangles representing cytokine-binding domains, and white circles representing fibronectin III domains. Mapping studies, such as those described herein, define the specific regions (major individual amino acid contacts) responsible for the binding of human OSM to OSMR. Hereinafter, homologous canid and feline OSMR protein regions used to immunize mice for the purpose of generating anti-OSMR antibodies are also shown. None of the antibodies selected for binding to canid and / or feline OSMR described herein bind to human OSMR, indicating that binding occurs in regions on canid and / or feline OSMR that are diversified from human OSMR. Specifically, the amino acids in these regions of canid and feline OSMR differ from those in human OSMR. The fact that these antibodies are known to bind to canid and / or feline OSMR and inhibit pSTAT3-mediated signaling induced by canid and / or feline OSMR (and / or IL-31) suggests that they bind to distinct regions of OSM and / or IL-31 that bind to OSMR, activating the receptor, and that these regions have different amino acid sequences in canids and felines compared to human OSMR.

[0221] There are three types of mouse anti-OSMR antibodies described herein: 1) an antibody that preferentially binds to feline OSMR, 2) an antibody that preferentially binds to canine OSMR, and 3) three types of antibodies that bind to both canine and feline OSMR. Based on these three binding phenotypes, the antibody that specifically binds to canine OSMR must bind to a region of OSMR near the binding male parent of OSM (and IL-31), whose amino acid sequence is unique to canine OSMR compared to feline OSMR. The antibody that preferentially binds to feline OSMR over canine OSMR will bind to a region that is unique to feline OSMR and near the binding site of OSM (and IL-31). The antibody that binds to both canine and feline OSMR and inhibits OSM (and / or) IL-31 mediated signaling will bind to a region on OSMR with an amino acid sequence that is conserved between canine and feline OSMR. Based on the modeling described herein and the functional data determined for these antibodies, these data define the epitopes of the anti-OSMR antibodies described herein.

[0222] Example 12. Docking of anti-OSMR antibodies against OSMR receptor models This specification describes homology models generated for five selected candidate mouse anti-OSMR antibodies that bind to canid and / or feline OSMR. Furthermore, variations in the structure-function relationship between these antibodies are described in relation to the CDR structure, differential binding, and efficacy for neutralizing canid and / or feline IL-31 and / or OSM pSTAT3 signaling in canid and / or feline cells. In addition, homology models of the OSM:OSMR binding interface, defining OSM cytokines and receptor regions crucial for receptor activation, are described. The use of these models, and knowledge of specific amino acid residues involved in their interactions, can be translated into homologous regions in canid and feline OSM and OSMR proteins.

[0223] Protein docking to another protein (or protein complex) is performed using software such as MOE or other relevant software currently available. The antibody homology model (or resolved antibody structure) is docked to the receptor using user input, defining regions on the protein (e.g., specific amino acid residues) and guiding the software. These regions are defined from knowledge of experimental data that defines residues important for contact between cytokines and the receptors to which they bind. This data may originate from homologous protein structures and / or protein structures with similar functional characteristics and similar architectures (not necessarily similar amino acid sequences). Antibody docking to anti-OSMR structures on OSMR structures is performed in MOE software using knowledge of human OSM:OSMR interactions, combined with knowledge that mouse anti-canid and / or feline CDRs are important for species specificity. The placement of antibody models against relevant regions of OSM:OSMR interaction is further supported by the fact that a) the mouse anti-OSMR antibodies described herein bind to canid and / or feline OSMR, and b) these antibodies block the ability of IL-31 and / or OSM to induce pSTAT3 signaling in canid and / or feline cells. These facts define distinct regions of the OSMR protein to which these antibodies logically bind and which can restrict the region to define where CDR docking should occur.

[0224] Example 13. Analysis of OSMR gene expression in skin tissue biopsies from felines with allergic dermatitis using in situ hybridization (ISH). Twenty skin biopsies from felines with allergic skin disease were formalin-fixed and paraffin-embedded (FFPE) before sectioning for ISH analysis. Nineteen biopsies from normal feline skin without signs of allergic disease were FFPE-treated and sectioned for comparative analysis. An automated single-chromogenic ISH protocol from Advanced Cell Diagnostics, RNAscope 2.5 LS Reagent Kit-Red (ACD, 322750-USM) was performed using a Leica Bond-RX. Prior to probe hybridization, tissue samples were subjected to thermal and enzymatic epitope activation using ACD's proprietary protease reagent. Each sample was stained with a positive control, and RNA integrity was determined using a probe for the cyclophyllin B (PPIB) gene (Advanced Cell Diagnostics, Inc., Newark, CA). A custom probe was designed to detect feline OSMR gene expression (Advanced Cell Diagnostics, Inc., Newark, CA). Each slide was visualized and quantified using Leica Aperio ImageScope 12.3.2.8013. Each image was annotated, and RNA expression was analyzed using the Leica RNA ISH v2 macro algorithm embedded in the ImageScope software. Data was extracted from the ImageScope software and reported as the ratio of RNA signals to the analyzed tissue region. Significance of the signals was determined using Student's one-sided test.

[0225] Figure 7 shows the quantitative results after imaging OSMR mRNA using ISH from normal and allergic feline skin biopsies. ISH of mRNA using RNAScope is highly specific for the detection of targeted transcripts, in this case feline OSMR mRNA. Clear and significant overexpression of OSMR mRNA is present in the skin of felines with allergic diseases. These results demonstrate a correlation between the clinically relevant diagnosis of allergic skin diseases in felines and the overexpression of the OSMR gene, which represents a link between OSMR and the pathophysiology of the disease. These results support the hypothesis that OSMR is involved in feline skin disorders and that targeting OSMR with antibodies capable of blocking cytokine-mediated signaling via receptors may be useful in treating such disorders, including, but not limited to, allergic dermatitis and atopic dermatitis.

[0226] Example 14. Analysis of OSMR protein in skin tissue biopsies from felines with allergic dermatitis using immunohistoscience (IHC). Five skin biopsies from felines with allergic skin disease were formalin-fixed and paraffin-embedded (FFPE) before sectioning for IHC analysis. Five biopsies from normal feline skin without signs of allergic disease were FFPE-treated and sectioned for comparative analysis. An automated single-color IHC refine red protocol was performed using the Leica Bond-RX system (Leica Biosystems, Buffalo Grove, IL). Sample epitopes were exposed to heat-inducible epitope activation in EDTA buffer before antibody staining. Feline OSMR protein expression was performed using polyclonal rabbit anti-OSMR antibody (LSBio LS-B11477, Seattle, WA) and detected with Leica Bond refine red AP-linked polymer and fast red chromogen (Leica Biosystems, Buffalo Grove, IL). Qualitative analysis was performed by examining each image for red staining indicating OSMR protein expression.

[0227] Figure 8 shows representative images comparing skin sections from a normal feline with those from a feline with an allergic disease. The colored skin slides include light brown epidermal areas, dark purple spots representing Dapi staining of the nuclei, and red colorimetric staining of OSMR protein after development with alkaline phosphatase (AP)-labeled secondary antibody. Figure 8 shows the staining of feline OSMR protein in control and allergic skin tissue images after converting the colored images to grayscale. In the control (non-allergic) skin sample, there is low expression of OSMR protein in the subepidermal space and a normal distribution of cells near the epidermal layer. Images from allergic skin samples clearly show elevated OSMR protein expression throughout the subepidermal space and large infiltration of cells with minimal distinction between the epidermis and subepidermal space. In these images, OSMR protein is visible as gray to black shading in the space outside the black-defined nucleus. Although cell boundaries in these images are not annotated, it should be noted that staining appears in interstitial regions presumably distant from cells, suggesting the possibility of the presence of secreted soluble forms of the OSMR receptor. While we do not wish to be constrained by a single hypothesis, it is conceivable that such secreted receptors may play a regulatory role by acting as antagonists or carrier proteins for proteins with which OSMR interacts. If these soluble forms have the same amino acid sequence and structure as the cell-associated forms of the receptor, then anti-OSMR antibodies, such as those described herein, would also bind to the soluble forms. Visual examination of five control feline skin slides and five allergic skin slides shows patterns for OSMR staining similar to those described herein for two representative images. These results support the quantitative results described herein, which show overexpression of OSMR in the skin of felines with allergic diseases compared to feline skin samples without allergic diseases. These results provide evidence for a role for the OSMR protein in the skin of felines with skin disorders (including, but not limited to, allergic dermatitis and atopic dermatitis).

[0228] Example 15. A canine and feline model of IL-31-induced pruritus to determine the in vivo efficacy of anti-OSMR antibodies. While not wishing to be constrained by any particular hypothesis, the inventors present evidence of the binding of selected antibodies to canid and / or feline OSMR to canid and / or feline OSMR, suggesting that it is possible to block (or neutralize) the biological activity of pSTAT3-induced signaling by IL-31 and / or OSM, and that such IL-31 and / or OSM inhibition at the OSMR receptor level may be useful as a therapeutic agent for atopic disorders, allergic disorders, inflammatory disorders and other disorders described herein for canids and / or felines. The ability of an antibody to effectively neutralize its target can be evaluated in vivo using an appropriate model for efficacy in the host species. Such a model for determining in vivo efficacy is described.

[0229] To determine in vivo efficacy in dogs, experimental dogs were administered subcutaneously (SC) with an anti-canine OSMR antibody. Baseline responses were performed in all dogs, which were randomly divided into groups and accommodated based on their pruritus score index (PSI). Dogs were then administered the anti-canine OSMR antibody on day 7, and IL-31 challenges were performed on days 8, 14, and 22. The reduction in mean PSI on days 8 and 14 compared to day 1 in antibody-treated animals was considered the efficacy endpoint when compared to the PSI score of untreated animals. Daily variability in PSI related to pruritus behavior in dogs was assessed by controlling for variability using the 30-minute baseline PSI determined for each dog daily before the IL-31 challenge. Data from such in vivo models may provide evidence that 1) anti-canine OSMR monoclonal antibodies can neutralize IL-31's ability to induce pruritus in dogs, 2) inhibition of IL-31-mediated signaling by blocking OSMR in cell-based assays correlates with in vivo efficacy, and 3) parameters necessary for utilizing IL-31 models for antibody evaluation can be established for the evaluation of other candidate antibodies.

[0230] The efficacy of anti-feline OSMR in an IL-31-induced in vivo feline model was evaluated. Pre-challenge pruritic behavior was assessed in the vehicle-placebo and antibody groups from day 7 to day 28, where day 0 was the day of antibody administration. On day 0, cats were subcutaneously administered the anti-OSMR antibody 7 days before the first feline IL-31 challenge. After intravenous challenge with the IL-31 protein, pruritic behavior was assessed over 1 hour on days 7, 21, and 28. A reduction in pruritic behavior observed 7, 21, or 28 days after the IL-31 challenge, compared to the vehicle-placebo control, was considered efficacy. These data support the ability of the anti-OSMR antibody to neutralize feline IL-31-induced pruritic behavior in vivo, suggesting that the antibody may function as a therapeutic agent in the treatment of IL-31-mediated diseases (including, but not limited to, atopic dermatitis) in cats.

[0231] Example 16. Additional cell-based data using mice and mice:canid chimeric anti-OSMR antibodies. Additional cell-based experiments were conducted to evaluate the efficacy of anti-OSMR antibodies in cell challenge assays for IL-31 and OSM-mediated pSTAT3 signaling. These data were generated to support previous studies described in Examples 6 and 7. The sequences of the mouse and mouse:canine chimeric antibodies are described in Examples 6 and 7, respectively. These data demonstrate that both antibodies, 10F07 and 19F07, exhibit superior efficacy in inhibiting IL-31 and OSM-mediated pSTAT3 signaling in both canine and feline cells. The selective efficacy of antibody 14C04 for inhibiting canine-mediated signaling, and the selective efficacy of 02D09 for felines, which may indicate structural mutations in the OSMR receptor, are also of interest, resulting in differential specificity for the epitopes recognized by these two antibodies. [Table 5]

[0232] Example 17. Binding data for canine and feline anti-OSMR antibodies Canine (canid) and feline (feline) anti-OSMR antibodies were cloned, expressed, and purified. The sequences of each speciation-speciation antibody are shown in Table 6 and are described in Example 8. The affinity of these speciation-speciation antibodies against canine and feline OSMR was measured using Biacore. Table 5 describes the results of these Biacore experiments for speciation-speciation antibodies expressing and conjugating canine and / or feline OSMR. As shown in the table, this first round of speciation produced antibodies from the 19F07, 09E09, and 10F07 strains that expressed and conjugated their protein targets. For comparisons of mice and mouse:canine chimeras with the same CDR as these speciation-speciation embodiments, see Tables 1 and 3, respectively. The maximum affinity speciation for each of these three series of antibodies is the binding of Fel_09E09_1.1 to feline OSMR with a KD of 7.73E-9M, Fel_10F07_2.2 to feline OSMR with a KD of 1.21E-9M, Can_19F07_2.1 to canine OSMR with a KD of 8.30E-9M, and Fel_19F07_2.2 to feline OSMR with a KD of 3.32E-12M. The objective during the speciation process is to maintain the affinity of the speciation-specified antibody forms for their respective target proteins, in this case, canine and / or feline OSMRs. This specification describes in detail the antibody binding dynamics illustrating this objective. [Table 6] The experimental control shows only binding of anti-human OSMR antibodies to human OSMR. None of these mouse precursor mAbs with CDRs showed binding to human OSMR (see Table 1). Untransfected controls were grown and purified identically to these specified recombinant mAbs that did not contain plasmids for expressing functional antibodies. cOSM binding indicates that these three OSMR receptor morphologies are capable of binding to the canine morphology of the OSM protein. Control-HBS-EP is a buffered control for Biacore instruments.

[0233] Example 18. Mutational analysis of anti-OSMR CDRs using alanine substitution To establish the functional relationships between each amino acid residue in antibody CDRs and the involvement of these residues in antibody binding to target proteins, alanine substitution mutation analysis was performed. Individual mammalian expression plasmids containing heavy chain and light chain mouse variable sequences were synthesized for four of the five anti-OSMR antibodies listed in Table 1 (alanine substitution was not performed for antibody Mu_14C04). The variable heavy chain and variable light chain were cloned in the heavy chain and light chain canid constant regions, respectively, as described herein for the generation of chimeric antibodies (Example 7). Expression and purification of these chimeric antibodies were carried out according to the procedure described herein (Example 1). For comparative analysis, each chimera was generated without altering the initial mouse variable heavy chain and variable light chain sequences (wild type). For each mutant, the respective heavy chain variable and light chain variable CDR position Individual plasmids were generated to substitute alanine for each non-alanine residue. Each plasmid containing a single alanine substitution at a single position in each CDR was paired with the corresponding heavy-chain or light-chain wild-type plasmid and expressed using a transient CHO cell line.

[0234] Example 19. ELISA and Biacore binding results for alanine variants of anti-OSMR antibodies The purpose of alanine substitution mutation analysis is to further understand the involvement of individual CDR amino acid residues in binding to OSMR target proteins. A single substitution of alanine in a CDR amino acid residue can result in a functional change, thereby negatively impacting the antibody's binding to its target (meaning little to no binding, lower binding affinity, or weaker ELISA signal compared to a wild-type antibody without the CDR substitution). Binding for each alanine variant was evaluated using an indirect ELISA with feline OSMR protein (SEQ ID NO: 112) passively immobilized on a polystyrene ELISA plate. The ELISA was performed following a standard procedure of blockage and washing with a 1-hour incubation time between steps. To determine binding, each wild-type or variant antibody was added to individual wells of the ELISA plate as purified antibody (pure antibody) or, where indicated, as cell culture supernatant (supernatant). Since these antibodies were constructed as mouse:canid chimeras, the amount of antibody bound to the feline OSMR protein was detected after development with an HRP substrate using a goat anti-canid HRP-labeled secondary antibody. To determine whether antibodies were produced from transient CHO cultures, separate ELISAs were performed using mouse anti-canine antibodies as the capture reagent against ELISA plates. Each supernatant or pure antibody preparation was added to individual wells and bound. The presence of antibodies was determined using HRP-labeled goat anti-canine detection mAb (antibody expression control). The colorimetric signals generated by each ELISA plate were normalized to a background control lacking the analyte. Data were expressed as the ratio of optical density (OD) from the ELISA using binding to feline OSMR (ELISA signal) to the OD from the antibody control plate (antibody expression control). Alanine mutations resulting in an OD ratio of 1.3 or less (ELISA signal / mAb control) were considered to negatively affect binding. The OD ratio for each wild-type expression control was greater than 1.6. In general, most mutations that negatively affected binding gave an OD ratio of less than 0.5 (Table A). [Table A]

[0235] The ELISA data represent the ratio of signals generated by the binding of alanine substitution mutants to feline OSMR. This ratio is calculated by dividing the signal generated from binding to feline OSMR by the signal from the antibody expression control (indicating the presence of recombinant antibody). The Biacore binding kinetics to canid and feline OSMR, using the corresponding signals from the expression control, are also shown here. The data shown in this table are from experiments using the same antibody preparation (supernatant or pure antibody), and only those alanine substitution mutants to which binding occurred adversely affected the ELISA assay.

[0236] Table A shows the dynamic binding data for each alanine substitution mutation that negatively affected binding in feline OSMR ELISA experiments. The data from the ELISA represent the equilibrium state in which the antibody associates, dissociates, and leads to steady-state binding over a 1-hour period. Using Biacore, the target protein is immobilized on a surface, and the antibody passes over this surface in the mobile liquid phase. Protein-protein interactions are determined from changes in surface resonance, and association (k a ), dissociation (k d ), and the calculation of the affinity constant (KD) are made possible. Although we do not wish to be bound to one hypothesis, the inventors present data from ELISA and Biacore experiments using the same preparation of the antibody, which may not only be consistent between the two methods with respect to binding, but may also show contradictory results due to the nature of the binding kinetics and different assay dynamics. The data presented in Table A (combined with experimental results from yeast display mutation analysis of Example 21) enable the definition of the structure:function relationship between the anti-OSMR antibody described herein and its ability to bind to and block IL-31 and OSM-mediated signaling via the OSMR receptor.

[0237] Example 20. A method for yeast surface display (YSD) combined with mutation scanning to determine the structure-function relationship of anti-OSMR CDR residues. To further define the amino acid residues in the anti-OSMR antibody 19F07 that are important for interaction with OSMR protein targets, an independent experimental method using the presentation of antibody variable domains on the surface of the yeast Saccharomyces cerevisiae was used. Mutational tolerance epitope mapping was performed on antibody Mu_19F07 using the approach defined by Klesmith et al. (2019) Biochemistry 58, 4869-4881. The heavy and light chain variable domains were purchased as gBlocks (IDT) and ligated via a (G4S)4 linker to construct the 19F07 single-stranded variable domain fragment (scFv). Using HiFi assembly, the gene was cloned into a minimal bacterial plasmid containing a BbvCI nicking restriction site (New England Biolabs). Using Nicking mutagenesis, two single-site saturated libraries were constructed using this vector, which has two different oligopools (IDTs) encoding NNK codons for each codon position in the VH and VL domains separately (Wrenbeck et al. (2016) Nature Methods 13, 928-930). For all cloning steps, a sufficient number of transformants were present to oversample the designed libraries (theoretical library sizes of 3744 and 3424 variants for the VH and VL libraries, respectively). The parental non-mutant scFv gBlock (wild-type), as well as the VH and VL mutant libraries, were PCR amplified and electroporated into EBY100 yeast using a linear yeast surface display vector with homologous recombination. The linear yeast display vector has a 5' (PAS) of the gene insertion. 40This is a c-terminal display vector characterized by an HA-tag (G4S)3 linker and a myc tag at the 3' of the gene insertion. Yeast cultures were grown for 1 day at 30°C in 50 mL of a synthetic medium of known composition containing glucose and casamino acid (SDCAA), and then subcultured once in SDCAA on day 2. The cell pellet was then transferred to 50 mL of SGCAA (galactose) culture at 18°C ​​for 2-3 days.

[0238] Sortable conditions were identified by titrating recombinant canine OSMR-hFc1 to yeast cells that presented the parent sequence and whose bound fragments were read using a flow cell analyzer (BD Accuri). Yeast cells presenting the parent scFv construct were titrated using a constant protein-to-presenting ligand approach, varying in volume over a sufficient number of labeling days until the expected label fraction reached 0.9. For all concentrations, the protein-to-presenting ligand ratio was at least 10:1 and remained constant across conditions. Cells were stained with chicken anti-myc FITC antibody (ICLlab) and protein A-AlexaFlour 647 to visualize full-length display and recombinant protein binding, respectively. Ligand binding profiles against concentration were calculated using the median 647 fluorescence of full-length presenting cells calculated via FlowJo. For the two library sorts, ligands with a labeling concentration of 100 nM were used for both recombinant canine and feline OSMR. Using a 100 μm chip, the library was sorted on an SH800 (Sony Biotechnology) in purity mode. In short, cells were gated to scattering, single-cell, and full-length display, and then bound to presentation. Cells below the diagonal of binding to presentation were collected (i.e., variants with reduced binding to the bulk population normalized for display). The number of cells collected for all sorts exceeded 67 times the theoretical library size for each.

[0239] For Illumina sequencing, a sorted reference library was prepared and sequenced to a read depth of at least 75 times oversampling for non-synonymous variants (Kowalsky et al. (2015) PLOS ONE 10, e0118193). The variant fitness z-score of the sorted variants against the reference library was calculated using PACT, with a reference library read count threshold of 12, and by removing undesigned variants from the total read counts (Klesmith et al. (2019) Bioinformatics 35, 2707-2712). The Z-score is defined as the ratio of the log2 abundance of each variant to the log2 abundance of the wild type, normalized by the variance of the wild-type synonymous codon abundance ([ε i -ε wt ] / σ wt,synon Using a Z-score threshold of 2.0, enriched mutations collected by sorting with reduced binding from tolerable mutations were separated. Thus, tolerable mutations are defined as those with a z-score less than 2.0.

[0240] Example 21. Results from YSD for determining amino acid residues on anti-OSMR antibodies important for binding to canid and feline OSMR. The results from these yeast display experiments illustrate an additional approach for determining relevant amino acid residues in the CDR binding domain of antibody 19F07. As described in Example 19, each independent method for analyzing changes in antibody affinity follows the experimental dynamics that occur during binding and dissociation events. Yeast display mutation analysis of antibody paratopes is performed in a fluid phase environment using the antibody binding portion (ScFv) presented on the surface of yeast cells, allowing access to the target antigen protein while linking genotype information encoding intracellular mutations. This allows for a correlation between the physical properties of the presented binding fragment and the DNA encoding the variable heavy and light chain fragments. This method not only allows for the combination of individual mutations at individual positions in the CDR with binding to the target protein, but also allows for the analysis of all amino acid mutations at individual positions in each CDR. These data make it possible to determine acceptable (enabling binding), unacceptable (detrimental to binding), or, in some situations, advantageous for binding (gain-of-function mutations) mutations.

[0241] Table B lists the mutations in the CDR of antibody Mu_19F07 that are acceptable according to the results of yeast surface display experiments. While data for all amino acid substitutions at each position were derived from this experiment, this table shows only those mutations at the listed positions where a smaller subset of amino acids is acceptable instead of the wild-type amino acid. These amino acid positions, where amino acid substitutions are rarely (or never) acceptable, indicate that these positions are more relevant by linking the structure of the antibody CDR to its functional outcome (binding to OSMR) (by binding to the target OSMR protein). Considering the results from ELISA and Biacore experiments for alanine substitutions, a more complete picture of the structural elements required for these anti-OSMR antibodies is observed. [Table B] Table B. Results from yeast display mutation analysis of each CDR against antibody 19F07, showing acceptable (binding-enabling) substitutions at each indicated position. The results described herein are from those mutations at amino acid positions where a limited number of acceptable substitutions affecting binding occurred, rather than from those positions where many acceptable substitutions occur.

[0242] Example 22. Results from YSD to determine amino acid residues on OSMR involved in anti-OSMR antibody binding. Example 20 describes a method used to determine the amino acid residues on the binding paratope of antibody 19F07 that are important for interaction with canid and feline OSMRs. Using a similar method, amino acid residues on feline OSMR that interact with feline IL-31 were determined. Feline OSMR (SEQ ID NO: 112 (Feline_OSMR_hIgG1_Fc)) was presented on the surface of yeast in its wild-type form, and the conditions for binding to feline IL-31 (SEQ ID NO: 125) were optimized. A variant library of feline OSMR was generated as described, and flow cytometry sorting combined with deep sequencing was performed to determine the mutations on feline OSMR that define the relevant amino acid contacts created using feline IL-31. These data reveal that the epitope for the binding of feline IL-31 to feline OSMR is a region on feline OSMR located between leucine 157 (L157) and phenylalanine 229 (F229) in SEQ ID NO: 112 (data not shown). It is important to note that these data support the homology model presented in Example 10 of this application, which describes the regions on canine and feline OSM that interact with OSMR. Functional data from cell-based assays described herein show that these selected anti-OSMR antibodies have inhibitory properties against both IL-31 and OSM-mediated pSTAT3 signaling, indicating that the binding sites of IL-31 and OSM on the OSMR protein are closely located. These mapping data certainly support the similar binding sites of feline IL-31 on feline OSMR that were proposed by the homology models of canine and feline OSM to their respective OSMR proteins. In summary, these results support knowledge of the relevant epitope spaces on canine and feline OSMRs necessary for antibody binding to inhibit OSMR signaling function in vivo.

[0243] Example 23. The pro-inflammatory role of canine open-synovial membrane (OSM) in canine synovial cells. Antagonism of OSMR signaling has been proven effective in inhibiting downstream activation of monocyte chemotactic protein-1 (MCP-1) in canine chondrocytes and canine arthrodetic synovial cells, significantly reducing oncostatin M (OSM)-induced cell proliferation in arthrodetic synovial cells in vitro, indicating potential novel therapeutics for reducing pain and inflammation in vivo. Anti-OSMR monoclonal antibodies represent a new class of novel therapeutics that block OSM signaling to alleviate pain and inflammation associated with osteoarthritis (OA) in canines and felines. To determine in vitro efficacy, anti-OSMR antibodies were evaluated for functional activity in arthrodetic synovial cells in cell-based assays for proliferation and inhibition. The effect of anti-OSMR mAbs on canine OSM-induced cell proliferation was evaluated using the CellTiter-GLO luminescent cell viability assay kit. Synovial cells derived from primary canid joints were isolated and determined to respond to OSM stimulation by activating the STAT-3 pathway, and to a lesser extent, by activating STAT-5 and STAT-1. Canid joint-derived synovial cells responded to OSM stimulation by rapid proliferation at 24 and 72 hours when plated on collagen type 1-coated plates (Figure 9a). The anti-OSMR antibody Mu_10F07 potently inhibited OSM-induced cell proliferation in canid joint-derived synovial cells (Figure 9b). Canid synovial cells also respond to OSM stimulation by rapidly synthesizing MCP-1, which recruits monocytes, macrophages, dendritic cells, and T cells to active inflammatory sites (Figure 10a). The anti-OSMR antibody 10F07 dose-dependently reduced canid OSM-induced MCP-1 synthesis. Overall, the results of these in vitro studies confirm the functional activity associated with the anti-inflammatory role of blocking OSMR signaling using novel anti-OSMR antibodies.

[0244] Example 24. The role of promoting fibrosis in OSM To investigate the role of OSM in renal fibrosis associated with chronic kidney disease in dogs and cats, activation of MCP-1 in primary canine renal fibroblasts was determined. Canine OSM (SEQ ID NO: 109) showed a dose-dependent increase in MCP-1 levels over a 72-hour period (Figure 11). Blocking this chemotactic protein in renal fibroblasts using an anti-OSMR antibody may be beneficial in altering the progression of fibrotic disease.

[0245] Example 25. Evaluation of anti-canine OSMR mAbs in a canine model of IL-31-induced pruritus. On day 7, the IL-31 challenge procedure was performed to establish baseline pruritus scores and day 7 pruritus scores. Intravenous solutions were prepared from stock concentrations of recombinant canine IL-31. Pruritus behavior was recorded for all dogs on day 7 of the study and after the last dog received the challenge on day 7. Pruritus behavior could also be recorded for individuals receiving a masked IL-31 challenge for treatment allocation.

[0246] The following classification scoring system was used to determine the pruritus score for each dog (post-challenge period). Specifically, each dog was judged as "yes" or "no" at consecutive 1-minute intervals regarding whether it exhibited pruritus behavior. The following presentations of pruritus behaviors were sufficient to elicit a "yes" response over each separate 1-minute time interval within the observation period. These behaviors included licking or biting (e.g., paws, flanks, tail, anal area), scratching (e.g., flanks or neck), shaking the head or body, and rubbing any part of the body (e.g., against the cage floor / walls). In the space provided for a particular 1-minute interval, a "yes" response was indicated by marking "1" and a "no" response by marking "0". The pruritus score was determined by the cumulative number of "yes" responses. Live feed cameras located directly above each pen in the monitoring room allowed scorers to observe the dogs from a separate room. Each scorer observed four dogs simultaneously, with real-time images of each dog displayed on a single monitor. The maximum possible itching score for the animals after the 120-minute challenge was 120. After the observation period ended, the dogs were returned to their normal enclosures.

[0247] The pruritus score for each animal was calculated by observing the total number of 1-minute time segments in which pruritus behavior was observed at each time point (-7 days and 7 days). A paired t-test was used to examine the mean difference in pruritus scores between -7 days and 7 days. Mice: Canid chimeric anti-OSMR antibodies 19F07(T01) and 10F07(T02) were administered as a single SC injection at 12.0 mg / kg, and the pruritus response was observed 7 days after administration. Figure 12 shows the post-challenge scores for a total observation period of 2 hours for the study's -7 days and study's 7 days. These results indicate that a single subcutaneous dose of 12 mg / kg of chimeric 19F07 and 10F07 resulted in a significantly lower total pruritus score on day 7 of the study compared to the pruritus score reported on day 7 of the study in a canine animal model of IL-31-induced pruritus.

Claims

1. 1) 02D09: Variable heavy chain (VH)-CDR1 (DYGMH) of SEQ ID NO: 1, VH-CDR2 (YISSGSRAVFFADTVKG) of SEQ ID NO: 2, VH-CDR3 (DRYDGRGFAY) of SEQ ID NO: 3, Variable light chain (VL)-CDR1 (RASQSISNNLH) of SEQ ID NO: 4, VL-CDR2 (YASQSIS) of SEQ ID NO: 5, and VL-CDR3 (QQSNSWPLT) of SEQ ID NO: 6 2) 09E09: Sequence ID 7 VH-CDR1 (SYAMS), Sequence ID 8 VH-CDR2 (YISSGGDYIYYADTVKG), Sequence ID 9 VH-CDR3 (DPITGTFAY), Sequence ID 10 VL-CDR1 (RASQDINNYLN), Sequence ID 11 VL-CDR2 (YTSTLHS), and Sequence ID 12 VL-CDR3 (QQGNTLPWT), 3) 10F07: Sequence ID 13 VH-CDR1 (SYAMS), Sequence ID 14 VH-CDR2 (YISSGGDYFYYADTVKG), Sequence ID 15 VH-CDR3 (DPITGTFAY), Sequence ID 16 VL-CDR1 (RASQDITNYLN), Sequence ID 17 VL-CDR2 (YTSTLHS), and Sequence ID 18 VL-CDR3 (QQGHMLPWT), 4) 14C04: VH-CDR1 (NYWMN) of SEQ ID NO: 19, VH-CDR2 (QIYPGHVNTNYNGNFKD) of SEQ ID NO: 20, VH-CDR3 (SADNSGFVLFAY) of SEQ ID NO: 21, VL-CDR1 (RASKSVSTSGYSYLH) of SEQ ID NO: 22, VL-CDR2 (LASNLES) of SEQ ID NO: 23, and VL-CDR3 (QHSRELPLT) of SEQ ID NO: 24, or 5) 19F07: Sequence ID 25 VH-CDR1 (DYYMA), Sequence ID 26 VH-CDR2 (NINYDGSSTYYLDSLKS), Sequence ID 27 VH-CDR3 (GLTWDFDV), Sequence ID 28 VL-CDR1 (KASQDVDTAVA), Sequence ID 29 VL-CDR2 (LASTRHT), and Sequence ID 30 VL-CDR3 (QQYSRFPLT) An isolated antibody, or its antigen-binding moiety, comprising a combination of complementarity-determining region (CDR) sequences selected from the group consisting of the following, wherein the antibody binds to at least one of canid or feline oncostatin M receptor beta (OSMR-β), and the antibody antagonistizes IL-31-mediated signaling, OSM-mediated signaling, or both, in canid and / or feline cells.

2. (a) Variable heavy chains including the following: Sequence ID 31 (MU_02D09_VH)EVQLVESGGGLLVKPGGSLTLSCAASGFTFFSDYGMHWLRQAPEKGLEWVAYISSGSRAVFFADTVKGRFTISRDNAKNTLFLQMTSLRSDDTAMYYYCARDRYDGRGFAYWGQGTLVTVSA, and Variable light chain including the following: Sequence ID 33 (MU_02D09_VL) DIVLTQSPATLSVTPGDSVSLSCLASQSISNNNLHWYQQTSHESPRLITYASQSISGIPSRFSGSGSGTDFFTLSINSVETEDDFGMYFCQQSNSWPLTFGAGTKLELK, (b) Variable heavy chains including the following: Sequence ID 35 (MU_09E09_VH)DVKLVESGEGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVAYISSGGDYIYYADTVKGRFTISRDNARNTLYLQMSSLKSEDTAMYYYCTRDPITGTFAYWGQGTLVTVSA, and Variable light chain including the following: Sequence ID 37 (MU_09E09_VL)DLQMTQTTSSLSASLGDRVTISCRASQDINNYLNWYQQKPDGTVKLLIYYTSTLHSGVPSRFSGSGSGTDYSLTISNLEEQEDIAATYFCQQGNTLLPWTFGGGTKLEIK, (c) Variable heavy chains including the following: Sequence ID 39 (MU_10F07_VH)DVKLVESGEGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVTYISSGGDYFYYADTVKGRFTISRDNARNTLYLQMSSLKSEDTAMYYYCTRDPITGTFAYWGQGTLVTVSA, and Variable light chain including the following: Sequence ID 41 (MU_10F07_VL)DIQMTQTTSSLSASLGDRVTISCRASQDITNYLNWYQQKPDGTVKLLIYYTSTLHSGVPSRFSGSGSGTDFSLTISNLEEQEDIATYFCQQGHMLPWTFGGGTKLEIK, (d) Variable heavy chains including the following: Sequence ID 43 (MU_14C04_VH) EVQLQESGAELVKPGASVKISCKASGYAFSNYWMNWMKQRPGKGLEWIGQIYPGHVNTNYNGNFKDKATLTADK SSSTAYMQLSSLTSEDSAVYFCARSADNSGFVLFAYWGQGTLVTVS, and Variable light chain including the following: Sequence ID 45 (MU_14C04_VL) DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIFLASNLESGVPPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLELK, (e) Variable heavy chains including the following: Sequence ID 47 (MU_19F07_VH) EVKLVESEGGLVQPGSSMKLSCTASGFTFSDYYMAWVRQVPEKGLEWVANINYDGSSTYYLDSLKSRFIISRDNAKNILY LQMSSLKSEDTATYYCARGLTWWDFDVWGTGTTVTVSS, and Variable light chain including the following: Sequence ID 49 (MU_19F07_VL) DIVMTQSHKFMSPSVGGDRVSITCKASQDVDTAVAWYQQKPGQSPKLLIYLASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSRFPLTFGAGTKLELK, (f) Variable heavy chains including the following: Sequence ID 51 (FEL_02D09_VH1)DVQLVESGGDLVKPGGSLRLTCVASGFTYSDYGMHWVRQAPGKGLQWVAYISSGSSRAVFFADTVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCVRDRYDGRGFAYWGQGTLVTVSS, and Variable light chain including the following: Sequence ID 55 (FEL_02D09_VL1)EIQMTQSPSSLSASPGDRVTITCRASQSISNNLHWYQQKPGKVPKLLLIYYASQSISGVPSRFSGSGSGTDFTLTISSLEPEDAATYYCQQSNSWPLTFGQGT, Variable heavy chains containing (g) or less: Sequence ID 53 (FEL_02D09_VH2)DVQLVESGGDLVKPGGSLRLTCVASGFTFSDYGMHWVRQAPGKGLQWVAYISSGSRAVFFADTVKGRFTISRDNAKNTLYLQMNGLRTEDTATYYCARDRYDGRGFAYWGQGTLVTVSS, and Variable light chain including the following: Sequence ID 57 (FEL_02D09_VL2) DIVMTQTPPLSLSVTPPGESASISCRASQSISNNLHWYLQKSGQSPRRLIYYASQSISGVPDRFSGSGSGTDFTTLRISRVEADDVGVYYCQQSNSWPLTFGQGT, Variable heavy chains including (h) below: Sequence ID 59 (CAN_09E09_VH1)EVQLVESGGDLLVKPGGSLRLSCVASGFTFSSSYAMSWVRQAPGKGLQWVAYISSGGDYIYYADTVKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYYCVRDPITGTFAYWGQGTLVTVSS, and Variable light chain including the following: Sequence ID 63 (CAN_09E09_VL1) EIVMTQSPASLSLSQEEKVTITCRASQDINNYLNWYQQKPGQAPKLLLIYYTSTLHSGVPSRFSGSGSGTDFSFTISSLEPEDVAVYYCQQGNTLLPWTFGQGT, (i) Variable heavy chains including the following: Sequence ID 61 (CAN_09E09_VH2)EVQLVESGGDLLVKPAGSLTLSCLASGFTFSSSYAMSWVRQTPEKGLQWVAYISSGGDYIYYADTVKGRFTISRDNAKNTLYLQMNSLRRDEDTAVYYCARDPITGTFAYWGQGTLVTVSS, and Variable light chain including the following: Sequence ID 65 (CAN_09E09_VL2) DIVLTQPTSVSGSLGQRVTISCRASQDINNYLNWYQQLPGKAAPKLLLVYYTSTLHSGVPDRFSGSNSGSATLTITGLQAEDEADYYCQQGNTLLPWTFGQGT, Variable heavy chains including (j) and below: Sequence ID 67 (FEL_09E09_VH1)DVQLVESGGDLVKPGGSLRLTCVVASGFTYSSYAMSWVRQAPGKGLQWVAYISSGGDYIYYADTVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCVRD PITGTFAYWGQGTLVTVSS, and Variable light chain including the following: Sequence ID 71 (FEL_09E09_VL1)EIQMTQSPSSLSASPGDRVTITCRASQDINNYLNWYQQKPGKVPKLLLIYYTSTLHSGVPSRFSGSGSGTDFTLTISSLEPEDAATYYCQQGNTLLPWTFGQGT, Variable heavy chains including (k) and below: Sequence ID 69 (FEL_09E09_VH2)DVQLVESGGGNLVKPGGSLRLTCVASGFTFSSYAMSWVRQAPGKGLQWVAYISSGGDYIYYADTVKGRFTISKDNAKNTLYLQMNSLKTEDTATYYCARDPITGTFAYWGQGTLVTVSS, and Variable light chain including the following: Sequence ID 73 (FEL_09E09_VL2) DITMTQSPGSLAGSPGQQVTMNCRASQDINNYLNWYQQKPGQHPKLLLIYYTSTLHSGVPDRFSGSGSGTDFTLTISNLQAEDVASYYCQQGNTLLPWTFGQGT, (l) Variable heavy chains including the following: Sequence ID 75 (CAN_10F07_VH1)EVQLVESGGDLLVKPGGSLRLSCVASGFTFSSSYAMSWVRQAPGKGLQWVAYISSGGDYFYYADTVKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCVRDPITGTFAYWGQGTLVTVSS, and Variable light chain including the following: Sequence ID 77 (CAN_10F07_VL1) EIVMTQSPASLSLSQEEKVTITCRASQDITNYLNWYQQKPGQAPKLLLIYYTSTLHSGVPSRFSGSGSGTDFSFTISSLEPEDVAVYYCQQGHMLLPWTFGQGT, Variable heavy chains including (m) or less: Sequence ID 79 (CAN_10F07_VH2)EVQLVESGGDLLVKPAGSLTLSCLASGFTFSSSYAMSWVRQTPEKGLQWVAYISSGGDYFYYADTVKGRFTISRDNAKNTLYLQMNSLRDEDTAVYYCARDPITGTFAYWGQGTLVTVSS, and Variable light chain including the following: Sequence ID 81 (CAN_10F07_VL2) DIVLTQPTSVSGSLGQRVTISCRASQDITNYLNWYQQLPGKAAPKLLLVYYTSTLHSGVPDRFSGSNSGSATLTITITGLQAEDEADYYCQQGHMLLPWTFGQGT, Variable heavy chains including (n) and below: Sequence ID 83 (FEL_10F07_VH1)DVQLVESGGDLVKPGGSLRLTCVASGFTYSSYAMSWVRQAPGKGLQWVAYISSGGDYFYYADTVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCVRDPITGTFAYWGQGTLVTVSS, and Variable light chain including the following: Sequence ID 85 (FEL_10F07_VL1)EIQMTQSPSSLSASPGDRVTITCRASQDITNYLNWYQQKPGKVPKLLLIYYTSTLHSGVPSRFSGSGSGTDFTLTISSLEPEDAATYYCQQGHMLLPWTFGQGT, (o) Variable heavy chains including the following: Sequence ID 87 (FEL_10F07_VH2)DVQLVESGGDLVKPGGSLRLTCVVASGFTFSSYAMSWVRQAPGKGLQWVAYISSGGDYFYYADTVKGRFTISRDDAKNTLYLQMSSLKTEDTATYYCTGD PITGTFAYWGQGTLVTVSS, and Variable light chain including the following: Sequence ID 89 (FEL_10F07_VL2) DITMTQSPGSLAGSPGQQVTMNCRASQDITNYLNWYQQKPGQHPKLLLIYYTSTLHSGVPDRFSGSGSGTDFTLTISNLQAEDVASYYCQQGHMLPWTFGQGT, Variable heavy chains including (p) and below: Sequence ID 91 (CAN_19F07_VH1)EVQLVESGGDLVKPGGSLRLSCVASGFTFFSDYYMAWVRQAPGKGLQWVANINYDGSSTYYLDSLKSRFTISRDNAKNTLYLQMNSLRAEDTAMYYCVRGLLTWDFDDVWGQGTLVTVSS, and Variable light chain including the following: Sequence ID 93 (CAN_19F07_VL1) EIVMTQSPASLSLSQEEKVTITCKASQDVDTAVAWYQQKPGQAPKLLLIYLASTRHTGVPSRFSGSGSGTDFSFTISSLEPEDVAVYYCQQYSRFPLTFGQGT, Variable heavy chains including (q) and below: Sequence ID 95 (CAN_19F07_VH2)EVQLVESGGDLLVKPAGSLTLSCLASGFTFFSDYYMAWVRQTPEKGLQWVANINYDGSSTYYLDSLKSRFTISRDNAKNTLYLQMNSLRRDEDTAVYYCARGLTWWDFDVWGQGTLVTVSS, and Variable light chain including the following: Sequence ID 97 (CAN_19F07_VL2) DIVMTQTPPLSLSVSPGETASISCKASQDVDTAVAWFRQKPGQSPQRLIYLASTRHTGVPDRFSGSGSGTDFTTLRISRVEADDTGVYYCQQYSRFPLTFGQGT, Variable heavy chains including (r) below: Sequence ID 99 (FEL_19F07_VH1)DVQLVESGGDLVKPGGSLRLTCVASGFTYSDYYMAWVRQAPGKGLQWVANINYDGSSTYYLDSLKSRFTISRDNAKNTLYLQMNSLKTEDTATYYCVRGLLTWDDFDVWGQGTLVTVSS, and Variable light chain including the following: Sequence ID 101 (FEL_19F07_VL1)EIQMTQSPSSLSASPGDRVTITCKASQDVDTAVAWYQQKPGKVPKLLLIYLASTRHTGVPSRFSGSGSGTDFTLTISSLEPEDAATYYCQQYSRFPLTFGQGT, Variable heavy chains including (s) and below: Sequence ID 103 (FEL_19F07_VH2)DVQLVESGGGNLVKPGGSLRLTCVASGFTFSDYYMAWVRQAPGKGLQWVANINYDGSSTYYLDSLKSRFTISRDNAKNTLYLQMNSLKTEDTATYYCARGLTWWDFDVWGQGTLVTVSS, and Variable light chain including the following: Sequence ID 105 (FEL_19F07_VL2) DITMTQSPGSLAGSPGQQVTMNCKASQDVDTAVAWYQQKPGQHPKLLLIYLASTRHTGVPDRFSGSGSGTDFTLTISNLQAEDVASYYCQQYSRFPLTFGQGT, Variable heavy chains including (t) or less: Sequence ID 127. (CAN_14C04_VH1) EVQLVESGGDLVKPGGSLRLSCVASGFTFSNYWMNWVRQAPGKGLQWVAQIYPGHVNTNYNGNFKDRFTISRDNARNTVY LQMNSLRAEDTAVYYCARSADNNSGFVLFAYWGQGTLVTVSS, and Variable light chain including the following: Sequence ID 131. (CAN_14C04_VL1) EIVMTQSPASLSLSQEEKVTITCRASKSVSSTSGYSYLHWYQQKPGQAPKLLLIYLASNLESGVPSRFSGSGSGTTDFSFTISSLEPEDVAVYYCQHSRELPLTFGQGT, or Variable heavy chains including (u) below: Sequence ID 129. (CAN_14C04_VH2) EVQLVESGGDLVKPGGSLRLSCVASGFTFSNYWMNWVRQSPGKGLQWVAQIYPGHVNTNYNGNFKDRFTISRDNAKNTLYLQMNSLRAEDTAVYFCCARSADNSGFVLFAYWGQGTLVTVSS; and Variable light chain including the following: Sequence ID 133. (CAN_14C04_VL2) DIVMTQTPLSLSVSPGETASISCRASKSVSTSGYSYLHWYLQKPGQSPQLLIYLASNLESGVSKRFSGSGSGTDFTLRISRVEADDDTGIYYCQHSRELPLTFGQGT The antibody according to claim 1, comprising at least one from the group consisting of the following.

3. The antibody according to claim 1 or 2, wherein the antibody is a chimeric antibody.

4. The antibody according to claim 1 or 2, wherein the antibody is canine-like or feline-like.

5. The antibody according to claim 1 or 2, wherein the antibody inhibits or neutralizes IL-31-mediated or OSM-mediated pruritic or allergic conditions in dogs or cats.

6. The antibody according to claim 5, wherein the IL-31-mediated or OSM-mediated pruritic condition is selected from the group consisting of atopic dermatitis, eczema, psoriasis, scleroderma, and pruritus.

7. The antibody according to claim 5, wherein the IL-31-mediated or OSM-mediated allergic condition is selected from the group consisting of allergic dermatitis, summer eczema, urticaria, respiratory fatigue, inflammatory airway disease, recurrent airway obstruction, airway hypersensitivity, chronic obstructive pulmonary disease, and inflammatory processes resulting from autoimmunity.

8. The antibody according to claim 1 or 2, wherein the antibody inhibits IL-31-mediated or OSM-mediated fibrous or inflammatory disorders.

9. The antibody according to claim 8, wherein the IL-31-mediated or OSM-mediated fibrosis is selected from the group consisting of renal fibrosis, pulmonary fibrosis, and cutaneous fibrosis.

10. The antibody according to claim 8, wherein the IL-31-mediated or OSM-mediated inflammatory disorder is selected from the group consisting of autoimmune inflammatory processes, inflammation of the skin or joints of animals suffering from osteoarthritis, immune-mediated polyarthritis, chronic bronchitis, allergic asthma, atopic dermatitis, allergic dermatitis, suppurative traumatic dermatitis, atherosclerosis, and cardiovascular disease.

11. The antibody according to claim 1 or 2, wherein the antibody reduces IL-31-mediated or OSM-mediated inflammatory pain.

12. The antibody according to claim 11, wherein the IL-31-mediated or OSM-mediated inflammatory pain is osteoarthritis pain.

13. A veterinary composition comprising a therapeutically effective amount of the antibody according to claim 1 or 2.

14. A veterinary composition for use in a method for treating IL-31-mediated or OSM-mediated disorders in a subject, the veterinary composition according to claim 13.

15. The veterinary composition according to claim 14, wherein the IL-31-mediated or OSM-mediated disorder is selected from the group consisting of pruritic state, allergic state, fibrous disorder, inflammatory disorder, and inflammatory pain.

16. The veterinary composition according to claim 15, wherein the IL-31-mediated or OSM-mediated pruritic condition is selected from the group consisting of atopic dermatitis, eczema, psoriasis, scleroderma, and pruritus.

17. The veterinary composition according to claim 15, wherein the IL-31-mediated or OSM-mediated allergic condition is selected from the group consisting of allergic dermatitis, summer eczema, urticaria, respiratory fatigue, inflammatory airway disease, recurrent airway obstruction, airway hypersensitivity, chronic obstructive pulmonary disease, and inflammatory processes resulting from autoimmunity.

18. The veterinary composition according to claim 15, wherein the IL-31-mediated or OSM-mediated fibrosis is selected from the group consisting of renal fibrosis, pulmonary fibrosis, and cutaneous fibrosis.

19. The veterinary composition according to claim 15, wherein the IL-31-mediated or OSM-mediated inflammatory disorder is selected from the group consisting of autoimmune inflammatory processes, inflammation of the skin or joints of animals suffering from osteoarthritis, immune-mediated polyarthritis, chronic bronchitis, allergic asthma, atopic dermatitis, allergic dermatitis, suppurative traumatic dermatitis, atherosclerosis, and cardiovascular disease.

20. The veterinary composition according to claim 15, wherein the IL-31-mediated or OSM-mediated inflammatory pain is osteoarthritis pain.

21. The veterinary composition according to any one of claims 14 to 20, wherein the subject is a dog or a cat.

22. A method for inhibiting IL-31 and / or OSM activity in dogs or cats, comprising administering the antibody described in claim 1 or 2 to the dogs or cats.

23. A method for detecting OSMR beta in a sample, a) Incubating a sample containing OSMR beta in the presence of the antibody described in claim 1 or 2, b) A method comprising detecting the antibody that binds to OSMR beta in the sample.

24. The method according to claim 23, wherein the antibody includes a label.

25. The method according to claim 23 or 24, further comprising quantifying the OSMR beta in the sample.

26. Host cells with the following combinations of complementarity-determining region (CDR) sequences: 1) 02D09: Variable heavy chain (VH)-CDR1 (DYGMH) of SEQ ID NO: 1, VH-CDR2 (YISSGSRAVFFADTVKG) of SEQ ID NO: 2, VH-CDR3 (DRYDGRGFAY) of SEQ ID NO: 3, Variable light chain (VL)-CDR1 (RASQSISNNLH) of SEQ ID NO: 4, VL-CDR2 (YASQSIS) of SEQ ID NO: 5, and VL-CDR3 (QQSNSWPLT) of SEQ ID NO: 6 2) 09E09: Sequence ID 7 VH-CDR1 (SYAMS), Sequence ID 8 VH-CDR2 (YISSGGDYIYYADTVKG), Sequence ID 9 VH-CDR3 (DPITGTFAY), Sequence ID 10 VL-CDR1 (RASQDINNYLN), Sequence ID 11 VL-CDR2 (YTSTLHS), and Sequence ID 12 VL-CDR3 (QQGNTLPWT), 3) 10F07: Sequence ID 13 VH-CDR1 (SYAMS), Sequence ID 14 VH-CDR2 (YISSGGDYFYYADTVKG), Sequence ID 15 VH-CDR3 (DPITGTFAY), Sequence ID 16 VL-CDR1 (RASQDITNYLN), Sequence ID 17 VL-CDR2 (YTSTLHS), and Sequence ID 18 VL-CDR3 (QQGHMLPWT), 4) 14C04: VH-CDR1 (NYWMN) of SEQ ID NO: 19, VH-CDR2 (QIYPGHVNTNYNGNFKD) of SEQ ID NO: 20, VH-CDR3 (SADNSGFVLFAY) of SEQ ID NO: 21, VL-CDR1 (RASKSVSTSGYSYLH) of SEQ ID NO: 22, VL-CDR2 (LASNLES) of SEQ ID NO: 23, and VL-CDR3 (QHSRELPLT) of SEQ ID NO: 24, or 5) 19F07: Sequence ID 25 VH-CDR1 (DYYMA), Sequence ID 26 VH-CDR2 (NINYDGSSTYYLDSLKS), Sequence ID 27 VH-CDR3 (GLTWDFDV), Sequence ID 28 VL-CDR1 (KASQDVDTAVA), Sequence ID 29 VL-CDR2 (LASTRHT), and Sequence ID 30 VL-CDR3 (QQYSRFPLT), A host cell that produces an isolated antibody, or its antigen-binding moiety, containing the specified substance.

27. ​​An isolated antibody according to Claim 1, or an isolated nucleic acid encoding the antigen-binding portion thereof, comprising the following combination of variable heavy chain (VH) and variable light chain (VL) complementarity-determining region (CDR) sequences: 1) 02D09: Sequence ID 1 VH-CDR1 (DYGMH), Sequence ID 2 VH-CDR2 (YISSGSRAVFFADTVKG), Sequence ID 3 VH-CDR3 (DRYDGRGFAY), Sequence ID 4 VL-CDR1 (RASQSISNNLH), Sequence ID 5 VL-CDR2 (YASQSIS), and Sequence ID 6 VL-CDR3 (QQSNSWPLT); 2) 09E09: VH-CDR1 (SYAMS) of SEQ ID NO: 7, VH-CDR2 (YISSGGDYIYYADTVKG) of SEQ ID NO: 8, VH-CDR3 (DPITGTFAY) of SEQ ID NO: 9, VL-CDR1 (RASQDINNYLN) of SEQ ID NO: 10, VL-CDR2 (YTSTLHS) of SEQ ID NO: 11, and VL-CDR3 (QQGNTLPWT) of SEQ ID NO: 12; 3) 10F07: Sequence ID 13 VH-CDR1 (SYAMS), Sequence ID 14 VH-CDR2 (YISSGGDYFYYADTVKG), Sequence ID 15 VH-CDR3 (DPITGTFAY), Sequence ID 16 VL-CDR1 (RASQDITNYLN), Sequence ID 17 VL-CDR2 (YTSTLHS), and Sequence ID 18 VL-CDR3 (QQGHMLPWT); 4) 14C04: VH-CDR1 (NYWMN) of SEQ ID NO: 19, VH-CDR2 (QIYPGHVNTNYNGNFKD) of SEQ ID NO: 20, VH-CDR3 (SADNSGFVLFAY) of SEQ ID NO: 21, VL-CDR1 (RASKSVSTSGYSYLH) of SEQ ID NO: 22, VL-CDR2 (LASNLES) of SEQ ID NO: 23, and VL-CDR3 (QHSRELPLT) of SEQ ID NO: 24; or 5) 19F07: VH-CDR1 (DYYMA) of SEQ ID NO: 25, VH-CDR2 (NINYDGSSTYYLDSLKS) of SEQ ID NO: 26, VH-CDR3 (GLTWDFDV) of SEQ ID NO: 27, VL-CDR1 (KASQDVDTAVA) of SEQ ID NO: 28, VL-CDR2 (LASTRHT) of SEQ ID NO: 29, and VL-CDR3 (QQYSRFPLT) of SEQ ID NO: 30; A nucleic acid sequence comprising encoding at least one of the following: Isolated nucleic acids containing the VH and VL CDR sequences 1) to 5) on the same vector or on separate vectors.

28. A method for producing an antibody, comprising: culturing the host cells described in claim 26 under conditions that result in the production of the antibody; and isolating the antibody from the host cells or the culture medium of the host cells.

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