Interleukin-31 monoclonal antibodies for veterinary use

Abstract

To provide antibodies in the form of veterinary compositions useful for treating IL-31-mediated disorders in cats, dogs, or horses.SOLUTION: A monoclonal antibody or antigen binding portion thereof is provided that specifically binds to a region on a mammalian IL-31 protein involved with interaction of the IL-31 protein with its co-receptor, where the binding of the antibody to the region is impacted by mutations in a 15H05 epitope binding region selected from: a region between about amino acid residues 124 and 135 of a feline IL-31 sequence represented by a specific sequence (Feline_IL31_wildtype); a region between about amino acid residues 124 and 135 of a canine IL-31 sequence represented by a specific sequence (Canine_IL31); and a region between about amino acid residues 118 and 129 of an equine IL-31 sequence represented by a specific sequence (Equine_IL31).SELECTED DRAWING: Figure 26

Description

[Technical Field]

[0001] The present invention relates to the field of recombinant monoclonal antibodies and their use in clinical and scientific procedures, including diagnostic procedures. The present invention also provides isolated anti-IL31 antibodies in the form of veterinary compositions useful for treating IL-31 disorders in mammals, such as cats, dogs, or horses. [Background technology]

[0002] Atopic dermatitis has been defined by the American College of Veterinary Dermatology Task Force as "a genetically predisposed inflammatory and pruritic allergic skin disease with distinctive clinical features" (Olivry, et al. Veterinary Immunology and Immunopathology 2001;81:143-146). The Task Force also recognizes that the canine disease is associated with allergen-specific IgE (Olivry, et al. al. 2001 (ibid.); Marsella & Olivry Clinics in Dermatology 2003;21:122-133). Severe pruritus, along with secondary alopecia and erythema, are among the most noticeable and worrisome symptoms for pet owners.

[0003] The potential factors involved in allergic dermatitis are numerous and poorly understood. In some cases, atopic dermatitis may be triggered by food components (Picco, et al. Vet Dermatol. 2008;19:150-155), while in other cases, it may be triggered by environmental allergens such as fleas, dust mites, ragweed, or plant extracts. Genetic factors also play an important role. While no breed bias has been identified, certain genetic patterns are thought to predispose to atopic dermatitis (Sousa & Marsella Veterinary Immunology and Immunopathology 2001;81:153-157; Schwartzman, et al. Clin. Exp. Immunol. 1971;9:549-569).

[0004] The prevalence of atopic dermatitis is estimated to be 10% of the total canine population (Marsella & Olivry 2003, supra; Scott, et al. Canadian Veterinary Journal 2002;43:601-603; Hillier Veterinary Immunology and Immunopathology 2001;81:147-151). Approximately 4.5 million dogs worldwide are affected by this chronic, lifelong condition. The incidence appears to be increasing. A breed and sex predilection is suspected, but there may be significant variation depending on geographic region (Hillier, 2001, supra; Picco, et al. 2008, supra).

[0005] Feline allergic dermatitis is an inflammatory and pruritic skin condition thought to be caused by an abnormal immune system response to substances that do not induce a reaction in healthy cats. The most consistent feature of feline allergic dermatitis is chronic recurrent pruritus. Common clinical signs of feline allergic dermatitis include self-induced alopecia, miliary dermatitis, eosinophilic granulomatous complex lesions (including plaques, granulomas, and painless ulcers), and pruritus localized on the head and neck characterized by excoriation, erosions, and / or ulcers. Although no breed or gender predilection has been demonstrated, young cats appear to be more susceptible to the disease (Hobi et al. Vet Dermatol 2011 22:406-413; Ravens et al.Vet Dermatol 2014;25:95-102;Buckely In Practice 2017;39:242-254).

[0006] Current treatments for cats diagnosed with allergic dermatitis depend on the severity and duration of clinical signs, and owner preference, and include allergen-specific immunotherapy and antipruritic medications such as glucocorticoids and cyclosporine (Buckley, supra). Immunotherapy treatments are effective in some patients, but require frequent injections and clinical improvement may not occur for 6 to 9 months (Buckley, supra). Immunosuppressants such as glucocorticoids and cyclosporine are generally effective, but long-term use often results in undesirable adverse effects.

[0007] Equine atopic dermatitis (AD) is recognized as a potential cause of pruritus. The role of environmental allergens in Equine atopic dermatitis is becoming more fully understood. The disease can be seasonal or nonseasonal, depending on the allergen(s) involved. Age, breed, and sex predilection have not been widely reported. A preliminary study from the School of Veterinary Medicine, University of California, Davis (SVM-UCD) found that the median age at onset was 6.5 years, Thoroughbreds were the most common breed, accounting for 25% of horses, and males (usually geldings) were nearly twice as prevalent as females. However, these data are from only 24 horses, and comparisons with the overall hospital population have not yet been performed. Pruritus, often directed toward the face, distal limbs, or trunk, is the most common clinical sign in Equine atopic dermatitis. Alopecia, erythema, urticaria, and papules may all be present. Urticarial lesions are very severe but not pruritic. There may be a familial predisposition to urticarial atopic dermatitis in horses. Horses may have secondary pyoderma, characterized by excessive scaling, small epidermal ringlets, or crusted papules ("miliary dermatitis"). Diagnosis of atopic dermatitis is based on clinical signs and the exclusion of other diagnoses, particularly insect (Culicoides) hypersensitivity (White Clin Tech Equine Pract 2005;4:311-313; Fadok Vet Clin Equine 2013;29 541-550). Currently, management of equine atopic dermatitis is achieved symptomatically by suppressing the inflammation and pruritus induced by the allergic response, as well as by addressing the specific cause (i.e., by identifying the causative allergen and formulating an allergen-specific vaccine). A symptomatic approach is typically required short-term to provide patient comfort and minimize self-trauma. This approach relies on a combination of topical and systemic therapies, including antihistamines, essential fatty acids, pentoxifylline, and glucocorticoids. The primary approach to environmental allergy control involves identifying the allergen that elicits the hypersensitivity reaction.It is generally accepted by dermatologists that allergen-specific immunotherapy can be useful in atopic horses, although as a rule, most horses only show improvement after the first six months of immunotherapy (Marsella Vet. Clin Equine 2013;29:551-557). Additionally, long-term use of immunosuppressive drugs in horses may result in unwanted adverse effects.

[0008] Interleukin-31 (IL-31), a cytokine produced by type 2 helper T cells, has been shown to induce pruritus in humans, mice, and dogs (Bieber N Engl J Med 2008;358:1483-1494;Dillon et al. al.Nat Immunol 2004;5:752-60;Bammert et al. (U.S. Patent No. 8,790,651 to Gonzalez et al.; Gonzalez et al. Vet Dermatl. 2013;24(1):48-53). IL-31 binds to a coreceptor composed of IL-31 receptor A (IL-31RA) and oncostatin M receptor (OSMR) (Dillon et al. 2004 (ibid.) and Bilsborough et al. J Allergy Clin Immunol. 2006 117(2):418-25). Receptor activation results in the phosphorylation of STATs via JAK receptor(s). Coreceptor expression is increased in macrophages, keratinocytes, and dorsal root canals. Shown in ganglia.

[0009] Recently, IL-31 has been found to be involved in dermatitis, pruritic skin lesions, allergies, and airway hyperresponsiveness. Cytopoint®, a canine anti-IL-31 monoclonal antibody manufactured by Zoetis Inc. (Parsippany, NJ), has been shown to reduce pruritus and skin lesions in dogs with atopic dermatitis (Gonzalez et al. 2013 (supra); Michels et al. Vet Dermatol. 2016; Dec; 27(6):478-e129). It would be desirable to provide additional anti-IL-31 antibodies for preventing and treating IL-31-mediated disorders in veterinary mammals. Given the current unmet need for safe and effective alternative treatments for atopic and allergic dermatitis in cats and horses, it would be particularly desirable to provide feline and equine anti-IL-31 antibodies for reducing pruritus and skin lesions in cats and horses with atopic dermatitis. Summary of the Invention

[0010] In one embodiment, the invention provides a monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to a region on a mammalian IL-31 protein that is involved in the interaction of the mammalian IL-31 protein with its co-receptor, wherein binding of the antibody to the region is affected by mutations in the 15H05 epitope binding region selected from at least one of the following: a) a region between about amino acid residues 124 and 135 of the feline IL-31 sequence represented by SEQ ID NO: 157 (feline_IL31_wildtype); b) a region between about amino acid residues 124 and 135 of the canine IL-31 sequence represented by SEQ ID NO: 155 (canine_IL31); and c) a region between about amino acid residues 118 and 129 of equine IL-31 represented by SEQ ID NO: 165 (equine_IL31).

[0011] In one embodiment, the mutations in the 15H05 epitope binding region are selected from at least one of the following: (a) a mutation in which positions 126 and 128 of SEQ ID NO: 157 are changed to alanine; (b) a mutation in which positions 126 and 128 of SEQ ID NO: 155 are changed to alanine; and (c) a mutation in which positions 120 and 122 of SEQ ID NO: 165 are changed to alanine.

[0012] In one embodiment, a monoclonal antibody according to the invention binds to the 15H05 epitope region. That is, in one embodiment, the invention provides a monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to a region on a mammalian IL-31 protein involved in the interaction of the mammalian IL-31 protein with its co-receptor, wherein the binding region is a 15H05 epitope binding region selected from at least one of the following: a) a region between about amino acid residues 124 and 135 of the feline IL-31 sequence represented by SEQ ID NO: 157 (feline_IL31_wildtype), b) a region between about amino acid residues 124 and 135 of the canine IL-31 sequence represented by SEQ ID NO: 155 (canine_IL31), and c) a region between about amino acid residues 118 and 129 of equine IL-31 represented by SEQ ID NO: 165 (equine_IL31).

[0013] In one embodiment, the mammalian IL-31 to which the antibody or antigen-binding portion thereof specifically binds is feline IL-31, wherein the antibody binds to a region between about amino acid residues 125 and 134 of the feline IL-31 sequence represented by SEQ ID NO: 157 (feline_IL31_wildtype). In some embodiments, the antibody that binds to this region on feline IL-31 comprises a VL chain comprising framework 2 (FW2) alterations selected from the following: a lysine to asparagine substitution at position 42, a valine to isoleucine substitution at position 43, a leucine to valine substitution at position 46, a lysine to asparagine substitution at position 49, and combinations thereof, wherein these positions are represented by SEQ ID NO: 127 (FEL_15H05_VL1). It conforms to the numbering system.

[0014] In one embodiment, the mammalian IL-31 to which the antibody or antigen-binding portion thereof specifically binds is canine IL-31, wherein the antibody binds to a region between about amino acid residues 125 and 134 of the canine IL-31 sequence represented by SEQ ID NO: 155 (canine_IL31).

[0015] In another embodiment, the mammalian IL-31 to which the antibody or antigen-binding portion thereof specifically binds is equine IL-31, wherein the antibody binds to a region between about amino acid residues 117 and 128 of equine IL-31 represented by SEQ ID NO: 165 (equine_IL31).

[0016] In one embodiment, the monoclonal antibody or antigen-binding portion thereof comprises the following combination of complementarity determining region (CDR) sequences: 1) Antibody 15H05: variable heavy chain (VH)-CDR1 is SYTIH (SEQ ID NO: 1), VH-CDR2 is NINPTSGYTENNQRFKD (SEQ ID NO: 2), VH-CDR3 is WGFKYDGEWSFDV (SEQ ID NO: 3), variable light chain (VL)-CDR1 is RASQGISIWLS (SEQ ID NO: 4), VL-CDR2 is KASNLHI (SEQ ID NO: 5), VL-CDR3 is LQSQTYPLT (SEQ ID NO: 6), or 2) A variant of 1) that differs from the parent antibody 15H05 by the addition, deletion, and / or substitution of one or more amino acid residues in at least one of CDR1, CDR2, or CDR3 of VH or VL. Includes.

[0017] In one embodiment, a variant of antibody 15H05 / 1505 comprises substitutions at one or more of the following positions within the CDRs: residue 4 (I) of SEQ ID NO: 1, residues 1-3 (NIN), 5-7 (TSG), 9-11 (TEN), and 13 (Q) of SEQ ID NO: 2, and residues 4 (K), 6 (D), and 13 (V) of SEQ ID NO: 3 in heavy chain CDRs 1, 2, and 3, respectively, and residues 3-7 (SQGIS) of SEQ ID NO: 4, residues 3 (S) and 5 (L) of SEQ ID NO: 5, and residues 4 (Q), 5 (T), and 9 (T) of SEQ ID NO: 6 in CDRLs 1, 2, and 3, respectively. In one embodiment, one or more of these substitutions are conservative amino acid substitutions.

[0018] In one embodiment, the monoclonal antibody 15H05 is selected from the group consisting of: a)FEL_15H05_VL1_FW2: a variable light chain comprising: EIQMTQSPSSLSASPGDRVTITCRASQGISIWLSWYQQKPGNIPKVLINKASNLHIGVPSRFSGSGSGTDFTLTISSLEPEDAATYYCLQSQTYPLTFGGGTKLEIK (SEQ ID NO: 135), and b) FEL_15H05_VH1: a variable heavy chain comprising QVLLVQSGAEVRTPGASVKIFCKASGYSFTSYTIHWLRQAPAQGLEWMGNINPTSGYTENNQRFKDRLTLTADTSTNTAYMELSSLRSADTAMYYCARWGFKYDGEWSFDVWGAGTTVTVSS (SEQ ID NO: 121) It includes at least one of the following:

[0019] The present invention also provides a combination of the following complementarity determining region (CDR) sequences: 1) Antibody ZIL1: variable heavy chain (VH)-CDR1 is SYGMS (SEQ ID NO: 13), VH-CDR2 is HINSGGSSTYYADAVKG (SEQ ID NO: 14), VH-CDR3 is VYTTLAAFWTDNFDY (SEQ ID NO: 15), variable light chain (VL)-CDR1 is SGSTNNIGILAAT (SEQ ID NO: 16), and VL-CDR2 is SDGNRPS (SEQ ID NO: 1 7), and VL-CDR3 is QSFDTTLDAYV (SEQ ID NO: 18), 2) antibody ZIL8: VH-CDR1 is DYAMS (SEQ ID NO: 19), VH-CDR2 is GIDSVGSGTSYADAVKG (SEQ ID NO: 20), VH-CDR3 is GFPGSFEH (SEQ ID NO: 21), VL-CDR1 is TGSSSNIGSGYVG (SEQ ID NO: 22), VL-CDR2 is YNSDRPS (SEQ ID NO: 23), VL-CDR3 is SVYDRTFNAV (SEQ ID NO: 24), 3) antibody ZIL9: VH-CDR1 is SYDMT (SEQ ID NO: 25), VH-CDR2 is DVNSGGTGTAYAVAVKG (SEQ ID NO: 26), VH-CDR3 is LGVRDGLSV (SEQ ID NO: 27), VL-CDR1 is SGESLNEYYTQ (SEQ ID NO: 28), VL-CDR2 is RDTERPS (SEQ ID NO: 29), VL-CDR3 is ESAVDTGTLV (SEQ ID NO: 30), 4) antibody ZIL11: VH-CDR1 is TYVMN (SEQ ID NO: 31), VH-CDR2 is SINGGGSSPTYADAVRG (SEQ ID NO: 32), VH-CDR3 is SMVGPFDY (SEQ ID NO: 33), VL-CDR1 is SGESLSNYYAQ (SEQ ID NO: 34), VL-CDR2 is KDTERPS (SEQ ID NO: 35), VL-CDR3 is ESAVSSDTIV (SEQ ID NO: 36), 5) antibody ZIL69: VH-CDR1 is SYAMK (SEQ ID NO: 37), VH-CDR2 is TINNDGTRTGYADAVRG (SEQ ID NO: 38), VH-CDR3 is GNAESGCTGDHCPPY (SEQ ID NO: 39), VL-CDR1 is SGESLNKYYAQ (SEQ ID NO: 40), VL-CDR2 is KDTERPS (SEQ ID NO: 41), VL-CDR3 is ESAVSSETNV (SEQ ID NO: 42), 6) antibody ZIL94: VH-CDR1 is TYFMS (SEQ ID NO: 43), VH-CDR2 is LISSDGSGTYYADAVKG (SEQ ID NO: 44), VH-CDR3 is FWRAFND (SEQ ID NO: 45), VL-CDR1 is GLNSGSVSTSNYPG (SEQ ID NO: 46), VL-CDR2 is DTGSRPS (SEQ ID NO: 47), VL-CDR3 is SLYTDSDILV (SEQ ID NO: 48), 7) antibody ZIL154: VH-CDR1 is DRGMS (SEQ ID NO: 49), VH-CDR2 is YIRYDGSRTDYADAVEG (SEQ ID NO: 50), VH-CDR3 is WDGSSFDY (SEQ ID NO: 51), VL-CDR1 is KASQSLLHSDGNTYLD (SEQ ID NO: 52), VL-CDR2 is KVSNRDP (SEQ ID NO: 53), and VL-CDR3 is MQAIHFPLT (SEQ ID NO: 54); 8) antibody ZIL159: VH-CDR1 is SYVMT (SEQ ID NO: 55), VH-CDR2 is GINSEGSRTAYADAVKG (SEQ ID NO: 56), VH-CDR3 is GDIVATGTSY (SEQ ID NO: 57), VL-CDR1 is SGETLNRFYTQ (SEQ ID NO: 58), VL-CDR2 is KDTERPS (SEQ ID NO: 59), VL-CDR3 is KSAVSIDVGV (SEQ ID NO: 60), 9) Antibody ZIL171: VH-CDR1 is TYVMN (SEQ ID NO: 61), VH-CDR2 is SINGGGSSPTYADAVRG (SEQ ID NO: 62), VH-CDR3 is SMVGPFDY (SEQ ID NO: 63), VL-CDR1 is SGKSLSYYYAQ (SEQ ID NO: 64), VL-CDR2 is KDTERPS (SEQ ID NO: 65), and VL-CDR3 is ESAVSSDTIV (SEQ ID NO: 66); 10) Antibody 04H07: VH-CDR1 is SYWMN (SEQ ID NO: 200), VH-CDR2 is MIDPSDSEIHYNQVFKD (SEQ ID NO: 201), VH-CDR3 is QDIVTTVDY (SEQ ID NO: 202), VL-CDR1 is KSSQSLLYSINQKNHLA (SEQ ID NO: 203), VL-CDR2 is WASTRES (SEQ ID NO: 204), and VL-CDR3 is QQGYTYPFT (SEQ ID NO: 205); 11) Antibody 06A09: VH-CDR1 is SYWMN (SEQ ID NO: 206), VH-CDR2 is MIDPSDSETHYNQIFRD (SEQ ID NO: 207), VH-CDR3 is QDIVTTVDY (SEQ ID NO: 208), VL-CDR1 is KSSQSLLYSINQKNFLA (SEQ ID NO: 209), VL-CDR2 is WASTRES (SEQ ID NO: 210), VL-C DR3 is QQHYGYPFT (SEQ ID NO: 211), or 12) A variant of 1) to 11) that differs from the respective parent antibody ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07, or 06A09 by the addition, deletion, and / or substitution of one or more amino acid residues in at least one of CDR1, CDR2, or CDR3 of VH or VL. Also included is a monoclonal antibody or antigen-binding portion thereof comprising:

[0020] In some embodiments of the present invention, 1) Antibody ZIL1: a)CAN-ZIL1_VL: a variable light chain comprising QSVLTQPTSVSGSLGQRVTISCSGSTNNIGILAATWYQQLPGKAPKVLVYSDGNRPSGVPDRFSGSSKSGNSATLTITGLQAEDEADYYCQSFDTTLDAYVFGSGTQLTVL (SEQ ID NO: 77), and b)CAN-ZIL1_VH: A variable heavy chain comprising: EVQLVESGGDLVKPGGSLRLSCVASGFTFSSYGMSWVRQAPGKGLQWVAHINSGGSSTYYYADAVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCVEVYTTLAAFWTDNFDYWGQGTLVTVSS (SEQ ID NO: 75) and 2) The antibody ZIL8 is: a)CAN-ZIL8_VL: a variable light chain comprising: QSVLTQPASVSGSLGQKVTISCTGSSSNIGSGYVGWYQQLPGTGPRTLIYYNSDRPSGVPDRFSGSRSGTTATLTISGLQAEDEADYYCSVYDRTFNAVFGGGT (SEQ ID NO: 81); and b)CAN-ZIL8_VH: variable heavy chain comprising EVQLVESGGDLVKPAGSLRLSCVASGFTFSDYAMSWVRQAPGRGLQWVAGIDSVGSGTSYADAVKGRFTISRDDAKNTLYLQMFNLRAEDTAIYYCASGFPGSFEHWGQGTLVTVSS (SEQ ID NO: 79) or below: c) ZTS_5864_VL: a variable light chain comprising QSVLTQPSSVSGTLGQRITISCTGSSSNIGSGYVGWYQQVPGMGPKTVIYYNSDRPSGVPDRFSGSKSGSSGTLTITGLQAEDEADYYCSVYDRTFNAVFGGGTHLTVLGQPKSAPPRSHSSRPISYAVFCL (SEQ ID NO: 230), and d) ZTS_5864_VH: a variable heavy chain comprising DVQLVESGGDLVKPGGSLRLTCVASGFTFSDYAMSWVRQAPGKGLQWVAGIDSVGSGTSYADSVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCASGFPGSFEHWGQGALVTVSS (SEQ ID NO: 228) or below: e)ZTS_5865_VL: Q a variable light chain comprising SVLTQPSSVSGTLGQRITISCTGSSSNIGSGYVGWYQQVPGMGPKTVIYYNSDRPSGVPDRFSGSKSGSSGTLTITGLQAEDEADYYCSVYDRTFNAVFGGGTHLTVLGQPKSAPPRSHSSRPISYAVFCL (SEQ ID NO: 234), and f)ZTS_5865_VH: a variable heavy chain comprising: DVQLVESGGDLVKPGGSLRLTCVASGFTFSDYAMNWVRQAPGKGLQWVAGIDSVGSGTSYADSVKGRFTISRDNAKNTLYLQMSGLKTEDTATYYCASGFPGSFEHWGQGTLVTVSS (SEQ ID NO: 232) and 3) The antibody ZIL9 is: a) CAN-ZIL9_VL: a variable light chain comprising SSVLTQPPSVSVSLGQTATISCSGESLNEYYTQWFQQKAGQAPVLVIYRDTERPSGIPDRFSGSSSGNTHTLTISGARAEDEADYYCESAVDTGTLVFGGGTHLAVL (SEQ ID NO: 85), and b)CAN-ZIL9_VH: A variable heavy chain comprising: EVQLVESGGDLVKPPGSLRLSCVASGFTFSSYDMTWVRQAPGKGLQWVADVNSGGTGTAYAVAVKGRFTISRDNAKKTLYLQMNSLRAEDTAVYYCAKLGVRDGLSVWGQGTLVTVSS (SEQ ID NO: 83) and 4) Antibody ZIL11: a) CAN-ZIL11_VL: a variable light chain comprising SSVLTQPPSVSVSLGQTATISCSGESLSNYYAQWFQQKAGQAPVLVIYKDTERPSGIPDRFSGSSSGNTHTLTISGARAEDEADYYCESAVSSDTIVFGGGT (SEQ ID NO: 89), and b) CAN-ZIL11_VH: a variable heavy chain comprising EVQLVESGGDLVKPAGSLRLSCVASGFTFRTYVMNWVRQAPGKGLQWVASINGGGSSPTYADAVRGRFTVSRDNAQNSLFLQMNSLRAEDTAVYFCVVSMVGPFDYWGQGTLVTVSS (SEQ ID NO: 87) and 5) The antibody ZIL69: a) CAN-ZIL69_VL: a variable light chain comprising SSVLTQPPSVSVSLGQTATISCSGESLNKYYAQWFQQKAGQAPVLVIYKDTERPSGIPDRFSGSSAGNTHTLTISGARAEDEADYYCESAVSSETNVFGSGTQLTVL (SEQ ID NO: 93), and b) CAN-ZIL69_VH: a variable heavy chain comprising EVQLVESGGDLVKPAGSLRLSCVASGFTFSSYAMKWVRQAPGKGLQWVATINNDGTRTGYADAVRGRFTISKDNAKNTLYLQMDSLRADDTAVYYCTKGNAESGCTGDHCPPYWGQGTLVTVSS (SEQ ID NO: 91) and 6) Antibody ZIL94: a) CAN-ZIL94_VL: a variable light chain comprising QTVVIQEPSLSVSPGGTVTLTCGLNSGSVSTSNYPGWYQQTRGRTPRTIIYDTGSRPSGVPNRFSGSISGNKAALTITGAQPEDEADYYCSLYTDSDILVFGGGTHLTVL (SEQ ID NO: 97), and b) CAN-ZIL94_VH: a variable heavy chain comprising EVQLVDSGGDLVKPGGSLRLSCVASGFTFSTYFMSWVRQAPGRGLQWVALISSDGSGTYYADAVKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCAIFWRAFNDWGQGTLVTVSS (SEQ ID NO: 95) and 7) Antibody ZIL154: a)CAN-ZIL154_VL:DIVVTQTPLSLSSVSPGETASFSCKASQSLLHSDGNTYLDWFRQKPGQSPQRLIYKVSNRDPGVP a variable light chain comprising DRFSGSGSGTDFTLRISGVEADDAGLYYCMQAIHFPLTFGAGTKVELK (SEQ ID NO: 101), and b) CAN-ZIL154_VH: a variable heavy chain comprising EVHLVESGGDLVKPWGSLRLSCVASGFTFSDRGMSWVRQSPGKGLQWVAYIRYDGSRTDYADAVEGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARWDGSSFDYWGQGTLVTVSS (SEQ ID NO: 99) and 8) The antibody ZIL159: a) CAN-ZIL159_VL: a variable light chain comprising SNVLTQPPSVSVSLGQTATISCSGETLNRFYTQWFQQKAGQAPVLVIYKDTERPSGIPDRFSGSSSGNIHTLTISGARAEDEAAYYCKSAVSIDVGVFGGGTHLTVF (SEQ ID NO: 105), and b) CAN-ZIL159_VH: a variable heavy chain comprising EVQLVESGGDLVKPAGSLRLSCVASGFTFSSYVMTWVRQAPGKGLQWVAGINSEGSRTAYADAVKGRFTISRDNAKNTLYLQIDSLRAEDTAIYYCATGDIVATGTSYWGQGTLVTVSS (SEQ ID NO: 103) and 9) Antibody ZIL171: a) CAN-ZIL171_VL: a variable light chain comprising SSVLTQPPSVSVSLGQTATISCSGKSLSYYYAQWFQQKAGQAPVLVIYKDTERPSGIPDRFSGSSSGNTHTLTISGARAEDEADYYCESAVSSDTIVFGGGTHLTVL (SEQ ID NO: 109), and b) CAN-ZIL171_VH: a variable heavy chain comprising EVQLVESGGDLVKPAGSLRLSCVASGFTFRTYVMNWVRQAPGKGLQWVASINGGGSSPTYADAVRGRFTVSRDNAQNSLFLQMNSLRAEDTAIYFCVVSMVGPFDYWGHGTLVTVSS (SEQ ID NO: 107) and 10) Antibody 04H07: a)Mu_04H07_VL: a variable light chain comprising: DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSINQKNHLAWFQQKPGQSPKLLIYWASTRESGVPARFTGSGSGTDFTLTISSVKTEDLAVYYCQQGYTYPFTFGSGTKLEIK (SEQ ID NO: 214), and b) Mu_04H07_VH: a variable heavy chain comprising QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWMNWAKQRPGQGLEWIGMIDPSDSEIHYNQVFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARQDIVTTVDYWGQGTTLTVSS (SEQ ID NO: 212) and 11) Antibody 06A09: a) a variable light chain comprising Mu_06A09_VL:DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSINQKNFLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKSEDLAVYYCQQHYGYPFTFGSGTKLEIK (SEQ ID NO: 218), and b) Mu_06A09_VH: a variable heavy chain comprising QVQLQQPGAELVRPGASVKLSCKAYGYTFTSYWMNWVKQRPGQGLEWIGMIDPSDSETHYNQIFRDKATLTIDKSSSTAYMQLSSLTSEDSAVYFCARQDIVTTVDYWGQGTTLTVSS (SEQ ID NO: 216) It includes at least one of the following:

[0021] In one embodiment, a monoclonal antibody or antigen-binding portion thereof in accordance with the invention reduces, inhibits, or neutralizes an IL-31-mediated pruritic or allergic condition in a mammal. In one embodiment, such mammal is selected from a dog, a cat, or a horse.

[0022] In some embodiments, the monoclonal antibody is chimeric. In further embodiments, the antibody is caninized, felineized, equineized, fully canine, fully feline, or fully equine.

[0023] The present invention also provides veterinary compositions comprising a therapeutically effective amount of at least one antibody or antigen-binding portion thereof described above.

[0024] Also provided is a method of treating an IL-31-mediated disorder in a subject, comprising administering to the subject a therapeutically effective amount of at least one antibody or antigen-binding portion thereof described above.

[0025] In one embodiment, the IL-31-mediated disorder is a pruritic condition or an allergic condition. In some embodiments, the pruritic condition or allergic condition is selected from atopic dermatitis, eczema, psoriasis, scleroderma, and pruritus. In other embodiments, the pruritic condition or allergic condition is selected from allergic dermatitis, summer eczema, urticaria, heaves, inflammatory airway disease, recurrent airway obstruction, airway hyperresponsiveness, chronic obstructive pulmonary disease, and an inflammatory process of autoimmune origin.

[0026] In other embodiments, the IL-31 mediated disorder is tumor progression. In some embodiments, the IL-31 mediated disorder is eosinophilic disease or mastocytoma.

[0027] Additionally provided is a method of inhibiting IL-31 activity in a mammal by administering to the mammal an antibody or antigen-binding portion thereof described above.

[0028] Also provided is the above antibody, or antigen-binding portion thereof, for use in treating a mammal having an IL-31-mediated disorder.

[0029] Further provided is the use of the above-described antibody or antigen-binding portion thereof to treat a mammal having an IL-31-mediated disorder.

[0030] Also provided is a method for detecting IL-31, comprising incubating a sample containing IL-31 in the presence of the antibody or antigen-binding portion thereof, and detecting the antibody bound to IL-31 in the sample. In one embodiment, the method further comprises quantifying IL-31 in the sample.

[0031] The present invention also provides a combination of the following complementarity determining region (CDR) sequences: 1) Antibody 15H05: variable heavy chain (VH)-CDR1 is SYTIH (SEQ ID NO: 1), VH-CDR2 is NINPTSGYTENNQRFKD (SEQ ID NO: 2), VH-CDR3 is WGFKYDGEWSFDV (SEQ ID NO: 3), variable light chain (VL)-CDR1 is RASQGISIWLS (SEQ ID NO: 4), VL-CDR2 is KASNLHI (SEQ ID NO: 5), VL-CDR3 is LQSQTYPLT (SEQ ID NO: 6), 2) Antibody ZIL1: variable heavy chain (VH)-CDR1 is SYGMS (SEQ ID NO: 13), VH-CDR2 is HINSGGSSTYYADAVKG (SEQ ID NO: 14), VH-CDR3 is VYTTLAAFWTDNFDY (SEQ ID NO: 15), variable light chain (VL)-CDR1 is SGS TNNIGILAAT (SEQ ID NO: 16), VL-CDR2 is SDGNRPS (SEQ ID NO: 17), and VL-CDR3 is QSFDTTLDAYV (SEQ ID NO: 18), 3) antibody ZIL8: VH-CDR1 is DYAMS (SEQ ID NO: 19), VH-CDR2 is GIDSVGSGTSYADAVKG (SEQ ID NO: 20), VH-CDR3 is GFPGSFEH (SEQ ID NO: 21), VL-CDR1 is TGSSSNIGSGYVG (SEQ ID NO: 22), VL-CDR2 is YNSDRPS (SEQ ID NO: 23), VL-CDR3 is SVYDRTFNAV (SEQ ID NO: 24), 4) antibody ZIL9: VH-CDR1 is SYDMT (SEQ ID NO: 25), VH-CDR2 is DVNSGGTGTAYAVAVKG (SEQ ID NO: 26), VH-CDR3 is LGVRDGLSV (SEQ ID NO: 27), VL-CDR1 is SGESLNEYYTQ (SEQ ID NO: 28), VL-CDR2 is RDTERPS (SEQ ID NO: 29), VL-CDR3 is ESAVDTGTLV (SEQ ID NO: 30), 5) antibody ZIL11: VH-CDR1 is TYVMN (SEQ ID NO: 31), VH-CDR2 is SINGGGSSPTYADAVRG (SEQ ID NO: 32), VH-CDR3 is SMVGPFDY (SEQ ID NO: 33), VL-CDR1 is SGESLSNYYAQ (SEQ ID NO: 34), VL-CDR2 is KDTERPS (SEQ ID NO: 35), VL-CDR3 is ESAVSSDTIV (SEQ ID NO: 36), 6) antibody ZIL69: VH-CDR1 is SYAMK (SEQ ID NO: 37), VH-CDR2 is TINNDGTRTGYADAVRG (SEQ ID NO: 38), VH-CDR3 is GNAESGCTGDHCPPY (SEQ ID NO: 39), VL-CDR1 is SGESLNKYYAQ (SEQ ID NO: 40), VL-CDR2 is KDTERPS (SEQ ID NO: 41), VL-CDR3 is ESAVSSETNV (SEQ ID NO: 42), 7) antibody ZIL94: VH-CDR1 is TYFMS (SEQ ID NO: 43), VH-CDR2 is LISSDGSGTYYADAVKG (SEQ ID NO: 44), VH-CDR3 is FWRAFND (SEQ ID NO: 45), VL-CDR1 is GLNSGSVSTSNYPG (SEQ ID NO: 46), VL-CDR2 is DTGSRPS (SEQ ID NO: 47), VL-CDR3 is SLYTDSDILV (SEQ ID NO: 48), 8) antibody ZIL154: VH-CDR1 is DRGMS (SEQ ID NO: 49), VH-CDR2 is YIRYDGSRTDYADAVEG (SEQ ID NO: 50), VH-CDR3 is WDGSSFDY (SEQ ID NO: 51), VL-CDR1 is KASQSLLHSDGNTYLD (SEQ ID NO: 52), VL-CDR2 is KVSNRDP (SEQ ID NO: 53), and VL-CDR3 is MQAIHFPLT (SEQ ID NO: 54); 9) Antibody ZIL159: VH-CDR1 is SYVMT (SEQ ID NO: 55), VH-CDR2 is GINSEGSRTAYADAVKG (SEQ ID NO: 56), VH-CDR3 is GDIVATGTSY (SEQ ID NO: 57), VL-CDR1 is SGETLNRFYTQ (SEQ ID NO: 58), VL-CDR2 is KDTERPS (SEQ ID NO: 59), VL-CDR3 is KSAVSIDVGV (SEQ ID NO: 60), 10) antibody ZIL171: VH-CDR1 is TYVMN (SEQ ID NO: 61), VH-CDR2 is SINGGGSSPTYADAVRG (SEQ ID NO: 62), VH-CDR3 is SMVGPFDY (SEQ ID NO: 63), VL-CDR1 is SGKSLSYYYAQ (SEQ ID NO: 64), VL-CDR2 is KDTERPS (SEQ ID NO: 65), VL-CDR3 is ESAVSSDTIV (SEQ ID NO: 66), 11) Antibody 04H07: VH-CDR1 is SYWMN (SEQ ID NO: 200), VH-CDR2 is MIDPSDSEIHYNQVFKD (SEQ ID NO: 201), VH-CDR3 is QDIVTTVDY (SEQ ID NO: 202), VL-CDR1 is KSSQSLLYSINQKNHLA (SEQ ID NO: 203), VL-CDR2 is WASTRES (SEQ ID NO: 204), and VL-CDR3 is QQGYTYPFT (SEQ ID NO: 205); 12) Antibody 06A09: VH-CDR1 is SYWMN (SEQ ID NO: 206), VH-CDR2 is MIDPSDSETHYNQIFRD (SEQ ID NO: 207), VH-CDR3 is QDIVTTVDY (SEQ ID NO: 208), and VL-CDR1 is KSSQSLLYSINQKNFL A (SEQ ID NO: 209), VL-CDR2 is WASTRES (SEQ ID NO: 210), and VL-CDR3 is QQHYGYPFT (SEQ ID NO: 211), or 13) A variant of 1) to 12) that differs from the respective parent antibody 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07, or 06A09 by the addition, deletion, and / or substitution of one or more amino acid residues in at least one of CDR1, CDR2, or CDR3 of VH or VL. Also provided is a host cell that produces a monoclonal antibody or antigen-binding portion thereof comprising at least one of:

[0032] Also provided is a method of producing an antibody, comprising culturing the host cell described above under conditions that result in the production of the antibody, and isolating the antibody from the host cell or the host cell medium.

[0033] The isolated nucleic acids according to the present invention are described below. Such nucleic acids may contain nucleic acid sequences encoding the CDR sequences of the variable heavy chain or variable light chain described above. Alternatively, the isolated nucleic acids according to the present invention may contain nucleic acid sequences encoding both the CDRs of the variable heavy chain and the variable light chain.

[0034] In one embodiment, the present invention provides a combination of the following variable heavy chain complementarity determining region (CDR) sequences: 1) 15H05: variable heavy chain (VH)-CDR1 is SYTIH (SEQ ID NO: 1), VH-CDR2 is NINPTSGYTENNQRFKD (SEQ ID NO: 2), VH-CDR3 is WGFKYDGEWSFDV (SEQ ID NO: 3); 2) ZIL1: VH-CDR1 is SYGMS (SEQ ID NO: 13), VH-CDR2 is HINSGGSSTYYADAVKG (SEQ ID NO: 14), and VH-CDR3 is VYTTLAAFWTDNFDY (SEQ ID NO: 15); 3) ZIL8: VH-CDR1 is DYAMS (SEQ ID NO: 19), VH-CDR2 is GIDSVGSGTSYADAVKG (SEQ ID NO: 20), and VH-CDR3 is GFPGSFEH (SEQ ID NO: 21); 4) ZIL9: VH-CDR1 is SYDMT (SEQ ID NO: 25), VH-CDR2 is DVNSGGTGTAYAVAVKG (SEQ ID NO: 26), and VH-CDR3 is LGVRDGLSV (SEQ ID NO: 27); 5) ZIL11: VH-CDR1 is TYVMN (SEQ ID NO: 31), VH-CDR2 is SINGGGSSPTYADAVRG (SEQ ID NO: 32), and VH-CDR3 is SMVGPFDY (SEQ ID NO: 33); 6) ZIL69: VH-CDR1 is SYAMK (SEQ ID NO: 37), VH-CDR2 is TINNDGTRTGYADAVRG (SEQ ID NO: 38), and VH-CDR3 is GNAESGCTGDHCPPY (SEQ ID NO: 39); 7) ZIL94: VH-CDR1 is TYFMS (SEQ ID NO: 43), VH-CDR2 is LISSDGSGTYYADAVKG (SEQ ID NO: 44), and VH-CDR3 is FWRAFND (SEQ ID NO: 45); 8) ZIL154: VH-CDR1 is DRGMS (SEQ ID NO: 49), VH-CDR2 is YIRYDGSRTDYADAVEG (SEQ ID NO: 50), and VH-CDR3 is WDGSSFDY (SEQ ID NO: 51); 9) ZIL159: VH-CDR1 is SYVMT (SEQ ID NO: 55), VH-CDR2 is GINSEGSRTAYADAVKG (SEQ ID NO: 56), and VH-CDR3 is GDIVATGTSY (SEQ ID NO: 57); 10) ZIL171: VH-CDR1 is TYVMN (SEQ ID NO: 61), VH-CDR2 is SINGGGSSPTYADAVRG (SEQ ID NO: 62), and VH-CDR3 is SMVGPF DY (SEQ ID NO: 63), 11) 04H07: VH-CDR1 is SYWMN (SEQ ID NO: 200), VH-CDR2 is MIDPSDSEIHYNQVFKD (SEQ ID NO: 201), and VH-CDR3 is QDIVTTVDY (SEQ ID NO: 202); 12) 06A09: VH-CDR1 is SYWMN (SEQ ID NO: 206), VH-CDR2 is MIDPSDSETHYNQIFRD (SEQ ID NO: 207), and VH-CDR3 is QDIVTTVDY (SEQ ID NO: 208), or 13) A variant of 1) to 12) that differs from the CDRs of the respective parent antibodies 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07, or 06A09 by addition, deletion, and / or substitution of one or more amino acid residues in at least one of CDR1, CDR2, or CDR3 of VH. An isolated nucleic acid is provided, comprising a nucleic acid sequence encoding at least one of:

[0035] In one embodiment, the isolated nucleic acid further comprises the following combination of variable light chain complementarity determining region (CDR) sequences: 1) 15H05: variable light chain (VL)-CDR1 is RASQGISIWLS (SEQ ID NO: 4), VL-CDR2 is KASNLHI (SEQ ID NO: 5), VL-CDR3 is LQSQTYPLT (SEQ ID NO: 6); 2) ZIL1: VL-CDR1 is SGSTNNIGILAAT (SEQ ID NO: 16), VL-CDR2 is SDGNRPS (SEQ ID NO: 17), and VL-CDR3 is QSFDTTLDAYV (SEQ ID NO: 18); 3) ZIL8: VL-CDR1 is TGSSSNIGSGYVG (SEQ ID NO: 22), VL-CDR2 is YNSDRPS (SEQ ID NO: 23), and VL-CDR3 is SVYDRTFNAV (SEQ ID NO: 24); 4) ZIL9: VL-CDR1 is SGESLNEYYTQ (SEQ ID NO: 28), VL-CDR2 is RDTERPS (SEQ ID NO: 29), and VL-CDR3 is ESAVDTGTLV (SEQ ID NO: 30); 5) ZIL11: VL-CDR1 is SGESLSNYYAQ (SEQ ID NO: 34), VL-CDR2 is KDTERPS (SEQ ID NO: 35), and VL-CDR3 is ESAVSSDTIV (SEQ ID NO: 36); 6) ZIL69: VL-CDR1 is SGESLNKYYAQ (SEQ ID NO: 40), VL-CDR2 is KDTERPS (SEQ ID NO: 41), and VL-CDR3 is ESAVSSETNV (SEQ ID NO: 42); 7) ZIL94: VL-CDR1 is GLNSGSVSTSNYPG (SEQ ID NO: 46), VL-CDR2 is DTGSRPS (SEQ ID NO: 47), and VL-CDR3 is SLYTDSDILV (SEQ ID NO: 48); 8) ZIL154: VL-CDR1 is KASQSLLHSDGNTYLD (SEQ ID NO: 52), VL-CDR2 is KVSNRDP (SEQ ID NO: 53), and VL-CDR3 is MQAIHFPLT (SEQ ID NO: 54); 9) ZIL159: VL-CDR1 is SGETLNRFYTQ (SEQ ID NO: 58), VL-CDR2 is KDTERPS (SEQ ID NO: 59), and VL-CDR3 is KSAVSIDVGV (SEQ ID NO: 60); 10) ZIL171: VL-CDR1 is SGKSLSYYYAQ (SEQ ID NO: 64), VL-CDR2 is KDTERPS (SEQ ID NO: 65), and VL-CDR3 is ESAVSSDTIV (SEQ ID NO: 66); 11) 04H07: VL-CDR1 is KSSQSLLYSINQKNHLA (SEQ ID NO: 203), VL-CDR2 is WASTRES (SEQ ID NO: 204), and VL-CDR3 is QQGYTYPFT (SEQ ID NO: 205); 12) 06A09: VL-CDR1 is KSSQSLLYSINQKNFLA (SEQ ID NO: 2 09), VL-CDR2 is WASTRES (SEQ ID NO: 210), and VL-CDR3 is QQHYGYPFT (SEQ ID NO: 211), or 13) A variant of 1) to 12) that differs from the CDRs of the respective parent antibodies 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07, or 06A09 by addition, deletion, and / or substitution of one or more amino acid residues in at least one of CDR1, CDR2, or CDR3 of VH or VL. The nucleic acid sequence may include a nucleic acid sequence encoding at least one of:

[0036] In one embodiment, the present invention provides a combination of the following variable light chain complementarity determining region (CDR) sequences: 1) 15H05: variable light chain (VL)-CDR1 is RASQGISIWLS (SEQ ID NO: 4), VL-CDR2 is KASNLHI (SEQ ID NO: 5), VL-CDR3 is LQSQTYPLT (SEQ ID NO: 6); 2) ZIL1: VL-CDR1 is SGSTNNIGILAAT (SEQ ID NO: 16), VL-CDR2 is SDGNRPS (SEQ ID NO: 17), and VL-CDR3 is QSFDTTLDAYV (SEQ ID NO: 18); 3) ZIL8: VL-CDR1 is TGSSSNIGSGYVG (SEQ ID NO: 22), VL-CDR2 is YNSDRPS (SEQ ID NO: 23), and VL-CDR3 is SVYDRTFNAV (SEQ ID NO: 24); 4) ZIL9: VL-CDR1 is SGESLNEYYTQ (SEQ ID NO: 28), VL-CDR2 is RDTERPS (SEQ ID NO: 29), and VL-CDR3 is ESAVDTGTLV (SEQ ID NO: 30); 5) ZIL11: VL-CDR1 is SGESLSNYYAQ (SEQ ID NO: 34), VL-CDR2 is KDTERPS (SEQ ID NO: 35), and VL-CDR3 is ESAVSSDTIV (SEQ ID NO: 36); 6) ZIL69: VL-CDR1 is SGESLNKYYAQ (SEQ ID NO: 40), VL-CDR2 is KDTERPS (SEQ ID NO: 41), and VL-CDR3 is ESAVSSETNV (SEQ ID NO: 42); 7) ZIL94: VL-CDR1 is GLNSGSVSTSNYPG (SEQ ID NO: 46), VL-CDR2 is DTGSRPS (SEQ ID NO: 47), and VL-CDR3 is SLYTDSDILV (SEQ ID NO: 48); 8) ZIL154: VL-CDR1 is KASQSLLHSDGNTYLD (SEQ ID NO: 52), VL-CDR2 is KVSNRDP (SEQ ID NO: 53), and VL-CDR3 is MQAIHFPLT (SEQ ID NO: 54); 9) ZIL159: VL-CDR1 is SGETLNRFYTQ (SEQ ID NO: 58), VL-CDR2 is KDTERPS (SEQ ID NO: 59), and VL-CDR3 is KSAVSIDVGV (SEQ ID NO: 60); 10) ZIL171: VL-CDR1 is SGKSLSYYYAQ (SEQ ID NO: 64), VL-CDR2 is KDTERPS (SEQ ID NO: 65), and VL-CDR3 is ESAVSSDTIV (SEQ ID NO: 66); 11) 04H07: VL-CDR1 is KSSQSLLYSINQKNHLA (SEQ ID NO: 203), VL-CDR2 is WASTRES (SEQ ID NO: 204), and VL-CDR3 is QQGYTYPFT (SEQ ID NO: 205); 12) 06A09: VL-CDR1 is KSSQSLLYSINQKNFLA (SEQ ID NO: 209), VL-CDR2 is WASTRES (SEQ ID NO: 210), and VL-CDR3 is QQHYGYPFT (SEQ ID NO: 211), or 13) Each of the parent antibodies 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL1 by adding, deleting, and / or substituting one or more amino acid residues in at least one of CDR1, CDR2, or CDR3 of the VL. Variants 1) to 12) that differ from the CDRs of 54, ZIL159, ZIL171, 04H07, or 06A09 An isolated nucleic acid is provided, comprising a nucleic acid sequence encoding at least one of:

[0037] The present invention further provides a vector comprising at least one of the above nucleic acids.

[0038] The present invention also provides a method for improving the consistency and / or quality of a feline antibody, comprising producing a feline antibody by expressing in a host cell a nucleotide sequence encoding a feline IgG kappa light chain and a nucleotide sequence encoding a feline IgG heavy chain, wherein the nucleotide sequence encoding the feline IgG kappa light chain comprises a kappa light chain constant nucleotide sequence in which the sequence encoding the C-terminal QRE sequence otherwise present in the wild-type feline IgG light chain constant region has been modified and / or deleted. Such modifications can include, for example, modifications to the nucleotide sequence resulting in the deletion, substitution, or addition of one or more amino acids at the C-terminus.

[0039] In one embodiment, the method for improving the consistency and / or quality of a feline antibody comprises: a) providing a nucleotide sequence encoding a wild-type feline IgG kappa light chain constant region of a feline antibody, wherein the wild-type feline kappa light chain constant region comprises a C-terminal amino acid sequence of a QRE; b) removing and / or modifying the sequence encoding the C-terminal QRE within the nucleotide sequence in a) to form a modified kappa light chain constant nucleotide sequence; c) combining the modified kappa light chain constant nucleotide sequence from b) with a nucleotide sequence encoding the feline IgG kappa light chain variable region to form a nucleotide sequence encoding the complete feline IgG kappa light chain; d) expressing in a host cell the nucleotide sequence encoding the complete feline IgG kappa light chain from c) and the nucleotide sequence encoding the feline IgG heavy chain to produce a feline antibody in which the C-terminal QRE sequence otherwise present in the wild-type feline IgG kappa light chain constant region has been modified and / or deleted.

[0040] In one embodiment, improving the consistency and / or quality of a feline antibody comprises reducing the level of free IgG kappa light chains, thereby increasing the percentage of intact feline IgG antibody monomers.

[0041] In one embodiment, the nucleotide sequence encoding the feline IgG kappa light chain and the nucleotide sequence encoding the feline IgG heavy chain are carried on the same vector used to transform the host cell, while in another embodiment, the nucleotide sequence encoding the feline IgG kappa light chain and the nucleotide sequence encoding the feline IgG heavy chain are carried on separate vectors used to transform the host cell.

[0042] In one embodiment of the method for improving the consistency and / or quality of a feline antibody, the feline antibody is induced by cytokines and / or growth factors. Specifically binds to a target involved in a factor-mediated disorder. In one particular embodiment, the feline antibody specifically binds to feline IL-31 or feline NGF.

[0043] In one embodiment, the feline antibody comprises a kappa light chain constant region having the sequence: RSDAQPSVFLFQPSLDELHTGSASIVCILNDFYPKEVNVKWKVDGVVQNKGIQESTTEQNSKDSTYSLSSTLTMSSTEYQSHEKFSCEVTHKSLASTLVKSFQRSEC (SEQ ID NO: 186) or a variant thereof. Such variants include, for example, a 1′ amino acid sequence relative to the c-terminus of SEQ ID NO: 186. The addition or modification of one or more amino acid residues is included.

[0044] The present invention further provides a method for improving the consistency and / or quality of a canine antibody, comprising expressing in a host cell a nucleotide sequence encoding a canine IgG kappa light chain and a nucleotide sequence encoding a canine IgG heavy chain to produce a canine antibody, wherein the nucleotide sequence encoding the canine IgG kappa light chain comprises a kappa light chain constant nucleotide sequence in which a sequence encoding the C-terminal QRVD sequence otherwise present in the wild-type canine IgG light chain (canine LC kappa wt, SEQ ID NO: 194) constant region has been modified and / or deleted. Such modifications can include, for example, modifications to the nucleotide sequence that result in the deletion, substitution, or addition of one or more amino acids to the C-terminus.

[0045] In one embodiment, the method for improving the consistency and / or quality of a canine antibody comprises: a) providing a nucleotide sequence encoding a wild-type canine IgG kappa light chain constant region of a canine antibody, wherein the wild-type canine kappa light chain constant region comprises a C-terminal amino acid sequence of QRVD; b) removing and / or modifying the C-terminal QRVD coding sequence within the nucleotide sequence of a) to form a modified kappa light chain constant nucleotide sequence; c) combining the modified kappa light chain constant nucleotide sequence from b) with a nucleotide sequence encoding a canine IgG kappa light chain variable region to form a nucleotide sequence encoding a complete canine IgG kappa light chain; d) expressing the nucleotide sequence encoding the complete canine IgG kappa light chain from c) and the nucleotide sequence encoding the canine IgG heavy chain in a host cell to produce a canine antibody in which the C-terminal QRVD sequence otherwise present in the wild-type canine IgG kappa light chain constant region has been modified and / or deleted.

[0046] In one embodiment, improving the consistency and / or quality of the canine antibody comprises reducing the level of free IgG kappa light chains, thereby increasing the percentage of intact canine IgG antibody monomers.

[0047] In one embodiment, the nucleotide sequence encoding the canine IgG kappa light chain and the nucleotide sequence encoding the canine IgG heavy chain are carried on the same vector used to transform the host cell, hi another embodiment, the nucleotide sequence encoding the canine IgG kappa light chain and the nucleotide sequence encoding the canine IgG heavy chain are carried on separate vectors used to transform the host cell.

[0048] In one embodiment of the method for improving the consistency and / or quality of a canine antibody, the canine antibody specifically binds to a target involved in a cytokine- and / or growth factor-mediated disorder. In one particular embodiment, the canine antibody specifically binds to canine IL-31.

[0049] In one embodiment of a method for improving the consistency and / or quality of a canine antibody, the canine antibody comprises a kappa light chain constant region having the sequence: RNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSEC (SEQ ID NO: 179), or a variant thereof. Such variants include, for example, the addition or modification of one or more amino acid residue(s) to the c-terminus of SEQ ID NO: 179.

[0050] The modified kappa light chain constant regions described herein may be used in any of the canine or feline antibodies described and claimed in the present specification, including, but not limited to, The modified kappa light chain constant region of the present invention can be used with any number of feline and canine antibodies, including those described herein. Canine or feline antibodies with targets other than IL-31 are also contemplated as being suitable for combination with the modified kappa light chain constant region disclosed herein. The present invention includes any feline or canine antibody that includes such a modified kappa light chain constant region, such that such antibodies can be reasonably expected to have improved consistency and / or quality based on the disclosure in the present specification and claims. [Brief explanation of the drawings]

[0051] [Figure 1] 1 is an alignment showing the amino acid sequence conservation between IL-31 from different species. Specifically, a comparison is shown between SEQ ID NO: 155 (canine IL-31), SEQ ID NO: 157 (feline IL-31), SEQ ID NO: 165 (canine IL-31), and SEQ ID NO: 181 (human IL-31). The percent amino acid sequence identity between canine, feline, equine, and human IL-31 is also shown. [Figure 2] Surface plasmon resonance (SPR) on a Biacore system (Biacore Life Sciences (GE Healthcare), Uppsala, Sweden) was used to detail the binding affinity of candidate Abs with CDRs from murine origin to canine and feline IL-31. [Figure 3] 1 is a table showing the potency (IC50 (μg / ml)) of candidate antibodies with CDRs from murine origin as measured in canine and feline cell assays. Specifically, candidate antibodies were evaluated for their ability to inhibit IL-31-mediated STAT phosphorylation in canine DH-82 or feline FCWF4 macrophage-like cells.

[0052] [Figure 4]shows the results obtained using both indirect ELISA and Biacore methods for the binding of candidate monoclonal antibodies with canine CDRs to various proteins. For indirect ELISA, binding to wild-type feline IL-31 and to the feline IL-31 15H05 variant, which has a mutation within the monoclonal antibody 15H05 epitope region, was assessed (ELISA OD). To confirm binding, Biacore analysis was performed using canine, feline, equine, human, feline 15H05 variant, and feline 11E12 variant IL-31 proteins as surfaces, using a single test concentration of antibody. The feline IL-31 11E12 variant has a mutation within the monoclonal antibody 11E12 epitope region. [Figure 5] A shows an alignment of the murine antibody 11E12 VL sequence (SEQ ID NO: 73) comparing previously disclosed caninized 11E12 sequences designated Can_11E12_VL_cUn_1 (SEQ ID NO: 182) and CAN_11E12_VL_cUn_FW2 (SEQ ID NO: 184) with felineized versions designated FEL_11E12_VL1 (SEQ ID NO: 113) and FEL_11E12_VL1_FW2 (SEQ ID NO: 117). Dots below the alignment in A indicate the positions of the relevant changes in Fel_11E12_VL1 that were required to restore affinity of this antibody for the IL-31 protein. B shows an alignment of the murine antibody 15H05 VL sequence, designated herein as MU_15H05_VL (SEQ ID NO: 69), with the felinized 15H05 VL sequences designated herein as FEl_15H05_VL1 (SEQ ID NO: 127) and FEl_15H05_VL_FW2 (SEQ ID NO: 135). The dots below the alignment in B indicate the changes required in the felinized 15H05 VL (Fel_15H05_VL1) to not only restore but also improve affinity for canine and feline IL-31 compared to the murine and chimeric forms of this antibody. [Figure 6](A) shows the alignment of wild-type feline IL-31 (SEQ ID NO: 157) with mutants 15H05 (SEQ ID NO: 163) and 11E12 (SEQ ID NO: 161), with the positions where alanine substitutions occur highlighted. (B) shows the feline IL-31 homology model, with the two amino acid positions involved in the binding of antibodies 11E12 (site 1) and 15H05 (site 2) highlighted. (C) is a graph showing the results obtained in the binding of monoclonal antibodies 11E12 and 15H05 to wild-type feline IL-31 and to the mutant IL-31 proteins 15H05 (SEQ ID NO: 163) and 11E12 (SEQ ID NO: 161), with wild-type and these mutants used as coating antigens. [Figure 7] 1 shows graphs depicting competitive binding evaluation of mAbs 15H05 and 11E12 using Biacore. (A) shows competitive binding data for murine 15H05 and 11E12 antibodies with canine IL-31. (B) shows competitive binding data for antibodies 15H05 and 11E12 on the surface of feline IL-31. [Figure 8] Graph showing the results obtained on the binding of the individual receptor subunits of OSMR and IL-31Ra to wild-type feline IL-31 and to the mutant IL-31 proteins 15H05 (SEQ ID NO: 163) and 11E12 (SEQ ID NO: 161), where wild-type and these mutants were used as coating antigens. [Figure 9] 1 is a graph showing the preliminary efficacy of mouse:feline 11E12 chimera, mouse:feline 15H05 chimera, and felineized 11E12 (feline 11E12 1.1) in a feline IL-31-induced pruritus model. [Figure 10]1A-B are graphs showing in vivo evaluation of the efficacy of the felineized 15H05 anti-IL-31 antibody, designated ZTS-361, in the feline pruritus challenge model. (A) Baseline pre-challenge pruritus behavior from day -7 to day 28 (day zero being the day of antibody administration to the T02 group) is shown in the T01 vehicle placebo group and the T02 antibody ZTS-361 group. (B) Efficacy of antibody ZTS-361 is shown, demonstrating significant reductions in pruritus compared to vehicle placebo controls on days 7 (p<0.0001), 21 (p<0.0027), and 28 (p<0.0238) following IL-31 challenge. [Figure 11] (A) is a graph showing plasma levels of IL-31 in client-owned dogs with atopic and allergic dermatitis compared to normal laboratory dogs. (B) is a graph showing the results of a recent study quantifying serum IL-31 levels in cats with a presumptive diagnosis of allergic dermatitis (AD) from several different geographic regions within the United States. (C) is a graph showing the pharmacokinetic profile of canine IL-31 in dogs following subcutaneous administration of 1.75 μg / kg of canine IL-31. [Figure 12]Shown is a 4-12% non-reducing SDS PAGE comparing lane 1, which is ZTS-361, whose heavy chain is a combination of (SEQ ID NO: 121; FEL_15H05_VH1) and a feline IgG heavy chain constant region (SEQ ID NO: 173; feline_HC_allele A_1). In ZTS-361, the light chain is a combination of (SEQ ID NO: 135; FEL-15H05-VL1_FW2) and a feline IgG kappa light chain constant region (SEQ ID NO: 175; feline_LC_kappa_G_minus). Lane 2 is mouse 15H05, whose heavy chain is a combination of (SEQ ID NO: 67; MU_15H05_VH) and a mouse IgG heavy chain constant region (SEQ ID NO: 188; mouse_HC_IgG1). In mouse 15H05, the light chain is a combination of (SEQ ID NO: 69; FMU_15H05_VL) and a mouse IgG light chain constant region (SEQ ID NO: 190; mouse_LC_kappa). Intact refers to an IgG with a predicted molecular weight of approximately 150 kDa, in which two heavy chains and two light chains are held together by interchain disulfide bonds. HHL refers to "heavy-heavy-light" and refers to an IgG lacking one light chain with a predicted molecular weight of approximately 125 kDa. HH refers to "heavy-heavy" and refers to an IgG lacking both light chains with a predicted molecular weight of approximately 100 kDa. HL refers to "heavy-light" and refers to an IgG with one heavy chain and one light chain with a predicted molecular weight of approximately 75 kDa. L refers to "light" and refers to an IgG with one light chain with a predicted molecular weight of approximately 25 kDa, also referred to herein as a free light chain. [Figure 13]A shows the results of non-reducing capillary gel electrophoresis (NR-CGE) comparing IgG from stable cell lines expressing ZTS-361 or murine 15H05 (each described above). The percentage of monomer and subspecies is calculated from the NR-CGE experimental output shown as an electropherogram in B. Time-corrected area (TCA) is defined as the area of ​​each individual peak from the instrument output divided by the migration time. Total TCA is defined as the sum of the TCAs for all peaks greater than 0.3%. The percent monomeric intact IgG (monomer %) and the percentage of individual fragments (HHL% and L%) are calculated based on these individual TCAs as a percentage of the total TCA. Fragment % is the sum of all peak areas migrating at lower molecular weights than intact IgG. [Figure 14] Panel A shows a 4-12% non-reducing SDS PAGE comparing IgG from individual stable CHO clones of ZTS-361 (described above and in section 1.9 of the Examples section). Lanes 1 and 8 are comparative IgG reference standards. Percent monomer was calculated from densitometric analysis of each band migrating at the expected molecular weight of approximately 150 kDa using BioRad VersaDoc software. Fragment % is the sum of individual bands with lower molecular weights. Panel B shows the results of non-reducing capillary gel electrophoresis (NR-CGE) comparing IgG from individual stable CHO clones of ZTS-361. Time-corrected area (TCA) is defined as the area of ​​each individual peak from the instrument output divided by the migration time. Total TCA is defined as the sum of the TCA for all peaks ≥ 0.3%. Percent monomeric intact IgG (%monomer) and percent fragment (%fragment) are calculated based on these individual TCAs as a percentage of total TCA. The % fragment is the sum of all peak areas migrating at lower molecular weights than intact IgG. [Figure 15]The amino acids located at the C-terminus of the Ig kappa light chain constant protein of the indicated species are shown. For canine LC kappa wt, the C-terminal amino acid residues shown are positions 103-109 of SEQ ID NO:194, and the nucleotide residue numbers shown are residues 307-330 of SEQ ID NO:195. For feline LC kappa G minus (G-), the C-terminal amino acid residues shown are positions 105-110 of SEQ ID NO:175, and the nucleotide residue numbers shown are residues 313-330 of SEQ ID NO:176. For porcine LC kappa, the C-terminal amino acid residues shown are positions 104-108 of SEQ ID NO:196, and the nucleotide residue numbers shown are residues 310-327 of SEQ ID NO:197. For mink LC kappa, the C-terminal amino acid residues shown are positions 105-108 of SEQ ID NO:198, and the nucleotide residue numbers shown are residues 313-327 of SEQ ID NO:199. For human LC kappa, the C-terminal amino acid residues shown are positions 102 to 106 of SEQ ID NO: 192, and the nucleotide residue numbers shown are residues 304 to 321 of SEQ ID NO: 193. For mouse LC kappa, the C-terminal amino acid residues shown are positions 102 to 106 of SEQ ID NO: 190, and the nucleotide residue numbers shown are residues 304 to 321 of SEQ ID NO: 191. The gray box encloses the position of the C-terminal cysteine ​​that forms the interchain disulfide bond with the IgG heavy chain constant region required for the intact antibody. Grey triangles highlight the percentage increase in kappa light chain utilization by IgG from the indicated species, following the utilization patterns of dog, cat, and pig (Arun et al. 1996 Zentralbl Veterinarmed. Nov;43(9):573-6), mink (Bovkun et al. 1993 Eur J Immunol. Aug;23(8):1929-34), mouse (Woloschak et al. 1987 Mol Immunol. Jul;24(7):751-7), and human (Barandun et al. 1976 Blood. Jan;47(1):79-89). Nucleotides shown in light grey highlight codons where a single nucleotide change at that position results in a stop codon. [Figure 16] Figure A is a diagrammatic representation of feline IgG, with the relative positions of predicted intra- and interchain disulfide bonds highlighted. The indicated amino acid residue CYS15 is position 15 in feline HC allele A wt (SEQ ID NO: 171) and feline HC allele A1 (SEQ ID NO: 173), with the indicated nucleotide residue numbers being 43-45 in feline HC allele A wt (SEQ ID NO: 172) and 43-45 in feline HC allele A1 (SEQ ID NO: 174), respectively. The indicated amino acid residue CYS107 is position 107 in feline LC kappa G minus (SEQ ID NO: 175), with the indicated nucleotide residue numbers being 319-321 in feline LC kappa G minus (SEQ ID NO: 176). Figure B is a homology model of ZTS-361, with the positions of CYS15 and CYS107 highlighted, as described above. C is an enlargement of the boxed region in B, again highlighting the positions of the two cysteines responsible for interchain pairing of the feline heavy and light chains. The wireframe surface shell shown is the calculated electrostatic contribution at the kappa light chain constant residue QRE immediately following CYS107 as described in Figure 15 for cat LCkappaG-. [Figure 17]Panel A lists the SEQ ID NOs corresponding to the heavy and light chains used to generate stable CHO cell lines producing antibodies ZTS-361 and ZTS-1505 (as described above and / or in section 1.9 of the Examples section). Highlighted is cat LC kappa G minus QRE minus (SEQ ID NO: 186), the corresponding nucleotide sequence of which is cat LC kappa G minus QRE minus (SEQ ID NO: 187). Panel B shows the results of non-reducing capillary gel electrophoresis (NR-CGE) comparing IgG from individual stable CHO clones of ZTS-1505. Time-corrected area (TCA) is defined as the area of ​​each individual peak from the instrument output divided by the migration time. Total TCA is defined as the sum of the TCAs for all peaks greater than or equal to 0.3%. Percent monomeric intact IgG (%monomer) and percent fragments (%fragment) can be calculated based on each individual TCA as a percentage of the total TCA. Fragment % is the sum of all peak areas migrating at a lower molecular weight than intact IgG. C shows a comparison of a single stable CHO clone producing antibody ZTS-361 with a single stable CHO clone producing ZTS-1505. The comparison of the two stable clones was performed in eight independent culture conditions, labeled A through G. The percent viability of each culture after 14 days of culture is shown. The titer indicates the amount of antibody produced by each stable clone under each culture condition after 14 days of culture. The percent monomer calculated from NR-CGE from each clone grown using various culture conditions is shown. [Figure 18] A shows the percent identity of the variable regions of these anti-feline NGF antibodies compared to anti-IL-31, calculated using ClustallW software. B and C show the alignment of the variable heavy and light chains of the anti-feline IL-31 and NGF antibodies, respectively, with the CDRs boxed. [Figure 19] 1 shows the results from NR CGE comparing anti-feline IL-31 and anti-feline NGF antibodies with and without modifications at the C-terminus of the kappa constant region. [Figure 20] 1 shows a ClustallW sequence alignment of feline IL-31 to equine IL-31. [Figure 21] Alignment of the variable heavy (Figure 21A) and light (Figure 21B) chains of antibodies 04H07 and 06A09 compared to murine antibody 15H05 using ClustallW is shown. The positions of each of the six CDRs are boxed for comparison. [Figure 22] Biacore sensorgram showing the average profile of anti-IL31 antibody ZTS-1505, with response thresholds defined using + / - 3 standard deviations to screen for alanine substitution CDR variants. [Figure 23] 1 shows the results of alanine substitution mutagenesis of the heavy chain CDR of antibody ZTS-1505, comparing binding and IL-31-mediated pSTAT signaling inhibition to the wild-type antibody. [Figure 24] 1 shows the results of alanine substitution mutagenesis of the light chain CDR of antibody ZTS-1505, comparing binding and IL-31-mediated pSTAT signaling inhibition to the wild-type antibody. [Figure 25] A and B show the binding affinity and cellular potency, respectively, of two felinized antibodies, designated herein as ZTS-5864 and ZTS-5865. [Figure 26] 1 is a graph showing the results of an in vivo evaluation of the efficacy of felinized ZTS-5864 anti-IL-31 antibody in a feline pruritus challenge model.

[0053] The antibodies described in Figures 12, 13, and 14 were grown in culture conditions equivalent to culture condition A from Figure 17C.

[0054] A brief description of arrays SEQ ID NO: 1 is the variable heavy chain CDR1, referred to herein as MU_15H05_VH_CDR1.

[0055] SEQ ID NO: 2 is the variable heavy chain CDR2, referred to herein as MU_15H05_VH_CDR2.

[0056] SEQ ID NO: 3 is the variable heavy chain CDR3, referred to herein as MU_15H05_VH_CDR3.

[0057] SEQ ID NO: 4 is the variable light chain CDR1, referred to herein as MU_15H05_VL_CDR1.

[0058] SEQ ID NO: 5 is the variable light chain CDR2, referred to herein as MU_15H05_VL_CDR2.

[0059] SEQ ID NO: 6 is the variable light chain CDR3, referred to herein as MU_15H05_VL_CDR3.

[0060] SEQ ID NO: 7 is the variable heavy chain CDR1, referred to herein as 11E12-VH-CDR1.

[0061] SEQ ID NO: 8 is the variable heavy chain CDR2, referred to herein as 11E12-VH-CDR2.

[0062] SEQ ID NO: 9 is the variable heavy chain CDR3 referred to herein as 11E12-VH-CDR3.

[0063] SEQ ID NO: 10 is the variable light chain CDR1, referred to herein as 11E12-VL-CDR1.

[0064] SEQ ID NO: 11 is the variable light chain CDR2, referred to herein as 11E12-VL-CDR2.

[0065] SEQ ID NO: 12 is the variable light chain CDR3, referred to herein as 11E12-VL-CDR3.

[0066] SEQ ID NO: 13 is the variable heavy chain CDR1, referred to herein as CAN_ZIL1_VH_CDR1.

[0067] SEQ ID NO: 14 is the variable heavy chain CDR2, referred to herein as CAN_ZIL1_VH_CDR2.

[0068] SEQ ID NO: 15 is the variable heavy chain CDR3, referred to herein as CAN_ZIL1_VH_CDR3.

[0069] SEQ ID NO: 16 is the variable light chain CDR1, referred to herein as CAN_ZIL1_VL_CDR1.

[0070] SEQ ID NO: 17 is the variable light chain CDR2, referred to herein as CAN_ZIL1_VL_CDR2.

[0071] SEQ ID NO: 18 is the variable light chain CDR3, referred to herein as CAN_ZIL1_VL_CDR3.

[0072] SEQ ID NO: 19 is the variable heavy chain CDR1, referred to herein as CAN_ZIL8_VH_CDR1.

[0073] SEQ ID NO: 20 is the variable heavy chain CDR2, referred to herein as CAN_ZIL8_VH_CDR2.

[0074] SEQ ID NO: 21 is the variable heavy chain CDR3 referred to herein as CAN_ZIL8_VH_CDR3.

[0075] SEQ ID NO: 22 is the variable light chain CDR1, referred to herein as CAN_ZIL8_VL_CDR1.

[0076] SEQ ID NO: 23 is the variable light chain CDR2, referred to herein as CAN_ZIL8_VL_CDR2.

[0077] SEQ ID NO: 24 is the variable light chain CDR3, referred to herein as CAN_ZIL8_VL_CDR3.

[0078] SEQ ID NO: 25 is the variable heavy chain CDR1, referred to herein as CAN_ZIL9_VH_CDR1.

[0079] SEQ ID NO: 26 is the variable heavy chain CDR2, referred to herein as CAN_ZIL9_VH_CDR2.

[0080] SEQ ID NO: 27 is the variable heavy chain CDR3 referred to herein as CAN_ZIL9_VH_CDR3.

[0081] SEQ ID NO: 28 is the variable light chain CDR1, referred to herein as CAN_ZIL9_VL_CDR1.

[0082] SEQ ID NO: 29 is the variable light chain CDR2, referred to herein as CAN_ZIL9_VL_CDR2.

[0083] SEQ ID NO: 30 is the variable light chain CDR3, referred to herein as CAN_ZIL9_VL_CDR3.

[0084] SEQ ID NO: 31 is the variable heavy chain CDR1, referred to herein as CAN_ZIL11_VH_CDR1.

[0085] SEQ ID NO: 32 is the variable heavy chain CDR2, referred to herein as CAN_ZIL11_VH_CDR2.

[0086] SEQ ID NO: 33 is the variable heavy chain CDR3 referred to herein as CAN_ZIL11_VH_CDR3.

[0087] SEQ ID NO: 34 is the variable light chain CDR1, referred to herein as CAN_ZIL11_VL_CDR1.

[0088] SEQ ID NO: 35 is the variable light chain CDR2, referred to herein as CAN_ZIL11_VL_CDR2.

[0089] SEQ ID NO: 36 is the variable light chain CDR3, referred to herein as CAN_ZIL11_VL_CDR3.

[0090] SEQ ID NO: 37 is the variable heavy chain CDR1, referred to herein as CAN_ZIL69_VH_CDR1.

[0091] SEQ ID NO: 38 is the variable heavy chain CDR2, referred to herein as CAN_ZIL69_VH_CDR2.

[0092] SEQ ID NO: 39 is the variable heavy chain CDR3 referred to herein as CAN_ZIL69_VH_CDR3.

[0093] SEQ ID NO: 40 is the variable light chain CDR1, referred to herein as CAN_ZIL69_VL_CDR1.

[0094] SEQ ID NO: 41 is the variable light chain CDR2, referred to herein as CAN_ZIL69_VL_CDR2.

[0095] SEQ ID NO: 42 is the variable light chain CDR3, referred to herein as CAN_ZIL69_VL_CDR3.

[0096] SEQ ID NO: 43 is the variable heavy chain CDR1 referred to herein as CAN_ZIL94_VH_CDR1.

[0097] SEQ ID NO: 44 is the variable heavy chain CDR2, referred to herein as CAN_ZIL94_VH_CDR2.

[0098] SEQ ID NO: 45 is the variable heavy chain CDR3 referred to herein as CAN_ZIL94_VH_CDR3.

[0099] SEQ ID NO: 46 is the variable light chain CDR1, referred to herein as CAN_ZIL94_VL_CDR1.

[0100] SEQ ID NO: 47 is the variable light chain CDR2, referred to herein as CAN_ZIL94_VL_CDR2.

[0101] SEQ ID NO: 48 is the variable light chain CDR3, referred to herein as CAN_ZIL94_VL_CDR3.

[0102] SEQ ID NO: 49 is a variable weight sequence designated herein as CAN_ZIL154_VH_CDR1. The CDR1 of the ribosomal chain is

[0103] SEQ ID NO: 50 is the variable heavy chain CDR2, referred to herein as CAN_ZIL154_VH_CDR2.

[0104] SEQ ID NO: 51 is the variable heavy chain CDR3 referred to herein as CAN_ZIL154_VH_CDR3.

[0105] SEQ ID NO: 52 is the variable light chain CDR1, referred to herein as CAN_ZIL154_VL_CDR1.

[0106] SEQ ID NO: 53 is the variable light chain CDR2, referred to herein as CAN_ZIL154_VL_CDR2.

[0107] SEQ ID NO: 54 is the variable light chain CDR3, referred to herein as CAN_ZIL154_VL_CDR3.

[0108] SEQ ID NO: 55 is the variable heavy chain CDR1 referred to herein as CAN_ZIL159_VH_CDR1.

[0109] SEQ ID NO: 56 is the variable heavy chain CDR2, referred to herein as CAN_ZIL159_VH_CDR2.

[0110] SEQ ID NO: 57 is the variable heavy chain CDR3 referred to herein as CAN_ZIL159_VH_CDR3.

[0111] SEQ ID NO: 58 is the variable light chain CDR1, referred to herein as CAN_ZIL159_VL_CDR1.

[0112] SEQ ID NO: 59 is the variable light chain CDR2, referred to herein as CAN_ZIL159_VL_CDR2.

[0113] SEQ ID NO: 60 is the variable light chain CDR3, referred to herein as CAN_ZIL159_VL_CDR3.

[0114] SEQ ID NO: 61 is the variable heavy chain CDR1, referred to herein as CAN_ZIL171_VH_CDR1.

[0115] SEQ ID NO: 62 is the variable heavy chain CDR2, referred to herein as CAN_ZIL171_VH_CDR2.

[0116] SEQ ID NO: 63 is the variable heavy chain CDR3 referred to herein as CAN_ZIL171_VH_CDR3.

[0117] SEQ ID NO: 64 is the variable light chain CDR1, referred to herein as CAN_ZIL171_VL_CDR1.

[0118] SEQ ID NO: 65 is the variable light chain CDR2, referred to herein as CAN_ZIL171_VL_CDR2.

[0119] SEQ ID NO: 66 is the variable light chain CDR3, referred to herein as CAN_ZIL171_VL_CDR3.

[0120] SEQ ID NO: 67 is the variable heavy chain designated herein as MU_15H05_VH.

[0121] SEQ ID NO: 68 is the nucleotide sequence encoding the variable heavy chain designated herein as MU_15H05_VH.

[0122] SEQ ID NO: 69 is the variable light chain designated herein as MU_15H05_VL.

[0123] SEQ ID NO: 70 is the nucleotide sequence encoding the variable light chain designated herein as MU_15H05_VL.

[0124] SEQ ID NO: 71 is the variable heavy chain designated herein as MU-11E12-VH.

[0125] SEQ ID NO: 72 is the nucleotide sequence encoding the variable heavy chain designated herein as MU-11E12-VH.

[0126] SEQ ID NO: 73 is the variable light chain designated herein as MU-11E12-VL.

[0127] SEQ ID NO: 74 is the nucleotide sequence encoding the variable light chain designated herein as MU-11E12-VL.

[0128] SEQ ID NO: 75 is the variable heavy chain referred to herein as CAN-ZIL1_VH.

[0129] SEQ ID NO: 76 is the nucleotide sequence encoding the variable heavy chain designated herein as CAN-ZIL1_VH.

[0130] SEQ ID NO: 77 is the variable light chain referred to herein as CAN-ZIL1_VL.

[0131] SEQ ID NO: 78 is the nucleotide sequence encoding the variable light chain designated herein as CAN-ZIL1_VL.

[0132] SEQ ID NO: 79 is the variable heavy chain referred to herein as CAN-ZIL8_VH.

[0133] SEQ ID NO: 80 is the nucleotide sequence encoding the variable heavy chain designated herein as CAN-ZIL8_VH.

[0134] SEQ ID NO: 81 is the variable light chain referred to herein as CAN-ZIL8_VL.

[0135] SEQ ID NO: 82 is the nucleotide sequence encoding the variable light chain designated herein as CAN-ZIL8_VL.

[0136] SEQ ID NO: 83 is the variable heavy chain referred to herein as CAN-ZIL9_VH.

[0137] SEQ ID NO: 84 is the nucleotide sequence encoding the variable heavy chain designated herein as CAN-ZIL9_VH.

[0138] SEQ ID NO: 85 is the variable light chain referred to herein as CAN-ZIL9_VL.

[0139] SEQ ID NO: 86 is the nucleotide sequence encoding the variable light chain designated herein as CAN-ZIL9_VL.

[0140] SEQ ID NO: 87 is the variable heavy chain referred to herein as CAN-ZIL11_VH.

[0141] SEQ ID NO: 88 is the nucleotide sequence encoding the variable heavy chain designated herein as CAN-ZIL11_VH.

[0142] SEQ ID NO: 89 is the variable light chain referred to herein as CAN-ZIL11_VL.

[0143] SEQ ID NO: 90 is the nucleotide sequence encoding the variable light chain designated herein as CAN-ZIL11_VL.

[0144] SEQ ID NO: 91 is the variable heavy chain referred to herein as CAN-ZIL69_VH.

[0145] SEQ ID NO: 92 is the nucleotide sequence encoding the variable heavy chain designated herein as CAN-ZIL69_VH.

[0146] SEQ ID NO: 93 is the variable light chain referred to herein as CAN-ZIL69_VL.

[0147] SEQ ID NO: 94 is the nucleotide sequence encoding the variable light chain designated herein as CAN-ZIL69_VL.

[0148] SEQ ID NO: 95 is the variable heavy chain referred to herein as CAN-ZIL94_VH.

[0149] SEQ ID NO: 96 is the nucleotide sequence encoding the variable heavy chain designated herein as CAN-ZIL94_VH.

[0150] SEQ ID NO: 97 is the variable light chain referred to herein as CAN-ZIL94_VL.

[0151] SEQ ID NO: 98 is the nucleotide sequence encoding the variable light chain designated herein as CAN-ZIL94_VL.

[0152] SEQ ID NO: 99 is the variable heavy chain referred to herein as CAN-ZIL154_VH.

[0153] SEQ ID NO: 100 is the nucleotide sequence encoding the variable heavy chain designated herein as CAN-ZIL154_VH.

[0154] SEQ ID NO: 101 is the variable light chain referred to herein as CAN-ZIL154_VL.

[0155] SEQ ID NO: 102 is the nucleotide sequence encoding the variable light chain designated herein as CAN-ZIL154_VL.

[0156] SEQ ID NO: 103 is the variable heavy chain referred to herein as CAN-ZIL159_VH.

[0157] SEQ ID NO: 104 is the nucleotide sequence encoding the variable heavy chain designated herein as CAN-ZIL159_VH.

[0158] SEQ ID NO: 105 is the variable light chain referred to herein as CAN-ZIL159_VL.

[0159] SEQ ID NO: 106 is the nucleotide sequence encoding the variable light chain designated herein as CAN-ZIL159_VL.

[0160] SEQ ID NO: 107 is the variable heavy chain referred to herein as CAN-ZIL171_VH.

[0161] SEQ ID NO: 108 is the nucleotide sequence encoding the variable heavy chain designated herein as CAN-ZIL171_VH.

[0162] SEQ ID NO: 109 is the variable light chain referred to herein as CAN-ZIL171_VL.

[0163] SEQ ID NO: 110 is the nucleotide sequence encoding the variable light chain designated herein as CAN-ZIL171_VL.

[0164] SEQ ID NO: 111 is the variable heavy chain designated herein as FEL_11E12_VH1.

[0165] SEQ ID NO: 112 is the nucleotide sequence encoding the variable heavy chain designated herein as FEL_11E12_VH1.

[0166] SEQ ID NO: 113 is the variable light chain referred to herein as FEL_11E12_VL1.

[0167] SEQ ID NO: 114 is the nucleotide sequence encoding the variable light chain designated herein as FEL_11E12_VL1.

[0168] SEQ ID NO: 115 is the variable light chain referred to herein as FEL_11E12_VL2.

[0169] SEQ ID NO: 116 is the nucleotide sequence encoding the variable light chain designated herein as FEL_11E12_VL2.

[0170] SEQ ID NO: 117 is the variable light chain referred to herein as FEL_11E12_VL1_FW2.

[0171] SEQ ID NO: 118 is the nucleotide sequence encoding the variable light chain designated herein as FEL_11E12_VL1_FW2.

[0172] SEQ ID NO: 119 is the variable light chain designated herein as FEL_11E12_VL1_K46Q.

[0173] SEQ ID NO: 120 is the nucleotide sequence encoding the variable light chain designated herein as FEL_11E12_VL1_K46Q.

[0174] SEQ ID NO: 121 is the variable heavy chain designated herein as FEL_15H05_VH1.

[0175] SEQ ID NO: 122 is the nucleotide sequence encoding the variable heavy chain designated herein as FEL_15H05_VH1.

[0176] SEQ ID NO: 123 is the variable heavy chain designated herein as FEL_15H05_VH2.

[0177] SEQ ID NO: 124 is the nucleotide sequence encoding the variable heavy chain designated herein as FEL_15H05_VH2.

[0178] SEQ ID NO: 125 is the variable heavy chain designated herein as FEL_15H05_VH3.

[0179] SEQ ID NO: 126 is the nucleotide sequence encoding the variable heavy chain designated herein as FEL_15H05_VH3.

[0180] SEQ ID NO: 127 is the variable light chain designated herein as FEL_15H05_VL1.

[0181] SEQ ID NO: 128 is the nucleotide sequence encoding the variable light chain designated herein as FEL_15H05_VL1.

[0182] SEQ ID NO: 129 is the variable light chain designated herein as FEL_15H05_VL2.

[0183] SEQ ID NO: 130 is the nucleotide sequence encoding the variable light chain designated herein as FEL_15H05_VL2.

[0184] SEQ ID NO: 131 is the variable light chain designated herein as FEL_15H05_VL3.

[0185] SEQ ID NO: 132 is the nucleotide sequence encoding the variable light chain designated herein as FEL_15H05_VL3.

[0186] SEQ ID NO: 133 is the variable light chain referred to herein as FEL_15H05_VL1_FW1.

[0187] SEQ ID NO: 134 is the nucleotide sequence encoding the variable light chain designated herein as FEL_15H05_VL1_FW1.

[0188] SEQ ID NO: 135 is the variable light chain designated herein as FEL_15H05_VL1_FW2.

[0189] SEQ ID NO: 136 is the nucleotide sequence encoding the variable light chain designated herein as FEL_15H05_VL1_FW2.

[0190] SEQ ID NO: 137 is the variable light chain designated herein as FEL_15H05_VL1_FW3.

[0191] SEQ ID NO: 138 identifies a variable light chain, designated herein as FEL_15H05_VL1_FW3. The nucleotide sequence encoding the strand.

[0192] SEQ ID NO: 139 is the variable light chain referred to herein as FEL_15H05_VL1_FW1_FW2.

[0193] SEQ ID NO: 140 is the nucleotide sequence encoding the variable light chain designated herein as FEL_15H05_VL1_FW1_FW2.

[0194] SEQ ID NO: 141 is the variable light chain referred to herein as FEL_15H05_VL1_FW1_FW3.

[0195] SEQ ID NO: 142 is the nucleotide sequence encoding the variable light chain designated herein as FEL_15H05_VL1_FW1_FW3.

[0196] SEQ ID NO: 143 is the variable light chain referred to herein as FEL_15H05_VL1_FW2_FW3.

[0197] SEQ ID NO: 144 is the nucleotide sequence encoding the variable light chain designated herein as FEL_15H05_VL1_FW2_FW3.

[0198] SEQ ID NO: 145 is the variable light chain designated herein as FEL_15H05_VL1_FW2_K42N.

[0199] SEQ ID NO: 146 is the nucleotide sequence encoding the variable light chain designated herein as FEL_15H05_VL1_FW2_K42N.

[0200] SEQ ID NO: 147 is the variable light chain designated herein as FEL_15H05_VL1_FW2_V43I.

[0201] SEQ ID NO: 148 is the nucleotide sequence encoding the variable light chain designated herein as FEL_15H05_VL1_FW2_V43I.

[0202] SEQ ID NO: 149 is the variable light chain designated herein as FEL_15H05_VL1_FW2_L46V.

[0203] SEQ ID NO: 150 is the nucleotide sequence encoding the variable light chain designated herein as FEL_15H05_VL1_FW2_L46V.

[0204] SEQ ID NO: 151 is the variable light chain designated herein as FEL_15H05_VL1_FW2_Y49N.

[0205] SEQ ID NO: 152 is the nucleotide sequence encoding the variable light chain designated herein as FEL_15H05_VL1_FW2_Y49N.

[0206] SEQ ID NO: 153 is the variable light chain referred to herein as FEL_15H05_VL1_FW2_K42N_V43I.

[0207] SEQ ID NO: 154 is the nucleotide sequence encoding the variable light chain designated herein as FEL_15H05_VL1_FW2_K42N_V43I.

[0208] SEQ ID NO: 155 is the amino acid sequence of the canine IL-31 protein, referred to herein as canine_IL31.

[0209] SEQ ID NO: 156 is the nucleotide sequence encoding the canine IL-31 protein, referred to herein as canine_IL31.

[0210] SEQ ID NO: 157 is the amino acid sequence corresponding to the wild-type feline IL-31 protein with a C-terminal His tag, referred to herein as feline_IL31_wildtype.

[0211] SEQ ID NO: 158 is the nucleotide sequence encoding the amino acid sequence referred to herein as feline_IL31_wildtype.

[0212] SEQ ID NO: 159 is the amino acid sequence corresponding to the feline IL-31 protein with an N-terminal His tag, referred to herein as feline_IL_31_E_coli.

[0213] SEQ ID NO: 160 is the nucleotide sequence encoding the amino acid sequence designated herein as feline_IL_31_E_coli.

[0214] SEQ ID NO: 161 is the amino acid sequence corresponding to the mutant feline IL-31 11E12 protein with a C-terminal His tag, referred to herein as feline_IL31_11E12_mutant.

[0215] SEQ ID NO: 162 is the nucleotide sequence encoding the amino acid sequence referred to herein as feline_IL31_11E12_variant.

[0216] SEQ ID NO: 163 is the amino acid sequence corresponding to the mutant feline IL-31 15H05 protein with a C-terminal His tag, referred to herein as feline_IL31_15H05_mutant.

[0217] SEQ ID NO: 164 is the nucleotide sequence encoding the amino acid sequence referred to herein as feline_IL31_15H05_variant.

[0218] SEQ ID NO: 165 is the amino acid sequence of the equine IL-31 protein, referred to herein as equine_IL31.

[0219] SEQ ID NO: 166 is the nucleotide sequence encoding the equine IL-31 protein, referred to herein as equine_IL31.

[0220] SEQ ID NO: 167 is the amino acid sequence corresponding to the extracellular domain of feline OSMR fused to human IgG1 Fc, designated herein as feline_OSMR_hIgG1_Fc.

[0221] SEQ ID NO: 168 is the nucleotide sequence encoding the amino acid sequence designated herein as feline_OSMR_hIgG1_Fc.

[0222] SEQ ID NO: 169 is the amino acid sequence corresponding to feline IL-31Ra fused to human IgG1 Fc, designated herein as feline_IL31Ra_HIgG1_Fc_X1_Fn3.

[0223] SEQ ID NO: 170 is the nucleotide sequence encoding the amino acid sequence designated herein as feline_IL31Ra_HIgG1_Fc_X1_Fn3.

[0224] SEQ ID NO: 171 is the feline heavy chain referred to herein as feline_HC_allele A_wt.

[0225] SEQ ID NO: 172 is the nucleotide sequence encoding the feline heavy chain referred to herein as feline_HC_allele A_wt.

[0226] SEQ ID NO: 173 is a feline heavy chain, referred to herein as feline_HC_allele A_1, which has been engineered by replacing M, L, and G at positions 120, 121, and 123, respectively, of the wild-type sequence of SEQ ID NO: 171 with alanine (A) to eliminate antibody effector function.

[0227] SEQ ID NO: 174 is the nucleotide sequence encoding the feline heavy chain referred to herein as feline_HC_allele A_1.

[0228] SEQ ID NO: 175 is a feline kappa light chain, referred to herein as feline_LC_kappa_G_minus, which has been engineered with a glycosylation knockout (G-) at position 103, such that the N normally present at this position in wild-type feline kappa light chain is changed to a Q.

[0229] SEQ ID NO: 176 is the nucleotide sequence encoding the amino acid sequence of the feline kappa light chain, referred to herein as feline_LC_kappa_G_minus.

[0230] SEQ ID NO: 177 is the canine heavy chain designated herein as Canine_HC_65_1.

[0231] SEQ ID NO: 178 is the nucleotide sequence encoding the canine heavy chain designated herein as canine_HC_65_1.

[0232] SEQ ID NO: 179 is the canine kappa light chain, referred to herein as canine_LC_kappa.

[0233] SEQ ID NO: 180 is the nucleotide sequence encoding the canine kappa light chain, referred to herein as canine_LC_kappa.

[0234] SEQ ID NO: 181 is the amino acid sequence of human IL-31.

[0235] SEQ ID NO: 182 is the variable light chain mAb sequence designated herein as Can_11E12_VL_cUn_1.

[0236] SEQ ID NO: 183 is the nucleotide sequence encoding the variable light chain mAb sequence designated herein as Can_11E12_VL_cUn_1.

[0237] SEQ ID NO: 184 is the variable light chain mAb sequence designated herein as Can_11E12_VL_cUn_FW2.

[0238] SEQ ID NO: 185 is the nucleotide sequence encoding the variable light chain mAb sequence designated herein as Can_11E12_VL_cUn_FW2.

[0239] SEQ ID NO: 186 is a feline kappa light chain referred to herein as feline_LC_kappa_G_minus_QRE_minus, which contains i) a glycosylation knockout at position 103, so that the N normally present at this position in wild-type feline kappa light chain is changed to a Q. (G-) and ii) engineered with a deletion of the C-terminal QRE relative to the wild type.

[0240] SEQ ID NO: 187 is the nucleotide sequence encoding the feline kappa light chain, referred to herein as feline_LC_kappa_G_minus_QRE_minus.

[0241] SEQ ID NO: 188 is the mouse heavy chain referred to herein as Mouse_HC_IgG1.

[0242] SEQ ID NO: 189 is the nucleotide sequence encoding the mouse heavy chain designated herein as Mouse_HC_IgG1.

[0243] SEQ ID NO: 190 is the mouse kappa light chain, referred to herein as mouse_LC_kappa.

[0244] SEQ ID NO: 191 is the nucleotide sequence encoding the mouse kappa light chain, referred to herein as mouse_LC_kappa.

[0245] SEQ ID NO: 192 is the human kappa light chain, referred to herein as human_LC_kappa.

[0246] SEQ ID NO: 193 is the nucleotide sequence encoding the human kappa light chain, referred to herein as human_LC_kappa.

[0247] SEQ ID NO: 194 is the wild-type canine kappa light chain, referred to herein as canine_LC_kappa_wt.

[0248] SEQ ID NO: 195 is the nucleotide sequence encoding the wild-type canine kappa light chain, referred to herein as canine_LC_kappa_wt.

[0249] SEQ ID NO: 196 is the porcine kappa light chain, referred to herein as porcine_LC_kappa.

[0250] SEQ ID NO: 197 is the nucleotide sequence encoding the porcine kappa light chain, referred to herein as porcine_LC_kappa.

[0251] SEQ ID NO: 198 is the mink kappa light chain, referred to herein as mink_LC_kappa.

[0252] SEQ ID NO:199 is the nucleotide sequence encoding the mink kappa light chain, referred to herein as mink_LC_kappa.

[0253] SEQ ID NO: 200 is the variable heavy chain CDR1 designated herein as Mu_04H07_VH_CDR1.

[0254] SEQ ID NO: 201 is the variable heavy chain CDR2 referred to herein as Mu_04H07_VH_CDR2.

[0255] SEQ ID NO: 202 is the variable heavy chain CDR3 referred to herein as Mu_04H07_VH_CDR3.

[0256] SEQ ID NO: 203 is the variable light chain CDR1, referred to herein as Mu_04H07_VL_CDR1.

[0257] SEQ ID NO: 204 is the variable light chain CDR2, referred to herein as Mu_04H07_VL_CDR2.

[0258] SEQ ID NO: 205 is the variable light chain CDR3 designated herein as Mu_04H07_VL_CDR3.

[0259] SEQ ID NO: 206 is the variable heavy chain CDR1 designated herein as Mu_06A09_VH_CDR1.

[0260] SEQ ID NO: 207 is the variable heavy chain CDR2 designated herein as Mu_06A09_VH_CDR2.

[0261] SEQ ID NO: 208 is the variable heavy chain CDR3 designated herein as Mu_06A09_VH_CDR3.

[0262] SEQ ID NO: 209 is the variable light chain CDR1, referred to herein as Mu_06A09_VL_CDR1.

[0263] SEQ ID NO: 210 is the variable light chain CDR2, referred to herein as Mu_06A09_VL_CDR2.

[0264] SEQ ID NO: 211 is the variable light chain CDR3 designated herein as Mu_06A09_VL_CDR3.

[0265] SEQ ID NO: 212 is the variable heavy chain designated herein as Mu_04H07_VH.

[0266] SEQ ID NO: 213 is the nucleotide sequence encoding the variable heavy chain designated herein as Mu_04H07_VH.

[0267] SEQ ID NO: 214 is the variable light chain designated herein as Mu_04H07_VL.

[0268] SEQ ID NO: 215 is the nucleotide sequence encoding the variable light chain designated herein as Mu_04H07_VL.

[0269] SEQ ID NO: 216 is the variable heavy chain designated herein as Mu_06A09_VH.

[0270] SEQ ID NO: 217 is the nucleotide sequence encoding the variable heavy chain designated herein as Mu_06A09_VH.

[0271] SEQ ID NO: 218 is the variable light chain designated herein as Mu_06A09_VL.

[0272] SEQ ID NO: 219 is the nucleotide sequence encoding the variable light chain designated herein as Mu_06A09_VL.

[0273] SEQ ID NO: 220 is the variable heavy chain designated herein as ZTS_768_VH.

[0274] SEQ ID NO: 221 is the nucleotide sequence encoding the variable heavy chain designated herein as ZTS_768_VH.

[0275] SEQ ID NO: 222 is the variable light chain designated herein as ZTS_768_VL.

[0276] SEQ ID NO: 223 is the nucleotide sequence encoding the variable light chain designated herein as ZTS_768_VL.

[0277] SEQ ID NO: 224 is the variable heavy chain designated herein as ZTS_943_VH.

[0278] SEQ ID NO: 225 is the nucleotide sequence encoding the variable heavy chain designated herein as ZTS_943_VH.

[0279] SEQ ID NO: 226 is the variable light chain designated herein as ZTS_943_VL.

[0280] SEQ ID NO: 227 is the nucleotide sequence encoding the variable light chain designated herein as ZTS_943_VL.

[0281] SEQ ID NO: 228 is the variable heavy chain designated herein as ZTS_5864_VH.

[0282] SEQ ID NO: 229 is the nucleotide sequence encoding the variable heavy chain designated herein as ZTS_5864_VH.

[0283] SEQ ID NO: 230 is the variable light chain designated herein as ZTS_5864_VL.

[0284] SEQ ID NO: 231 is the nucleotide sequence encoding the variable light chain designated herein as ZTS_5864_VL.

[0285] SEQ ID NO: 232 is the variable heavy chain designated herein as ZTS_5865_VH.

[0286] SEQ ID NO: 233 is the nucleotide sequence encoding the variable heavy chain designated herein as ZTS_5865_VH.

[0287] SEQ ID NO: 234 is the variable light chain designated herein as ZTS_5865_VL.

[0288] SEQ ID NO: 235 is the nucleotide sequence encoding the variable light chain designated herein as ZTS_5865_VL.

[0289] SEQ ID NO: 236 is the amino acid sequence of the feline kappa light chain, referred to herein as feline_LC_lambda.

[0290] SEQ ID NO: 237 is the nucleotide sequence encoding the feline kappa light chain, referred to herein as feline_LC_lambda.

[0291] definition Before describing the present invention in detail, some terms used in the context of the present invention will be defined. 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 their art-recognized meanings.

[0292] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "an antibody" includes a plurality of such antibodies.

[0293] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements.

[0294] As used herein, an epitope refers to an antigenic determinant recognized by an antibody's CDR. In other words, an epitope refers to that portion of any molecule that can be recognized and bound by an antibody. Unless otherwise specified, the term "epitope" as used herein refers to the region of IL-31 with which an anti-IL-31 agent reacts.

[0295] An "antigen" is a molecule or portion of a molecule capable of being bound by an antibody, which in turn is capable of being recognized and bound by the antibody (the binding region of the corresponding antibody is sometimes referred to as the paratope). Generally, an epitope consists of a chemically active surface grouping of molecules (e.g., amino acids or sugar side chains) with specific three-dimensional structural and charge characteristics. An epitope is an antigenic determinant on a protein that is recognized by the immune system. Components of the immune system that recognize epitopes are antibodies, T cells, and B cells. T cell epitopes are displayed on the surface of antigen-presenting cells (APCs) and are typically 8–11 amino acids long (MHC class I) or 15 or more amino acids long (MHC class II). Recognition of displayed MHC-peptide complexes is essential for T cell activation. These mechanisms enable proper recognition of self proteins relative to "non-self" proteins such as bacteria and viruses. Individual, not necessarily adjacent, amino acid residues contribute to interaction with the APC binding groove and subsequent recognition by the T cell receptor (Janeway, Travers, Walport, Immunobiology: The Immune System in Health and Disease. 5 th edition New York: Garland Science; 2001). Epitopes recognized by soluble antibodies and cell surface-associated B cell receptors vary greatly in length and degree of contiguousness (Sivalingam and Shepherd, Immunol. 2012;51(3-4):304-309). Linear epitopes, or even epitopes found within a continuous protein sequence, often have noncontiguous amino acids that correspond to key contact points with the antibody paratope or B cell receptor. Epitopes recognized by antibodies and B cells may adopt a conformational structure in three-dimensional space, with amino acids constituting common contact regions on the protein, depending on the tertiary and quaternary structural features of the protein. Such residues are often found within spatially distinct regions of the primary amino acid sequence.

[0296] As used herein, a "mimotope" is a linear or constrained peptide that mimics an epitope of an antigen. A mimotope can have a primary amino acid sequence capable of eliciting a T cell effector response and / or the three-dimensional structure necessary to bind to B cells that mature an adaptive immune response in an animal. Antibodies against a given epitope antigen recognize mimotopes that mimic that epitope.

[0297] The term "specifically" in the context of antibody binding refers to high avidity and / or high affinity binding of an antibody to a particular antigen, i.e., polypeptide, or epitope. In many embodiments, the specific antigen is the antigen (or a fragment or subfraction of the antigen) used to immunize the animal host from which antibody-producing cells are isolated. Specific binding of an antibody to an antigen is stronger than the binding of the same antibody to other antigens. An antibody that specifically binds to a polypeptide may also be able to bind other polypeptides at weak, but detectable, levels (e.g., 10% or less of the binding shown to the polypeptide of interest). Such weak binding, i.e., background binding, is readily distinguishable from the specific binding of the antibody to the polypeptide of interest, e.g., by use of appropriate controls. Generally, specific antibodies can be produced in 10 -7 M or less, e.g. 10 -8 M or less (e.g., 10 -9 M or less, 10 -10 Below, 10 -11 Below, 10 -12 Less than or equal to 10 -13 (e.g., K) D It binds with a binding affinity of

[0298] 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 can be a polyclonal antibody, a monoclonal antibody, a synthetic antibody, a recombinant antibody, a chimeric antibody, a heterochimeric antibody, a caninized antibody, a feline antibody, a whole canine antibody, a whole feline antibody, or a whole equine antibody. The term "antibody" preferably refers to monoclonal antibodies and fragments thereof, as well as immunologically binding equivalents thereof capable of binding to the IL-31 protein and fragments thereof. The term antibody is used to refer to both homogeneous molecules or mixtures, such as serum products, composed of multiple different molecular entities.

[0299] "Native antibodies" and "native immunoglobulins" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced interchain disulfide bridges. Each heavy chain contains at one end a variable domain (V H ), followed by multiple constant domains. Each light chain has one end (V L ) and a constant domain at the other end, with the constant domain of the light chain aligned with the first constant domain of the heavy chain, and the variable domain of the light chain aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain variable domain and the heavy-chain variable domain.

[0300] The term "antibody fragment" refers to less than an 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, and the like.

[0301] The term "variable" region includes framework and CDRs (also known as hypervariable regions), and refers to the fact that certain portions of the variable domains differ significantly in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. Variability is concentrated in three segments called hypervariable regions within the variable domains of both the light and heavy chains. The more highly conserved portions of the variable domains are called framework regions (FR). Native heavy and light chain variable domains each contain multiple FR regions that largely adopt a β-sheet configuration, connected by three hypervariable regions, which form loops that connect and, in some cases, form part of the β-sheet structure. The hypervariable regions in each chain are held in close proximity to the hypervariable regions of the other chain by the FRs and contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), pp. 647-669). The constant domains are not directly involved in binding an antibody to an antigen, but exhibit various effector functions, such as allowing the antibody to participate in antibody-dependent cellular cytotoxicity.

[0302] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen-binding. The hypervariable region includes the "complementarity determining regions" or "CDRs" (Kaba). (1991) (supra)) and / or amino acid residues from the "hypervariable loops" (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0303] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments. Each Fab contains a single antigen-binding site, plus a residual "Fc" fragment, the Fc designation reflecting its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that contains two antigen-binding sites and is still capable of cross-linking antigen.

[0304] "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In this configuration, the three hypervariable regions of each variable domain interact to form a V H -V L The six hypervariable regions define an antigen-binding site on the surface of the dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0305] Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of several residues to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteine(s) from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical linkages for antibody fragments are also known.

[0306] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0307] Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chains. 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 designations). The heavy chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of various classes of immunoglobulins are well known in multiple species. The prevalence of distinct isotypes and functional activities associated with these constant domains is species-specific and must be experimentally defined.

[0308] As defined herein, a "monoclonal antibody" is an antibody produced by a single cell clone (particularly a single cell clone such as a hybridoma cell) and is therefore a single, pure, homogeneous type of antibody. All monoclonal antibodies produced from the same clone are identical and have the same antigen specificity. The term "monoclonal" refers to a single cell clone, a single cell, and the progeny of that cell.

[0309] As defined herein, a "whole dog antibody" is an antibody produced by a cell clone (typically a CHO cell line ), and therefore a single, pure, homogeneous type of antibody. Antibodies identified from a single B cell of an immunized mammal, such as a dog, are made as recombinant IgG proteins after identification of their variable domain sequences. When such variable domains are grafted onto dog constant domains (heavy and light chain kappa or lambda constant), recombinant whole dog antibodies are produced. All whole dog monoclonal antibodies produced from the same clone are identical and have the same antigen specificity. The term "monoclonal" refers to a single cell clone, a single cell, and the progeny of that cell.

[0310] As defined herein, a "whole feline antibody" is a monoclonal antibody produced by a cell clone (typically a CHO cell line) and is therefore a single, pure, homogeneous type of antibody. Antibodies identified from a single B cell of an immunized mammal, such as a dog, are produced as recombinant IgG proteins after identification of their variable domain sequences. Grafting of such variable domains into feline constant domains (heavy and light chain kappa or lambda constant) produces recombinant whole feline antibodies. All whole feline monoclonal antibodies produced from the same clone are identical and have the same antigen specificity. The term "monoclonal" refers to a single cell clone, a single cell, and the progeny of that cell.

[0311] As defined herein, a "whole horse antibody" is a monoclonal antibody produced by a cell clone (typically a CHO cell line) and is therefore a single, pure, homogeneous type of antibody. An antibody identified from a single B cell of an immunized mammal, such as a dog, is made as a recombinant IgG protein after identification of its variable domain sequence. When such variable domains are grafted onto equine constant domains (heavy and light chain kappa or lambda constant), a recombinant whole horse antibody is produced. All whole horse monoclonal antibodies produced from the same clone are identical and have the same antigen specificity. The term "monoclonal" refers to a single cell clone, a single cell, and the progeny of that cell.

[0312] Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which portions of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies from a particular species, while the remainder of the chain(s) are identical or homologous to corresponding sequences in antibodies from another species, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. Typically, chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species. For example, the variable segments of genes from a mouse monoclonal antibody can be linked to canine constant segments. Chimeric mouse: In one embodiment of a canine IgG, the antigen-binding site is derived from a mouse and the F C The part is from a dog.

[0313] "Caninized" forms of non-canine (e.g., murine) antibodies are genetically engineered antibodies that contain minimal sequence derived from non-canine immunoglobulins. Caninized antibodies are canine immunoglobulin sequences (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-canine species (donor antibody), such as a mouse, having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the canine immunoglobulin sequences are replaced by corresponding non-canine residues. Furthermore, caninized antibodies can comprise residues that are not found in either the recipient antibody or the donor antibody. Such modifications are made to further refine antibody performance. Generally, caninized antibodies will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable regions corresponding to those of the non-canine immunoglobulin sequence and all or substantially all of the FRs being those of the canine immunoglobulin sequence. The caninized antibody also optionally comprises a complete or at least a portion of an immunoglobulin constant region (Fc), typically a complete or at least a portion of a canine immunoglobulin sequence. In one embodiment of speciation or caninization of a murine IgG, In this case, the murine CDRs are grafted onto a canine framework.

[0314] "Fennised" forms of non-feline (e.g., murine) antibodies are genetically engineered antibodies containing minimal sequence derived from non-feline immunoglobulins. Fenised antibodies are feline immunoglobulin sequences (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-feline species (donor antibody), such as mouse, having the desired specificity, affinity, and capacity. In some cases, framework region (FR) residues of the feline immunoglobulin sequence are replaced by corresponding non-feline residues. Furthermore, fenised antibodies can include residues that are not found in either the recipient antibody or the donor antibody. Such modifications are made to further refine antibody performance. Generally, fenised antibodies comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-feline immunoglobulin sequence and all or substantially all of the FRs are those of a feline immunoglobulin sequence. The felineized antibody also optionally comprises a complete or at least a portion of an immunoglobulin constant region (Fc), typically a complete or at least a portion of a feline immunoglobulin constant region (Fc).

[0315] "Equined" forms of non-equine (e.g., murine) antibodies are genetically engineered antibodies that contain minimal sequence derived from a non-equine immunoglobulin. Equined antibodies are equine immunoglobulin sequences (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-equine species (donor antibody), such as a mouse, having the desired specificity, affinity, and capacity. In some cases, framework region (FR) residues of the equine immunoglobulin sequence are replaced by corresponding non-equine residues. Furthermore, equine-enhanced antibodies can contain residues that are not found in either the recipient or donor antibody. Such modifications are made to further refine antibody performance. Generally, equine-enhanced antibodies comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-equine immunoglobulin sequence and all or substantially all of the FRs are those of an equine immunoglobulin sequence. The equine-modified antibody also optionally comprises a complete or at least a portion of an immunoglobulin constant region (Fc), typically a complete or at least a portion of an equine immunoglobulin constant region (Fc).

[0316] A "fully canine" antibody is a genetically engineered antibody that does not contain sequences derived from non-canine immunoglobulins. A fully canine antibody is one in which hypervariable region residues (recipient antibody) are derived from a naturally occurring canine antibody (donor antibody) having the desired specificity, affinity, and capacity. In some cases, framework region (FR) residues of the canine immunoglobulin sequence are replaced with corresponding non-canine residues. Furthermore, fully canine antibodies can contain residues that are not found in either the recipient antibody or the donor antibody, including, but not limited to, CDR changes to modify affinity. Such modifications are made to further refine antibody performance. Generally, fully canine antibodies contain substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable regions corresponding to those of a canine immunoglobulin sequence and all or substantially all of the FRs being those of a canine immunoglobulin sequence. A whole canine antibody optionally also comprises a complete or at least a portion of an immunoglobulin constant region (Fc), typically a complete or at least a portion of a canine immunoglobulin sequence.

[0317] A "full feline" antibody is a genetically engineered antibody that does not contain sequences derived from non-feline immunoglobulins. A full feline antibody is one in which hypervariable region residues are derived from feline immunoglobulin sequences (recipient antibody) from a natural feline antibody dog ​​(donor antibody) with the desired specificity, affinity, and capacity. In some cases, framework region (FR) residues of the feline immunoglobulin sequences are replaced with corresponding non-feline residues. Furthermore, a full feline antibody can contain residues that are not found in either the recipient antibody or the donor antibody, e.g., limited These modifications include, but are not limited to, alterations of the CDRs to modify affinity. Such modifications are made to further refine antibody performance. Generally, a full feline antibody will contain substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable regions corresponding to those of a feline immunoglobulin sequence, and all or substantially all of the FRs being those of a feline immunoglobulin sequence. Optionally, a full feline antibody will also contain a complete or at least a portion of an immunoglobulin constant region (Fc), typically a complete or at least a portion of a feline immunoglobulin sequence.

[0318] A "fully equine" antibody is a genetically engineered antibody that does not contain sequences derived from non-equine immunoglobulins. A fully equine antibody is an equine immunoglobulin sequence (recipient antibody) in which hypervariable region residues are derived from a natural equine antibody dog ​​(donor antibody) having the desired specificity, affinity, and capacity. In some cases, framework region (FR) residues of the equine immunoglobulin sequence are replaced with corresponding non-equine residues. Furthermore, fully equine antibodies can contain residues that are not found in either the recipient antibody or the donor antibody, including, for example, but not limited to, CDR changes to modify affinity. Such modifications are made to further refine antibody performance. Generally, fully equine antibodies contain substantially all of at least one, and typically two, variable domains in which all or substantially all of the hypervariable regions correspond to those of an equine immunoglobulin sequence and in which all or substantially all of the FRs are those of an equine immunoglobulin sequence. A fully equine antibody optionally also comprises a complete or at least a portion of an immunoglobulin constant region (Fc), typically a complete or at least a portion of an equine immunoglobulin sequence.

[0319] As defined herein, the term "heterochimeric" refers to an antibody in which one of the antibody chains (heavy or light) is caninized, felineized, or equineized, and the other is chimeric. In one embodiment, a felineized variable heavy chain (all CDRs are murine and all FRs are feline) is paired with a chimeric variable light chain (all CDRs are murine and all FRs are murine). In this embodiment, both the variable heavy and light chains are fused to feline constant regions.

[0320] As used herein, a "variant" anti-IL-31 antibody refers to a molecule whose amino acid sequence differs from that of a "parent" anti-IL-31 antibody due to the addition, deletion, and / or substitution of one or more amino acid residue(s) in the parent antibody sequence, but which retains at least one desired activity of the parent anti-IL-31 antibody. Desired activities may include the ability to specifically bind to an antigen, the ability to reduce, inhibit, or neutralize IL-31 activity in an animal, and the ability to inhibit IL-31-mediated pSTAT signaling in a cell-based assay. In one embodiment, the variant contains one or more amino acid substitution(s) in one or more hypervariable and / or framework region(s) of the parent antibody. For example, the variant may contain at least one substitution, e.g., about 1 to about 10, preferably about 2 to about 5, in one or more hypervariable and / or framework regions of the parent antibody. Typically, a variant has an amino acid sequence that has at least 50% amino acid sequence identity to the heavy or light chain variable domain sequence of the parent antibody, 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. Identity or homology to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical to the parent antibody residues after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Neither N-terminal, C-terminal, nor internal extensions, deletions, or insertions into the antibody sequence shall be considered to affect sequence identity or homology. Variants retain the ability to bind to IL-31 and preferably have a desired activity that is superior to that of the parent antibody. For example, a variant may have a stronger binding affinity, yet still reduce, inhibit, or neutralize IL-31 activity in an animal. In some cases, the ability of the antibody to inhibit IL-31-mediated pSTAT signaling in a cell-based assay may be enhanced.

[0321] By "variant" nucleic acid herein is meant a molecule that differs in sequence from a "parent" nucleic acid. Differences in polynucleotide sequence can arise from mutational changes such as deletion, substitution, or addition of one or more nucleotides. Each of these changes can occur alone or in combination one or more times within a given sequence.

[0322] A "parent" antibody herein is an antibody encoded by an amino acid sequence used to prepare a variant. In one embodiment, the parent antibody has canine framework regions and, if present, canine antibody constant region(s). For example, the parent antibody can be a caninized or canine antibody. As another example, the parent antibody can be a felineized or feline antibody. As yet another example, the parent antibody can be an equineized or equine antibody. In yet another example, the parent antibody is a murine monoclonal antibody.

[0323] As used throughout this specification and claims, the terms "antigen-binding region," "antigen-binding portion," and the like refer to the portion of an antibody molecule comprising amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen. The antibody-binding region includes "framework" amino acid residues necessary to maintain the proper conformation of the antigen-binding residues. Alternatively, the antigen-binding portion of an antibody in accordance with the invention may be referred to herein as, for example, an IL-31-specific peptide or polypeptide, or an anti-IL-31 peptide or polypeptide.

[0324] The term "isolated" means that a material (e.g., an antibody or nucleic acid) has been separated and / or recovered from components of its natural environment. Contaminant components of the material's natural environment are materials that may interfere with diagnostic or therapeutic uses of the material; such components may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. With respect to nucleic acids, isolated nucleic acids can include those separated from the 5' to 3' sequences with which they are normally associated within the chromosome. In preferred embodiments, the material is purified to greater than 95%, and most preferably greater than 99%, by weight of the material. Isolated material includes material in situ within recombinant cells, since at least one component of the material's natural environment will be absent. However, isolated material will usually be prepared by at least one purification step.

[0325] The term "label," as used herein, refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody or nucleic acid. The label itself may be detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.

[0326] The terms "nucleic acid," "polynucleotide," "nucleic acid molecule," and the like, may be used interchangeably herein and refer to a series of nucleotide bases (also called "nucleotides") in DNA and RNA. A nucleic acid can include deoxyribonucleotides, ribonucleotides, and / or their analogs. The term "nucleic acid" includes, for example, single-stranded and double-stranded molecules. A nucleic acid can be, for example, a gene or gene fragment, an exon, an intron, a DNA molecule (e.g., cDNA), an RNA molecule (e.g., mRNA), a recombinant nucleic acid, a plasmid, and other vectors, primers, and probes. Both 5' to 3' (sense) and 3' to 5' (antisense) polynucleotides are included.

[0327] "Subject" or "patient" refers to an animal in need of treatment that may be affected by a molecule of the invention. Animals that can be treated according to the invention include vertebrates, with mammals such as canines, felines, and equines being particularly preferred examples.

[0328] A "therapeutically effective amount" (or "effective amount") refers to an amount of an active ingredient (e.g., an agent according to the present invention) sufficient to produce a beneficial or desired result when administered to a subject or patient. An effective amount can be administered in one or more administrations, applications, or dosages. A therapeutically effective amount of a composition according to the present invention can be readily determined by one of ordinary skill in the art. In the context of the present invention, a "therapeutically effective amount" is an amount that produces an objectively measured change in one or more parameters associated with the treatment of a pruritic or allergic condition, including clinical improvement of symptoms. Of course, a therapeutically effective amount will vary depending on the particular subject and condition being treated, the subject's weight and age, the severity of the disease state, the particular compound selected, the dosing regimen to be followed, the timing of administration, the method of administration, etc., all of which can be readily determined by one of ordinary skill in the art.

[0329] As used herein, the term "therapeutic" encompasses the full range of treatments for a disease or disorder. "Therapeutic" agents of the invention can act in a prophylactic or preventative manner, or essentially ameliorative or curative manner, including those incorporating procedures designed to target animals that may be identified as at risk (pharmacogenetics), or can act to slow the rate or extent of progression of at least one symptom of the disease or disorder being treated.

[0330] "Treatment," "treating," and the like refer to both therapeutic treatment and prophylactic or preventative measures. Animals in need of treatment include animals already with the disorder as well as animals in which the disorder is to be prevented. The term "treatment" of or "treating" a disease or disorder includes preventing or protecting against the disease or disorder (i.e., preventing clinical symptoms from occurring), inhibiting the disease or disorder (i.e., preventing or suppressing the development of clinical symptoms), and / or alleviating the disease or disorder (i.e., reducing clinical symptoms). It will be understood that it is not always possible to distinguish between "prevention" and "suppression" of a disease or disorder, because the ultimate inciting event(s) may be unknown or latent. Thus, the term "prevention" should be understood to constitute a type of "treatment" that encompasses both "prevention" and "suppression." Thus, the term "treatment" includes "prevention."

[0331] The term "allergic condition" is defined herein as a disease or disorder caused by an interaction between the immune system and a substance foreign to the body. This foreign substance is referred to as an "allergen." Common allergens include airborne allergens, such as pollen, dust, mold, dust mite proteins, and saliva injected from insect bites. Examples of allergic conditions include, but are not limited to, the following: allergic dermatitis, summer eczema, urticaria, heaves, inflammatory airway disease, recurrent airway obstruction, airway hyperresponsiveness, chronic obstructive pulmonary disease, and autoimmune-driven inflammatory processes such as irritable bowel syndrome (IBS).

[0332] The term "pruritic condition" is defined herein as a disease or disorder characterized by an intense itchy sensation that results in the urge to rub or scratch the skin to find relief. Examples of pruritic conditions include, but are not limited to: atopic dermatitis, allergic dermatitis, eczema, psoriasis, scleroderma, and pruritus.

[0333] As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. All of these terms also include their progeny, which include any subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing heterologous nucleic acid sequences, "host cell" means a prokaryotic or eukaryotic cell (e.g., bacterial cell, yeast cell, mammalian cell, and insect cell) whether in vitro or in vivo. For example, the host cell may be in a transgenic animal. A host cell can be used as a recipient for a vector and can include any transformable organism capable of replicating the vector and / or expressing a heterologous nucleic acid encoded by the vector.

[0334] A "composition" is intended to mean a combination of an active agent with another compound or composition, which may be inert (e.g., a label) or active (e.g., an adjuvant).

[0335] Pharmaceutically acceptable carriers suitable for use in the present invention, as defined herein, are well known to those skilled in the art. Such carriers include, but are not limited to, water, saline, buffered saline, phosphate buffer, alcoholic / aqueous solutions, emulsions, or suspensions. Other conventionally used diluents, adjuvants, and excipients can be added according to conventional techniques. Examples of such carriers include ethanol, polyols, and suitable mixtures thereof, vegetable oils, and injectable organic esters. Buffers and pH adjusters can also be used. Buffers include, but are not limited to, salts prepared from organic acids or bases. Representative buffers include, but are not limited to, organic acid salts such as citric acid (e.g., citrate), ascorbic acid, gluconic acid, histidine-HCl, carbonate, tartaric acid, succinic acid, acetic acid, or phthalic acid, Tris, trimethanmine hydrochloride, or phosphate buffers. Parenteral carriers can include sodium chloride solution, Ringer's dextrose, dextrose, trehalose, sucrose, and sodium chloride, lactated Ringer's, or fixed oils. Intravenous carriers can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents (e.g., EDTA), inert gases, and the like, can also be provided in the pharmaceutical carrier. The present invention is not limited by the choice of carrier. It is within the skill of one in the art to prepare these pharmaceutically acceptable compositions from the above components with appropriate pH, isotonicity, stability, and other conventional characteristics. See, for example, Remington: The Science and Practice of Pharmacy, 20th Edition. See texts such as The Handbook of Pharmaceutical Excipients, 4th sup.th edit., eds. RC Rowe et al., APhA Publications, 2003.

[0336] The term "conservative amino acid substitution" refers to any amino acid substitution of a given amino acid residue, where the substituted residue is so chemically similar to the given residue that it does not substantially reduce polypeptide function (e.g., enzymatic activity). Conservative amino acid substitutions are generally known in the art, and examples are described, for example, in U.S. Pat. Nos. 6,790,639, 6,774,107, 6,194,167, or 5,350,576. In a preferred embodiment, the conservative amino acid substitution is any one occurring within one of the following six groups: 1. Small aliphatic, substantially non-polar residues: Ala, Gly, Pro, Ser, and Thr 2. High molecular weight aliphatic non-polar residues: Ile, Leu, and Val; Met 3. Polar, negatively charged residues and their amides: Asp and Glu 4. Amides of polar, negatively charged residues: Asn and Gln; His 5. Polar, positively charged residues: Arg and Lys; His 6. Polymeric aromatic residues: Trp and Tyr; Phe

[0337] In preferred embodiments, the conservative amino acid substitution is any one of the following listed as a native residue (conservative substitution) pair: 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).

[0338] Just as a polypeptide can contain conservative amino acid substitution(s), a polynucleotide of a polypeptide can also contain conservative codon substitution(s). A codon substitution is considered conservative if, when expressed, it results in a conservative amino acid substitution as described above. Degenerate codon substitutions that do not result in amino acid substitutions are also useful in polynucleotides in accordance with the present invention. Thus, for example, a polynucleotide encoding a selected polypeptide useful in embodiments of the present invention may be mutated by degenerate codon substitutions to approximate the codon usage exhibited by the expression host cell into which it is transformed, or to otherwise improve its expression. DETAILED DESCRIPTION OF THE INVENTION

[0339] It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein, as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined only by the claims.

[0340] Unless otherwise defined, scientific and technical terms used in connection with the antibodies described herein shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature utilized in connection with and techniques of cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transfection (e.g., electroporation, lipofection).

[0341] Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, or as commonly accomplished in the art, or as described herein. The techniques and procedures described above are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al. 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 nomenclature utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used in chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0342] Except in the working examples or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term "about."

[0343] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodology described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application.

[0344] The present invention provides recombinant monoclonal antibodies and peptides and their use in clinical and scientific procedures, including diagnostic procedures.

[0345] With the advent of molecular biology and recombinant technology methods, it is now possible to produce antibodies and antibody-like molecules by recombinant means, thereby generating gene sequences that encode the specific amino acid sequences found in the polypeptide structure of an antibody. Such antibodies can be produced by cloning the gene sequences encoding the antibody polypeptide chains or by directly synthesizing the polypeptide chains and assembling the synthetic chains to form an active tetrameric (H2L2) structure with affinity for a specific epitope and antigenic determinant. This allows for the facile production of antibodies from different species and sources with sequences characteristic of neutralizing antibodies.

[0346] Regardless of the source of the antibody, or how it is recombinantly constructed or synthesized—in vitro or in vivo, using transgenic animals, large-scale laboratory or commercial cell cultures, transgenic plants, or direct chemical synthesis without the use of any living organism at any stage of the process—all antibodies have a similar overall three-dimensional structure. This structure is often defined as H2L2, referring to the fact that antibodies generally contain two amino acid light chains (L) and two amino acid heavy chains (H). Both chains have regions capable of interacting with structurally complementary antigen targets. The target-interacting regions are referred to as "variable" or "V" regions and are characterized by differences in amino acid sequence between antibodies with different antigen specificities. The variable regions of the H or L chain contain amino acid sequences capable of specifically binding to antigen targets.

[0347] As noted above, the terms "antigen-binding region," "antigen-binding portion," and the like, as used throughout the specification and claims, refer to the portion of an antibody molecule comprising amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen. The antibody-binding region includes "framework" amino acid residues necessary to maintain the proper conformation of the antigen-binding residues. The antigen-binding portion of an antibody according to the invention may be referred to herein as, for example, an IL-31-specific peptide or polypeptide, or an anti-IL-31 peptide or polypeptide.

[0348] Within the variable regions of the heavy or light chains that provide the antigen-binding domain, there are smaller sequences called "hypervariable" because they vary greatly among antibodies of different specificities. These hypervariable regions are also called "complementarity-determining regions" or "CDR" regions. These CDR regions are responsible for the basic specificity of antibodies for particular antigenic determinant structures.

[0349] Although CDRs represent non-contiguous stretches of amino acids within the variable region, the positions of these essential amino acid sequences within the variable heavy and light chain regions are known to be similar within the amino acid sequences of the variable chains, regardless of species. The variable heavy and light chains of all antibodies each have three CDR regions, and each CDR region is not adjacent to the other CDR regions.

[0350] In all mammalian species, antibody peptides contain constant (i.e., highly conserved) regions and variable regions, within which are CDRs and so-called "framework regions," which are composed of amino acid sequences other than the CDRs in the variable region of the heavy or light chain.

[0351] The antigenic determinant recognized by the CDR region of an antibody is also called an "epitope." In other words, an epitope refers to that part of any molecule that can be recognized and bound by an antibody (the corresponding antibody-binding region is sometimes called a paratope).

[0352] An "antigen" is a molecule or portion of a molecule that can be bound by an antibody and that can induce an animal to produce antibodies capable of binding to an epitope of that antigen. An antigen can have one or more epitopes. The specific reaction referred to above is intended to indicate that the antibody reacts in a highly selective manner with the corresponding antibody and not with the many other antibodies that may be elicited by other antigens.

[0353] Antibodies of the present invention are intended to include intact immunoglobulin molecules, as well as portions, fragments, peptides, and derivatives thereof (e.g., Fab, Fab', F(ab')2, Fv, Fse, CDR regions, paratopes, or any portion (e.g., polypeptide) or peptide sequence of an antibody capable of binding to an antigen or epitope). An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to bind the molecule to the antibody.

[0354] The antibodies of the present invention also include chimeric antibodies, heterochimeric antibodies, caninized antibodies, feline antibodies, or equine antibodies, as well as fragments, portions, regions, peptides, or derivatives thereof, provided by any known technique (for example, but not limited to, enzymatic cleavage, peptide synthesis, or recombinant techniques). Such antibodies of the present invention are capable of specifically binding to at least one of canine IL-31 or feline IL-31. Antibody fragments or portions lack the Fc fragment of the intact antibody, may be cleared from the circulation more rapidly, and may have lower non-specific tissue binding than the intact antibody. Exemplary antibody fragments can be produced from intact antibodies using methods well known in the art, for example, by proteolytic cleavage with enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). See, e.g., Wahl et al., 24 J. Nucl. Med. 316-25 (1983). Portions of antibodies can be produced by any of the methods described above, or by expressing portions of recombinant molecules. For example, the CDR region(s) of a recombinant antibody can be isolated and subcloned into an appropriate expression vector. See, e.g., U.S. Patent No. 6,680,053.

[0355] As used throughout this specification and claims, the terms "antigen-binding region," "antigen-binding portion," and the like refer to the portion of an antibody molecule comprising amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen. The antibody-binding region includes "framework" amino acid residues necessary to maintain the proper conformation of the antigen-binding residues. Alternatively, the antigen-binding portion of an antibody in accordance with the invention may be referred to herein as, for example, an IL-31-specific peptide or polypeptide, or an anti-IL-31 peptide or polypeptide.

[0356] Clones 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07, and 06A09 Nucleotide and amino acid sequences In some embodiments, the present invention provides novel monoclonal antibodies that specifically bind to at least one of canine IL-31, feline IL-31, or equine IL-31. In one embodiment, the monoclonal antibodies of the present invention bind to canine IL-31, feline IL-31, or equine IL-31 and prevent its binding to and activation of co-receptors, including IL-31 receptor A (IL-31Ra) and oncostatin M-specific receptor (OsmR or IL-31Rb). The monoclonal antibodies of the present invention are identified herein as "15H05," "ZIL1," "ZIL8," "ZIL9," "ZIL11," "ZIL69," "ZIL94," "ZIL154," "ZIL159," "ZIL171," 04H07, and 06A09, which refer to the numbers assigned to the clones. As used herein, "15H05," "ZIL1," "ZIL8," "ZIL9," "ZIL11," "ZIL69," "ZIL94," "ZIL154," "ZIL159," "ZIL171," "04H07," and "06A09" also refer to portions, CDRs, or paratopes of monoclonal antibodies that have the ability to bind to the 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07, and 06A09 antibodies and therefore specifically bind to the IL-31 epitopes identified as 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07, and 06A09, respectively. Certain recombinant, chimeric, heterochimeric, caninized, felineized, equineized, fully canine, fully feline, and / or fully equine forms of 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07, and 06A09 described herein may be referred to by the same name. In some embodiments, 15H05 may be alternatively referred to herein as 1505, since it shares at least the same CDRs.

[0357] In one embodiment, the invention provides a monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to a region on a mammalian IL-31 protein that is involved in the interaction of the mammalian IL-31 protein with its co-receptor, wherein binding of the antibody to the region is affected by mutations in the 15H05 epitope binding region selected from at least one of the following: a) a region between about amino acid residues 124 and 135 of the feline IL-31 sequence represented by SEQ ID NO: 157 (feline_IL31_wildtype); b) a region between about amino acid residues 124 and 135 of the canine IL-31 sequence represented by SEQ ID NO: 155 (canine_IL31); and c) a region between about amino acid residues 118 and 129 of equine IL-31 represented by SEQ ID NO: 165 (equine_IL31). In one embodiment, the mutations in the 15H05 epitope binding region are selected from at least one of the following: (a) a mutation at positions 126 and 128 of SEQ ID NO: 157 that changes the amino acid sequence to alanine; (b) a mutation at positions 126 and 128 of SEQ ID NO: 155 that changes the amino acid sequence to alanine; and (c) a mutation at positions 120 and 122 of SEQ ID NO: 165 that changes the amino acid sequence to alanine.

[0358] In a specific embodiment, an antibody according to the invention binds to the above-mentioned 15H05 epitope-binding region. That is, in one embodiment, the invention provides a monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to a region on a mammalian IL-31 protein that is involved in the interaction of the mammalian IL-31 protein with its co-receptor, wherein the binding region is selected from at least one of the following: a) a region between about amino acid residues 124 and 135 of the feline IL-31 sequence represented by SEQ ID NO: 157 (feline_IL31_wildtype), b) a region between about amino acid residues 124 and 135 of the canine IL-31 sequence represented by SEQ ID NO: 155 (canine_IL31), and c) a region between about amino acid residues 118 and 129 of equine IL-31 represented by SEQ ID NO: 165 (equine_IL31).

[0359] In one embodiment, the mammalian IL-31 to which the antibody or antigen-binding portion thereof specifically binds is feline IL-31, wherein the antibody binds to a region between about amino acid residues 125 and 134 of the feline IL-31 sequence represented by SEQ ID NO: 157 (feline_IL31_wildtype). In some embodiments, the antibody that binds to feline IL31 comprises a VL chain comprising a framework 2 (FW2) alteration selected from the following: a lysine to asparagine substitution at position 42, a valine to isoleucine substitution at position 43, a leucine to valine substitution at position 46, a lysine to asparagine substitution at position 49, and combinations thereof, wherein these positions are numbered according to SEQ ID NO: 127 (FEL_15H05_VL1).

[0360] In one embodiment, the monoclonal antibody or antigen-binding portion thereof comprises the following combination of complementarity determining region (CDR) sequences: 1) Antibody 15H05: variable heavy chain (VH)-CDR1 is SYTIH (SEQ ID NO: 1), VH-CDR2 is NINPTSGYTENNQRFKD (SEQ ID NO: 2), VH-CDR3 is WGFKYDGEWSFDV (SEQ ID NO: 3), variable light chain (VL)-CDR1 is RASQGISIWLS (SEQ ID NO: 4), VL-CDR2 is KASNLHI (SEQ ID NO: 5), VL-CDR3 is LQSQTYPLT (SEQ ID NO: 6), or 2) A variant of 1) that differs from the parent antibody 15H05 by the addition, deletion, and / or substitution of one or more amino acid residues in at least one of CDR1, CDR2, or CDR3 of VH or VL. Includes.

[0361] In one embodiment, the above-mentioned antibody 15H05 has the following variable heavy and / or variable light chains: a) FEL_15H05_VL1_FW2: a variable light chain comprising EIQMTQSPSSLSASPGDRVTITCRASQGISIWLSWYQQKPGNIPKVLINKASNLHIGVPSRFSGSGSGTDFTLTISSLEPEDAATYYCLQSQTYPLTFGGGTKLEIK (SEQ ID NO: 135), and b) FEL_15H05_VH1: a variable heavy chain comprising QVLLVQSGAEVRTPGASVKIFCKASGYSFTSYTIHWLRQAPAQGLEWMGNINPTSGYTENNQRFKDRLTLTADTSTNTAYMELSSLRSADTAMYYCARWGFKYDGEWSFDVWGAGTTVTVSS (SEQ ID NO: 121) It includes at least one of the following:

[0362] In another embodiment, the monoclonal antibody or antigen-binding portion thereof comprises the following combination of complementarity determining region (CDR) sequences: 1) Antibody ZIL1: variable heavy chain (VH)-CDR1 is SYGMS (SEQ ID NO: 13), VH-CDR2 is HINSGGSSTYYADAVKG (SEQ ID NO: 14), VH-CDR3 is VYTTLAAFWTDNFDY (SEQ ID NO: 15), variable light chain (VL)-CDR1 is SGSTNNIGILAAT (SEQ ID NO: 16), VL-CDR2 is SDGNRPS (SEQ ID NO: 17), and VL-CDR3 is QSFDTTLDAYV (SEQ ID NO: 18); 2) antibody ZIL8: VH-CDR1 is DYAMS (SEQ ID NO: 19), VH-CDR2 is GIDSVGSGTSYADAVKG (SEQ ID NO: 20), VH-CDR3 is GFPGSFEH (SEQ ID NO: 21), VL-CDR1 is TGSSSNIGSGYVG (SEQ ID NO: 22), VL-CDR2 is YNSDRPS (SEQ ID NO: 23), VL-CDR3 is SVYDRTFNAV (SEQ ID NO: 24), 3) Antibody ZIL9: VH-CDR1 is SYDMT (SEQ ID NO: 25), VH-CDR2 is DVNSGGTGTAYAVAVKG (SEQ ID NO: 26), VH-CDR3 is LGVRDGLSV (SEQ ID NO: 27), VL-CDR1 is SGESLNEYYTQ (SEQ ID NO: 28), V L-CDR2 is RDTERPS (SEQ ID NO: 29), VL-CDR3 is ESAVDTGTLV (SEQ ID NO: 30), 4) antibody ZIL11: VH-CDR1 is TYVMN (SEQ ID NO: 31), VH-CDR2 is SINGGGSSPTYADAVRG (SEQ ID NO: 32), VH-CDR3 is SMVGPFDY (SEQ ID NO: 33), VL-CDR1 is SGESLSNYYAQ (SEQ ID NO: 34), VL-CDR2 is KDTERPS (SEQ ID NO: 35), VL-CDR3 is ESAVSSDTIV (SEQ ID NO: 36), 5) antibody ZIL69: VH-CDR1 is SYAMK (SEQ ID NO: 37), VH-CDR2 is TINNDGTRTGYADAVRG (SEQ ID NO: 38), VH-CDR3 is GNAESGCTGDHCPPY (SEQ ID NO: 39), VL-CDR1 is SGESLNKYYAQ (SEQ ID NO: 40), VL-CDR2 is KDTERPS (SEQ ID NO: 41), VL-CDR3 is ESAVSSETNV (SEQ ID NO: 42), 6) antibody ZIL94: VH-CDR1 is TYFMS (SEQ ID NO: 43), VH-CDR2 is LISSDGSGTYYADAVKG (SEQ ID NO: 44), VH-CDR3 is FWRAFND (SEQ ID NO: 45), VL-CDR1 is GLNSGSVSTSNYPG (SEQ ID NO: 46), VL-CDR2 is DTGSRPS (SEQ ID NO: 47), VL-CDR3 is SLYTDSDILV (SEQ ID NO: 48), 7) antibody ZIL154: VH-CDR1 is DRGMS (SEQ ID NO: 49), VH-CDR2 is YIRYDGSRTDYADAVEG (SEQ ID NO: 50), VH-CDR3 is WDGSSFDY (SEQ ID NO: 51), VL-CDR1 is KASQSLLHSDGNTYLD (SEQ ID NO: 52), VL-CDR2 is KVSNRDP (SEQ ID NO: 53), and VL-CDR3 is MQAIHFPLT (SEQ ID NO: 54); 8) antibody ZIL159: VH-CDR1 is SYVMT (SEQ ID NO: 55), VH-CDR2 is GINSEGSRTAYADAVKG (SEQ ID NO: 56), VH-CDR3 is GDIVATGTSY (SEQ ID NO: 57), VL-CDR1 is SGETLNRFYTQ (SEQ ID NO: 58), VL-CDR2 is KDTERPS (SEQ ID NO: 59), VL-CDR3 is KSAVSIDVGV (SEQ ID NO: 60), 9) Antibody ZIL171: VH-CDR1 is TYVMN (SEQ ID NO: 61), VH-CDR2 is SINGGGSSPTYADAVRG (SEQ ID NO: 62), VH-CDR3 is SMVGPFDY (SEQ ID NO: 63), VL-CDR1 is SGKSLSYYYAQ (SEQ ID NO: 64), VL-CDR2 is KDTERPS (SEQ ID NO: 65), and VL-CDR3 is ESAVSSDTIV (SEQ ID NO: 66); 10) Antibody 04H07: VH-CDR1 is SYWMN (SEQ ID NO: 200), VH-CDR2 is MIDPSDSEIHYNQVFKD (SEQ ID NO: 201), VH-CDR3 is QDIVTTVDY (SEQ ID NO: 202), VL-CDR1 is KSSQSLLYSINQKNHLA (SEQ ID NO: 203), VL-CDR2 is WASTRES (SEQ ID NO: 204), and VL-CDR3 is QQGYTYPFT (SEQ ID NO: 205); 11) Antibody 06A09: VH-CDR1 is SYWMN (SEQ ID NO: 206), VH-CDR2 is MIDPSDSETHYNQIFRD (SEQ ID NO: 207), VH-CDR3 is QDIVTTVDY (SEQ ID NO: 208), VL-CDR1 is KSSQSLLYSINQKNFLA (SEQ ID NO: 209), VL-CDR2 is WASTRES (SEQ ID NO: 210), and VL-CDR3 is QQHYGYPFT (SEQ ID NO: 211), or 12) Variants of 1) to 11) that differ from the respective parent antibodies ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07, or 06A09 by addition, deletion, and / or substitution of one or more amino acid residues in at least one of CDR1, CDR2, or CDR3 of VH or VL.

[0363] In some embodiments of the present invention, 1) Antibody ZIL1: a)CAN-ZIL1_VL: a variable light chain comprising QSVLTQPTSVSGSLGQRVTISCSGSTNNIGILAATWYQQLPGKAPKVLVYSDGNRPSGVPDRFSGSSKSGNSATLTITGLQAEDEADYYCQSFDTTLDAYVFGSGTQLTVL (SEQ ID NO: 77), and b)CAN-ZIL1_VH: A variable heavy chain comprising: EVQLVESGGDLVKPGGSLRLSCVASGFTFSSYGMSWVRQAPGKGLQWVAHINSGGSSTYYYADAVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCVEVYTTLAAFWTDNFDYWGQGTLVTVSS (SEQ ID NO: 75) and 2) The antibody ZIL8 is: a)CAN-ZIL8_VL: a variable light chain comprising: QSVLTQPASVSGSLGQKVTISCTGSSSNIGSGYVGWYQQLPGTGPRTLIYYNSDRPSGVPDRFSGSRSGTTATLTISGLQAEDEADYYCSVYDRTFNAVFGGGT (SEQ ID NO: 81); and b)CAN-ZIL8_VH: EVQLVESGGDLVKPAGSLRLSCVASGFTFSDYAMSWVRQAPGRGLQWVAGIDSVGSGTSYADAVKGRFTISRDDAKNTLYLQMFNLRAEDTAIYYCASGFPGSFEHWGQGTLVTVSS (SEQ ID NO: 79), or the antibody ZIL8 comprises at least one of the following: c) ZTS_5864_VL: a variable light chain comprising QSVLTQPSSVSGTLGQRITISCTGSSSNIGSGYVGWYQQVPGMGPKTVIYYNSDRPSGVPDRFSGSKSGSSGTLTITGLQAEDEADYYCSVYDRTFNAVFGGGTHLTVLGQPKSAPPRSHSSRPISYAVFCL (SEQ ID NO: 230), and d) ZTS_5864_VH: a variable heavy chain comprising DVQLVESGGDLVKPGGSLRLTCVASGFTFSDYAMSWVRQAPGKGLQWVAGIDSVGSGTSYADSVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCASGFPGSFEHWGQGALVTVSS (SEQ ID NO: 228) or The antibody ZIL8 is: e)ZTS_5865_VL: Q a variable light chain comprising SVLTQPSSVSGTLGQRITISCTGSSSNIGSGYVGWYQQVPGMGPKTVIYYNSDRPSGVPDRFSGSKSGSSGTLTITGLQAEDEADYYCSVYDRTFNAVFGGGTHLTVLGQPKSAPPRSHSSRPISYAVFCL (SEQ ID NO: 234), and f)ZTS_5865_VH: a variable heavy chain comprising: DVQLVESGGDLVKPGGSLRLTCVASGFTFSDYAMNWVRQAPGKGLQWVAGIDSVGSGTSYADSVKGRFTISRDNAKNTLYLQMSGLKTEDTATYYCASGFPGSFEHWGQGTLVTVSS (SEQ ID NO: 232) and 3) The antibody ZIL9 is: a) CAN-ZIL9_VL: a variable light chain comprising SSVLTQPPSVSVSLGQTATISCSGESLNEYYTQWFQQKAGQAPVLVIYRDTERPSGIPDRFSGSSSGNTHTLTISGARAEDEADYYCESAVDTGTLVFGGGTHLAVL (SEQ ID NO: 85), and b)CAN-ZIL9_VH: A variable heavy chain comprising: EVQLVESGGDLVKPPGSLRLSCVASGFTFSSYDMTWVRQAPGKGLQWVADVNSGGTGTAYAVAVKGRFTISRDNAKKTLYLQMNSLRAEDTAVYYCAKLGVRDGLSVWGQGTLVTVSS (SEQ ID NO: 83) and 4) Antibody ZIL11: a) CAN-ZIL11_VL: a variable light chain comprising SSVLTQPPSVSVSLGQTATISCSGESLSNYYAQWFQQKAGQAPVLVIYKDTERPSGIPDRFSGSSSGNTHTLTISGARAEDEADYYCESAVSSDTIVFGGGT (SEQ ID NO: 89), and b) CAN-ZIL11_VH: a variable heavy chain comprising EVQLVESGGDLVKPAGSLRLSCVASGFTFRTYVMNWVRQAPGKGLQWVASINGGGSSPTYADAVRGRFTVSRDNAQNSLFLQMNSLRAEDTAVYFCVVSMVGPFDYWGQGTLVTVSS (SEQ ID NO: 87) and 5) The antibody ZIL69: a) CAN-ZIL69_VL: a variable light chain comprising SSVLTQPPSVSVSLGQTATISCSGESLNKYYAQWFQQKAGQAPVLVIYKDTERPSGIPDRFSGSSAGNTHTLTISGARAEDEADYYCESAVSSETNVFGSGTQLTVL (SEQ ID NO: 93), and b) CAN-ZIL69_VH: a variable heavy chain comprising EVQLVESGGDLVKPAGSLRLSCVASGFTFSSYAMKWVRQAPGKGLQWVATINNDGTRTGYADAVRGRFTISKDNAKNTLYLQMDSLRADDTAVYYCTKGNAESGCTGDHCPPYWGQGTLVTVSS (SEQ ID NO: 91) and 6) Antibody ZIL94: a) CAN-ZIL94_VL: a variable light chain comprising QTVVIQEPSLSVSPGGTVTLTCGLNSGSVSTSNYPGWYQQTRGRTPRTIIYDTGSRPSGVPNRFSGSISGNKAALTITGAQPEDEADYYCSLYTDSDILVFGGGTHLTVL (SEQ ID NO: 97), and b) CAN-ZIL94_VH: a variable heavy chain comprising EVQLVDSGGDLVKPGGSLRLSCVASGFTFSTYFMSWVRQAPGRGLQWVALISSDGSGTYYADAVKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCAIFWRAFNDWGQGTLVTVSS (SEQ ID NO: 95) and 7) Antibody ZIL154: a) CAN-ZIL154_VL: a variable light chain comprising DIVVTQTPLSLSVSPGETASFSCKASQSLLHSDGNTYLDWFRQKPGQSPQRLIYKVSNRDPGVPDRFSGSGSGTDFTLRISGVEADDAGLYYCMQAIHFPLTFGAGTKVELK (SEQ ID NO: 101), and b) CAN-ZIL154_VH: a variable heavy chain comprising EVHLVESGGDLVKPWGSLRLSCVASGFTFSDRGMSWVRQSPGKGLQWVAYIRYDGSRTDYADAVEGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARWDGSSFDYWGQGTLVTVSS (SEQ ID NO: 99) and 8) The antibody ZIL159: a) CAN-ZIL159_VL: a variable light chain comprising SNVLTQPPSVSVSLGQTATISCSGETLNRFYTQWFQQKAGQAPVLVIYKDTERPSGIPDRFSGSSSGNIHTLTISGARAEDEAAYYCKSAVSIDVGVFGGGTHLTVF (SEQ ID NO: 105), and b) CAN-ZIL159_VH: a variable heavy chain comprising EVQLVESGGDLVKPAGSLRLSCVASGFTFSSYVMTWVRQAPGKGLQWVAGINSEGSRTAYADAVKGRFTISRDNAKNTLYLQIDSLRAEDTAIYYCATGDIVATGTSYWGQGTLVTVSS (SEQ ID NO: 103) and 9) Antibody ZIL171: a) CAN-ZIL171_VL: a variable light chain comprising SSVLTQPPSVSVSLGQTATISCSGKSLSYYYAQWFQQKAGQAPVLVIYKDTERPSGIPDRFSGSSSGNTHTLTISGARAEDEADYYCESAVSSDTIVFGGGTHLTVL (SEQ ID NO: 109), and b) CAN-ZIL171_VH: a variable heavy chain comprising EVQLVESGGDLVKPAGSLRLSCVASGFTFRTYVMNWVRQAPGKGLQWVASINGGGSSPTYADAVRGRFTVSRDNAQNSLFLQMNSLRAEDTAIYFCVVSMVGPFDYWGHGTLVTVSS (SEQ ID NO: 107) and 10) Antibody 04H07: a)Mu_04H07_VL: a variable light chain comprising: DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSINQKNHLAWFQQKPGQSPKLLIYWASTRESGVPARFTGSGSGTDFTLTISSVKTEDLAVYYCQQGYTYPFTFGSGTKLEIK (SEQ ID NO: 214), and b) Mu_04H07_VH: a variable heavy chain comprising QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWMNWAKQRPGQGLEWIGMIDPSDSEIHYNQVFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARQDIVTTVDYWGQGTTLTVSS (SEQ ID NO: 212) and 11) Antibody 06A09: a) a variable light chain comprising Mu_06A09_VL:DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSINQKNFLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKSEDLAVYYCQQHYGYPFTFGSGTKLEIK (SEQ ID NO: 218), and b) Mu_06A09_VH: a variable heavy chain comprising QVQLQQPGAELVRPGASVKLSCKAYGYTFTSYWMNWVKQRPGQGLEWIGMIDPSDSETHYNQIFRDKATLTIDKSSSTAYMQLSSLTSEDSAVYFCARQDIVTTVDYWGQGTTLTVSS (SEQ ID NO: 216) It includes at least one of the following:

[0364] In other embodiments, the present invention provides host cells that produce the above-described antibodies.

[0365] The present invention also includes within its scope nucleotide sequences encoding the light and heavy chain variable regions of the anti-IL-31 antibodies of the present invention, as well as any nucleotide sequence encoding the amino acid sequence of 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07, 06A09, or these IL-31-specific polypeptides or peptides.

[0366] In some embodiments, the present invention provides the following combinations of variable heavy chain complementarity determining region (CDR) sequences: 1) 15H05: variable heavy chain (VH)-CDR1 is SYTIH (SEQ ID NO: 1), VH-CDR2 is NINPTSGYTENNQRFKD (SEQ ID NO: 2), VH-CDR3 is WGFKYDGEWSFDV (SEQ ID NO: 3); 2) ZIL1: VH-CDR1 is SYGMS (SEQ ID NO: 13), VH-CDR2 is HINSGGSSTYYADAVKG (SEQ ID NO: 14), and VH-CDR3 is VYTTLAAFWTDNFDY (SEQ ID NO: 15); 3) ZIL8: VH-CDR1 is DYAMS (SEQ ID NO: 19), VH-CDR2 is GIDSVGSGTSYADAVKG (SEQ ID NO: 20), and VH-CDR3 is GFPGSFEH (SEQ ID NO: 21); 4) ZIL9: VH-CDR1 is SYDMT (SEQ ID NO: 25), VH-CDR2 is DVNSGGTGTAYAVAVKG (SEQ ID NO: 26), and VH-CDR3 is LGVRDGLSV (SEQ ID NO: 27); 5) ZIL11: VH-CDR1 is TYVMN (SEQ ID NO: 31), VH-CDR2 is SINGGGSSPTYADAVRG (SEQ ID NO: 32), and VH-CDR3 is SMVGPFDY (SEQ ID NO: 33); 6) ZIL69: VH-CDR1 is SYAMK (SEQ ID NO: 37), VH-CDR2 is TINNDGTRTGYADAVRG (SEQ ID NO: 38), and VH-CDR3 is GNAESGCTGDHCPPY (SEQ ID NO: 39); 7) ZIL94: VH-CDR1 is TYFMS (SEQ ID NO: 43), VH-CDR2 is LISSDGSGTYYADAVKG (SEQ ID NO: 44), and VH-CDR3 is FWRAFND (SEQ ID NO: 45); 8) ZIL154: VH-CDR1 is DRGMS (SEQ ID NO: 49), VH-CDR2 is YIRYDGSRTDYADAVEG (SEQ ID NO: 50), and VH-CDR3 is WDGSSFDY (SEQ ID NO: 51); 9) ZIL159: VH-CDR1 is SYVMT (SEQ ID NO: 55), VH-CDR2 is GINSEGSRTAYADAVKG (SEQ ID NO: 56), and VH-CDR3 is GDIVATGTSY (SEQ ID NO: 57); 10) ZIL171: VH-CDR1 is TYVMN (SEQ ID NO: 61), VH-CDR2 is SINGGGSSPTYADAVRG (SEQ ID NO: 62), and VH-CDR3 is SMVGPFDY (SEQ ID NO: 63); 11) 04H07: VH-CDR1 is SYWMN (SEQ ID NO: 200), VH-CDR2 is MIDPSDSEIHYNQVFKD (SEQ ID NO: 201), and VH-CDR3 is QDIVTTVDY (SEQ ID NO: 202); 12) 06A09: VH-CDR1 is SYWMN (SEQ ID NO: 206), VH-CDR2 is MIDPSDSETHYNQIFRD (SEQ ID NO: 207), and VH-CDR3 is QDIVTTVDY (SEQ ID NO: 208), or 13) A variant of 1) to 12) that differs from the CDRs of the respective parent antibodies 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07, or 06A09 by addition, deletion, and / or substitution of one or more amino acid residues in at least one of CDR1, CDR2, or CDR3 of VH. An isolated nucleic acid is provided, comprising a nucleic acid sequence encoding at least one of:

[0367] In one embodiment, the isolated nucleic acid further comprises the following combination of variable light chain complementarity determining region (CDR) sequences: 1) 15H05: variable light chain (VL)-CDR1 is RASQGISIWLS (SEQ ID NO: 4), VL-CDR2 is KASNLHI (SEQ ID NO: 5), VL-CDR3 is LQSQTYPLT (SEQ ID NO: 6); 2) ZIL1: VL-CDR1 is SGSTNNIGILAAT (SEQ ID NO: 16), VL-CDR2 is SDGNRPS (SEQ ID NO: 17), and VL-CDR3 is QSFDTTLDAYV (SEQ ID NO: 18), 3) ZIL8: VL-CDR1 is TGSSSNIGSGYVG (SEQ ID NO: 22), VL-CDR2 is YNSDRPS (SEQ ID NO: 23), and VL-CDR3 is SVYDRTFNAV (SEQ ID NO: 24); 4) ZIL9: VL-CDR1 is SGESLNEYYTQ (SEQ ID NO: 28), VL-CDR2 is RDTERPS (SEQ ID NO: 29), and VL-CDR3 is ESAVDTGTLV (SEQ ID NO: 30); 5) ZIL11: VL-CDR1 is SGESLSNYYAQ (SEQ ID NO: 34), VL-CDR2 is KDTERPS (SEQ ID NO: 35), and VL-CDR3 is ESAVSSDTIV (SEQ ID NO: 36); 6) ZIL69: VL-CDR1 is SGESLNKYYAQ (SEQ ID NO: 40), VL-CDR2 is KDTERPS (SEQ ID NO: 41), and VL-CDR3 is ESAVSSETNV (SEQ ID NO: 42); 7) ZIL94: VL-CDR1 is GLNSGSVSTSNYPG (SEQ ID NO: 46), VL-CDR2 is DTGSRPS (SEQ ID NO: 47), and VL-CDR3 is SLYTDSDILV (SEQ ID NO: 48); 8) ZIL154: VL-CDR1 is KASQSLLHSDGNTYLD (SEQ ID NO: 52), VL-CDR2 is KVSNRDP (SEQ ID NO: 53), and VL-CDR3 is MQAIHFPLT (SEQ ID NO: 54); 9) ZIL159: VL-CDR1 is SGETLNRFYTQ (SEQ ID NO: 58), VL-CDR2 is KDTERPS (SEQ ID NO: 59), and VL-CDR3 is KSAVSIDVGV (SEQ ID NO: 60); 10) ZIL171: VL-CDR1 is SGKSLSYYYAQ (SEQ ID NO: 64), VL-CDR2 is KDTERPS (SEQ ID NO: 65), and VL-CDR3 is ESAVSSDTIV (SEQ ID NO: 66); 11) 04H07: VL-CDR1 is KSSQSLLYSINQKNHLA (SEQ ID NO: 203), VL-CDR2 is WASTRES (SEQ ID NO: 204), and VL-CDR3 is QQGYTYPFT (SEQ ID NO: 205); 12) 06A09: VL-CDR1 is KSSQSLLYSINQKNFLA (SEQ ID NO: 209), VL-CDR2 is WASTRES (SEQ ID NO: 210), and VL-CDR3 is QQHYGYPFT (SEQ ID NO: 211), or 13) A variant of 1) to 12) that differs from the CDRs of the respective parent antibodies 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07, or 06A09 by addition, deletion, and / or substitution of one or more amino acid residues in at least one of CDR1, CDR2, or CDR3 of VH or VL. The nucleic acid sequence may include a nucleic acid sequence encoding at least one of:

[0368] In one embodiment, the present invention provides a combination of the following variable light chain complementarity determining region (CDR) sequences: 1) 15H05: variable light chain (VL)-CDR1 is RASQGISIWLS (SEQ ID NO: 4), VL-CDR2 is KASNLHI (SEQ ID NO: 5), VL-CDR3 is LQSQTYPLT (SEQ ID NO: 6); 2) ZIL1: VL-CDR1 is SGSTNNIGILAAT (SEQ ID NO: 16), VL-CDR2 is SDGNRPS (SEQ ID NO: 17), and VL-CDR3 is QSFDTTLDAYV (SEQ ID NO: 18); 3) ZIL8: VL-CDR1 is TGSSSNIGSGYVG (SEQ ID NO: 22), VL-CDR2 is YNSDRPS (SEQ ID NO: 23), and VL-CDR3 is SVYDRTFNAV (SEQ ID NO: 24); 4) ZIL9: VL-CDR1 is SGESLNEYYTQ (SEQ ID NO: 28), VL-CD R2 is RDTERPS (SEQ ID NO: 29), VL-CDR3 is ESAVDTGTLV (SEQ ID NO: 30), 5) ZIL11: VL-CDR1 is SGESLSNYYAQ (SEQ ID NO: 34), VL-CDR2 is KDTERPS (SEQ ID NO: 35), and VL-CDR3 is ESAVSSDTIV (SEQ ID NO: 36); 6) ZIL69: VL-CDR1 is SGESLNKYYAQ (SEQ ID NO: 40), VL-CDR2 is KDTERPS (SEQ ID NO: 41), and VL-CDR3 is ESAVSSETNV (SEQ ID NO: 42); 7) ZIL94: VL-CDR1 is GLNSGSVSTSNYPG (SEQ ID NO: 46), VL-CDR2 is DTGSRPS (SEQ ID NO: 47), and VL-CDR3 is SLYTDSDILV (SEQ ID NO: 48); 8) ZIL154: VL-CDR1 is KASQSLLHSDGNTYLD (SEQ ID NO: 52), VL-CDR2 is KVSNRDP (SEQ ID NO: 53), and VL-CDR3 is MQAIHFPLT (SEQ ID NO: 54); 9) ZIL159: VL-CDR1 is SGETLNRFYTQ (SEQ ID NO: 58), VL-CDR2 is KDTERPS (SEQ ID NO: 59), and VL-CDR3 is KSAVSIDVGV (SEQ ID NO: 60); 10) ZIL171: VL-CDR1 is SGKSLSYYYAQ (SEQ ID NO: 64), VL-CDR2 is KDTERPS (SEQ ID NO: 65), and VL-CDR3 is ESAVSSDTIV (SEQ ID NO: 66); 11) 04H07: VL-CDR1 is KSSQSLLYSINQKNHLA (SEQ ID NO: 203), VL-CDR2 is WASTRES (SEQ ID NO: 204), and VL-CDR3 is QQGYTYPFT (SEQ ID NO: 205); 12) 06A09: VL-CDR1 is KSSQSLLYSINQKNFLA (SEQ ID NO: 209), VL-CDR2 is WASTRES (SEQ ID NO: 210), and VL-CDR3 is QQHYGYPFT (SEQ ID NO: 211), or 13) A variant of 1) to 12) that differs from the CDRs of the respective parent antibodies 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07, or 06A09 by addition, deletion, and / or substitution of one or more amino acid residues in at least one of CDR1, CDR2, or CDR3 of the VL. An isolated nucleic acid is provided, comprising a nucleic acid sequence encoding at least one of:

[0369] The present invention further provides a vector comprising at least one of the above nucleic acids.

[0370] As described in more detail below, the nucleic acid sequence encoding at least one of the above combinations of variable heavy chain complementarity determining region (CDR) sequences may be contained in the same vector as the nucleic acid sequence encoding at least one of the above combinations of variable light chain CDR sequences. Alternatively, the nucleic acid sequence encoding at least one of the above combinations of variable light chain CDR sequences and the nucleic acid sequence encoding at least one of the above combinations of variable heavy chain CDR sequences may each be contained in a separate vector.

[0371] 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 potentially capable of coding for that amino acid. The likelihood that a particular oligonucleotide actually constitutes an actual XXX coding sequence can be estimated by considering unusual base pair relationships in eukaryotic or prokaryotic cells expressing an anti-IL-31 antibody or an IL-31-specific portion thereof and the frequency with which a particular codon is actually used (to code for a particular amino acid). Such "codon usage rules" were established by Lathe, et al., 183 J. Molec. Biol. 1-12 (1985) The "codon usage rules" of Lathe can be used to identify a single nucleotide sequence or set of nucleotide sequences that contain the theoretically "most likely" nucleotide sequence capable of encoding an anti-IL-31 sequence. It is also contemplated that antibody coding regions for use in the present invention can be provided by modifying existing antibody genes using standard molecular biology techniques to yield variants (agonists) of the antibodies and peptides described herein. Such variants include, but are not limited to, deletions, additions, and substitutions of amino acid sequences in anti-IL-31 antibodies or IL-31-specific polypeptides or peptides (including the CDR regions of antibodies). For example, residues known to be non-essential for antigen binding within the CDR regions or other regions of an antibody can be substituted. Examples of the types of experiments used to evaluate whether a particular residue is non-essential for antigen binding are described in section 1.21 of the Examples section below. In one embodiment, one or more of the substitutions are conservative amino acid substitutions, as described in further detail herein. However, antibody variants (including CDR variants) in accordance with the present invention are not limited to conservative amino acid substitutions.

[0372] For example, one class of substitutions is conservative amino acid substitutions. Such substitutions replace a given amino acid in an anti-IL-31 antibody peptide with another amino acid with similar properties. Typical conservative substitutions include substitutions between the aliphatic amino acids Ala, Val, Leu, and Ile for one another, exchanges of hydroxyl residues Ser and Thr, exchanges of acidic residues Asp and Glu, substitutions between amide residues Asn and Gln, exchanges of basic residues Lys and Arg, and substitutions between aromatic residues Phe, Tyr, etc. Guidance on which amino acid changes may be phenotypically silent can be found in Bowie et al., 247 Science 1306-10 (1990).

[0373] Variants or agonist anti-IL-31 antibodies or IL-31-specific polypeptides or peptides may be fully functional or may lack function in one or more activities. Fully functional variants typically contain only conservative variations or variations in non-essential residues or non-essential regions. Functional variants may also contain substitutions of similar amino acids that result in no or insignificant changes to function. Alternatively, such substitutions may have some positive or negative effect on function. Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncations, or substitutions, insertions, inversions, or deletions of essential residues or essential regions.

[0374] 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). In the latter procedure, single alanine mutations are introduced at every residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as epitope binding or in vitro ADCC activity. Sites essential 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).

[0375] Additionally, polypeptides often contain amino acids other than the 20 "natural" 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 that are well known in the art. Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, and the like. These include covalent conjugation, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA-mediated addition of amino acids to proteins (e.g., arginylation), and ubiquitination.

[0376] Such modifications are well known in the art and have been described in great detail in the scientific literature. Some particularly common modifications, such as glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation, are described in most basic texts, such as Proteins—Structure and Molecular Properties (2nd ed., TECreighton, W.H. Freeman & Co., NY, 1993). Many detailed reviews on this subject are available, such as those by Wold, Posttranslational Covalent Modification of Proteins, 1-12 (Johnson, ed., Academic Press, NY, 1983), Seifter et al. Meth. Enzymol. 626-46 (1990), and Rattan et al. Ann. NY Acad. Sci. 48-62 (1992).

[0377] Thus, the IL-31-specific antibodies, polypeptides, and peptides of the present invention also encompass derivatives or analogs in which the substituted amino acid residue is not encoded by the genetic code.

[0378] Similarly, the additions and substitutions of amino acid sequences, as well as the variations and modifications just described, may be equally applicable to the amino acid sequences of the IL-31 antigen and / or its epitopes or peptides, and are therefore also encompassed by the present invention. As described above, the genes encoding the monoclonal antibodies according to the present invention are particularly effective in recognizing IL-31.

[0379] antibody derivative Antibody derivatives are included within the scope of the present invention. A "derivative" of an antibody includes additional chemical moieties that are not normally part of the protein. Covalent modifications of proteins are included within the scope of the present invention. Such modifications can be introduced into the molecule by reacting targeted amino acid residues of the antibody with organic derivatizing agents capable of reacting with selected side chains or terminal residues. For example, derivatization with bifunctional agents well known in the art is useful for crosslinking antibodies or fragments to water-insoluble support matrices or other macromolecular carriers.

[0380] Derivatives also include radiolabeled monoclonal antibodies, e.g., radioactive iodine ( 125 I, 131 I), carbon ( 14 C), sulfur ( 35 S), indium ( 111 In), tritium ( 3 H); conjugates of monoclonal antibodies with biotin or avidin, enzymes such as horseradish peroxidase, alkaline phosphatase, beta-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, or glucose 6-phosphate dehydrogenase; and conjugates of monoclonal antibodies with bioluminescent agents (e.g., luciferase), chemiluminescent agents (e.g., acridine esters), or fluorescent agents (e.g., phycobiliproteins).

[0381] Another derivative bifunctional antibody of the present invention is a bispecific antibody, generated by combining portions of two separate antibodies that recognize two different antigenic groups. This can be achieved by crosslinking or recombinant techniques. In addition, moieties can be added to the antibody or portion thereof to increase its in vivo half-life (e.g., by increasing the time it takes for it to be cleared from the bloodstream). Such techniques include, for example, the addition of a PEG moiety (also known as PEGylation), and are well known in the art. See U.S. Patent Application Publication No. 20030031671.

[0382] Recombinant expression of antibodies In some embodiments, nucleic acids encoding a subject monoclonal antibody are directly introduced into host cells, and the cells are incubated under conditions sufficient to induce expression of the encoded antibody. After the cells have been introduced with the subject nucleic acid, they are typically incubated for a period of about 1 to 24 hours, usually at 37°C, optionally under selection, to allow for expression of the antibody. In one embodiment, the antibody is secreted into the supernatant of the medium in which the cells are growing.

[0383] Traditionally, monoclonal antibodies have been produced as native molecules in murine hybridoma systems. In addition to that technology, the present invention provides recombinant DNA expression of monoclonal antibodies, allowing the production of caninized, feline, equine, fully canine, fully feline, and fully equine antibodies, as well as a spectrum of antibody derivatives and fusion proteins in the host species of choice.

[0384] Nucleic acid sequences encoding at least one anti-IL-31 antibody, portion, or polypeptide of the present invention can be recombined with vector DNA according to conventional techniques, including blunt or staggered ends for ligation, restriction enzyme digestion to provide appropriate ends, filling in sticky ends as needed, alkaline phosphatase treatment to avoid undesired ligation, and ligation with an appropriate ligase. Techniques for such manipulations 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 (supra) can be used to construct nucleic acid sequences encoding monoclonal antibody molecules or antigen-binding regions thereof.

[0385] A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide when it contains a nucleotide sequence containing transcriptional and translational regulatory information, and such a sequence is "operably linked" to a nucleotide sequence encoding that polypeptide. An operably linked linkage is one in which the regulatory DNA sequence and the DNA sequence desired to be expressed are connected in a manner that allows for gene expression as recoverable amounts of an anti-IL-31 peptide or antibody portion. The precise nature of the regulatory regions required for gene expression can vary from organism to organism, as is well known in the art. See, e.g., Sambrook et al., 2001 (supra); Ausubel et al., 1993 (supra).

[0386] Thus, the present invention encompasses the expression of anti-IL-31 antibodies or IL-31-specific polypeptides or peptides in either prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including bacterial, yeast, insect, fungal, avian, and mammalian cells, either in vivo or in situ, or host cells of mammalian, insect, avian, or yeast origin. Mammalian cells or tissues can be of human, primate, hamster, rabbit, rodent, bovine, porcine, ovine, equine, caprine, canine, or feline origin, although any other mammalian cells may be used.

[0387] In one embodiment, the introduced nucleotide sequence is autonomous in the recipient host. The vector is incorporated into a replicable plasmid or viral vector. Any of a wide variety of vectors can be used for this purpose. See, e.g., Ausubel et al., 1993 (supra). Important factors in selecting a particular plasmid or viral vector include the ease of recognizing and selecting recipient cells containing the vector from those that do not, the copy number of the vector desired in a particular host, and whether it is desirable to be able to "shuffle" the vector between host cells of different species.

[0388] Examples of prokaryotic vectors known in the art include plasmids, such as those capable of replication in E. coli (e.g., pBR322, ColE1, pSC101, pACYC 184, pi.VX). Such plasmids are disclosed, for example, by Maniatis et al., 1989 (supra); Ausubel et al., 1993 (supra). Bacillus plasmids include pC194, pC221, pT127, and the like. Such plasmids are disclosed by Gryczan (THE MOLEC. BIO. OF THE BACILLI 307-329 (Academic Press, NY, 1982)). A suitable Streptomyces plasmid is pIJ101 (Kendall Examples of plasmids that can be used include Streptomyces bacteriophages such as phi.C31 (Chater et al., 169 J. Bacteriol. 4177-83 (1987)), and Streptomyces bacteriophages such as phi.C31 (Chater et al., SIXTH INT'L SYMPOSIUM ON ACTINOMYCETALES BIO. 45-54 (Akademiai Kaido, Budapest, Hungary 1986)). Pseudomonas plasmids are reviewed in John et al., 8 Rev. Infect. Dis. 693-704 (1986), Izaki, 33 Jpn. J. Bacteriol. 729-42 (1978), and Ausubel et al., 1993 (ibid.).

[0389] Alternatively, gene expression elements useful for expressing cDNA encoding an anti-IL-31 antibody or peptide include, but are not limited to, (a) viral transcription promoters and their enhancer elements, such as the SV40 early 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 murine leukemia virus LTR (Grosschedl et al., 41 Cell 885 (1985)), (b) splice regions and polyadenylation sites, such as the SV40 late region (Okayama et al., MCB, 3:280 (1983)), and (c) polyadenylation sites, such as those within SV40 (Okayama et al., 1983 (supra)).

[0390] Immunoglobulin cDNA genes were derived from Weidle et al., 51(1) Gene As described by W. Beck, 2002, pp. 21-29 (1987), expression can be achieved using the SV40 early promoter and enhancer, the mouse immunoglobulin heavy chain promoter enhancer, the SV40 late region mRNA splicing sequence, the rabbit S-globin intervening sequence, the immunoglobulin and rabbit S-globin polyadenylation sites, and the SV40 polyadenylation element as expression elements.

[0391] For immunoglobulin genes composed of part of cDNA and part of genomic DNA (Whittle et al., Protein Engin. 499-505 (1987)), the transcription promoter can be human cytomegalovirus, and the promoter enhancer can be cytomegalovirus and mouse / human immunoglobulin. and the mRNA splicing and polyadenylation regions can be those of the native chromosomal immunoglobulin sequences.

[0392] In one embodiment, for expression of cDNA genes in rodent cells, the transcription promoter is a viral LTR sequence, the transcription promoter enhancer is either or both of a mouse immunoglobulin heavy chain enhancer and a viral LTR enhancer, the splice region contains an intron of greater than 31 bp, and the polyadenylation and transcription termination regions are derived from the native chromosomal sequence corresponding to the immunoglobulin chain being synthesized. In other embodiments, cDNA sequences encoding other proteins are combined with the above expression elements to achieve expression of the proteins in mammalian cells.

[0393] Each fusion gene can be assembled within or inserted into an expression vector. Recipient cells capable of expressing the chimeric immunoglobulin chain gene products are then transfected with genes encoding the anti-IL-31 peptide or chimeric H or L chain, either alone or co-transfected with the chimeric H and L chain genes. The transfected recipient cells are cultured under conditions that allow expression of the integrated genes, and the expressed immunoglobulin chains or intact antibodies or fragments are recovered from the culture.

[0394] In one embodiment, fusion genes encoding anti-IL-31 peptides or chimeric heavy and light chains, or portions thereof, are assembled in separate expression vectors, which are then used to co-transfect recipient cells. Alternatively, fusion genes encoding chimeric heavy and light chains may be assembled on the same expression vector.

[0395] For transfection of expression vectors and production of chimeric antibodies, the recipient cell line may be a myeloma cell. Myeloma cells are capable of synthesizing, assembling, and secreting immunoglobulins encoded by the transfected immunoglobulin genes and possess the machinery for glycosylation of the immunoglobulins. Myeloma cells can be grown in culture or in the peritoneal cavity of mice, where the secreted immunoglobulins can be obtained from the ascites fluid. Other suitable recipient cells include lymphoid cells, such as B lymphocytes of human or non-human origin, hybridoma cells of human or non-human origin, or interspecies heterohybridoma cells.

[0396] The chimeric, caninized, felineized, equineized, fully canine, fully feline, or fully equine anti-IL-31 antibody constructs or IL-31-specific polypeptides or peptides (e.g., antigen-binding portions of the antibodies described herein) 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, calcium phosphate precipitation, application with polycations such as diethylaminoethyl (DEAE) dextran, and mechanical means such as electroporation, direct microinjection, and particle bombardment (Johnston et al., 240 Science 1538-1541 (1988)).

[0397] Yeast may offer considerable advantages over bacteria in the production of immunoglobulin heavy and light chains. Yeast performs post-translational peptide modifications, including glycosylation. Currently, several recombinant DNA strategies exist that utilize strong promoter sequences and high copy number plasmids that can be used to produce desired proteins in yeast. Yeast recognizes leader sequences in cloned mammalian gene products and secretes peptides bearing leader sequences (i.e., pre-peptides). Hitzman et al., 11th Int'l Conference on Yeast,Genetics & Mo lec. Biol. (Montpelier, France, 1982).

[0398] Yeast gene expression systems can be routinely used to evaluate the production, secretion, and stability levels of anti-IL-31 peptides, antibodies, and assembled mouse and chimeric, heterochimeric, caninized, feline, equine, fully canine, fully feline, or fully equine antibodies, fragments, and regions thereof. Any of a range of yeast gene expression systems incorporating promoter and termination elements from actively expressed genes encoding glycolytic enzymes that are produced in large amounts when yeast is grown in glucose-rich medium can be utilized. Known glycolytic genes can also provide highly efficient transcriptional regulatory signals. For example, the promoter and termination signal of the phosphoglycerate kinase (PGK) gene can be utilized. Several approaches can be taken to evaluate the optimal expression plasmid for expression of cloned immunoglobulin cDNA in yeast. See Vol. II DNA Cloning, pp. 45-66 (Glover, ed., IRL Press, Oxford, UK 1985).

[0399] Bacterial strains can also be utilized as hosts for the production of the antibody molecules or peptides described by this invention. In conjunction with such bacterial hosts, plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used. The vector carries a replication site as well as specific genes capable of providing phenotypic selection in transformed cells. Several approaches can be taken to evaluate expression plasmids for producing murine, chimeric, heterochimeric, caninized, feline, equine, fully canine, fully feline, or fully equine antibodies, fragments and regions, or antibody chains encoded by cloned immunoglobulin cDNAs in bacteria (see Glover, 1985, supra; Ausubel, 1993, supra; Sambrook, 2001, supra; 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)).

[0400] Host mammalian cells can be grown in vitro or in vivo and are responsible for post-translational modifications of immunoglobulin protein molecules, including removal of leader peptides, folding and assembly of heavy and light chains, glycosylation of antibody molecules, and secretion of functional antibody proteins.

[0401] Mammalian cells that may be useful as hosts for producing antibody proteins include, in addition to the cells of lymphoid origin described above, cells of fibroblastic origin, such as Vero cells (ATCC CRL 81) or CHO-K1 cells (ATCC CRL 61).

[0402] Many vector systems are available for expressing cloned anti-IL-31 peptide heavy and light chain genes in mammalian cells (see Glover, 1985, supra). Complete H2L2 antibodies can be obtained according to various approaches. H and L chains can be co-expressed in the same cell to achieve intracellular association and binding of the heavy and light chains to form complete tetrameric H2L2 antibodies and / or anti-IL-31 peptides. Co-expression can occur by using the same or different plasmids in the same host. Genes for both heavy and light chains and / or anti-IL-31 peptides can be placed in the same plasmid, and this plasmid can then be transfected into cells to directly select for cells expressing both chains. Alternatively, cells can be first transfected with a plasmid encoding one chain, e.g., the light chain, and then the resulting cell line can be transfected with a heavy chain plasmid containing a second selection marker. Producing anti-IL-31 peptides and / or H2L2 molecules by either route The cell lines can be transfected with plasmids encoding additional copies of the peptide, H, L, or H and L chains along with additional selectable markers to generate cell lines with enhanced properties (e.g., improved production of assembled H2L2 antibody molecules or enhanced stability of the transfected cell line).

[0403] Stable expression can be used for long-term, high-yield production of recombinant antibodies. For example, cell lines that stably express antibody molecules can be engineered. Instead of using expression vectors containing viral origins of replication, host cells can be transformed with an immunoglobulin expression cassette and a selectable marker. After introduction of the foreign DNA, engineered cells can be grown in an enriched medium for 1-2 days and then switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection, allowing cells to stably integrate the plasmid into their chromosomes and grow to form foci that can be cloned and expanded into cell lines. Such engineered cell lines can be particularly useful for screening and evaluating compounds / components that interact directly or indirectly with antibody molecules.

[0404] Once an antibody of the invention is produced, it can be purified by any method known in the art for purifying immunoglobulin molecules, for example, by chromatography (e.g., ion exchange, affinity, particularly for specific antigens followed by protein A, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. In many embodiments, the antibody is secreted from the cells into the culture medium and harvested from the culture medium.

[0405] Medical uses The anti-IL-31 antibodies or IL-31-specific polypeptides or peptides of the present invention can be used to treat, for example, pruritic or allergic conditions in companion animals such as dogs, cats, and horses. In one embodiment, such polypeptides or peptides comprise the antigen-binding portion of an anti-IL-31 antibody described herein. More specifically, the present invention further provides pharmaceutical compositions comprising, as an active ingredient, an antibody or polypeptide or peptide according to the present invention and a pharmaceutically acceptable carrier or diluent. The antibody can be a chimeric, heterochimeric, caninized, felineized, equinized, fully canine, fully feline, or fully equine antibody according to the present invention. Intact immunoglobulins or binding fragments thereof (e.g., Fabs) are also contemplated. The antibodies of the present invention and pharmaceutical compositions thereof are useful for parenteral administration, e.g., subcutaneous, intramuscular, or intravenous administration.

[0406] The anti-IL-31 antibodies and / or IL-31-specific polypeptides and / or IL-31-specific peptides of the present invention can be administered as individual therapeutic agents or in combination with other therapeutic agents, and while they can be administered alone, they will generally be administered with a pharmaceutical carrier selected based on the chosen route of administration and standard practice.

[0407] Administration of the antibodies disclosed herein can be by any suitable means, including parenteral injection (e.g., intraperitoneal, subcutaneous, or intramuscular injection), oral administration, or topical administration of the antibody to respiratory tract surfaces (typically delivered in a pharmaceutical formulation). Topical administration to respiratory tract surfaces can be by intranasal administration (e.g., by use of a dropper, swab, or inhaler). Topical administration of the antibody to respiratory tract surfaces can also be by inhalation administration, for example, by forming inhalable antibody-containing pharmaceutical formulation particles (including both solid and liquid particles) into an aerosol suspension and then having a subject inhale the inhalable particles. Methods and devices for administering inhalable pharmaceutical formulation particles are well known, and any conventional technique can be used. Oral administration can be in the form of, for example, an orally ingestible liquid or solid formulation.

[0408] In some desirable embodiments, the antibody is administered by parenteral injection. For parenteral administration, the anti-IL-31 antibody, polypeptide, or peptide can be formulated as a solution, suspension, emulsion, or lyophilized powder in a pharmaceutically acceptable parenteral vehicle. For example, the vehicle can be a solution of the antibody or a cocktail thereof dissolved in an acceptable carrier, such as an aqueous carrier. Such vehicles include water, saline, Ringer's solution, dextrose solution, trehalose, or sucrose solution, or 5% serum albumin, 0.4% saline, 0.3% glycine, or the like. Liposomes and non-aqueous vehicles (e.g., fixed oils) can also be used. Such solutions are sterile and generally free of particulate matter. Such compositions can be sterilized by conventional, well-known sterilization techniques. The compositions can contain pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of antibody in such formulations can vary widely, for example, from less than about 0.5% by weight, usually from about 1% or more to as much as 15% or 20% by weight, and is selected primarily based on liquid volume, viscosity, etc., depending on the particular mode of administration selected. The vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by commonly used techniques.

[0409] Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail, for example, in REMINGTON'S PHARMA. SCI. (15th ed., Mack Pub. Co., Easton, Pa., 1980).

[0410] The antibodies of the present invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective for conventional immunoglobulins. Any suitable lyophilization and reconstitution technique can be used. Those skilled in the art will recognize that lyophilization and reconstitution can lead to varying degrees of antibody activity loss, and that use levels may need to be adjusted to compensate.

[0411] Compositions containing the antibodies of the invention or a cocktail thereof can be administered to prevent recurrence and / or treat existing diseases. Suitable pharmaceutical carriers are described in the most recent edition of REMINGTON'S PHARMACEUTICAL SCIENCES, a standard reference text in the field.

[0412] In therapeutic applications, compositions can be administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest or ameliorate the disease and its complications. An amount sufficient to accomplish this is defined as a "therapeutically effective dose" or "therapeutically effective amount." Effective amounts for this use will depend on the severity of the disease and the general state of the subject's own immune system, but generally range from about 0.1 mg antibody / kg body weight to about 15 mg antibody / kg body weight, preferably about 0.3 mg antibody / kg body weight to about 12 mg antibody / kg body weight. In one embodiment, a therapeutically effective amount provides a duration of efficacy of at least one month at a dose of up to 12 mg / kg body weight. Given the minimization of foreign material and the reduced likelihood of "foreign material" rejection achieved with the canine, feline, and equine antibodies of the present invention, it is believed possible to administer substantial excesses of these antibodies.

[0413] The dosage administered will, of course, vary depending on known factors such as the pharmacodynamic properties of the particular agent, its mode and route of administration, the age, health, and weight of the recipient, the nature and extent of the condition, type of concurrent treatment, frequency of treatment, and the desired effect.

[0414] As a non-limiting example, treatment of an IL-31-associated condition in a dog, cat, or horse can be provided as a biweekly or monthly dose of an anti-IL-31 antibody of the invention within the dosage ranges described above.

[0415] Exemplary antibodies for canine, feline, or equine therapeutic use are potent in accordance with the present invention. High affinity (which may also be high avidity) antibodies having in vivo anti-IL-31 activity, as well as fragments, regions, and derivatives thereof. Antibody fragments and regions may alternatively be referred to herein as polypeptides or peptides of the invention comprising the antigen-binding portion of an anti-IL-31 antibody.

[0416] Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating veterinarian. In any event, the pharmaceutical formulation should provide a quantity of the antibody(ies) of the invention sufficient to effectively treat the subject.

[0417] Diagnostic Use The present invention also provides the above-described anti-IL-31 antibodies, polypeptides, and / or peptides for use in diagnostic methods for detecting IL-31 in companion animals known to have or suspected of having a pruritic and / or allergic condition.

[0418] The anti-IL-31 antibodies, polypeptides, and / or peptides of the present invention are useful in immunoassays for detecting or quantifying IL-31 or anti-IL-31 antibodies in a sample. Immunoassays for IL-31 typically involve incubating a clinical or biological sample in the presence of a detectably labeled, high-affinity (or high-avidity) anti-IL-31 antibody, polypeptide, or peptide of the present invention capable of selectively binding to IL-31, and detecting bound, labeled polypeptide, peptide, or antibody in the sample. Various clinical assay procedures are well known in the art. See, for example, IMMUNOASSAYS FOR THE 80'S (Voller et al., eds., Univ. Park, 1981). Such samples include tissue biopsies, blood, serum, fecal samples, or fluids collected from animal subjects and subjected to ELISA analysis, as described below.

[0419] In some embodiments, the binding of the antigen to the antibody is detected without the use of a solid support, for example, the binding of the antigen to the antibody can be detected in a liquid format.

[0420] In other embodiments, the anti-IL-31 antibody, polypeptide, or peptide can be immobilized, for example, on 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 then treated with a detectably labeled IL-31-specific polypeptide, peptide, or antibody. The solid support can then be washed again with buffer to remove unbound polypeptide, peptide, or antibody. The amount of bound label on the solid support can then be detected by known method steps.

[0421] "Solid phase support" or "carrier" refers to any support capable of binding a polypeptide, peptide, antigen, or antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, polyvinylidene fluoride (PVDF), dextran, nylon, amylase, natural and modified celluloses (e.g., nitrocellulose), polyacrylamide, agarose, and magnetite. The nature of the carrier can be soluble to some extent or insoluble for the purposes of the present invention. Any support material is contemplated as long as the binding molecule is capable of binding to IL-31 or an anti-IL-31 antibody. The support can have almost any structural configuration. Thus, the configuration can be spherical, such as a bead, or cylindrical, such as the interior surface of a test tube or the exterior surface of a rod. Alternatively, the surface can be flat, such as a sheet, culture dish, test strip, etc. For example, the support can comprise polystyrene beads. Those of skill in the art will know many other suitable carriers for binding antibodies, polypeptides, peptides, or antigens, or can ascertain suitable carriers by routine experimentation.

[0422] Well-known method steps can determine the binding activity of a given lot of anti-IL-31 polypeptides, peptides, and / or antibodies, and those skilled in the art can determine effective and optimal assay conditions by routine experimentation.

[0423] Detectable labeling of IL-31-specific polypeptides, peptides, and / or antibodies can be accomplished by linking them to an enzyme for use in enzyme immunoassays (EIAs) or enzyme-linked immunosorbent assays (ELISAs). The linked enzyme reacts with an exposed substrate to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric, or visual means. Enzymes that can be used to detectably label IL-31-specific antibodies of the present invention include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

[0424] By radioactively labeling IL-31-specific antibodies, it is possible to detect IL-31 using radioimmunoassay (RIA). Work et al., LAB. See TECHNIQUES & BIOCHEM. IN MOLEC. Bio. (No. Holland Pub. Co., NY, 1978). Radioactive isotopes can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography. Isotopes that are particularly useful for the purpose of the present invention include: 3 H, 125 I, 131 I, 35 S, 14 C, and 125 I can be mentioned.

[0425] IL-31-specific antibodies can also be labeled with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. The most commonly used fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine.

[0426] IL-31 specific antibodies may also be coupled to fluorescent metals, e.g. 125 Detectable labeling can also be achieved using Eu or other metals of the lanthanide series, which can be attached to IL-31-specific antibodies using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0427] An IL-31-specific antibody can also be detectably labeled by conjugation with a chemiluminescent compound. In this case, the presence of the chemiluminescently-tagged antibody is determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.

[0428] Similarly, a bioluminescent compound can be used to label an IL-31-specific antibody, portion, fragment, polypeptide, or derivative of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase, and aequorin.

[0429] Detection of IL-31-specific antibodies, portions, fragments, polypeptides, or derivatives can be accomplished, for example, by scintillation counters if the detectable label is a radioactive gamma emitter, or by fluorescence systems, for example, if the label is a fluorescent substance. In the case of an enzyme label, detection can be accomplished by colorimetric methods which employ a substrate for the enzyme. Detection can also be accomplished by visual comparison of the extent of enzymatic reaction of the substrate to similarly prepared standards.

[0430] For the purposes of the present invention, the IL-31 detected by the above assay may be present in a biological sample. Any sample containing IL-31 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, but those skilled in the art will be able to determine suitable conditions that allow the use of other samples in light of this specification.

[0431] In situ detection can be accomplished by removing a histological specimen from an animal subject and providing such specimen with a 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) on a biological sample. Using such a procedure, it is possible to determine not only the presence of IL-31, but also the distribution of IL-31 in the examined tissue. Those skilled in the art will readily appreciate that, using the present invention, any of a wide variety of histological methods (e.g., staining procedures) can be modified to achieve such in situ detection.

[0432] The antibodies, fragments, or derivatives of the invention can be adapted for use in immunometric assays, also known as "two-site" or "sandwich" assays. In a typical immunometric assay, a quantity of unlabeled antibody (or antibody fragment) is bound to an insoluble solid support in the liquid being tested, and a detectably labeled soluble antibody is added, allowing for detection and / or quantitation of the ternary complex formed between the solid-phase antibody, antigen, and labeled antibody.

[0433] Antibodies can be used to quantitatively or qualitatively detect IL-31 in a sample or to detect the presence of cells expressing IL-31. This can be accomplished by fluorescence microscopy, flow cytometry, or immunofluorescence techniques using fluorescently labeled antibodies (see below) with fluorometric detection. For diagnostic purposes, antibodies can be labeled or unlabeled. Unlabeled antibodies can be used in combination with other labeled antibodies (secondary antibodies) that react with the antibody (e.g., antibodies specific for canine or feline immunoglobulin constant regions). Alternatively, antibodies can be directly labeled. A wide variety of labels can be used, including radionuclides, fluorescent substances, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, and ligands (e.g., haptens). Many types of immunoassays, such as those discussed above, are available and well known to those skilled in the art.

[0434] In one embodiment, the diagnostic method for detecting IL-31 is a lateral flow immunoassay test, which is an immunochromatographic assay, Rapid Immunomigration (RIM®). Lateral flow immunoassays are also known as lateral flow (LAF) or strip tests. A lateral flow immunoassay is essentially an immunoassay adapted to operate along a single axis to fit a test strip format. Several variations of this technology 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) a sample pad (an absorbent pad to which the test sample is applied); (2) a conjugate or reagent pad (which contains an antibody specific for the target analyte conjugated to colored particles (usually colloidal gold particles or latex microspheres)); (3) a reaction membrane (typically a hydrophobic nitrocellulose or cellulose acetate membrane on which anti-target analyte antibodies are immobilized in a line across the membrane as a capture zone or test line (a control zone containing an antibody specific for the conjugate antibody may also be present); and (4) a wicking or waste reservoir (a further absorbent pad designed to draw and collect the sample across the reaction membrane by capillary action). The strip components are typically fixed to an inert backing material and are provided in a simple dipstick format or in a plastic case with a sample port and reaction window that reveals the capture and control zones.

[0435] There are two main types of lateral flow immunoassays used in microbiological testing: double-antibody sandwich assays and competitive assays. In the double-antibody sandwich format, the sample migrates from the sample pad through the conjugate pad, and any target analyte present binds to the conjugate. The sample then continues across the membrane until it reaches the capture zone, where the target / conjugate complex binds to the immobilized antibody and a line is visualized on the membrane. The sample then continues along the strip until it reaches the control zone, where excess conjugate binds and a second line is visualized on the membrane. This control line indicates that the sample migrated 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. Competitive assays differ from the double-antibody sandwich format in that the conjugate pad contains an antibody already bound to the target analyte or its analog. Therefore, if the target analyte is present in the sample, it will not bind to the conjugate and will remain unlabeled. As the sample migrates along the membrane and reaches the capture zone, excess unlabeled analyte binds to the immobilized antibody, blocking capture of the conjugate; therefore, no line is visible. Unbound conjugate then binds to the antibody in the control zone, visualizing the control line. A single control line on the membrane indicates a positive result. Visible lines in the capture and control zones indicate a negative result. However, a faint line may be produced in the capture zone in the absence of excess unlabeled target analyte, indicating an inconclusive result. Several variations of lateral flow technology exist. The capture zone on the membrane may contain immobilized antigens or enzymes rather than antibodies, depending on the target analyte. Multiple capture zones can also be applied to create multiplexed tests. For example, commercially available test strips have been developed that can separately detect both EHEC Shiga toxins ST1 and ST2 in the same sample.

[0436] Importantly, the antibodies of the present invention may be useful in diagnosing pruritic or allergic conditions in dogs, cats, or horses. More specifically, the antibodies of the present invention can identify overexpression of IL-31 in companion animals. Thus, the antibodies of the present invention may provide an important immunohistochemistry tool. In one embodiment, an assay design is contemplated herein in which an IL-31 mimotope (peptide) is used to capture a labeled antibody of the present invention for detection in an assay. The captured antibody would have a lower affinity for the bound mimotope than the affinity of native circulating IL-31 of the host species. In this embodiment, an incubation of a fluid derived from the host species is incubated with a labeled antibody:mimotope complex tethered to a solid surface. The test fluid derived from the host species Because IL-31 has a higher affinity for the antibody if present in the solid surface, the labeled antibody can be released from the solid surface and removed during the washing step. Therefore, the level of IL-31 in the test solution can be correlated with the lack of signal appearing on the mimotope-bound surface. Such an assay is believed to be useful for measuring IL-31 in research or clinical settings for use as a diagnostic test.

[0437] The antibodies of the present invention can be used on antibody arrays, which are highly suitable for measuring gene expression profiles.

[0438] kit Kits for carrying out the subject methods are also within the scope of the present invention. The kits include at least one or more of the following: an antibody of the present invention, a nucleic acid encoding the antibody of the present invention, or cells containing the antibody of the present invention. In one embodiment, the antibody of the present invention can be provided in a lyophilized form, typically in a container. The antibody, which may or may not be conjugated to a label or toxin, is typically included in the kit along with buffers (e.g., Tris, phosphate, carbonate, etc.), stabilizers, biocides, inert proteins (e.g., serum albumin), and the like. Generally, these materials are present in an amount of less than 5 wt% based on the amount of active antibody, and usually in a total amount of at least about 0.001 wt%, again based on the antibody concentration. It is often desirable to include an inert bulking agent or excipient to dilute the active ingredient, which can be present at about 1 wt% to 99 wt% of the total composition. If a secondary antibody capable of binding to the primary antibody is used in the assay, it is typically present in a separate vial. The secondary antibody is typically conjugated to 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.

[0439] In one embodiment, the kit according to the present invention is a test strip kit (lateral flow immunoassay kit) useful for detecting canine, feline, or equine IL-31 protein in a sample. Such a test strip typically includes a sample pad to which the test sample is applied, a conjugate or reagent pad containing an antibody specific for canine, feline, or equine IL-31 and conjugated to colored particles (usually colloidal gold particles), a reaction membrane on which an anti-IL-31 antibody is immobilized in a line across the membrane as a capture zone or test line (a control zone containing an antibody specific for the conjugate antibody may also be present), and an additional absorbent pad designed to draw and collect the sample across the reaction membrane by capillary action. Generally, the test strip kit also includes instructions for use.

[0440] Methods for improving the consistency and / or quality of feline or canine antibodies Such methods are described in the Summary of the Invention section above and in the Examples and Figures of this application.

[0441] The inventors' surprising discovery is that removing or modifying the C-terminus of the kappa light chain constant region from animal species whose native germline encodes additional residues other than the terminal light chain cysteine ​​is beneficial to the production of homogeneous recombinant antibodies in such species, as well as to the amount of antibody produced from stable cell lines (e.g., improved yields). The results described herein support the idea that additional amino acid residues other than the terminal cysteine ​​in feline (and canine) kappa light chains are detrimental to efficient pairing with heavy chains, leading to mispairing and poor production of antibodies.

[0442] In one embodiment, the present invention provides a method for improving the consistency and / or quality of feline antibodies, comprising expressing in a host cell a nucleotide sequence encoding a feline IgG kappa light chain and a nucleotide sequence encoding a feline IgG heavy chain to produce a feline antibody. The nucleotide sequence encoding the feline IgG kappa light chain comprises a kappa light chain constant nucleotide sequence in which the sequence encoding the C-terminal QRE sequence otherwise present in the wild-type feline IgG light chain constant region has been modified and / or deleted. With respect to the C-terminal amino acid residues of the feline Ig kappa light chain constant domain shown in FIG. 15, in one embodiment, the present invention provides for the removal of the C-terminal "QRE" immediately following CYS107 of the feline light chain kappa sequence of SEQ ID NO: 175. This modification has been found to improve the production of monomeric recombinant feline IgG. However, the present invention is not limited in this respect. For example, the addition of three additional consecutive amino acids C-terminal to cysteine ​​107 in place of the native QRE may be tolerated if the electrostatic charge of these three amino acids is minimally affected.

[0443] The present invention further provides a method for improving the consistency and / or quality of a canine antibody. The method comprises producing a canine antibody by expressing in a host cell a nucleotide sequence encoding a canine IgG kappa light chain and a nucleotide sequence encoding a canine IgG heavy chain, wherein the nucleotide sequence encoding the canine IgG kappa light chain comprises a kappa light chain constant nucleotide sequence in which a sequence encoding a C-terminal QRVD sequence otherwise present in the wild-type canine IgG light chain constant region has been modified and / or deleted. With respect to the C-terminal amino acid residues of the canine Ig kappa light chain constant domain shown in FIG. 15, in one embodiment, the present invention provides for the deletion of the c-terminal "QRVD" immediately following CYS105 of the canine light chain kappa sequence of SEQ ID NO: 194. However, the present invention is not limited in this respect. For example, the addition of three consecutive amino acids c-terminal to cysteine ​​105 in place of the native QRVD may be tolerated if the electrostatic charge of these three amino acids is minimally affected.

[0444] Because the results herein clearly demonstrate that the above method applies to structurally distinct antibodies that recognize completely distinct targets, it is possible that such modifications may also be applicable to a broader genera of feline antibodies, including, but not limited to, anti-IL-31 and anti-NGF antibodies, as well as other mammalian antibodies with additional C-terminal amino acids on the kappa light chain constant region. While not wishing to be bound by any one theory, this light chain modification appears to pair immunoglobulin chains with greater fidelity during induction production from stable CHO cell lines, resulting in higher amounts of monomeric IgG and, in some cases, higher overall antibody yields. Both of these attributes are highly desirable from the perspective of producing commercial-grade antibody therapeutics.

[0445] In one embodiment, any of the anti-IL-31 antibodies disclosed herein can include a deletion and / or modification of the kappa light chain constant region disclosed herein. For example, in one embodiment, a feline antibody according to the invention comprises a kappa light chain constant region in which the "QRE" normally present at the c-terminus of the kappa light chain constant region has been removed and, optionally, replaced with up to three additional amino acids that minimize the effect of electrostatic charge. In a specific embodiment, a feline antibody according to the invention comprises a feline kappa light chain having the sequence: RSDAQPSVFLFQPSLDELHTGSASIVCILNDFYPKEVNVKWKVDGVVQNKGIQESTTEQNSKDSTYSLSSTLTMSSTEYQSHEKFSCEVTHKSLASTLVKSFQRSEC (SEQ ID NO: 186) or a variant thereof.

[0446] The present invention will now be further described by the following non-limiting examples. In the Examples section below and in the Figures, any data presented for an antibody containing "11E12" in its name is for comparison with the antibodies of the present invention. [Example]

[0447] 1.1. Production of canine interleukin-31 (cIL-31) from Chinese hamster ovary (CHO) cells Although the amino acid sequence of the interleukin-31 protein varies among homologous species (Figure 1), it is believed to share a common structural architecture with other members of the type I cytokine family (Boulay et al. 2003, Immunity. Aug;19(2):159-63 2003; Dillon et al. 2004 Nat Immunol. Jul;5(7):752-60). This up-down bundle topology is important for the receptor recognition mode shared by these cytokines (Dillon et al. (ibid.), Cornelissen et al. 2012 Eur J Cell Biol. Jun-Jul;91(6-7):552-66). Because of the variability in IL-31 protein sequence identity among different species, it is impossible to predict whether antibodies raised against one species will cross-react with those from other species given the different epitope properties and local amino acid composition. Consequently, multiple forms of the IL-31 protein representing multiple species and expression systems have been investigated to address this issue. Canine IL-31 protein (cIL-31) was produced and used as an immunogen and reagent for affinity and potency testing of antibody hits. Recombinant cIL-31 was generated in CHO cells using CHROMOS ACE (artificial chromosome expression) (Chromos Molecular Systems, Inc., Burnaby, British Columbia) to generate secreted canine IL-31 protein with the sequence of (SEQ ID NO: 155; canine_IL31) (the corresponding nucleotide sequence is (SEQ ID NO: 156; canine_IL31)). Conditioned medium was obtained from a 400 ml cell culture (CHO cell line) and dialyzed against 10 volumes of QA buffer (20 mM Tris pH 8.0, 20 mM NaCl) for 4.5 hours. The dialyzed medium was 0.2 μm filtered and loaded at 1 ml / min onto a SOURCE™ Q column (GE Healthcare, Uppsala, Sweden) pre-equilibrated with QA buffer. The protein was eluted using a multi-step linear gradient, with the majority of cIL-31 remaining in the flow-through (FT) fraction and a small amount of cIL-31 eluting early in the gradient.Protein identity was confirmed by Western immunoblotting and mass spectrometry (MS) of a tryptic digest. Proteins in the FT fraction were concentrated 4-5 times and dialyzed overnight against phosphate-buffered saline (PBS) at 4°C. Protein stability was examined after dialysis against PBS. After several days at 4°C, no precipitation or proteolysis was observed. Deglycosylation experiments using N-glycosidase F resulted in the protein being reduced to a single band of approximately 15 kDa on SDS-PAGE. Protein concentration was determined using the bicinchoninic acid assay (BCA assay) with bovine serum albumin (BSA) as a standard (ThermoFisher Scientific, Inc., Rockford, IL). The protein solution was divided into aliquots, snap-frozen (liquid N2), and stored at -80°C.

[0448] 1.2. Transient expression of wild-type and mutant feline interleukin-31 (fIL-31) from CHO cells To aid in the identification of antibodies with appropriate epitope-binding properties, wild-type and mutant feline IL-31 proteins were expressed in mammalian expression systems and produced, purified, and evaluated in affinity and cell-based assays. The binding site of antibody 11E12 on IL-31 has been previously described (U.S. Patent No. 8,790,651 to Bammert, et al.). Characterization of the novel binding site on IL-31 recognized by antibody 15H05 is described herein. The wild-type designation refers to the full-length feline IL-31 protein, with no changes to the native amino acid residues. The mutant proteins are named by the corresponding antibody names (11E12 and 15H05), and refer to amino acid mutations in the IL-31 protein that (when modified) affect binding to each respective antibody. Identification of the appropriate mutations required for the feline IL-31 15H05 protein is described in Section 1.10. The goal was to change amino acids in the IL-31 epitope and observe the loss of binding phenotype for each respective antibody. Comparisons were then made during screening to see if the new candidate antibodies bound to the wild-type protein and not the mutant form. The new antibody hits can then be binned according to binding to the same or similar epitope as antibody 11E12 or 15H05.

[0449] Expression constructs were codon-optimized and synthesized for expression in Chinese hamster ovary (CHO) cells. The synthesized genes were cloned into pD2529 (ATUM vector) for transient expression. The wild-type feline IL-31 protein is represented by (SEQ ID NO: 157; feline_IL31_wildtype) (the corresponding nucleotide sequence is (SEQ ID NO: 158; feline_IL31_wildtype)). The mutant feline IL-31 11E12 protein is represented by (SEQ ID NO: 161; feline_IL31_11E12_mutant) (the corresponding nucleotide sequence is (SEQ ID NO: 162; feline_IL31_11E12_mutant)). The mutant feline IL-31 15H05 protein is represented by (SEQ ID NO: 163; feline_IL31_15H05_mutant) (the corresponding nucleotide sequence is (SEQ ID NO: 164; feline_IL31_15H05_mutant)). Recombinant feline IL-31 protein was expressed in ExpiCHO-S™ cells (ThermoFisher Scientific, Inc., Rockford, IL) by following the manufacturer's maximum titer protocol for transient CHO expression. Twelve days after transfection, cells were centrifuged and filtered to capture the secreted protein in the conditioned medium. For each construct (wild-type and mutant), 120 mL of conditioned medium (0.2 μm filtered from CHO cell culture) was adjusted to 30 mS / cm by adding NaCl, 5 mM imidazole, and pH 7.4. Each medium sample was combined with 5 mL of HisPur cobalt resin (ThermoFisher Scientific, Inc., Rockford, IL) equilibrated with 5 mM imidazole, 20 mM sodium phosphate, 300 mM NaCl, pH 7.4. Each sample and resin were mixed overnight at 4°C. The resin was collected (and separated from the unbound fraction) by pouring through a BioRad Econocolumn (Bio-Rad, Hercules, CA). The resin was washed with 5 x 5 mL of buffer (same as above) and then eluted with 5 x 5 mL of 500 mM imidazole in the same buffer. Fractions were assessed by SDS-PAGE. Concentration was determined by BCA protein assay using standard methods.

[0450] 1.3.Production of feline interleukin-31 (fIL-31) from E. coli Recombinant feline IL-31 protein was produced in an E. coli expression host for use as an assay reagent and in in vivo challenge studies to induce pruritic responses in cats. A gene corresponding to feline IL-31 was synthesized for optimal expression in E. coli. An expression construct was made with the full-length feline IL-31 gene including an N-terminal 6-His tag for detection and purification. This feline IL-31 protein is represented by (SEQ ID NO: 159; feline_IL-31_E_coli) (the corresponding nucleotide sequence is (SEQ ID NO: 160; feline_IL-31_E_coli)). The sequence-verified plasmid was used to transform E. coli BL21 DE3 (Invitrogen Corp., Carlsbad, CA), and the protein was then expressed.

[0451] Cell paste (262.3 g) from E. coli was lysed as follows: the cell paste was resuspended in 500 mL of 50 mM Tris, pH 8, filtered through a stainless steel mesh filter to remove particulates, and then lysed by passing it twice through a microfluidizer at 1300 psi. The lysate (approximately 1200 mL in volume) was divided into four bottles and centrifuged at 12,000 g for 20 minutes at 10°C. The supernatant was decanted and discarded. Each pellet was washed by suspending it in 300 mL of 5 mM EDTA, 0.5% Triton X-100, pH 9.0, followed by centrifugation at 12,000 g for 50 minutes at 10°C. The supernatant was decanted and discarded. The washed pellets were stored at -20°C until folding and isolation.

[0452] Prior to isolation, one of the pellets was washed with water to remove residual detergent and then centrifuged at 10,000 g for 20 minutes at 4°C. The supernatant was again decanted. Finally, the washed pellet was dissolved in 60 mL of 50 mM sodium phosphate, 300 mM NaCl, 6 M guanidine-HCl, 5 mM imidazole, pH 7.4. The pellet was mixed at room temperature for approximately 25 minutes and then centrifuged again at 10,000 g for 20 minutes at 4°C. This time, the supernatant was decanted and saved for further processing. The pellet (resuspended in water to the original volume) was reserved for SDS-PAGE analysis. Crude IMAC (immobilized metal affinity chromatography) was performed to enhance purity before folding. In this case, 15 mL of Ni-NTA Superflow (Qiagen Inc., Germantown, MD (catalog no. 30450, pre-equilibrated in the same buffer)) was added to the clarified supernatant and mixed at room temperature for approximately 90 minutes. The unbound fraction was decanted and saved for SDS-PAGE. The IMAC resin was washed with 5 mM imidazole, 50 mM sodium phosphate, 300 mM NaCl, 6 M guanidine-HCl, pH 7.4 (the same as the lysis buffer). The resin was eluted with 200 mM imidazole, 50 mM sodium phosphate, 300 mM NaCl, 6 M guanidine-HCl, pH 7.4 (initial 7.5 mL, then 15 mL aliquots; protein elution was monitored by Bradford assay). Protein-containing elution fractions (according to Bradford) were pooled (125 mL) for further processing.

[0453] IL-31 protein was folded as follows: IL-31 was reduced to a final 10 mM by adding dithiothreitol and mixed for 2 hours at room temperature. The diluted sample was then diluted dropwise into 2500 mL (20 volumes) of PBS + 1 M NaCl with rapid stirring. At this point, the theoretical concentration of urea was estimated to be approximately 0.4 M. Residual urea was slowly removed by dialysis against three changes of PBS (4 L each) overnight at 4°C. After dialysis, the sample was 0.2 μm filtered to remove any unfolded / precipitated protein.

[0454] The sample was further purified by a second round of IMAC, this time using a linear gradient elution. 15 mL of Ni-NTA Superflow resin was added to the sample and bound batchwise by stirring (with a stirring bar) overnight at 4°C. Again, the unbound fraction was decanted and retained. The Ni-NTA Superflow resin was packed into an XK16 column (GE Healthcare Lifesciences, Marlborough, MA) and connected to an AKTA brand chromatography system (GE Healthcare Lifesciences, Marlborough, MA). The column was then washed with 50 mM Tris, 300 mM NaCl, pH 8.2, and eluted with a 150 mL linear gradient of 0 to 500 mM imidazole, each in wash buffer. Fractions were analyzed by SDS-PAGE. Fractions with sufficient purity of IL-31 were pooled and the buffer was again exchanged by dialysis against three changes of PBS (2 L each) overnight at 4°C. Finally, the folded and purified sample was collected from dialysis, sterile filtered, the concentration determined, aliquoted, snap frozen in a dry ice / isopropanol bath, and stored at -80°C.

[0455] 1.4. Method for determining the affinity of anti-IL-31 antibodies to IL-31 using surface plasmon resonance The binding affinity of candidate mAbs to canine and feline IL-31 was determined using surface plasmon resonance (SPR) on a Biacore system (Biocore Life Sciences (GE Healthcare), Uppsala, Sweden). To avoid affinity differences associated with different surface preparations that may arise when immobilizing antibodies to surfaces, a strategy was adopted in which IL-31 was directly conjugated to the surface. Immobilization was performed using N-hydroxysuccinimide (NHS) / 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. 5 μg / mL of IL-31 was obtained by amine coupling using EDC chemistry. The chip was quenched with ethanolamine, and the affinity of all candidate mAbs bound to the immobilized IL-31 was evaluated. All curves were fitted to a 1:1 model. 1 × 10 -11 Affinity constants (KD) less than M(1E-11 M) are considered below the lower limit of quantitation for detection by the instrument. The results of affinity measurements are described herein.

[0456] 1.5. Methods for Quantifying the Potency of Anti-IL-31 Antibodies Assessed by Inhibition of Canine and Feline IL-31-Induced pSTAT3 Signaling in Canine and Feline Macrophage Cells To identify candidates with inhibitory activity, antibodies were assessed for their ability to affect IL-31-mediated STAT3 phosphorylation in either canine or feline cell-based assays. STAT3 phosphorylation was quantified in canine DH-82 (ATCC® CRL-10389™) or feline FCWF4 macrophage-like cells (ATCC CRL-2787). DH82 and FCWF4 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 IL-31-induced STAT3 phosphorylation at 1 μg / mL in dogs or 0.2 μg / mL in cats using two independent methods. Assays were also performed to demonstrate cross-reactivity between dog and cat cytokines and cross-functionality of the antibodies' ability to inhibit signaling in both species. To ensure complex formation, a 1-hour co-incubation of mAb and IL-31 cytokine was completed before cell stimulation. IL-31 cell stimulation was performed for 5 minutes. STAT3 phosphorylation was measured using AlphaLISA SureFire ULTRA™ technology (Perkin Elmer, Waltham, MA). When antibody concentration and purity were unknown, hybridoma supernatants were co-incubated with 1 mg / mL canine IL-31 or 0.2 mg / mL feline IL-31 for 1 hour, followed by qualitative determination of their ability to inhibit STAT3 phosphorylation. The potency of monoclonal antibodies, defined by the ability of individual monoclonal antibodies to inhibit IL-31-mediated STAT3 phosphorylation in these assays, was considered a key selection criterion for further antibody selection. The term potency refers to the IC50 value calculated from these assays, which is the concentration of antibody at which IL-31-induced signaling is reduced by half of its maximum. Increased potency, as described herein, correlates with a decrease in IC50 value.

[0457] 1.6. Identification of murine and canine monoclonal antibodies that recognize canine and feline interleukin-31 To identify antibodies, mice and dogs were immunized with recombinant canine IL-31 (SEQ ID NO: 155). Serum antibody titers from immunized animals were quantified using enzyme-linked immunosorbent assay (ELISA). Canine or feline IL-31 (50 ng / well) was immobilized on a polystyrene microplate and used as a capture antigen. Serum from immunized animals was diluted in phosphate-buffered saline containing 0.05% Tween-20 (PBST). The presence of anti-IL-31 antibodies was detected with an appropriate HRP-conjugated secondary antibody. A chromogenic substrate (SureBlue Reserve TMB 1-Component Microwell Peroxidase Substrate (KPL, Inc., Gaithersburg, MD)) was added and incubated for 10 minutes at room temperature (RT), after which the reaction was stopped by adding 100 μL of 0.1 N HCl. The absorbance of each well was quantified at an optical density (OD) of 450 nm. Antibodies were selected for their ability to bind to canine and feline IL-31 using ELISA. In some cases, further characterization was performed during selection using ELISA with mutant forms of feline IL-31 protein as capture antigens. The desired binding and Cells producing antibodies with inhibitory properties were selected.

[0458] For mouse antibodies, donor splenocytes from monoresponsive CF-1 mice were used for fusion, and hybridoma supernatants were screened by ELISA for antibodies that bound to either canine or feline IL-31 protein. This identified a single mouse antibody, Mu-15H05, with subnanomolar affinity for IL-31 from both species (Figure 2A). Mouse anti-IL-31 15H05 was further subcloned to generate hybridomas producing homogeneous antibodies, and the variable heavy and light chains were sequenced. The murine anti-IL-31 variable sequences determined for antibody 15H05 are as follows: 15H05 variable heavy chain (SEQ ID NO: 67; MU-15H05-VH) with the corresponding nucleotide sequence (SEQ ID NO: 68; MU-15H05-VH), and 15H05 variable light chain (SEQ ID NO: 69; MU-15H05-VL) with the corresponding nucleotide sequence (SEQ ID NO: 70; MU-15H05-VL).

[0459] In addition to the murine antibody 15H05, we also investigated the murine-derived antibody 11E12, previously described in U.S. Patent No. 8,790,651 to Bammert et al. Herein, we present data demonstrating the ability of antibody 11E12 to bind with high affinity to both canine and feline IL-31 proteins. The ability of 11E12 to bind to feline IL-31 makes this antibody a suitable candidate for felinization and potential therapeutic use in cats. The previously determined murine anti-IL-31 variable sequences for antibody 11E12 are as follows: 11E12 variable heavy chain (SEQ ID NO: 71; MU-11E12-VH) (the corresponding nucleotide sequence is (SEQ ID NO: 72; MU-11E12-VH)), and 11E12 variable light chain (SEQ ID NO: 73; MU-11E12-VL) (the corresponding nucleotide sequence is (SEQ ID NO: 74; MU-11E12-VL)).

[0460] Dogs that developed elevated anti-IL-31 titers after vaccination were selected for analysis of B cell populations producing antibodies with the desired phenotype. B cells were derived from PBMCs, bone marrow, spleen, or lymph nodes for further analysis. Single B cells were isolated into individual wells and assayed for the presence of secreted IgG capable of binding to wild-type, 11E12 mutant, and 15H05 mutant forms of canine IL-31 (AbCellera, Vancouver, BC) using methods described in US2012 / 0009671 A1, US2016 / 0252495 A1, US9,188,593, WO2015 / 176162 A9, and WO2016 / 123692 A1.

[0461] This screening strategy is based on known regions of the IL-31 protein that are essential for binding and signaling through the IL-31 protein's co-receptor complex. The selection of these mutant proteins for screening is described in Section 1.2 of this application. Sequencing of the IgG variable heavy and light chain domains was performed after RT-PCR reactions from individual candidate B cells. Nine canine antibodies were identified from these screens and selected for further evaluation. These canine anti-IL-31 variable sequences are as follows: ZIL1 variable heavy chain (SEQ ID NO: 75; CAN-ZIL1_VH) (the corresponding nucleotide sequence is (SEQ ID NO: 76; CAN-ZIL1_VH)), ZIL1 variable light chain (SEQ ID NO: 77; CAN-ZIL1_VL) (the corresponding nucleotide sequence is (SEQ ID NO: 78; CAN-ZIL1_VL)); ZIL8 variable heavy chain (SEQ ID NO: 79; CAN-ZIL8_VH) (the corresponding nucleotide sequence is (SEQ ID NO: 80; CAN-ZIL8_VH)); The nucleotide sequence is (SEQ ID NO: 80; CAN-ZIL8_VH)), ZIL8 variable light chain (SEQ ID NO: 81; CAN-ZIL8_VL) (the corresponding nucleotide sequence is (SEQ ID NO: 82; CAN-ZIL8_VL)); ZIL9 variable heavy chain (SEQ ID NO: 83; CAN-ZIL9_VH) (the corresponding nucleotide sequence is (SEQ ID NO: 84; CAN-ZIL9_VH)), ZIL9 variable light chain (SEQ ID NO: 85; CAN-ZIL9_VL) (the corresponding nucleotide sequence is (SEQ ID NO: 86; CAN-ZIL9_VH)), The nucleotide sequence is (SEQ ID NO: 86; CAN-ZIL9_VL); ZIL11 variable heavy chain (SEQ ID NO: 87; CAN-ZIL11_VH) (the corresponding nucleotide sequence is (SEQ ID NO: 88; CAN-ZIL11_VH)), ZIL11 variable light chain (SEQ ID NO: 89; CAN-ZIL11_VL) (the corresponding nucleotide sequence is (SEQ ID NO: 90; CAN-ZIL11_VL)); ZIL69 variable heavy chain (SEQ ID NO: 91; CAN-ZIL69_VH) (the corresponding nucleotide sequence is (SEQ ID NO: 92; ZIL69 variable light chain (SEQ ID NO: 93; CAN-ZIL69_VL) (the corresponding nucleotide sequence is (SEQ ID NO: 94; CAN-ZIL69_VL)); ZIL94 variable heavy chain (SEQ ID NO: 95; CAN-ZIL94_VH) (the corresponding nucleotide sequence is (SEQ ID NO: 96; CAN-ZIL94_VH)); ZIL94 variable light chain (SEQ ID NO: 97; CAN-ZIL94_VL) (the corresponding nucleotide sequence is (SEQ ID NO: 98; CAN-ZIL94_VL)). ZIL154 variable heavy chain (SEQ ID NO: 99; CAN-ZIL154_VH) (the corresponding nucleotide sequence is (SEQ ID NO: 100; CAN-ZIL154_VH)), ZIL154 variable light chain (SEQ ID NO: 101; CAN-ZIL154_VL) (the corresponding nucleotide sequence is (SEQ ID NO: 102; CAN-ZIL154_VL)); ZIL159 variable heavy chain (SEQ ID NO: 103; CAN-ZIL159_VH) (the corresponding nucleotide sequence is (SEQ ID NO: 104; CAN-ZIL159_VH)), ZIL154 variable light chain (SEQ ID NO: 105; CAN-ZIL154_VL) (the corresponding nucleotide sequence is (SEQ ID NO: 106; CAN-ZIL159_VH)), ZIL154 variable heavy chain (SEQ ID NO: 107; CAN-ZIL159_VH) (the corresponding nucleotide sequence is (SEQ ID NO: 108; CAN-ZIL159_VH)), ZIL154 variable light chain (SEQ ID NO: 109; CAN-ZIL159_VH) (the corresponding nucleotide sequence is (SEQ ID NO: 110; CAN-ZIL159_VH)), ZIL154 variable heavy chain (SEQ ID NO: 111; CAN-ZIL154_VL) (the corresponding nucleotide sequence is (SEQ ID NO: 112; CAN-ZIL154_VL)), ZIL159 variable heavy chain (SEQ ID NO: 113; CAN-ZIL159_VH) (the corresponding nucleotide sequence is (SEQ ID NO: 114; CAN-ZIL159_VH)), ZIL154 variable light chain (SEQ ID NO: 1 IL159 variable light chain (SEQ ID NO: 105; CAN-ZIL159_VL) (the corresponding nucleotide sequence is (SEQ ID NO: 106; CAN-ZIL159_VL)); ZIL171 variable heavy chain (SEQ ID NO: 107; CAN-ZIL171_VH) (the corresponding nucleotide sequence is (SEQ ID NO: 108; CAN-ZIL171_VH)); ZIL171 variable light chain (SEQ ID NO: 109; CAN-ZIL171_VL) (the corresponding nucleotide sequence is (SEQ ID NO: 110; CAN-ZIL171_VL)).

[0462] The nine monoclonal antibodies selected for further characterization may be referred to elsewhere in this specification, in the figures, or in the claims as ZIL1, ZIL8, ZIL8, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, and ZIL171.

[0463] 1.7. Construction of Recombinant Chimeric and Whole Canine Antibodies Antibody variable domains are responsible for antigen binding. Grafting complete variable domains onto the respective constant regions is expected to have little or no effect on the antibody's ability to bind to the IL-31 immunogen. To simultaneously confirm the identification of the correct heavy and light chain variable region sequences and generate homogeneous material, expression vectors were designed to generate recombinant chimeric or fully canine antibodies in a mammalian expression system. The chimeric antibodies described here consist of variable sequences (both CDRs and framework sequences) from a host species antibody grafted onto the respective heavy and light chain constant regions of a feline or canine IgG molecule (e.g., a mouse variable sequence:canine constant region is referred to as a mouse:canine chimera). The fully canine antibodies described here consist of variable sequences (both CDRs and framework sequences) from a host species antibody (canine) grafted onto the respective heavy and light chain constant regions of a canine IgG molecule. Synthetic DNA sequences were constructed for the variable heavy (VH) and variable light (VL) chain sequences of selected antibodies. These sequences contain unique restriction endonuclease sites, a Kozak consensus sequence, and an N-terminal secretory leader to facilitate expression and secretion of recombinant antibodies from mammalian cell lines.

[0464] For the mouse:cat chimeric type, each variable region was derived from a mammalian gene containing either a feline IgG heavy chain constant region (SEQ ID NO: 173; cat_HC_allele A_1) (the corresponding nucleotide sequence is (SEQ ID NO: 174; cat_HC_allele A_1)) or a light chain constant region (SEQ ID NO: 175; cat_LC_kappa_G_minus) (the corresponding nucleotide sequence is (SEQ ID NO: 176; cat_LC_kappa_G_minus)). For mouse:canine chimeric or fully expressed canine antibodies, the respective mouse or canine variable regions were cloned into mammalian expression plasmids containing either the canine IgG heavy chain constant region (SEQ ID NO: 177: canine_HC_65_1) (the corresponding nucleotide sequence is (SEQ ID NO: 178; canine_HC_65_1)) or the light chain constant region (SEQ ID NO: 179; canine_LC_kappa) (the corresponding nucleotide sequence is (SEQ ID NO: 180; canine_LC_kappa)). Plasmids encoding each heavy and light chain under the control of the CMV promoter were co-transfected into HEK293 cells using standard methods. After 6 days of expression, chimeric mAbs were purified from 50 ml of transiently transfected HEK293FS cell supernatant using MabSelect Sure Protein A resin (GE Healthcare, Uppsala, Sweden) according to standard methods for protein purification. The eluted fractions were pooled and dialyzed overnight at 4°C in 1x PBS, pH 7.2, up to approximately 500 µl using 10,000 nominal MW cutoff Nanosep Omega centrifugal devices (Pall Corp., Port Washington, NY) and stored at 4°C for further use. The affinity and cell-based potency of selected recombinant antibodies are described below.

[0465] Figure 2 details the affinities of antibodies with CDRs from murine origin using biacore. Figure 2a shows the affinities of the murine anti-IL-31 antibodies 11E12 and 15H05, as well as the corresponding affinities of their chimeric forms for both feline and canine IL-31 surfaces. These observations confirm the correct sequence of both murine antibodies and demonstrate that conversion to chimeric forms yields antibodies with similar or even better affinities compared to the parental murine antibodies, with the exception of the mouse:feline 15H05 chimeric form, which lost some affinity for both IL-31 species upon conversion to the chimeric form.

[0466] The fully murine and chimeric forms of antibodies 11E12 and 15H05 were also tested for activity in the canine and feline cell assays described in Section 1.5. Figure 3 shows the results of these assays. Murine antibodies 11E12 and 15H05 were tested for activity against canine and feline cell types using both canine and feline IL-31 to stimulate signaling. The potency of both murine antibodies was comparable against both canine and feline cells using the feline cytokine, with the exception of 15H05 against feline IL-31 in feline FCWF4 cells, which showed a slightly increased IC50. Murine 15H05 was able to block canine IL-31 signaling in both feline and canine cells and was slightly more potent in the canine assay. These results indicate that the respective epitopes recognized by these antibodies are present on both canine and feline IL-31 and that binding of these antibodies is capable of neutralizing receptor-mediated cell signaling in relevant cell lines from both species.

[0467] Figure 3 also describes the potency of select chimeras in both cell assays. In the feline potency assay, converting the feline antibody to feline and canine chimeric forms had minimal effect on potency against feline IL-31 (IC50 range, 1.15–3.45 μg / ml). Similar results were observed when these chimeric forms were tested against feline IL-31 signaling in the canine DH82 cell line, with a slight increase in potency (IC50 = 0.71 μg / ml) observed for the 15H05 mouse:canine chimeric form. Overall, IC50 values ​​against canine IL-31 increased in both canine and feline cell types. The mouse:cat 15H05 chimeric form was slightly less potent than the mouse:canine form in this assay format (IC50 12.49 μg / ml vs. 28.61 μg / ml). Consistent with the observations with the murine antibody, conversion to canine and feline chimeric forms showed minimal change in potency.

[0468] Antibodies identified from single B cells of the immunized dogs described above were constructed as recombinant IgG proteins after identifying their variable domain sequences. These variable domains were grafted onto a canine heavy chain Fc (65_1 isotype), resulting in the generation of recombinant, complete canine antibodies. Our interest was in identifying additional canine antibodies that bind to wild-type feline IL-31 and which canine antibodies show reduced binding to the feline IL-31 15H05 mutant (i.e., targeting the 15H05 epitope). These antibodies obtained from this alternative source (canine vs. mouse) recognize the 15H05 epitope, thus providing additional paratopes (portions of antibodies that recognize the IL-31 protein, including CDRs) to increase the diversity of antibodies with different physical properties from which to select.

[0469] Figure 4 shows the results obtained by binding these recombinant canine antibodies to various proteins using both ELISA and Biacore methods. Antibody binding to wild-type and feline IL-31 15H05 mutant proteins was assessed using indirect ELISA. All nine canine monoclonal antibodies (ZIL1, ZIL8, ZIL8, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, and ZIL171) were able to bind to wild-type feline IL-31, and binding was affected by the mutations in the 15H05 epitope region of the mAb, confirming the binding phenotype determined during the initial screening used to identify them. On the other hand, the 11E12 antibody bound to wild-type feline IL-31, and its binding was not affected by the mutations in the 15H05 epitope region, as supported by the data in Figure 4. To confirm binding, biacore analysis was performed using canine, feline, equine, human, feline 15H05 mutant, and feline 11E12 mutant IL-31 proteins as surfaces and a single test concentration of antibody. Similar to the ELISA observations, all tested antibodies bound to wild-type feline IL-31. Consistent with the data described earlier in this section, both murine antibodies 11E12 and 15H05 bound to canine and feline IL-31 surfaces. Three additional antibodies, ZIL69 (partial canine binding), ZIL94, and ZIL159, were shown to have this dual-binding property. From this group of nine fully canine antibodies, only ZIL1 and ZIL9 cross-reacted with equine IL-31. Notably, antibody 15H05 was the only antibody among all antibodies assayed herein that bound to canine, feline, and equine IL-31, indicating some level of epitope conservation across the three species. In contrast, none of the antibodies described herein bind to human IL-31. Additional biacore surfaces were used to verify the ELISA observations showing differential binding of the antibodies to wild-type feline IL-31 and two proteins with mutations in the 15H05 epitope (15H05 variant) or the 11E12 epitope (11E12 variant).As expected, the control mouse antibody 11E12 bound to the 15H05 IL-31 variant but not to the 11E12 IL-31 variant due to the epitope mutation. Similarly, mouse 15H05 did not bind to the 15H05 variant but retained binding to the 11E12 IL-31 variant, further distinguishing the distinct epitopes recognized by these two antibodies. Consistent with the ELISA results, all intact canine antibodies were affected by the 15H05 mutation, except for ZIL94, ZIL154, and ZIL171 (which were partially affected). The discrepancy in results is likely due to differences in the methodology of the two assays. In addition, the binding of three antibodies was also shown to be affected by the 11E12 variant (ZIL1 (partially affected), ZIL8, and ZIL159). These results indicate that the epitopes recognized by these antibodies are affected by changes in both regions of the IL-31 protein. Taken together, these results support the characterization of nine antibodies derived from canine B cells that share binding with the region on the feline IL-31 protein recognized by antibody 15H05.

[0470] 1.8. Fetinization of Murine 11E12 and 15H05 Antibodies and Optimization of Binding Affinity The generation of anti-drug antibodies (ADA) is a major risk factor for any biotherapeutic protein, including monoclonal antibodies. This may be associated with a loss of antibody efficacy. A comprehensive literature review has shown that speciation of monoclonal antibodies can reduce the tendency of mAbs to become immunogenic, although examples of immunogenic fully human mAbs and non-immunogenic chimeric mAbs can also be found. To help mitigate the risks associated with ADA formation for the anti-IL-31 monoclonal antibodies provided herein, a felinization strategy was employed. This strategy is based on identifying the most suitable feline germline antibody sequences for CDR grafting. After extensive analysis of all available feline germline sequences for both the variable heavy and light chains, germline candidates were selected based on their homology to mouse mAbs, and CDRs from mouse mAb precursor cells were used to replace the native cat CDRs. The goal was to retain high affinity and cell-based activity using the feline antibody framework and minimize the potential for in vivo immunogenicity. The felinized mAbs were expressed, and their affinity and potency for feline IL-31 were characterized in cell-based assays. When felinized antibodies lost their ability to bind to IL-31, systematic investigation was performed to identify 1) the chain responsible for the loss of function, 2) the framework responsible for the loss of function, and 3) the amino acid(s) responsible for the loss of function.

[0471] Synthetic nucleotide constructs corresponding to the felineized variable heavy and light chains of mAb 11E12 and 15H05 were generated. Each variable chain was subcloned into a plasmid containing the respective feline heavy or kappa constant region, and the plasmids were then co-transfected into HEK293 cells for antibody expression. Initial attempts at felinization of antibody 11E12 focused on utilizing a single feline VH framework (SEQ ID NO: 111; FEL_11E12_VH1) (the corresponding nucleotide sequence is (SEQ ID NO: 112; FEL_11E12_VH1)) paired separately with the VL framework (SEQ ID NO: 113; FEL_11E12_VL1) (the corresponding nucleotide sequence is (SEQ ID NO: 114; FEL_11E12_VL1)) and (SEQ ID NO: 115; FEL_11E12_VL2) (the corresponding nucleotide sequence is (SEQ ID NO: 116; FEL_11E12_VL2)), to form feline 11E12 1.1 and feline 11E12 1.2, respectively. This attempted speciation resulted in a loss of affinity of feline 11E12 1.1 for both feline and canine IL-31 proteins compared to the murine form of the antibody, and a complete loss of binding for feline 11E12 1.2 mAb (Figure 2b). The potency of these speciation antibodies was tested in canine DH82 and feline FCWF4 cell assays using feline IL-31 cytokine. The potency of felineized 11E12 1.1 for feline IL-31 was reduced approximately two-fold in the feline FCWF assay compared to the murine version of the antibody. Consistent with the loss of affinity in felineized 11E12 1.2, a complete loss of cellular potency was observed for this antibody (Figure 3). Based on previous experience during caninization of the mAb 11E12 orthologue, a similar strategy was employed to attempt to restore the loss of affinity upon felineization (Bammert, 2014). (U.S. Patent No. 8,790,651 to Muller et al.). The feline framework 2 (FW2) region of feline 11E12 VL1 was replaced with murine FW2 from (SEQ ID NO: 73; Mu_11E12_VL) (the corresponding nucleotide sequence is (SEQ ID NO: 74; Mu_11E12_VL)) to generate feline 11E12 VL1 FW2. In addition, a single substitution (K46Q) at position 46 of feline VL was made to generate (SEQ ID NO: 119; FEL_11E12_VL1_K46Q) (the corresponding nucleotide sequence is (SEQ ID NO: 120; FEL_11E12_VL1_K46Q)). Pairing of the above VL with Fel_11E12_VH1 yielded feline 11E12 1.1 FW2 and feline 11E12 1.1 K46Q, respectively. The FW2 modifications restored the affinity of feline 11E12 1.1 FW2 for the feline IL-31 protein, resulting in a KD comparable to that of the murine and chimeric forms (Figures 2A and 2B). However, these modifications had a detrimental effect on the affinity of feline 11E12 1.1 FW2 for the canine IL-31 protein, indicating distinct differences in the nature of the binding ability of antibody 11E12 to this epitope on the feline and canine cytokine. The single amino acid substitution 11E12 1.1 K46Q did not affect the affinity of this antibody. The increased affinity of antibody 11E12 1.1 FW2 for feline IL-31 protein resulted in increased potency against the feline cytokine in the canine DH82 assay (Figure 3).

[0472] Fetinization efforts on the murine antibody 15H05 focused on combining three feline VH and three feline VL frameworks in a total of nine felineized mAbs. FEL_15H05_VH1 (SEQ ID NO: 121; FEL_15H05_VH1) (the corresponding nucleotide sequence is (SEQ ID NO: 122; FEL_15H05_VH1)) was combined with (SEQ ID NO: 127; FEL_15H05_VL1) (the corresponding nucleotide sequence is (SEQ ID NO: 128; FEL_15H05_VL1)), (SEQ ID NO: 129; FEL_15H05_VL2) (the corresponding nucleotide sequence is (SEQ ID NO: 130; FEL_15H05_VL2)), and (SEQ ID NO: 131; FEL_15H05_VL3) (the corresponding nucleotide sequence is (SEQ ID NO: 132; FEL_15H05_VL3)) and used to generate Cat15H05 1.1, Cat15H05 1.2, and Cat15H05 1.3, respectively. FEL_15H05_VH2 (SEQ ID NO: 123; FEL_15H05_VH2) (the corresponding nucleotide sequence is (SEQ ID NO: 124; FEL_15H05_VH2)) was combined with (SEQ ID NO: 127; FEL_15H05_VL1) (the corresponding nucleotide sequence is (SEQ ID NO: 128; FEL_15H05_VL1)), (SEQ ID NO: 129; FEL_15H05_VL2) (the corresponding nucleotide sequence is (SEQ ID NO: 130; FEL_15H05_VL2)), and (SEQ ID NO: 131; FEL_15H05_VL3) (the corresponding nucleotide sequence is (SEQ ID NO: 132; FEL_15H05_VL3)) and used to generate feline 15H05 2.1, feline 15H05 2.2, and feline 15H05 2.3, respectively.FEL_15H05_VH3 (SEQ ID NO: 125; FEL_15H05_VH3) (the corresponding nucleotide sequence is (SEQ ID NO: 126; FEL_15H05_VH3)) was combined with (SEQ ID NO: 127; FEL_15H05_VL1) (the corresponding nucleotide sequence is (SEQ ID NO: 128; FEL_15H05_VL1)), (SEQ ID NO: 129; FEL_15H05_VL2) (the corresponding nucleotide sequence is (SEQ ID NO: 130; FEL_15H05_VL2)), and (SEQ ID NO: 131; FEL_15H05_VL3) (the corresponding nucleotide sequence is (SEQ ID NO: 132; FEL_15H05_VL3)) and used to generate Cat15H05 3.1, Cat15H05 3.2, and Cat15H05 3.3, respectively. Similar to the observations with antibody 11E12, initial attempts at felinization of antibody 15H05 resulted in a loss of affinity for feline IL-31 protein compared to mouse 15H05 and a neutral effect compared to the 15H05 mouse-cat chimeric form (Figures 2A and 2C). Similar to the observations with the binding of felinized antibody 11E12 to canine IL-31, certain VH and VL framework combinations of feline 15H05 had a neutral to positive effect on the affinity for canine IL-31 (Figure 2C: see feline 15H05 1.1, 2.2, and 3.2).

[0473] In an attempt to restore the affinity of felinized antibody 15H05, each felinized 15H05 VH was paired with a murine 15H05 VL to generate a heterochimeric antibody. FEL_15H05_VH1 (SEQ ID NO: 121; FEL_15H05_VH1) (the corresponding nucleotide sequence is (SEQ ID NO: 122; FEL_15H05_VH1)) was combined with MU_15H05_VL (SEQ ID NO: 69; MU_15H05_VL) (the corresponding nucleotide sequence is (SEQ ID NO: 70; MU_15H05_VL)) to generate the felinized 15H05 VH1 murine VL. FEL_15H05_VH2 (SEQ ID NO: 123; FEL_15H05_VH2) (the corresponding nucleotide sequence is (SEQ ID NO: 124; FEL_15H05_VH2)) was aliquoted into MU_15H05_VL (SEQ ID NO: 69; MU_15H FEL_15H05_VH3 (SEQ ID NO: 125; FEL_15H05_VH3) (the corresponding nucleotide sequence is (SEQ ID NO: 126; FEL_15H05_VH3)) was combined with MU_15H05_VL (SEQ ID NO: 69; MU_15H05_VL) (the corresponding nucleotide sequence is (SEQ ID NO: 70; MU_15H05_VL)) to generate the feline 15H05 VH2 mouse VL. FEL_15H05_VH3 (SEQ ID NO: 125; FEL_15H05_VH3) (the corresponding nucleotide sequence is (SEQ ID NO: 126; FEL_15H05_VH3)) was combined with MU_15H05_VL (SEQ ID NO: 69; MU_15H05_VL) (the corresponding nucleotide sequence is (SEQ ID NO: 70; MU_15H05_VL)) to generate the feline 15H05 VH3 mouse VL. These felineized VH mouse VL heterochimeras were analyzed for their affinity to canine and feline IL-31. Pairing felineized 15H05 VH1 and VH3 with murine 15H05 VL restored affinity for feline IL-31 to levels comparable to or better than those of the murine and chimeric forms, and this trend toward improved affinity was also observed for the canine IL-31 protein (Figures 2A and 2C).

[0474] To further investigate the location of the 15H05 framework responsible for the loss of affinity, a single feline VH of 15H05 (FEL_15H05_VH1) was used to compare the affinity of mouse 15H05 with that of mouse 15H05. FEL_15H05_VH1 (SEQ ID NO: 122; FEL_15H05_VH1) (the corresponding nucleotide sequence is (SEQ ID NO: 123; FEL_15H05_VH1)) was paired with individual framework substitutions from VL. FEL_15H05_VL1_FW1 (SEQ ID NO: 133; FEL_15H05_VL1_FW1) (the corresponding nucleotide sequence is (SEQ ID NO: 134; FEL_15H05_VL1_FW1)), FEL_15H05_VL1_FW2 (SEQ ID NO: 135; FEL_15H05_VL1_FW2) (the corresponding nucleotide sequence is (SEQ ID NO: 136; FEL_15H05_VL1_FW2)), and FEL_15H05_VL1_FW3 (SEQ ID NO: 137; FEL_15H05_VL1_FW3) (the corresponding nucleotide sequence is (SEQ ID NO: 138; FEL_15H05_VL1_FW3)) were combined separately to generate feline 15H05 1.1 FW1, feline 15H05 1.1 FW2, and feline 15H05 1.1 FW3, respectively. Substitution of FW1 was detrimental to affinity for both feline and canine IL-31, but substitution of mouse FW2 or FW3 for feline 15H05 1.1 achieved excellent affinity for canine and feline IL-31, with FW2 being superior in both species (Figure 2C). Additional pairwise framework substitutions were performed to quantify the degree of affinity modulation by this approach. FEL_15H05_VH1 (SEQ ID NO: 121; FEL_15H05_VH1) (the corresponding nucleotide sequence is (SEQ ID NO: 122; FEL_15H05_VH1)) was used in combination with FEL_15H05_VL1_FW1_2 (SEQ ID NO: 139; FEL_15H05_VL1_FW1_FW2) (the corresponding nucleotide sequence is (SEQ ID NO: 140; FEL_15H05_VL1_FW1_FW2)), FEL_15H05_VL1_FW2_3 (sequence 1.1 FW1_2, feline 15H05 1.1 FW2_3, and feline 15H05 1.1 FW1_3, respectively. Interestingly, substitution of mouse FW1 alone was detrimental to affinity, whereas combination of FW1 with FW2 or FW3 resulted in good affinity for both feline and canine IL-31 (Figure 2C).

[0475] Finally, an attempt was made to minimize the number of feline framework backmutations from the most promising felineized VH and VL sequence combinations. To this end, FEL_15H05_VH1 (SEQ ID NO: 121; FEL_15H05_VH1) (the corresponding nucleotide sequence is (SEQ ID NO: 122; FEL_15H05_VH1)) was combined with FEL_15H05_VL1 _FW2_K42N (SEQ ID NO: 145; FEL_15H05_VL1_FW2_K42N) (the corresponding nucleotide sequence is (SEQ ID NO: 146; FEL_15H05_VL1_FW2_K42N)), FEL_15H05_VL1_FW2_V43I (SEQ ID NO: 147; FEL_15H05_VL1_FW2_V43I) (the corresponding nucleotide sequence is (SEQ ID NO: 148; FEL_15H05_VL1_FW2_V43I)), FEL_15H05_VL1_FW2_L46V (SEQ ID NO: 149; FEL_15H05_VL1_FW2_L46V) (the corresponding nucleotide sequence is (SEQ ID NO: 150 ;FEL_15H05_VL1_FW2_L46V), FEL_15H05_VL1_FW2_Y49N (SEQ ID NO: 151; FEL_15H05_VL1_FW2_Y49N) (the corresponding nucleotide sequence is (SEQ ID NO: 152; FEL_15H05_VL1_FW2_Y49N)), and FEL_15H05_VL1_FW2_K42N_V43I (SEQ ID NO: 153; FEL_15H05_VL1_FW2_K42N_V43I) (the corresponding nucleotide sequence is (SEQ ID NO: 154; FEL_15H05_VL1_FW2_K42N_V43I)), We generated the following antibodies: 1.1 K42N, feline 15H05 1.1 V43I, feline 15H05 1.1 L46V, feline 15H05 1.1 Y49N, and feline 15H05 1.1 K42N_V43I. Substitution of the entire mouse FW2 framework into felineized 15H05 VL1 resulted in an antibody with excellent affinity for canine and feline IL-31 (Figure 2C, feline 15H05 1.1 FW2), whereas individual backmutations of FW2 amino acid residues had neutral or detrimental effects. This indicates that all four substitutions are necessary to maintain an optimal tertiary structure for positioning the CDRs at the IL-31 epitope. Because felineized 15H05 1.1 FW2 exhibited increased affinity for both feline and canine IL-31, this antibody was selected for further study.

[0476] Figure 5A shows an alignment of the murine antibody 11E12 VL sequence, comparing the caninized 11E12 sequence mentioned above with the felineized version. Dots below the alignment indicate the location of the relevant changes in Fel_11E12_VL1 that were required to restore this antibody's affinity for the IL-31 protein. Similarly, Figure 5B shows the changes required for the felineized 15H05 VL (Fel_15H05_VL1) that were required to not only restore but also improve affinity for canine and feline IL-31 compared to the murine and chimeric forms of this antibody.

[0477] 1.9. Generation of cell lines expressing felinized anti-IL-31 antibodies from a glutamine synthase (GS) plasmid Felineized 15H05 1.1 FW2 was selected as a candidate for generating a stable cell line homogeneously supplying antibody for further characterization. Genes encoding the felineized heavy and light chains for cell line production were cloned into GS plasmids pEE 6.4 and pEE 12.4 (Lonza, Basel, Switzerland), respectively. The resulting plasmids were digested and ligated together to form a single mammalian expression plasmid according to the manufacturer's protocol. In ZTS-927, the heavy chain is a combination of (SEQ ID NO: 121; FEL_15H05_VH1) (the corresponding nucleotide sequence is (SEQ ID NO: 122; FEL_15H05_VH1)) and the feline IgG heavy chain constant region (SEQ ID NO: 171; feline_HC_allele A_wt) (the corresponding nucleotide sequence is (SEQ ID NO: 172; feline_HC_allele A_wt)). In ZTS-927, the light chain is a combination of (SEQ ID NO: 135; FEL-15H05-VL1_FW2) (the corresponding nucleotide sequence is (SEQ ID NO: 136; FEL-15H05-VL1_FW2)) and a feline IgG light chain constant region (SEQ ID NO: 175; feline_LC_kappa_G_minus) (the corresponding nucleotide sequence is (SEQ ID NO: 176; feline_LC_kappa_G_minus)). In ZTS-361, the heavy chain is a combination of (SEQ ID NO: 121; FEL_15H05_VH1) (the corresponding nucleotide sequence is (SEQ ID NO: 12 In ZTS-361, the light chain is a combination of (SEQ ID NO: 135; FEL-15H05-VL1_FW2) (the corresponding nucleotide sequence is (SEQ ID NO: 136; FEL-15H05-VL1_FW2)) and a feline IgG light chain constant region (SEQ ID NO: 175; cat_LC_kappa_G_minus) (the corresponding nucleotide sequence is (SEQ ID NO: 176; cat_LC_kappa_G_minus)). In ZTS-1505, the heavy chain is a combination of (SEQ ID NO: 121; FEL_15H05_VH1) (the corresponding nucleotide sequence is (SEQ ID NO: 122; FEL_15H05_VH1)) and a feline IgG heavy chain constant region (SEQ ID NO: 173; feline_HC_allele A_1) (the corresponding nucleotide sequence is (SEQ ID NO: 174; feline_HC_allele A_1)). In ZTS-1505, the light chain is a combination of (SEQ ID NO: 135; FEL-15H05-VL1_FW2) (the corresponding nucleotide sequence is (SEQ ID NO: 136; FEL-15H05-VL1_FW2)) and a feline IgG light chain constant region (SEQ ID NO: 186; feline_LC_kappa_G_minus_QRE_minus) (the corresponding nucleotide sequence is (SEQ ID NO: 187; feline_LC_kappa_G_minus_QRE_minus)). Affinity and efficacy data for ZTS-1505 are provided below in section 1.18 of this Examples section.

[0478] To demonstrate transient antibody production, each plasmid was used to transfect HEK293 cells, and expression was carried out in cultures of various sizes. Following standard protein purification methods, protein was isolated from conditioned HEK medium using protein A affinity chromatography. The medium was loaded onto the chromatography resin and eluted by pH shift. The eluted protein was pH adjusted, dialyzed, and sterile filtered before use. ZTS-361 was then used to evaluate its in vivo efficacy in the cat pruritus model.

[0479] The affinity and potency of antibodies ZTS-927 and ZTS-361, produced from a single GS plasmid, were tested. Figure 2D shows the results of affinity evaluation of these antibodies using biacore. The affinity of ZTS-927 and ZTS-361 for feline IL-31 is highly consistent with that of the murine and chimeric forms of the precursor cell mouse mAb 15H05. The potency of these two antibodies for canine and feline IL-31 was quantified using both canine and feline cell assays (Figure 3). Consistent with previous observations, IC50 values ​​were proportionally higher when the canine form of IL-31 was used on both cell types. The IC50 values ​​of ZTS-927 and ZTS-361 for feline IL-31 were also highly consistent with those derived from the chimeric and murine forms of the antibody, indicating that the final felineized version of mAb 15H05, produced from a single GS plasmid, is suitable for cell line development.

[0480] To generate stable cell lines producing candidate antibodies, the GS plasmid was linearized and then transfected with the restriction enzyme PvuI, which cuts at a single site within the plasmid backbone. GS-CHOK1SV (clone 144E12) cells were transfected with the linearized plasmid DNA via electroporation. After transfection, cells were plated in 48-well plates (48WP) to generate stable pools. Once pools reached at least 50% confluence in 48WP, 100 μl of supernatant was analyzed for IgG expression using a ForteBio Octet and Protein A biosensor (Pall ForteBio, Fremont, CA). The best-expressing clones were scaled up to 6-well plates (6WP) and then to 125 mL shake flasks (SF). Once cells were adapted to suspension culture in 125 mL flasks, two vials of each cell line pool were banked for LN storage. Because cell line production must be clonal, the top three high-expressing pools were subcloned by limiting dilution in 96-well culture plates. To avoid two rounds of limiting dilution, 96-well plates were imaged using a Molecular Devices Clone-Select Imager (CSI) (Molecular Devices LLC, San Jose, CA), which captures images of single cells and their subsequent growth. Clones were selected based on successful CSI imaging, growth, and production in 96WP.

[0481] To assess cell culture growth and productivity, the top expressing pool was further evaluated in a 14-day fed-batch in 125 mL SF. Cells were seeded on platform medium and a feed consisting of Life Technologies' CD CHO plus four amino acids, the proprietary feed CDF v6.2, and 10% glucose. After 14 days of fed-batch, the pool was centrifuged and the CD CHO-produced mABs were isolated by filtering the supernatant through a 0.20 μm polyethersulfone (PES) membrane, followed by purification.

[0482] A typical purification consisted of loading 2 liters of conditioned medium (from CHO cell culture, 0.2 μm filtered) onto a 235 mL column of MabSelect (GE healthcare, catalog number 17-5199-02). The column was pre-equilibrated with PBS. The sample was loaded with a residence time of >2.5 min. After loading, the column was washed again with PBS and then with 25 mM sodium acetate (near-neutral pH). The column was eluted with 25 mM acetic acid, pH 3.6, and then stripped with 250 mM acetic acid, 250 mM sodium chloride, pH ∼2.2. Fractions (50 mL) were collected during the elution and stripping steps. UV absorbance at A280 was monitored throughout. Peak fractions were pooled, the pH adjusted to ∼5.5 by adding 20 mM sodium acetate, and dialyzed against three changes of buffer. The dialysate was collected, sterile filtered, and stored at 4 °C.

[0483] 1.10. Identification of the epitope of IL-31 recognized by antibody 15H05 Knowledge of the epitope of IL-31 recognized by an antibody is essential for understanding the mechanism by which the antibody neutralizes cytokine binding to the IL-31Ra:OSMR co-receptor. Additionally, knowledge of the epitope allows (but is not limited to) optimization of antibody binding affinity and the design of peptide epitope mimetics (mimotopes), which may be highly useful as analytical capture reagents and subunit vaccines to elicit relevant, focused immune responses. Using a multistep process using CLIPS (Chemical Linkage of Peptides onto Scaffolds) technology (Timmerman et al. J Mol Recognit. 2007;20(5):283-299), we identified and optimized peptides capable of binding to the paratope of mAb 15H05 (Pepscan, Lelystad, Netherlands). Given the high affinity of mAb 15H05 for canine and feline IL-31 proteins (Figure 2, MU-15H05), the primary sequences of both IL-31 species were considered relevant for this study. A peptide microarray library corresponding to canine IL-31 proteins was constructed and used to identify peptides capable of binding to mAb 15H05 using indirect ELISA. After identifying peptides whose primary amino acid sequences corresponded to the mAb 15H05-binding region on IL-31, a focused full substitution analysis was performed, using peptides corresponding to a segment of IL-31 and replacing each of the 12 amino acids in this mAb 15H05-binding region with 19 other possible amino acid residues at each position. This analysis was essential for identifying key amino acid residues on IL-31 involved in mAb 15H05 binding and also demonstrated substitution positions on the canine primary sequence that led to enhanced antibody binding.

[0484] The amino acids on the canine IL-31 protein recognized by antibody 11E12 have been previously described (U.S. Patent No. 8,790,651 to Bammert, et al. No. 11E12). This paper described a mutational analysis of the canine IL-31 protein that revealed positions on the protein that, when converted to alanine, affected the binding of mAb 11E12. Based on the complete substitution analysis described above for mAb 15H05 and previous knowledge of the binding epitope of 11E12, mutant forms of the feline IL-31 protein were created by substituting two key residues in the epitopes recognized by each antibody with alanine (mutants described in section 1.2 above). Each epitope mutation was named according to the antibody that recognizes the site of the mutation (mutants 11E12 and 15H05 relative to the native wt protein sequence).

[0485] Figure 6A shows the alignment of wild-type feline IL-31 (SEQ ID NO: 157) with mutants 15H05 (SEQ ID NO: 163) and 11E12 (SEQ ID NO: 161), with the positions where alanine substitutions occur highlighted. IL-31 belongs to the IL-6 family of cytokines and has a four helix bundle up-down architecture (CATH). database, Dawson et al. 2017 Nucleic Acids Res. 2017 Jan 4;45 (Database issue):D289-D295). The homology model was generated using MOE software (Chemical Computing Group, Montreal, QC, Canada) based on the human IL-6 structure 1P9M (Boulanger et al. 2003 Science. Jun 27;300(5628):2101-4). Figure 6B shows the feline IL-31 homology model, highlighting the amino acid positions involved in the binding of antibodies 11E12 (site 1) and 15H05 (site 2). The binding sites of each antibody appear to be located at distinct positions on the IL-31 protein.

[0486] To quantify the effect of mAbs 11E12 and 15H05 on their ability to bind these mutant forms of feline IL-31, an indirect ELISA was performed using the mutants coated directly onto immunoassay plates. Figure 6C shows the results of this ELISA, demonstrating that mAbs 11E12 and 15H05 are able to bind wild-type feline IL-31 in this assay format. When mutant 11E12 is used as the capture protein, binding of mAb 11E12 is greatly weakened, and binding of mAb 15H05 is partially weakened. Previous analysis of the 11E12 epitope on canine IL-31 (described in U.S. Patent No. 8,790,651 to Bammert et al.) showed that mutation of four amino acid residues to alanine affects mAb binding; therefore, in this case, mutation of two residues may not be sufficient to completely eliminate the high-affinity binding of mAb 11E12 using this ELISA format. The slightly reduced binding of mAb 15H05 to the 11E12 variant is likely due to a translational effect of the mutations, resulting in the displacement of the two front helices affecting the 15H05 binding site. Mutations designed to affect mAb 15H05 binding (mutant 15H05) completely abolished the ability of mAb 15H05 to bind to this IL-31 variant by ELISA. Unlike the 11E12 variant, altering the random coil recognized by mAb 15H05 (mutant 15H05) did not affect mAb 11E12 binding, further supporting the distinction between the two epitopes (Figure 6C).

[0487] 1.11. Competitive Binding Assessment of mAbs 15H05 and 11E12 Using Biacore To further characterize the IL-31 epitopes bound by mAbs 15H05 and 11E12, blocking experiments were performed using biacore to generate surfaces containing IL-31 protein, followed by sequential addition of the antibodies. Figure 7 shows the relative binding of each antibody to IL-31 after capture with 11E12 or 15H05. The column labeled HBS-EP (assay buffer) shows the maximum signal obtained from each antibody binding to the IL-31 surface alone, without competition. Figure 7A shows the competitive binding data for murine 15H05 and 11E12 antibodies with canine IL-31. These results clearly demonstrate the binding of antibodies 15H05 and 11E12 to canine IL-31. 1E12 were able to bind to canine IL-31 in the presence of each other, indicating that these antibodies recognized different epitopes on the protein. The sensorgrams associated with Figure 7A show that the dissociation rates of both antibodies on this newly formed biacore surface were very slow, so that addition of the same antibody could not result in additional occupancy of the binding sites (data not shown).

[0488] Figure 7B shows the competitive binding data of antibodies 15H05 and 11E12 on the feline IL-31 surface, again demonstrating no overlap in the epitopes they recognize. Binding of additional antibodies in the presence of the same antibody is a result of increased off-rates due to the lower quality of the surface used. The increased off-rates can be seen and compared to the KD values ​​from the newly formed feline IL-31 surface in Figure 2.

[0489] These results further support the epitope mapping data in section 1.10, which demonstrates that the CDRs contained in antibody MU-15H05 recognize a distinct epitope when compared to antibody MU-11E12. The epitope recognized by antibody 15H05 is distinct from that of antibody 11E12 described in (U.S. Patent No. 8,790,651 to Bammert, et al.) and represents a novel target on the IL-31 protein for neutralizing this cytokine's activity in multiple species. These findings highlight the distinct spatial relationship of the binding sites described in the feline IL-31 homology model (Figure 6B) and support the hypothesis that this face of the cytokine is essential for its interaction with the IL-31Ra:OSMR receptor complex.

[0490] 1.12. Synthesis and Characterization of Soluble Feline IL-31 Co-receptors (IL-31RA and OSMR) The human IL-31 heteromeric receptor co-complex, consisting of IL31Ra and OSMR subunits, is required for IL31-mediated intracellular activation of the JAK-STAT pathway and has been shown to be involved in atopic skin disease (Dillon et al. 2004 Nat. Immunol. Jul;5(7):752-60; Dreuw et al.2004 J Biol Chem.279:36112-36120; and Diveu et al.2004 Eur Cytokine Netw.15:291-302). The human IL-31 Ra subunit was subsequently described as the initial binding event that occurs when IL-31 contacts the cell surface receptor, and this event is a prerequisite for the recruitment of OSMR and the subsequent formation of a high-affinity coreceptor complex (Le Saux et al.2010 J Biol Chem.Jan 29;285(5):3470-7). Herein, we describe the evidence that feline IL-31 protein can bind to both OSMR and IL-31 Ra independently. This observation is novel and has important implications for our understanding of how IL-31 proteins interact with the IL-31Ra:OSMR co-receptor, and for the biological role of IL-31 as it interacts with individual subunits independently.

[0491] To be able to understand how IL-31 binds to its co-receptor and to characterize the inhibitory properties of the identified antibodies, two receptor forms were synthesized. The individual IL-31 receptor subunits, IL-31Ra (SEQ ID NO: 169; feline_IL31Ra_HIgG1_Fc_X1_Fn3) (the corresponding nucleotide sequence is (SEQ ID NO: 170; feline_IL31Ra_HIgG1_Fc_X1_Fn3)) and OSMR-(SEQ ID NO: 167; feline_OSMR_hIgG1_Fc) (the corresponding nucleotide sequence is (SEQ ID NO: 168; feline_OSMR_hIgG1_Fc)), were both constructed as human IgG1 Fc fusions. Homology with the human homologues identified the cytokine-binding, fibronectin III, and Ig-like domains. To evaluate the individual receptor subunits, the extracellular domains (and predicted nucleotide sequences) of OSMR and IL-31Ra were analyzed. The N-terminal proximal fibronectin III domain (associated with the N-terminal proximal fibronectin III domain) was produced as a human IgG1 Fc fusion, all with their native signal peptides. All synthetic cassettes were cloned into pcDNA3.1, expressed in the ExpiCHO system, and purified as described above.

[0492] To analyze the binding ability of these receptor forms to wild-type and mutant IL-31 proteins, indirect ELISA was performed by coating 100 μl of each respective protein onto immulon 2HB plates (1 μg / ml) overnight in 4C carbonate / bicarbonate buffer (Sigma C3041-100CAP). The ELISA plates were then blocked with 5% NFDM blocking buffer in PBST for 1 hour at room temperature, and then multiple concentrations of each receptor construct were allowed to bind for 1 hour at room temperature. After washing with PBST, the presence of bound receptor (Fc fusion) was identified using mouse anti-human IgG1 (Lifetech A10684, 1:500 dilution) for 1 hour at room temperature. The wells were washed again with PBST and developed with KPL sureblue 3,3',5,5'-tetramethylbenzidine (TMB) microwell substrate. Figure 8 shows the results of this indirect ELISA using wild-type and mutant forms of feline IL-31 proteins as capture agents. These data demonstrate that wild-type feline IL-31 can bind independently to the IL-31Ra and OSMR receptor subunits. These observations contrast with previous reports showing that the IL-31 protein first binds to the IL-31Ra subunit and then recruits OSMR to that site. Because the biological role of IL-31 is still being determined, understanding the kinetics of receptor binding and the potential consequences of its compromised role in diseases such as atopic dermatitis is crucial. Therefore, these observations prompted further investigation into the characterization of antibodies binding to epitopes that could interfere with IL-31's ability to recognize IL-31Ra and OSMR.

[0493] Section 1.2 describes the weakened binding of antibodies 11E12 and 15H05 to mutant forms in which key amino acids within their binding sites were converted to alanine (mutants 11E12 and 15H05, respectively). Therefore, it was of great interest to understand the effect of these mutations on their ability to bind to the individual IL-31Ra and OSMR receptor subunits. Figure 8 shows that mutations in either the 11E12 or 15H05 binding sites completely abolish the ability to bind IL-31Ra and OSMR, indicating that both antibodies bind to epitopes required for the interaction of IL-31 with both receptor subunits. While the lack of binding could be due to altered IL-31 conformation resulting from the mutations, the fact that these mutant forms are still able to bind the antibodies suggests this is not the case. This key finding supports the ability of both antibodies 11E12 and 15H05 (and derivatives) to recognize epitopes on IL-31 that neutralize cytokine signaling through its co-receptors and block cellular association of the cytokine with either receptor during this process. These data support the identification of antibodies that can remove IL-31 from the circulation and render it unable to bind to cell surface or soluble receptor forms.

[0494] 1.13. In vivo evaluation of chimeric antibodies in a feline IL-31 pruritus challenge model The ability of an antibody to effectively neutralize its target can be assessed in vitro by examining binding to a relevant epitope on the target protein with appropriate affinity and potency in cell-based assays that allow extrapolation to in vivo efficacy. Above are the steps taken to characterize two series of antibodies generated from mouse progenitor cells, mAbs 11E12 and 15H05. In Section 1.7, we report the development of mAbs that have affinity for feline and canine IL-31 comparable to that of the original mouse monoclonal antibody. The generation of mouse:cat chimeric forms of 11E12 and 15H05 is described (Figure 2A). The mouse:cat chimeric forms of 11E12 and 15H05 also yielded comparable IC50 values ​​for the inhibition of feline IL-31-induced pSTAT3 signaling in canine and feline macrophage cells (Figure 3). During the felinization process described in Section 1.8, mouse mAb 11E12 was converted to a felinized version (feline 11E12 1.1), which subsequently lost affinity for canine and feline IL-31 (Figure 3) and potency against feline IL-31 signaling in canine and feline cells (Figure 3). Before optimizing the felinized 11E12 and 15H05 antibodies described in Section 1.8, we were interested in understanding the ability of these preliminary felinized and chimeric forms to neutralize the pruritic activity of feline IL-31 in a feline challenge model. Of interest was understanding the pharmacodynamic effects of these different antibodies on neutralizing pruritus, and any correlation with affinity, cellular potency, or epitope recognition that may affect efficacy. By developing a range of cellular potencies that correlate with in vivo efficacy in pruritus challenge models, in vitro assays could be used to predict further necessary optimization.

[0495] A feline IL-31-induced pruritus model was developed to test the preliminary efficacy of mouse:feline 11E12 chimera, mouse:feline 15H05 chimera, and felineized 11E12 (feline 11E12 1.1). After intravenous administration of 0.5 μg / kg of feline IL-31 (SEQ ID NO: 159; feline_IL-31_E_coli) (the corresponding nucleotide sequence is (SEQ ID NO: 160; feline_IL-31_E_coli)), cats exhibited transient pruritic behaviors, including (but not limited to) licking, biting, scratching, and head or body shaking. Cage rubbing was not considered pruritic activity. Pruritus observations were conducted by trained researchers 30 minutes before and 1 hour after administration of IL-31 protein. In this study, a baseline challenge with feline IL-31 was performed up to 1 month before antibody administration. On day 0, a 0.5 mg / kg antibody dose was combined with 0.5 μg / kg feline IL-31 for 60 minutes at room temperature, after which the pre-bound complex was injected into each animal. Controls included a "no mAb" control. The mAb dose represented a total molar excess of antibody over cytokine. Pruritus activity was monitored as described on days 0, 7, and 21. The results in Figure 9 show that the pruritus scores of the mouse:cat 15H05 chimera were significantly improved (p<0.05) on days 0, 7, and 21 compared with the placebo control. The mouse:cat 11E12 chimera showed an initial trend toward efficacy on day 0 but did not achieve a significant reduction in pruritus at any time point compared with vehicle placebo. Because feline 11E12 1.1 did not reduce pruritus on day 0 and did not demonstrate a trend toward efficacy compared with vehicle placebo, an IL-31 challenge was not performed on days 7 and 21.

[0496] Taken together, these results demonstrate that the lack of efficacy of 11E12 1.1 in preventing IL-31-induced scratching in cats clearly demarcates the activity of these antibodies. The lack of in vivo efficacy likely resulted from a loss of affinity and potency for feline 11E12 1.1. The distinction becomes more subtle when comparing the efficacy results of the mouse:feline 11E12 chimeric and mouse:feline 15H05 chimeric forms. The K values ​​of the chimeric forms of both mAbs are comparable to their mouse precursor cells, demonstrating the slightly superior affinity of mouse:feline 11E12 for both feline and canine IL-31 (Figure 2A). However, this increased affinity does not directly translate to increased potency, as the IC50 of the mouse:feline 15H05 chimeric form for feline IL-31-induced pSTAT3 signaling in feline FCWF4 cells is approximately twofold higher than that of the mouse:feline 11E12 chimeric form (Figure 3). These data suggest that the way in which antibody 15H05 CDR recognizes feline IL-31 is superior in neutralizing the cytokine's ability to signal through its co-receptor, making it more effective in blocking pruritus in cats. The differences in IC50 observed in these cellular assays suggest that i This provides a promising tool for predicting in vivo efficacy and distinguishing subtle differences in epitope recognition within and between antibody series.

[0497] 1.14. In vivo evaluation of the efficacy of felineized 15H05 anti-IL-31 antibody in a feline pruritus challenge model Based on the positive efficacy results using the mouse:feline 15H05 chimera described above, further work was done to increase the affinity and potency of felineized 15H05 (described in section 1.8 above). Systematic substitution of the feline variable light chain framework within the feline 15H05 1.1 antibody led to the identification of feline 15H05 1.1 FW2, which has increased affinity for both feline and canine IL-31 compared to mouse 15H05 (Figure 2). Combining the heavy and light chains of feline 15H05 1.1 FW2 within a single plasmid resulted in the formation of the ZTS-927 and ZTS-361 antibodies following production from HEK and CHO expression systems. The affinity and potency of both antibodies resulting from expression from a single plasmid are also shown in Figures 2 and 3, respectively.

[0498] The efficacy of the fully feline anti-feline IL-31 mAb ZTS-361 was evaluated in an IL-31-induced in vivo cat model by assessing its ability to neutralize scratching behavior. Figure 10A shows baseline pre-challenge scratching behavior from day -7 to day 28 (day 0 being the day of antibody administration to group T02) in the vehicle placebo group (T01) and the antibody ZTS-361 group (T02). As shown in this graph, the variance of scratching behavior scored before IL-31 challenge in both groups T01 and T02 remained largely unchanged, with the number of scratching events observed ranging from 0 to 10 within the 30-minute observation period. This study differed from the preliminary cat challenge model described in section 1.13 above in that 4 mg / kg of ZTS-361 was administered subcutaneously to cats on day 0 without forming a pre-bound complex with feline IL-31. This study provides a more rigorous efficacy evaluation because the antibody ZTS-361 circulates for 7 days before the initial IL-31 challenge, which requires sufficient exposure for the antibody to bind and neutralize circulating IL-31.

[0499] In this study, pruritus behavior was assessed for 1 hour after intravenous challenge with 0.5 μg / kg of IL-31 protein on days 7, 21, and 28. Figure 10B shows the efficacy of antibody ZTS-361, demonstrating a significant reduction in pruritus compared to vehicle placebo control on days 7 (p<0.0001), 21 (p<0.0027), and 28 (p<0.0238) after IL-31 challenge. Data from this challenge model support previous observations demonstrating the efficacy of the mouse:feline 15H05 chimera and also support the cell-based efficacy and relevance of the epitope on feline IL-31 recognized by the 15H05 CDR. These data further support the ability of antibody ZTS-361 to neutralize feline IL-31-induced pruritus and suggest that this antibody may function as a therapeutic agent for IL-31-mediated diseases, including atopic dermatitis, in cats.

[0500] Recent data examining plasma levels of IL-31 in client-owned animals indicate increased amounts of the cytokine in circulation among dogs with atopic and allergic dermatitis compared with normal laboratory beagles (Figure 11A). A recent study was conducted to quantify serum IL-31 levels in cats with a presumptive diagnosis of allergic dermatitis (AD) from several different geographic regions within the United States. Figure 11B shows the results from this evaluation, demonstrating that the mean circulating IL-31 levels in 73 cats studied with this presumptive diagnosis, as well as dogs with atopic and allergic dermatitis, were 8799 fg / ml, compared with 205 fg / ml in 17 age-matched control cats. To understand canine IL-31 levels in previous model development studies, the pharmacokinetic profile of canine IL-31 was analyzed in dogs following a subcutaneous dose of 1.75 μg / kg. Figure 11C shows peak plasma levels, with a maximum of approximately 30 ng / ml within the first hour. / ml, with a sustained level of approximately 400 pg / ml at 3 hours. Based on these findings, it is reasonable to assume that the intravenous administration of 0.5 μg / kg of feline IL-31 used in this cat model results in circulating amounts far in excess of those observed in naturally occurring disease states in dogs and cats.

[0501] 1.15. Analytical Methods Used to Advance Lead Feline Anti-IL-31 Antibodies During the cell line development process, various analytical methods are employed to ensure that antibody therapeutics can be manufactured in a consistent manner. Careful attention to analytical methods that ensure (but are not limited to) product identity, purity, and potency is essential for consistent production of lead monoclonal antibodies and a strong correlation with efficacy and safety outcomes in the target animal species. Because antibodies are homodimers of two heterodimeric units held together via interchain disulfide bonds, any disruption to the pairing process can lead to heterogeneity of the protein drug. Two analytical methods well suited to monitoring antibody dissociation are nonreducing (NR) sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and nonreducing (NR) capillary gel electrophoresis (CGE).

[0502] NR SDS-PAGE provides a simple, qualitative method for quantifying the mass of individual protein species in a test sample. SDS hydrophobically associates with proteins, imparting a uniform net negative charge to them, allowing for the separation of individual components based on mass. After electrophoretic separation on a polyacrylamide gel, proteins are stained with dyes such as Coomassie Blue to enable detection. Although the nonlinearity of the staining prevents absolute quantification of individual protein bands, it can be estimated using software capable of densitometric analysis (VersaDoc, Bio-Rad). Capillary gel electrophoresis (commonly known as CGE) involves subjecting proteins to SDS again, resulting in a uniform negative charge, dissociating noncovalently bound protein complexes. In the presence of an electric field, SDS-coated proteins migrate toward the anode and are detected using ultraviolet light absorbance at a fixed wavelength of 220 nm. Separation is based on the size of the components in the sample within a capillary filled with an exchangeable SDS-polymer gel sieving matrix. In non-reducing CGE, the alkylating agent iodoacetamide (IAM) is added to minimize disulfide bond shuffling during sample preparation. Intact IgG is separated from any fragmented species, allowing for quantification of purity. Software for CGE analysis (e.g., Empower or 32 Karat) utilizes time-corrected area (TCA), which is defined as the area of ​​an individual peak by its migration time. Total TCA is defined as the sum of TCA for all peaks greater than or equal to 0.3%. Therefore, the percentage of monomeric intact IgG and subspecies can be calculated based on individual TCA as a percentage of total TCA.

[0503] Given the promising in vivo efficacy data for ZTS-361 described for the feline pruritus model (Section 1.14), we decided to further characterize various lots of the antibody using these described analytical methods. During this process, we found that stable pools expressing felineized anti-IL-31 (ZTS-361) antibodies had higher levels of low molecular weight species (containing free light chains), visualized by Coomassie staining of SDS-PAGE, compared to the mouse progenitor hybridoma 15H05 (Figure 12). As used herein, intact, or intact monomer, refers to an IgG with a predicted molecular weight of approximately 150 kDa, in which two heavy chains and two light chains are held together by interchain disulfide bonds. HHL, as used herein, refers to "heavy-heavy-light" IgG, lacking one light chain and with a predicted molecular weight of approximately 125 kDa. HH, as used herein, refers to "heavy-heavy" and refers to an IgG lacking both light chains and having a predicted molecular weight of approximately 100 kDa. HL, as used herein, refers to "heavy-light" and refers to an IgG having one heavy chain and one light chain and a predicted molecular weight of approximately 75 kDa. L, as used herein, refers to "light" and refers to an IgG having one light chain and a predicted molecular weight of approximately 25 kDa, also referred to herein as a free light chain. Quantitative evaluation of this same material using NR CGE revealed significantly less intact monomeric IgG in ZTS-361 (83%) compared to mouse progenitor cell 15H05 (94.7%) (Figure 13a). Figure 13B shows the electropherograms after sample digestion by NR CGE. Data from these peaks at different retention times were used to quantify the total TCA percentage for Sample 1 (ZTS-361) and Sample 2 (mouse 15H05). The sum of minor peaks with molecular weights smaller than the major intact IgG peak was used to calculate the fragment percentage (fragment %) shown in Figure 13A. In the stable CHO pool producing ZTS-361, 17% of the final antibody product was obtained as a fragmented form of felineized IgG, compared to only 5.3% for mouse hybridoma 15H05.

[0504] To further our understanding of this phenomenon, we derived single-clonal CHO cell isolates from the ZTS-361 clone pool and examined whether the percentage of intact IgG monomer varied among individual clones. Figure 14A shows Coomassie-stained NR SDS-PAGE using purified antibodies from cultures of eight individual clones stably expressing ZTS-361. For comparison, lanes labeled 1 and 8 show reference standard antibodies known to have a high percentage of intact IgG monomer (approximately 97%). Qualitative densitometric quantification of band intensity is shown on the right side of the figure. Clonal variation in percent intact monomer ranged from 80.2% to 86%, averaging approximately 82%. Similarly, quantification of percent fragments ranged from 14.0% to 19.5% among individual clones, averaging 17.5%. Figure 14B shows quantitative evaluation of these individual clones using NR-CGE. Using this method, less variability was observed, with an average percentage of intact monomer of 86.3% and a percentage of lower molecular weight species of 13.7% across the eight clones tested. While a high level of consistency was observed in the percentage of intact IgG monomer for ZTS-361 among individual clones, our interest lay in understanding why the overall level of intact IgG monomer was lower than that observed with the murine version of the antibody. It is important to note that antibodies (including, but not limited to, felinized antibodies produced from transient expression systems (e.g., HEK and CHO cells)) produced IgG with high levels of percent monomeric form (approximately 88% to approximately 92%) (data not shown). Correspondingly, the amount of antibody produced from these transient cultures is significantly lower than that produced from stable CHO systems. Without wishing to be bound by any one theory, the occurrence of fragmented antibody species seen in feline and other species may be observed under conditions where host cells produce exceptionally large amounts of antibody and where inherent constraints in culture conditions and / or molecular composition of the antibody are observed.

[0505] 1.16. Examination of the primary amino acid sequences of IgG kappa light chain constant domains from several mammalian species Analysis of potential limitations of felinized antibody ZTS-361 began with an examination of the primary antibody sequence. ZTS-361 is composed of a heavy chain comprising a variable region (SEQ ID NO: 121; FEL_15H05_VH1) (the corresponding nucleotide sequence is (SEQ ID NO: 122; FEL_15H05_VH1)) in combination with a feline IgG heavy chain constant region (SEQ ID NO: 173; feline_HC_allele A_1) (the corresponding nucleotide sequence is (SEQ ID NO: 174; feline_HC_allele A_1)), and a light chain comprising a variable region (SEQ ID NO: 135; FEL-15H05-VL1_FW2) (the corresponding nucleotide sequence is (SEQ ID NO: 136; FEL-15H05-VL1_FW2)) in combination with a feline IgG light chain constant region (SEQ ID NO: 175; feline_LC_kappa_G_minus) (the corresponding nucleotide sequence is (SEQ ID NO: 176; feline_LC_kappa_G_minus)). The functional characteristics of the heavy chain constant region of natural feline antibodies have been previously reported by Strietzel et al. (2014 Veterinary Immunology and Immunopathol This has been described by Strietzel et al. (2014) in Biochemistry April 15;158(3-4):214-223. Here, we describe the cloning and expression of felinized antibody ZTS-361 using a heavy chain constant region (SEQ ID NO: 171; feline_HC_allele A_1). The feline HC allele A 1 of SEQ ID NO: 171 corresponds to feline IgG1a according to Strietzel et al. (2014) (ibid.) and appears to be functionally equivalent to human IgG1. Comparison of these functional attributes and alignment of this heavy chain constant region with other constant regions from a diverse set of species did not reveal any obvious regions of interest that would result in inefficient formation of intact IgG monomers (data not shown).

[0506] Similar analysis of the kappa constant chain used in ZTS-361 (SEQ ID NO: 175; cat_LC_kappa_G_minus) reveals unique aspects of the kappa constant light chain diversity observed in at least feline and canine sequences. Different species utilize kappa light chains in their immunoglobulin repertoires at different frequencies. Figure 15 shows representative c-terminal amino acids and corresponding nucleotides in several kappa light chain constant regions from the indicated species. The percentage of kappa light chain utilization by IgG from the indicated species is comparable for dog, cat, and pig (Arun et al. 1996 Zentralbl Veterinarmed. Nov;43(9):573-6), mink (Bovkun et al. 1993 Eur J Immunol. Aug;23(8):1929-34), and mouse (Woloschak et al. 1987 Mol Immunol. Jul;24(7):751-7), and humans (Barandun et al. 1976 Blood. Jan;47(1):79-89). Conventional production of monoclonal antibodies is performed using mice. As shown in Figure 15, mice utilize kappa light chains approximately 95% of the time compared to lambda light chains. In contrast, dogs and cats primarily use lambda light chains in their immunoglobulin repertoires (9% and 8%, respectively). In comparison, two non-human mammals (pig and mink) and human mammals show balanced utilization of kappa and lambda light chains (50%, 46%, and approximately 50%, respectively). In this figure, the position of the most c-terminal cysteine ​​is annotated and aligned across different species. This cysteine ​​is essential for the formation of the quaternary complex that is generated to form the intact IgG structure, as it participates in the formation of an interchain covalent disulfide bond with the heavy chain constant region. While not wishing to be bound by any one theory, it is of great interest to note that at least canine and feline light chains contain several amino acids after the terminal cysteine. These additional amino acid residues are both polar (glutamine) and charged (arginine, aspartic acid, and glutamic acid). These residues are typically found in environments where they participate in hydrogen-bonding interactions, including but not limited to, with the external aqueous environment. The nature and location of these additional residues other than the terminal cysteine ​​may interfere with the formation of interchain disulfide bonds necessary for the formation of IgG heterodimers. Two mammalian species (pig and mink) have a low number of additional residues other than the c-terminal cysteine ​​and use kappa and lambda light chains in approximately equal proportions (50% and 46%, respectively). Recombinant forms of ZTS-361 with variations and deletions of amino acids at the c-terminus of the kappa light chain constant region immediately following the cysteine ​​at amino acid position 107 of SEQ ID NO: 175 were generated using transient expression from HEK cells and tested for percent monomeric IgG by NR-CGE (data not shown).As mentioned above, the production of unpaired light chains and lower molecular weight IgG complexes is more evident under conditions in which antibodies are overproduced from stable clonal cells. However, NR-CGE can detect differences in the amount of intact monomeric IgG and lower molecular weight impurities from cultures produced by transient transfection of cells (e.g., HEK and CHO). This allows for a qualitative assessment of the percentage of monomeric IgG produced from transient cultures. Qualitative use of NR-CGE from such transient cultures allows assaying multiple modifications and deletions to the C-terminus of the kappa constant chain of ZTS-361 for the presence of the percentage of monomeric IgG. This allowed for the production of monomeric recombinant feline IgG (data not shown). While not wishing to be bound by any one theory, deletion of residues QRE from the C-terminus of the feline kappa constant chain appears to be most optimal for the production of monomeric recombinant feline IgG. Other additions to the C-terminus appear to be tolerated. In general, one or two additional amino acids added beyond the cysteine ​​at position 107 of feline LC kappa G minus (SEQ ID NO: 175) appear to be tolerated using qualitative assessment of the percent monomer from transiently produced IgG. Using these same qualitative assays, the addition of three additional amino acids consecutively adjacent to the C-terminus of the cysteine ​​at position 107 in place of the native QRE amino acid residue appears to be tolerated if the electrostatic charge of these three amino acids has minimal impact. It is noted herein that the number and chemical characteristics of the additional amino acids after the cysteine ​​at position 107 of feline LC kappa G minus (SEQ ID NO: 175) affect the efficient formation of interchain disulfide bonds between the kappa light chain constant region and the corresponding Ig heavy chain constant region. It is conceivable that modifications and / or deletions to this region may have beneficial effects on the production of homogeneous intact IgG from stable recombinant cell lines.

[0507] Because the c-terminal amino acid of the human and mouse kappa light chain is a terminal cysteine, no additional amino acid residues are available for interaction with the external environment during disulfide bond formation with the heavy chain constant region. Therefore, it is hypothesized herein that these additional c-terminal residues, at least in the feline and canine kappa light chain constant regions, provide constraints on disulfide bond formation that are not typically observed in nature but are highly relevant to the overproduction of these speciation forms in laboratory settings. Such constraints may include the lack of disulfide bond formation between the c-terminus of the kappa light chain and the heavy chain constant chain, which results in the presence of the previously described HHL, HH, HL, and L species when recombinant antibodies are produced from stable cell lines. These lower molecular weight species are undesirable for producing homogeneous pharmaceuticals, and their presence may be problematic from the standpoint of quality and safety.

[0508] Added to this observation are the codons encoding the amino acid residues within this region, shown below each amino acid letter in Figure 15. In mammals, there are three stop codons (TAA, TGA, and TAG) that signal the termination of polypeptide translation within the ribosome. While not wishing to be bound by any one theory, it has been observed that the majority of codons encoding the c-terminal cysteine ​​and subsequent amino acid are one nucleotide away from the stop codon (Figure 15, light gray text among the codons). Herein, we hypothesize that somatic mutations at various nucleotide positions may have led to the selection of an optimal immunoglobulin kappa constant chain length and amino acid composition that allows efficient expression and correct chain pairing of IgG kappa antibodies. Nuances cannot be overlooked when evaluating the tolerance of appropriate amino acids that may be present at the c-terminus adjacent to the terminal cysteine. Herein, we note the observation that the addition of residues EA to pig light chains or Q to mink light chains appears to have little or no detrimental effect on interchain disulfide formation and expression of intact IgG molecules. These species show comparable usage of kappa and lambda light chains, and the tolerance of these additions is described within the tolerance range. Without limitation, with respect to the C-terminal kappa light chain residues in dog and cat, the distance from the C-terminal cysteine ​​and the charged nature of the arginine (as well as aspartic acid and glutamic acid) residues appear to significantly influence the ability of cat and dog antibodies to form efficient and accurate covalent disulfide bonds with their respective heavy chains.

[0509] 1.17. Observation of the quaternary structure at the chain interface of feline heavy chains and kappa light chains Figure 16A is a diagrammatic representation of the predicted structure of a feline IgG with heavy and light chains equivalent to ZTS-361 (sup...

Claims

1. 1. A method for improving the consistency and / or quality of a feline antibody, comprising: The method comprises producing a feline antibody by expressing in a host cell a nucleotide sequence encoding a feline IgG kappa light chain and a nucleotide sequence encoding a feline IgG heavy chain, wherein the nucleotide sequence encoding the feline IgG kappa light chain comprises a kappa light chain constant nucleotide sequence that is absent from a sequence encoding a C-terminal QRE sequence present in a wild-type feline IgG light chain constant region, and improving the consistency and / or quality of the feline antibody comprises reducing the level of free IgG kappa light chain, thereby increasing the percentage of intact feline IgG antibody monomers.

2. The method comprises: a) providing a nucleotide sequence encoding a wild-type feline IgG kappa light chain constant region of a feline antibody, wherein the wild-type feline kappa light chain constant region comprises a C-terminal amino acid sequence of a QRE; b) removing the sequence encoding the C-terminal QRE from the nucleotide sequence in a) to form a modified kappa light chain constant nucleotide sequence; c) combining the modified kappa light chain constant nucleotide sequence from b) with a nucleotide sequence encoding a feline IgG kappa light chain variable region to form a nucleotide sequence encoding a complete feline IgG kappa light chain; d) expressing the nucleotide sequence encoding the complete feline IgG kappa light chain from c) and a nucleotide sequence encoding a feline IgG heavy chain in a host cell to produce a feline antibody that does not have the C-terminal QRE sequence present in the wild-type feline IgG kappa light chain constant region.

3. The method according to any one of claims 1 to 2, wherein the nucleotide sequence encoding the feline IgG kappa light chain and the nucleotide sequence encoding the feline IgG heavy chain are carried on the same vector used to transform the host cell.

4. The method according to any one of claims 1 to 2, wherein the nucleotide sequence encoding the feline IgG kappa light chain and the nucleotide sequence encoding the feline IgG heavy chain are carried on separate vectors used to transform the host cell.

5. The method according to any one of claims 1 to 4, wherein said feline antibody specifically binds to a target involved in a cytokine and / or growth factor mediated disorder.

6. The method of claim 5, wherein the feline antibody specifically binds to feline IL-31 or feline NGF.

7. The method of any one of claims 1 to 6, wherein the feline antibody comprises a kappa light chain constant region having the sequence RSDAQPSVFLFQPSLDELHTGSASIVCILNDFYPKEVNVKWKVDGVVQNKGIQESTTEQNSKDSTYSLSSTLTMSTEYQSHEKFSCEVTHKSLASTLVKSFQRSEC (SEQ ID NO: 186).

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