Bispecific canylated antibodies and bispecific binding partners for treatment of atopic dermatitis

Abstract

The present invention provides a composition for the treatment of canine atopic dermatitis comprising a fusion protein that binds to canine IL-22, a canine anti-pruritus antibody, and a canine anti-inflammatory antibody. The invention further provides a composition comprising a bispecific binding partner comprising the fusion protein and a canine anti-pruritus antibody or a canine anti-inflammatory antibody, or, a bispecific antibody comprising a canine anti-pruritus antibody and a canine anti-inflammatory antibody, which further comprises a fusion protein that binds to canine IL-22.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202080087151.9, filed December 18, 2020, and entitled “Bispecific Caninized Antibodies and Bispecific Binding Partners for Treatment of Atopic Dermatitis.”

[0002] Cross Reference to Related Applications

[0003] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application Serial No. 63 / 092,294, filed October 15, 2020, U.S. Provisional Patent Application Serial No. 63 / 092,296, filed October 15, 2020, U.S. Provisional Patent Application Serial No. 63 / 015,209, filed April 24, 2020, U.S. Provisional Patent Application Serial No. 63 / 015,220, filed April 24, 2020, U.S. Provisional Patent Application Serial No. 62 / 951,778, filed December 20, 2019, and U.S. Provisional Patent Application Serial No. 62 / 951,793, filed December 20, 2019, the contents of all of the above applications are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0004] The present invention relates to a composition for treating atopic dermatitis in a canine comprising a fusion protein that binds canine IL-22, a caninized anti-pruritic antibody and one caninized anti-inflammatory antibody. The present invention further relates to a composition comprising a bispecific antibody comprising the fusion protein and the caninized anti-pruritic antibody or the caninized anti-inflammatory antibody, or alternatively, a bispecific antibody comprising the caninized anti-pruritic antibody and the caninized anti-inflammatory antibody, which additionally comprises the fusion protein that binds canine IL-22. BACKGROUND

[0005] The immune system comprises a network of resident and recirculating specialized cells that act in concert to protect the host from infectious diseases and cancer. The ability of the immune system to perform this function depends in large part on the biological activities of a group of proteins secreted by white blood cells, collectively known as interleukins. Among the well-studied interleukins, three important molecules have been identified as interleukin 4 (IL 4), interleukin 31 (IL-31), and interleukin 22 (IL-22). While IL-4, IL-31, and IL-22 are key cytokines of the immune response necessary to produce protection from extracellular pathogens (e.g., tissue- or lumen- parasitizing parasites), these cytokines are also implicated in the pathogenesis of allergic diseases, including atopic dermatitis, in humans and animals.

[0006] Atopic dermatitis (AD) is a relapsing pruritic and chronic inflammatory skin disease characterized by dysregulation of the immune system and abnormalities of the epidermal barrier in humans. The pathological and immunological attributes of atopic dermatitis have been the subject of extensive research [reviewed in Rahman et al. Inflammation & Allergy-drug target 10:486-496 (2011) and Harskamp et al., Seminar in Cutaneous Medicine and Surgery 32:132-139 (2013)]. Atopic dermatitis is also a common disease in companion animals, particularly dogs, with an estimated prevalence of approximately 10-15% of the canine population. The pathogenesis of atopic dermatitis in dogs and cats [reviewed in Nuttall et al., Veterinary Records 172(8):201-207 (2013)] is significantly similar to the pathogenesis of atopic dermatitis in humans, including skin infiltration of multiple immune cells and a dominant CD4 + Th2-polarized cytokine environment. In addition, IL-22 is associated with excessive epithelial proliferation leading to epidermal hyperplasia, a characteristic of atopic dermatitis.

[0007] For example, antibodies against canine IL-31 have been shown to have a significant effect on pruritus associated with atopic dermatitis in dogs [US 8,790,651 B2; US 10,093,731 B2]. In addition, antibodies against human IL-31 receptor alpha (IL31RA) have been tested and found to have a significant effect on pruritus associated with atopic dermatitis in humans [Ruzicka et al., New England Journal of Medicine, 376(9), 826-835 (2017)]. Thus, blocking the binding of IL-31 to its receptor IL31RA leads to the relief of pruritus associated with atopic dermatitis.

[0008] Monoclonal antibodies against human interleukin 4 receptor alpha have been developed and some of these antibodies have been extensively tested for their therapeutic effect on the treatment of human atopic dermatitis [see, e.g., US 2015 / 0017176 Al]. Recently, caninized antibodies against canine interleukin 4 receptor alpha (canine IL-4R alpha, canine IL-4R α binding have also been disclosed [US 2018 / 0346580 Al, the entirety of which is hereby incorporated by reference herein]. Since the type II IL-4 receptor consists of an IL-4 receptor alpha chain and an IL-13 receptor alpha 1 chain, caninized antibodies against canine IL-4R α , cIL-4R alpha or cIL-4R alpha) have been obtained [US 2018 / 0346580 Al, the entirety of which is hereby incorporated by reference herein]. Since the type II IL-4 receptor consists of an IL-4 receptor alpha chain and an IL-13 receptor alpha 1 chain, caninized antibodies against canine IL-4R αAntibodies that can block canine IL-4 and canine IL-13 binding to the type II canine IL-4 receptor, thereby serving to help block inflammation associated with atopic dermatitis [US2018 / 0346580A1].

[0009] Interleukin 22 (IL-22) belongs to the IL-10 cytokine family. IL-22 specifically binds to a receptor complex composed of a heterodimeric complex of IL-10R2 (also known as IL-10Rβ) and interleukin 22 receptor (IL-22R) and signals through this receptor complex [see Lee et al., Pharmacology Research & Perspectives, pp. 1-13 (2018: e00434)]. The interleukin 22 receptor is also known as interleukin 22R, alpha 1; IL-22RA1; IL-22R1; zcytor11; and CRF2-9 [Xu et al., Proc. Nat. Acad. Sci. 98(17) 9511-9516 (2001); Gelebart and Lai, Atlas of Genetics and Cytogenetics 14(12): 1106-1110 (2010)]. IL-22 induces epithelial cell proliferation in the wound healing process, and its absence can promote tumor development [Huber et al., Nature 491: 259 263 (2012)].

[0010] Interleukin 22 binding protein (IL-22BP), also known as IL-22RA2, CRF2-10, and CRF2-X, is critical for limiting epidermal hyperplasia that is characteristic of atopic dermatitis by binding to IL-22, thereby blocking the interaction of IL-22 with IL-22R and thus blocking the signaling pathway that leads to epidermal proliferation. IL-22BP is a soluble class II cytokine receptor that is a naturally occurring antagonist of IL-22 [Xu et al., Proc. Nat. Acad. Sci. 98(17) 9511-9516 (2001)]. In vivo, IL-22BP modulates the proinflammatory effects of IL-22 (e.g., neutrophil infiltration), but not the effects of IL-10 [Weber et al., Infect. Immun. 75: 1690-1697 (2007)]. Thus, IL-22BP acts as an anti-proliferative agent by blocking the interaction of IL-22 with IL-22R.

[0011] In addition, a biosynthetic binding partner for IL-22, a fusion protein comprising the extracellular domain of human IL-22RA1 [AAH29273.1; Pro-18-Thr228] fused to the N-terminus of a mouse IgG2a Fc region [purchased from Creative BioMart 45-1 Ramsey Road, Shirley NY 11967], has been shown to inhibit IL-22-induced IL-10 secretion by COLO 205 human colorectal adenocarcinoma cells [data sheet from Creative BioMart].

[0012] Medicaments that have proven useful in the treatment of atopic dermatitis and / or that have shown promise in the treatment of atopic dermatitis include: Janus kinase (JAK) inhibitors [see, e.g., US 8,133,899; US 8,987,283; WO 2018 / 108969], spleen tyrosine kinase (SYK) inhibitors [see, e.g., US 8,759,366], and antagonists of chemokine receptor homolog molecules expressed on TH2 cells [see, e.g., US 7,696,222; US 8,546,422; US 8,637,541; and US 8,546,422].

[0013] However, despite recent successes in treating atopic dermatitis, none of the therapies currently employed produce a rapid onset of anti-pruritic effects that are accompanied by a significant effect on skin inflammation and improvement in skin barrier function. Thus, there is a need for better therapies that can address all three symptoms of atopic dermatitis at once.

[0014] The citation of any reference in this document should not be construed as an admission that such reference is available as "prior art" to the present application. SUMMARY

[0015] The present application relates to bispecific binding partners, bispecific antibodies, and compositions comprising bispecific binding partners and / or bispecific antibodies for use in the treatment of atopic dermatitis in canids. In particular embodiments, the present application relates to compositions for use in the treatment of atopic dermatitis in canids comprising a fusion protein that binds canine interleukin IL-22 (IL-22), an anti-pruritic antibody, and an anti-inflammatory antibody. In certain embodiments, the anti-pruritic antibody and / or the anti-inflammatory antibody is a chimeric rodent (i.e., mouse or rat)-canine antibody. In particular embodiments of this type, both the anti-pruritic antibody and the anti-inflammatory antibody are chimeric rodent-canine antibodies. In other embodiments, the anti-pruritic antibody and / or the anti-inflammatory antibody is a caninized antibody. In particular embodiments of this type, both the anti-pruritic antibody and the anti-inflammatory antibody are caninized antibodies. In other embodiments, the anti-pruritic antibody and / or the anti-inflammatory antibody is a canine antibody. In particular embodiments of this type, both the anti-pruritic antibody and the anti-inflammatory antibody are canine antibodies.

[0016] Accordingly, the present application includes compositions for treating atopic dermatitis in a canine comprising a fusion protein that binds canine interleukin IL-22 (IL-22), a caninized anti-pruritic antibody, and a caninized anti-inflammatory antibody. In particular embodiments, the fusion protein that binds canine IL-22 is a canine IL-22RA1-Fc fusion protein. In related embodiments, the fusion protein that binds canine IL-22 is a canine IL-22RA2-Fc fusion protein.

[0017] In specific embodiments, the compositions comprise a caninized anti-inflammatory antibody and a bispecific binding partner comprising a canine IL-22RA1-Fc fusion protein and a monomer of a caninized anti-pruritic antibody. In certain embodiments, the caninized anti-pruritic antibody is a caninized interleukin 31 (cIL-31) antibody. In particular embodiments, the caninized IL-31 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 12 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 18. In other embodiments, the caninized IL-31 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 11 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 15. In specific embodiments, the light chain of the caninized IL-31 antibody is modified to comprise a heavy chain constant region 1 (CH1) from the heavy chain of the caninized IL-31 antibody in place of the constant light chain domain (CL), and the heavy chain of the caninized IL-31 antibody is modified to comprise a constant light chain domain (CL) from the light chain of the caninized IL-31 antibody in place of the CH1. In specific embodiments, the canine IL-22RA1-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the caninized anti-inflammatory antibody is a caninized interleukin 4 receptor alpha (IL-4Rα) antibody. In particular embodiments, the caninized IL-4Rα antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 4 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 5. In other embodiments, the caninized IL-4Rα antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 6 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 7.

[0018] In related embodiments, the composition comprises a caninized anti-inflammatory antibody and a bispecific binding partner comprising a canine IL-22RA2-Fc fusion protein and a monomer of a caninized anti-pruritic antibody. In certain embodiments, the caninized anti-pruritic antibody is a caninized interleukin 31 (cIL-31) antibody. In particular embodiments, the caninized IL-31 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 12 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 18. In other embodiments, the caninized IL-31 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 11 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 15. In specific embodiments, the light chain of the caninized IL-31 antibody is modified to comprise a heavy chain constant region 1 (CH1) from the heavy chain of the caninized IL-31 antibody in place of the constant light chain domain (CL) and the heavy chain of the caninized IL-31 antibody is modified to comprise a constant light chain domain (CL) from the light chain of the caninized IL-31 antibody in place of the CH1. In specific embodiments, the canine IL-22RA2-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 17. In certain embodiments of this type, the caninized anti-inflammatory antibody is a caninized interleukin 4 receptor alpha (IL-4Rα) antibody. In particular embodiments, the caninized IL-4Rα antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 4 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 5. In other embodiments, the caninized IL-4Rα antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 6 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 7.

[0019] In alternative embodiments, the composition comprises a canine IL-22RA1-Fc fusion protein and a bispecific antibody comprising a monomer of a caninized anti-pruritic antibody comprising a heavy chain and a light chain and a monomer of a caninized anti-inflammatory antibody comprising a heavy chain and a light chain. In certain embodiments, the caninized anti-pruritic antibody is a caninized interleukin 31 receptor alpha (IL-31RA) antibody. In other embodiments, the caninized anti-inflammatory antibody is a caninized IL-4R alpha antibody. In particular embodiments, the caninized anti-pruritic antibody is a caninized IL-31RA antibody and the caninized anti-inflammatory antibody is a caninized IL-4R alpha antibody. In specific embodiments, the caninized IL-4R alpha antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 19 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 20. In related embodiments, the caninized IL-4R alpha antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 21 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 22. In particular embodiments, the light chain of the caninized IL-4R alpha antibody is modified to comprise a heavy chain constant region 1 (CH1) from the heavy chain of the caninized IL-4R alpha antibody in place of the constant light chain domain (CL) and the heavy chain of the caninized IL-4R alpha antibody is modified to comprise a constant light chain domain (CL) from the light chain of the caninized IL-4R alpha antibody in place of the CH1. In certain embodiments, the caninized anti-pruritic antibody of the bispecific antibody is a monomer of a caninized interleukin 31 receptor alpha (IL-31RA) antibody. In more particular embodiments, the caninized IL-31RA antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 52 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 53. In more particular embodiments, the canine IL-22RA1-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 2.

[0020] In related embodiments, the composition comprises a canine IL-22RA2-Fc fusion protein and a bispecific antibody comprising a monomer of a caninized anti-pruritic antibody comprising a heavy chain and a light chain and a monomer of a caninized anti-inflammatory antibody comprising a heavy chain and a light chain. In certain embodiments of this type, the caninized anti-pruritic antibody is a caninized interleukin-31 receptor alpha (IL-31RA) antibody. In other embodiments, the caninized anti-inflammatory antibody is a caninized IL-4R alpha antibody. In specific embodiments, the caninized anti-pruritic antibody is a caninized IL-31RA antibody and the caninized anti-inflammatory antibody is a caninized IL-4R alpha antibody. In particular embodiments, the caninized IL-4R alpha antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 19 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 20. In related embodiments, the caninized IL-4R alpha antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 21 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 22. In specific embodiments, the light chain of the caninized IL-4R alpha antibody is modified to comprise a heavy chain constant region 1 (CH1) from the heavy chain of the caninized IL-4R alpha antibody in place of the constant light chain domain (CL) and the heavy chain of the caninized IL-4R alpha antibody is modified to comprise a constant light chain domain (CL) from the light chain of the caninized IL-4R alpha antibody in place of the CH1. In certain embodiments, the caninized anti-pruritic antibody of the bispecific antibody is a monomer of a caninized interleukin 31 receptor alpha (IL-31RA) antibody. In more specific embodiments, the caninized IL-31RA antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 52 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 53. In more specific embodiments, the canine IL-22RA2-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 3.

[0021] The present application still further provides a bispecific binding partner of the present application. In certain embodiments, the bispecific binding partner comprises a monomer of a canine IL-22RA1-Fc fusion protein and a caninized anti-pruritic antibody. In particular embodiments of this type, the caninized anti-pruritic antibody is a caninized interleukin 31 (cIL-31) antibody. In more particular embodiments, the caninized IL-31 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 12 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 18. In other embodiments, the caninized IL-31 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 11 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 15. In specific embodiments, the light chain of the caninized IL-31 antibody is modified to comprise a heavy chain constant region 1 (CH1) from the heavy chain of the caninized IL-31 antibody in place of a constant light chain domain (CL) and the heavy chain of the caninized IL-31 antibody is modified to comprise a constant light chain domain (CL) from the light chain of the caninized IL-31 antibody in place of CH1. In more particular embodiments, the canine IL-22RA1-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 16.

[0022] In related embodiments, the bispecific binding partner comprises a monomer of a canine IL-22RA2-Fc fusion protein and a caninized anti-pruritic antibody. In particular embodiments of this type, the caninized anti-pruritic antibody is a caninized interleukin 31 (cIL-31) antibody. In more particular embodiments, the caninized IL-31 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 12 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 18. In other embodiments, the caninized IL-31 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 11 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 15. In specific embodiments, the light chain of the caninized IL-31 antibody is modified to comprise a heavy chain constant region 1 (CH1) from the heavy chain of the caninized IL-31 antibody in place of a constant light chain domain (CL) and the heavy chain of the caninized IL-31 antibody is modified to comprise a constant light chain domain (CL) from the light chain of the caninized IL-31 antibody in place of CH1. In more particular embodiments, the canine IL-22RA2-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 17.

[0023] In other embodiments, the compositions comprising the antibodies and / or bispecific antibodies and / or bispecific binding partners and / or fusion proteins of the present application further comprise one or more additional therapeutic components. In one such embodiment, the therapeutic component is a Janus kinase (JAK) inhibitor. In a specific embodiment of this type, the JAK inhibitor is oclacitinib and pharmaceutically acceptable salts thereof. In alternative embodiments, the JAK inhibitor is: 1-[(3R,4S)-4-cyanotetrahydropyran-3-yl]-3-[(2-fluoro-6-methoxy-4-pyridyl)amino]pyrazole-4-carboxamide, and pharmaceutically acceptable salts thereof. In another embodiment, the therapeutic component is a spleen tyrosine kinase (SYK) inhibitor. In a specific embodiment of this type, the SYK inhibitor is (1S,4R)-4-hydroxy-2,2-dimethyl-4-{5-[3-methyl-5-(4-methyl- pyrimidin-2-ylamino)-phenyl]-1,3-thiazol-2-yl}-cyclohexanecarboxylic acid or a pharmaceutically acceptable salt thereof. In another embodiment, the therapeutic component is an antagonist of a chemokine receptor homolog molecule expressed on TH2 cells.

[0024] The present application further provides a method of treating atopic dermatitis in a canine, comprising administering one of the above compositions to a canine having atopic dermatitis.

[0025] In another aspect, the present application provides an isolated mammalian antibody or antigen binding fragment thereof that specifically binds canine interleukin 4 receptor alpha (IL-4Rα). In one such embodiment, the IL-4R α The antibody comprises a set of six complementarity determining regions (CDRs), three of which are heavy chain CDRs: CDR heavy 1 (CDRH1), CDR heavy 2 (CDRH2), and CDR heavy 3 (CDRH3), and three of which are light chain CDRs: CDR light 1 (CDRL1), CDR light 2 (CDRL2), and CDR light 3 (CDRL3); wherein the set of six CDRs are: CDRH1 comprising the amino acid sequence of SEQ ID NO: 28; CDRH2 comprising the amino acid sequence of SEQ ID NO: 30; CDRH3 comprising the amino acid sequence of SEQ ID NO: 32; CDRL1 comprising the amino acid sequence of SEQ ID NO: 34; CDRL2 comprising the amino acid sequence of SEQ ID NO: 36; and CDRL3 comprising the amino acid sequence of SEQ ID NO: 38.

[0026] In another embodiment, the IL-4R αThe antibody comprises a set of six complementarity determining regions (CDRs), three of which are heavy chain CDRs: CDR heavy 1 (CDRH1), CDR heavy 2 (CDRH2), and CDR heavy 3 (CDRH3), and three of which are light chain CDRs: CDR light 1 (CDRL1), CDR light 2 (CDRL2), and CDR light 3 (CDRL3); wherein the set of six CDRs is: CDRH1 comprising the amino acid sequence of SEQ ID NO: 40; CDRH2 comprising the amino acid sequence of SEQ ID NO: 42; CDRH3 comprising the amino acid sequence of SEQ ID NO: 44; CDRL1 comprising the amino acid sequence of SEQ ID NO: 46; CDRL2 comprising the amino acid sequence of SEQ ID NO: 48; and CDRL3 comprising the amino acid sequence of SEQ ID NO: 50.

[0027] In particular embodiments, the isolated mammalian antibody or antigen-binding fragment thereof binds canine IL-4R α and blocks canine IL-4R α binding to canine interleukin 4. In more particular embodiments, the isolated mammalian antibody IL-4R α or antigen-binding fragment thereof is a caninized antibody or caninized antigen-binding fragment thereof.

[0028] In one particular embodiment, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a light chain comprising the amino acid sequence of SEQ ID NO: 4, or an antigenic fragment of such an antibody. In another such embodiment, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 20 and a light chain comprising the amino acid sequence of SEQ ID NO: 19, or an antigenic fragment of such an antibody. In another embodiment, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the amino acid sequence of SEQ ID NO: 6, or an antigenic fragment of such an antibody. In yet another embodiment, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 22 and a light chain comprising the amino acid sequence of SEQ ID NO: 21, or an antigenic fragment of such an antibody.

[0029] These and other aspects of the present application will be better appreciated by reference to the following Figures and Detailed Description. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1is a graph showing binding of canine IL-22 to canine IL-22RA1-Fc fusion protein. The canine IL-22RA1-Fc fusion protein used was a fusion protein of (i) the extracellular domain of canine IL-22RA1 and (ii) canine IgG-B Fc (canine IL-22RA1-FcB). The canine IL-22RA1-Fc fusion protein used was a fusion protein of (i) the extracellular domain of canine IL-22RA1 and (ii) canine IgG-B Fc. The canine IL-22RA1-FcB fusion protein was tested for its ability to bind to canine IL-22, and the results showed that the canine IL-22RA1-FcB fusion protein specifically and in a dose-dependent manner binds to canine IL-22.

[0031] Figure 2 is a graph showing binding of canine IL-22 to canine IL-22BP (IL-22RA2). The canine IL-22RA2-Fc fusion protein used was a fusion protein of (i) canine IL-22BP (IL-22RA2) and (ii) canine IgG-B Fc, canine IL-22BP-FcB (IL-22RA2-FcB). The canine IL-22BP-FcB fusion protein was tested for its ability to bind to canine IL-22, and the results showed that the canine IL-22BP-FcB fusion protein specifically and in a dose-dependent manner binds to canine IL-22.

[0032] Figure 3 is a graph showing inhibition of IL-4 mediated STAT-6 phosphorylation by IL-4R α ) antibodies. Three different caninized monoclonal anti-canine IL-4R α antibodies (designated c4H3, c146E2-H3L3, and c152H11-H3L3) were evaluated for their ability to inhibit aSTAT-6 phosphorylation. The data show that in the presence of IL-4, all three antibodies resulted in dose-dependent inhibition of STAT-6 phosphorylation. IL-4 control in the absence of IL-4R α ) antibodies is shown in the upper left portion of the graph.

[0033] Figure 4is a graph showing inhibition of IL-13 mediated STAT-6 phosphorylation by IL-4Ra antibodies. Three different caninized monoclonal anti-canine IL-4Ra antibodies (designated c4H3, cl46E2-H3L3, and cl52Hl l-H3L3) were evaluated for their ability to inhibit STAT-6 phosphorylation. The data show that in the presence of IL-13, all three antibodies resulted in a dose-dependent inhibition of STAT-6 phosphorylation. The IL-13 control in the absence of IL-4Ra (IL-4Ra) antibodies is shown in the upper left portion of the graph.

[0034] Figure 5 is a graph showing binding of IL-31 to the IL-31R (IL-31RA) extracellular domain. The extracellular domain (ECD) of canine IL-31RA was tested for its ability to bind to caninized IL-31. The results indicate that IL-31RA ECD binds to biotinylated canine IL-31 in a dose-dependent manner with an EC50 of 0.3679 μg / ml. DETAILED DESCRIPTION

[0035] In response to the need for a better therapy for atopic dermatitis, the present invention provides formulations and methods that can achieve simultaneous modulation of the canine IL-4 (cIL-4), canine IL-31 (cIL-31), and canine IL-22 (cIL-22) signaling pathways involved in atopic dermatitis to produce a rapid onset of an anti-pruritic effect accompanied by a significant effect on skin inflammation and improvement in skin barrier function.

[0036] ABBREVIATIONS

[0037] Throughout the detailed description and examples of the present invention, the following abbreviations will be used:

[0038] ADCC antibody-dependent cellular cytotoxicity

[0039] CDC complement-dependent cellular cytotoxicity

[0040] CDR complementarity determining region in an immunoglobulin variable region, defined using the Kabat numbering system

[0041] CHO Chinese hamster ovary

[0042] EC50 concentration that results in 50% potency or binding

[0043] ELISA enzyme-linked immunosorbent assay

[0044] FR antibody framework region: an immunoglobulin variable region not including the CDR regions

[0045] HRP horseradish peroxidase

[0046] IFN Interferon

[0047] IC50 Concentration that results in 50% inhibition

[0048] IgG Immunoglobulin G

[0049] Kabat Immunoglobulin alignment and numbering system initiated by Elvin A. Kabat [Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)]

[0050] mAb Monoclonal antibody (also referred to as Mab or MAb)

[0051] MOA Mechanism of action

[0052] NHS Normal human serum

[0053] PCR Polymerase chain reaction

[0054] PK Pharmacokinetics

[0055] SEB Staphylococcal enterotoxin B

[0056] TT Tetanus toxoid

[0057] VH Immunoglobulin heavy chain variable region

[0058] VL Immunoglobulin light chain variable region

[0059] VK Immunoglobulin kappa light chain variable region

[0060] Definitions

[0061] For the purposes of the present invention, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used have the meanings that are commonly understood by one of ordinary skill in the art to which this invention belongs.

[0062] As used herein, including the appended claims, the singular forms "a," "an," and "the" include their corresponding plural references unless the context clearly dictates otherwise.

[0063] "Activation" when applied to a cell or receptor means to activate or treat the cell or receptor with a ligand, unless the context indicates otherwise or clearly dictates otherwise. "Ligand" includes natural and synthetic ligands, such as cytokines, cytokine variants, analogs, muteins, and antibody-derived binding compounds. "Ligand" also includes small molecules, such as peptide mimetics of cytokines and peptide mimetics of antibodies. "Activation" can refer to cellular activation by internal mechanisms as well as external or environmental factors.

[0064] "Activity" of a molecule can describe or refer to binding of the molecule to a ligand or to a receptor, catalytic activity; ability to stimulate gene expression or cell signaling, differentiation or maturation; antigenic activity, activity to modulate other molecules, etc. "Activity" of a molecule can also refer to activity to modulate or maintain cell-cell interactions (e.g., adhesion), or to maintain cell structure (e.g., cell membrane or cytoskeleton). "Activity" can also refer to specific activity, such as [catalytic activity] / [mg protein] or [immunological activity] / [mg protein], concentration in a biological compartment, etc. "Activity" can refer to modulation of components of the innate or adaptive immune system.

[0065] "Administering" and "treatment" when applied to an animal (e.g., a canine subject, a cell, a tissue, an organ, or a biological fluid) means contacting an exogenous drug, therapeutic, diagnostic reagent, or composition with the animal (e.g., a canine subject, a cell, a tissue, an organ, or a biological fluid). Treatment of a cell includes contacting the reagent with the cell, as well as contacting the reagent with a fluid that is in contact with the cell.

[0066] "Administering" and "treatment" also mean in vitro and ex vivo treatment, e.g., by a reagent, diagnostic, binding compound, or by another cell. The term "subject" includes any organism, preferably an animal, more preferably a mammal (e.g., a canine, feline, or human) and most preferably a canine.

[0067] "Treatment" or "treating" means to administer a therapeutic agent (such as a composition containing any of the antibodies and / or bispecific antibodies and / or fusion proteins of the present application) internally or externally to, e.g., a canine subject or patient having one or more symptoms or suspected of having a condition for which the agent has therapeutic activity.

[0068] Generally, an agent is administered in an amount effective to reduce and / or ameliorate one or more symptoms of a disease / condition in a subject or population being treated, whether by inducing regression of such symptoms or by inhibiting progression of such symptoms, as measured by any clinically measurable metric. The amount of a therapeutic agent that is effective to alleviate any particular symptom of a disease / condition (also referred to as a “therapeutically effective amount”) can vary according to factors such as the disease state, age, and weight of the patient (e.g., canine), and the ability of the pharmaceutical composition to elicit a desired response in the subject. Whether a disease / condition symptom has been reduced or ameliorated can be assessed by any clinical assessment typically used by a veterinarian or other skilled healthcare provider to assess the severity or state of progression of the symptom. While an embodiment of the present application (e.g., a method of treatment or article of manufacture) can not be effective to reduce the target disease / condition symptom in every subject, it should reduce the target disease / condition symptom in a statistically significant number of subjects, as determined by any statistical test known in the art, such as Student’s t-test, Chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0069] “Treatment” when applied to human, veterinary (e.g., canine) or research subjects, refers to therapeutic treatment as well as research and diagnostic applications. “Treatment” when applied to human, veterinary (e.g., canine) or research subjects, or cells, tissues or organs, includes the contacting of antibodies and / or bispecific antibodies and / or fusion proteins of the present application with, for example, a canine or other animal subject, cell, tissue, physiological compartment or physiological fluid.

[0070] As used herein, the term “canine” includes all domestic dogs, Canis lupus familiaris or Canis familiaris, unless otherwise indicated.

[0071] As used herein, the term “feline” refers to any member of the family Felidae. Members of this family include wild, zoo, and domestic members, including domestic cats, purebred and / or hybrid companion cats, show cats, laboratory cats, cloned cats, and wild cats or feral cats.

[0072] As used herein, the term "canine framework" refers to the amino acid sequences of the heavy and light chains of a canine antibody other than the hypervariable region residues defined herein as CDR residues. With regard to caninized antibodies, in most embodiments, the amino acid sequences of native canine CDRs are replaced by corresponding exogenous CDRs (e.g., CDRs from a mouse or rat antibody) in both chains. Optionally, the heavy and / or light chains of a canine antibody can comprise some exogenous non-CDR residues, e.g., to maintain the conformation of the exogenous CDRs within the canine antibody, and / or to alter Fc function, as exemplified below and / or disclosed in US 10,106,607 B2, the entire contents of which are hereby incorporated by reference herein.

[0073] A canine antibody (also known as immunoglobulin G or IgG) is a large tetrameric protein of about 150 Kd. Each IgG protein is composed of two identical light chains (each about 25 Kd) and two identical heavy chains (each about 50 Kd). There are four known subclasses of IgG heavy chains in canines, designated IgGA, IgGB, IgGC, and IgGD. There are two types of light chains; kappa and lambda chains. Each kappa or lambda light chain is composed of one variable domain (VL) and one constant domain (CL). Each of the two heavy chains is composed of one variable domain (VH) and three constant domains designated heavy chain constant region 1 (CH1 or CH 1), heavy chain constant region 2 (CH2 or CH 2), and heavy chain constant region 3 (CH3 or CH3). The CH1 domain is connected to the CH2 domain by an amino acid sequence called the "hinge" or alternatively "hinge region". In the present invention, the amino acid sequences of each of the four canine IgG Fc fragments are based on the identified boundaries of the CH1 and CH2 domains determined by Tang et al. [Vet. Immunol. Immunopathol. 80: 259-270 (2001)]. In humans, IgG exists in one of four subclasses designated IgG1, IgG2, IgG3, and IgG4. The subclass of IgG is determined primarily by the sequence of the hinge region, which differs among the four subclasses of IgG. The two heavy chains are connected to each other by disulfide bonds, and each heavy chain is also connected to a light chain by disulfide bonds.

[0074] IgG antibody with papain breaks the antibody molecule at the hinge region and results in the formation of three fragments. Two of these fragments are identical, each consisting of the light chain and the VH and CHI domains of the heavy chain joined together. These fragments are called "Fab" fragments and they contain the antigen binding site of the antibody. The Fab fragments are attached to the VH-CHI chain by disulfide bonds. The third fragment, which results from papain digestion, is called "Fc" and it comprises the rest of each heavy chain that is held together by disulfide bonds. Thus, the Fc comprises a dimer of CH2 and CH3 domains from each of the two heavy chains. While the Fab enables the antibody to bind to its cognate epitope, the Fc enables the antibody to mediate immune effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). A "Fab fragment" consists of one light chain (VL and CL domains) and the CHI and variable region (VH) of one heavy chain. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0075] The "Fragment crystallizable region" abbreviated as "Fc" corresponds to the CH3-CH2 portion of an antibody that interacts with cell surface receptors called Fc receptors. As used herein, and particularly with respect to being part of a fusion protein with canine IL-22RA1 or canine IL-22RA2, "Fc" is the monomer of the CH3-CH2 portion of an antibody. Tang et al. first described the canine fragment crystallizable region (cFc) for each of the four canine IgGs [Vet. Immunol. Immunopathol. 80:259-270 (2001); see also, Bergeron et al., Vet. Immunol. Immunopathol. 157:31-41 (2014)].

[0076] As used herein, canine Fc“IgG-Bm” is a canine IgG-B Fc comprising two (2) amino acid residue substitutions, D31A and N63A (see below), in the amino acid sequence of SEQ ID NO: 14 of IgG-B. The aspartic acid residue (D) at position 31 of SEQ ID NO: 14 and the asparagine residue (N) at position 63 of SEQ ID NO: 14 are substituted with alanine residues (A) in IgG-Bm. These two amino acid residue substitutions serve to significantly reduce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of naturally occurring canine IgG-B [see US 10,106,607 B2, the contents of which are incorporated by reference in their entirety herein]. Further amino acid substitutions to IgG-Bm are also contemplated, similar to those that can be made in IgG-B, and can include amino acid substitutions that favor the formation of heterodimers in bispecific antibodies and / or bispecific binding partners, as described below.

[0077] Interleukin 22 (IL-22) specifically binds to and signals through a receptor complex consisting of a heterodimer complex of interleukin 22 receptor and IL-10R2. The interleukin 22 receptor is also known as interleukin 22RA1 (IL-22RA1). As used herein, a “canine IL-22RA1-Fc fusion protein” comprises the extracellular domain of canine IL-22RA1 fused to the N-terminus of canine IgG Fc. A canine IL-22RA1-Fc fusion protein can be produced by genetic engineering from a chemically synthesized nucleic acid encoding the IL-22RA1 extracellular domain fused to the N-terminus of canine IgG Fc.

[0078] Interleukin 22 binding protein (also known as IL-22RA2 or IL-22BP) acts as an anti-proliferative agent by blocking the interaction of IL-22 with its receptor (see above). As used herein, a “canine IL-22RA2-Fc fusion protein” comprises canine interleukin 22 binding protein fused to canine IgG Fc. A canine IL-22RA2-Fc fusion protein can be produced by genetic engineering from a chemically synthesized nucleic acid encoding canine IL-22RA2 fused to the N-terminus of canine IgG Fc.

[0079] As used herein, for example, "substituting an amino acid residue" with another amino acid residue in an amino acid sequence of an antibody is equivalent to "replacing an amino acid residue" with another amino acid residue and means that the particular amino acid residue at a particular position in the amino acid sequence has been replaced (or substituted) with a different amino acid residue. Such substitutions can be specifically designed, i.e., by replacing an alanine with a serine at a particular position in the amino acid sequence by, for example, recombinant DNA technology. Alternatively, a particular amino acid residue or string of amino acid residues of an antibody can be replaced with one or more amino acid residues through a more natural selection process, for example, based on the ability of a cell-produced antibody to bind a given region on the antigen (e.g., a region containing an epitope or portion thereof), and / or to cause the antibody to comprise a particular CDR that retains the same canonical structure as the CDR it is replacing. Such substitutions / replacements can result in a "variant" CDR and / or a variant antibody.

[0080] As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological activity. An antibody can be a monomer, a dimer, or a larger multimer. Thus, it is used in the broadest sense, and specifically encompasses, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), caninized antibodies, fully canine antibodies, chimeric antibodies, and camelized single-domain antibodies. A "parental antibody" is an antibody obtained by exposing the immune system to an antigen, which is then modified for the intended use, for example, caninizing the antibody for use as a canine therapeutic antibody.

[0081] As used herein, "blocks" or "prevents" or "precludes" the binding of, for example, a canine receptor to its binding partner (ligand) is an antibody and / or bispecific antibody and / or fusion protein and / or bispecific binding partner of the present application that (partially or completely) blocks the binding of a canine receptor to its canine ligand, and vice versa, as determined in a standard binding assay (e.g., ELISA or flow cytometry). As used herein, "blocks" or "prevents" or "precludes" the binding of, for example, a canine receptor to its binding partner (ligand) is an antibody and / or bispecific antibody and / or fusion protein and / or bispecific binding partner of the present application that (partially or completely) blocks the binding of a canine receptor to its canine ligand, and vice versa, as determined in a standard binding assay (e.g., ELISA or flow cytometry).

[0082] A "bivalent antibody" comprises two antigen binding sites. In some instances, the two binding sites have the same antigen specificity.

[0083] As used herein, the terms "bispecific binding partner" and "bispecific binding protein" are used interchangeably and refer to an artificial protein molecule that can target two different antigens simultaneously, and wherein the artificial protein molecule is formed by association of a monomeric heavy chain and light chain of a monoclonal antibody with a fusion protein comprising a canine IgG Fc (or a modified canine IgG Fc, such as IgG-Bm as defined below), fused by genetic engineering to another protein portion that binds to a target protein. When the bispecific binding partner targets two different antigens, the two binding sites of the bispecific binding partner are different. One example of a bispecific binding partner is a caninized monomer of an antibody to canine IL-31 associated with a fusion protein comprising the extracellular domain of canine IL-22RA1 fused by genetic engineering to a canine IgG Fc, such as exemplified herein, and wherein the bispecific binding partner can target canine IL-31 and canine IL-22 simultaneously.

[0084] As used herein, a "bispecific antibody" is an artificial protein that can target two different antigens simultaneously. One preferred type of bispecific antibody is an IgG-like antibody that consists of four different polypeptide chains. Thus, a bispecific antibody can be a heterodimer comprising two monomers, each monomer comprising a heavy chain and a light chain. The first monomer can be from an antibody to one particular antigen, while the second monomer can be from an antibody to a different antigen. For example, certain bispecific antibodies of the present application can target IL-31RA and IL-4R α simultaneously. Such antibodies are formed by combining one heavy chain and one light chain having specificity for IL-4R α with one heavy chain and one light chain having specificity for IL-31RA. Furthermore, each heavy chain and light chain can be modified with specific mutations in its amino acid sequence in order to favor the association of the heavy and light chains of the IL-4R α antibody and the heavy and light chains of the IL-31RA antibody, but at the same time favor the association of the heavy chain of the IL-4R α antibody with the other IL-4R α heavy chain or the IL-31RA heavy chain with the other IL-31RA heavy chain, over the association of the heavy chain of the IL-4R α antibody with the heavy chain of the IL-31RA antibody.

[0085] In the context of bispecific antibodies and bispecific binding partners, a "monomer" of an antibody consists of one heavy chain and one light chain of the antibody.

[0086] As used herein, "artificial protein" and "artificial protein molecule" are used interchangeably and refer to a protein (or multimer of proteins, such as dimers, heterodimers, tetramers, and heterotetramers, etc.) that does not naturally occur in nature, such as a man-made fusion protein or a heterodimer of monomers from two different antibodies.

[0087] Generally, the antibodies or antigen-binding fragments of the present application retain at least 10% of their activity (when compared to the parent antibody) when canine antigen binding activity is expressed on a molar basis. Preferably, the antibodies or antigen-binding fragments of the present application retain at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the canine antigen binding affinity of the parent antibody. It is also intended that the antibodies or antigen-binding fragments of the present application can include conservative or non-conservative amino acid substitutions that do not significantly alter their biological activity (referred to as "conservative variants" or "functionally conservative variants" of the antibodies).

[0088] "Isolated antibody" refers to the state of purification and in this context means that the molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials such as cellular debris and growth media. In general, the term "isolated" does not mean the complete absence of such materials or the absence of water, buffers, or salts, unless they are present in amounts that substantially interfere with the experimental or therapeutic uses of the binding compounds described herein.

[0089] As used herein, a "chimeric antibody" is an antibody having variable domains from a first antibody and constant domains from a second antibody, wherein the first and second antibodies are from different species. [US 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1984)]. Typically, the variable domains are obtained from an antibody from an experimental animal (e.g., a rodent) (parental antibody), while the constant domain sequences are obtained from an animal subject antibody (e.g., human or canine), thus producing a chimeric antibody that is less likely to elicit an adverse immune response in a human or canine subject, respectively, compared to the parental (e.g., rodent) antibody.

[0090] As used herein, the term "caninized antibody" refers to a form of antibody comprising sequences from both canine and non-canine (e.g., murine or rat) antibodies. Generally, a caninized antibody will comprise substantially all of at least one or more, usually two, variable domains in which all or substantially all of the hypervariable loops correspond to those of a non-canine immunoglobulin (e.g., comprising the six CDRs as exemplified below) and all or substantially all of the framework (FR) regions (and generally all or substantially all of the remaining framework) are those of a canine immunoglobulin sequence. As exemplified herein, a caninized antibody comprises three heavy chain CDRs and three light chain CDRs from a murine or rat anti-canine antigen antibody and a canine framework or a modified canine framework. The modified canine framework comprises one or more amino acid changes as exemplified herein that further optimize the effectiveness of the caninized antibody, e.g., increase its binding to its canine antigen and / or its ability to block the canine antigen from binding to its natural binding partner.

[0091] The variable regions of each light / heavy chain pair to form the antibody binding site. Thus, generally, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, these two binding sites are typically identical.

[0092] Generally, the variable domains of both the heavy and light chains contain three hypervariable regions, also called complementarity determining regions (CDRs), within the overall framework region (FR). The CDRs are typically aligned with the framework regions, enabling binding to a particular epitope. Typically, both light and heavy chain variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, from N- to C-terminus. Generally, the assignment of amino acids to each domain is according to the definition of Sequences of Proteins of Immunological Interest, Kabat et al.; National Institutes of Health, Bethesda, Md.; 5th Ed.; NIH Publ. No. 91-3242 (1991); Kabat, Adv. Prot. Chem. 32: 1-75 (1978); Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Chothia et al., J. Mol. Biol. 196: 901-917 (1987) or Chothia et al., Nature 342: 878-883 (1989)].

[0093] As used herein, the term "hypervariable region" refers to amino acid residues in an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" [i.e., CDR1, CDR2, and CDR3 in the light chain variable domain (LCDR1, LCDR2, and LCDR3) and CDR1, CDR2, and CDR3 in the heavy chain variable domain (HCDR1, HCDR2, and HCDR3)]. [See Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) for definition of CDR regions of antibodies in terms of sequence; see also Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987) for definition of CDR regions of antibodies in terms of structure].

[0094] As used herein, the term "framework" or "FR" residues refer to those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0095] There are four known IgG heavy chain subtypes of dog IgG and are referred to as IgG-A, IgG-B, IgG-C, and IgG-D. There are two known light chain subtypes referred to as lambda and kappa. In particular embodiments of the application, in addition to binding and activation of canine immune cells, the canine or caninized antibodies of the application against their antigen optimally have two attributes:

[0096] 1. lack of effector functions such as antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC), and

[0097] 2. ease of large scale purification using industry standard techniques such as protein A chromatography based techniques.

[0098] None of the naturally occurring canine IgG isotypes meet both criteria. For example, IgG-B can be purified using protein A, but has high levels of ADCC activity. IgG-A, on the other hand, binds weakly to protein A, but also shows ADCC activity. In addition, neither IgG-C nor IgG-D can be purified on a protein A column, although IgG-D shows no ADCC activity. (There is considerable ADCC activity with IgG-C). One way in which the present application addresses these issues is by providing a modified canine IgG-B antibody of the application specific for the antigen of the application, and / or a bispecific antibody, and / or a bispecific binding partner, and / or a fusion protein that lacks effector functions such as ADCC and can be easily purified using industry standard protein A chromatography.

[0099] In alternative embodiments of the application, the canine IgG-B or IgG-C antibodies of the application, and / or bispecific antibodies, and / or bispecific binding partners, and / or fusion proteins of the application specific for the antigens of the application are intentionally not modified to remove / substantially reduce effector functions such as ADCC, and thus retain effector functions such as ADCC.

[0100] As used herein, the terms "anti-pruritic agent" and "anti-pruritic agent" are used interchangeably and are a compound, macromolecule, and / or formulation that acts to inhibit, alleviate, and / or prevent pruritus. Anti-pruritic agents are also known as anti-itch drugs.

[0101] As used herein, the terms "anti-pruritic agent" and "anti-pruritic agent" are used interchangeably and are a compound, macromolecule, and / or formulation that acts to inhibit, alleviate, and / or prevent pruritus. Anti-pruritic agents are also known as anti-itch drugs.

[0102] As used herein, an "anti-inflammatory agent" is a compound, macromolecule, and / or formulation that reduces inflammation by blocking the interaction of certain substances that cause inflammation in the body.

[0103] As used herein, an "anti-inflammatory agent" is a compound, macromolecule, and / or formulation that reduces inflammation by blocking the interaction of certain substances that cause inflammation in the body. α As used herein, an "anti-inflammatory agent" is a compound, macromolecule, and / or formulation that reduces inflammation by blocking the interaction of certain substances that cause inflammation in the body. α As used herein, an "anti-inflammatory agent" is a compound, macromolecule, and / or formulation that reduces inflammation by blocking the interaction of certain substances that cause inflammation in the body. α As used herein, an "anti-inflammatory agent" is a compound, macromolecule, and / or formulation that reduces inflammation by blocking the interaction of certain substances that cause inflammation in the body.

[0104] As used herein, an “anti-proliferative agent” is a compound, macromolecule, and / or agent that counteracts the induction of epithelial cell proliferation, particularly the induction of keratinocyte proliferation, particularly for atopic dermatitis. Interleukin 22 binding protein (IL-22BP) is an example of a naturally occurring anti-proliferative agent. Canine IL-22RA2-Fc fusion protein is an example of an artificial anti-proliferative agent for canines. Canine IL-22RA1-Fc fusion protein is another example of an artificial anti-proliferative agent for canines.

[0105] As used herein, an “anti-proliferative antibody” is an antibody that can act as an anti-proliferative agent in an animal, including a mammal such as a human, canine, and / or feline, particularly for atopic dermatitis. In particular embodiments, an anti-proliferative antibody binds to a particular protein in the IL-22 signaling pathway, such as IL-22 or receptor IL-22 receptor (IL-22R). Binding of an anti-proliferative antibody to its corresponding antigen (e.g., IL-22 or IL-22R) inhibits, for example, the binding of IL-22 to IL-22R and interferes with and / or prevents signaling of the pathway, thereby interfering with or preventing keratinocyte proliferation associated with atopic dermatitis.

[0106] “Homology” refers to the sequence similarity between two polynucleotide sequences or between two polypeptide sequences when aligned for maximum correspondence. Two molecules are homologous at a position if they are occupied by the same base or amino acid monomer subunit, e.g., if one position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology is the number of homologous positions shared by the two sequences divided by the total number of positions compared x 100. For example, if 6 of 10 positions in two sequences are matched or homologous when the sequences are optimally aligned, the two sequences are 60% homologous. Typically, the comparison is made when the two sequences are aligned for maximum percent homology.

[0107] “Isolated nucleic acid molecule” refers to a DNA or RNA, mRNA, cDNA, or synthetic origin, or some combination thereof, of a genome that is not associated with all or a portion of the polynucleotide as it exists in nature, or linked to polynucleotides with which it is not linked in nature. For purposes of the present disclosure, it is understood that “a nucleic acid molecule comprising (a specified nucleotide sequence)” does not include an entire chromosome. An isolated nucleic acid molecule comprising a specified nucleotide sequence can include, in addition to the specified sequence, up to ten or even up to twenty or more additional protein-encoding sequences of other proteins or portions or fragments thereof, or can include operably linked regulatory sequences that control expression of the coding region of the nucleic acid sequence, and / or can include vector sequences.

[0108] The phrase "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are appropriate for a given organism can vary, but they include promoters, optional operator sequences, and ribosome binding sites. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.

[0109] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretion leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretion leader, contiguous and in reading phase. Enhancers, however, need not be contiguous. Linking is accomplished over a distance of about five base pairs to upwards of several thousand base pairs, depending on the overlap of the hybridization sequences.

[0110] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. The words "transformant" and "transformed cell" therefore include the primary subject cell and cultures derived from it, regardless of the number of transfers. It is also understood that, due to natural

[0111] The present application provides methods of using the isolated caninized antibodies and / or bispecific antibodies and / or bispecific binding partners and / or fusion proteins of the present application for treating a disorder, e.g., treating atopic dermatitis in a dog. In canines, there are four IgG heavy chains, designated A, B, C, and D. These heavy chains represent four different subclasses of canine IgG, designated IgG-A (or IgGA), IgG-B (or IgGB), IgG-C (or IgGC), and IgG-D (or IgGD). Each of the two heavy chains consists of one variable domain (VH) and three constant domains designated CH-1, CH-2, and CH-3. The CH-1 domain is connected to the CH-2 domain by an amino acid sequence designated a "hinge" or, alternatively, a "hinge region."

[0112] The DNA and amino acid sequences of these four heavy chains were first identified by Tang et al. [Vet. Immunol. Immunopathol. 80: 259-270 (2001)]. The amino acid and DNA sequences of these heavy chains are also available from the GenBank database. For example, the accession number for the amino acid sequence of IgG A heavy chain is AAL35301.1, for IgG B is AAL35302.1, for IgG C is AAL35303.1, and for IgG D is (AAL35304.1). Canine antibodies also comprise two types of light chains, kappa or lambda. The DNA and amino acid sequences of these light chains are available from the GenBank database. For example, the accession number for the kappa light chain amino acid sequence is ABY 57289.1, and for the lambda light chain is ABY 55569.1.

[0113] Caninized murine or rat anti-canine antibodies that bind canine IL-31, IL-31Rα, IL-22, or IL-4Rα include, but are not limited to, antibodies and / or bispecific antibodies and / or bispecific binding partners and / or fusion proteins of the application comprising canine IgG-A, IgG-B, IgG-C, and IgG-D heavy chains and / or canine kappa or lambda light chains and murine or rat anti-canine antigen CDRs. Accordingly, the application provides isolated caninized murine or rat anti-canine antibodies, and / or bispecific antibodies, and / or bispecific binding partners, and / or fusion proteins of the application that bind to their respective canine antigen and block the binding of the canine antigen to its natural binding partner.

[0114] Accordingly, the application further provides caninized murine or rat antibodies and methods of using antibodies and / or bispecific antibodies and / or bispecific binding partners and / or fusion proteins of the application to treat a disorder, for example, atopic dermatitis in a canine.

[0115] The application further provides full-length canine heavy chains that can be matched with respective light chains to make caninized antibodies. Accordingly, the application further provides caninized murine or rat anti-canine antigen antibodies (including isolated caninized murine or rat anti-canine antibodies) and / or bispecific antibodies and / or bispecific binding partners comprising caninized antibodies and / or fusion proteins of the application, and methods of using antibodies and / or bispecific antibodies and / or bispecific binding partners and / or fusion proteins of the application to treat a disorder, for example, atopic dermatitis in a canine.

[0116] The present application also provides antibodies and / or bispecific antibodies and / or bispecific binding partners and / or fusion proteins of the present application comprising a canine fragment crystallizable region (cFc region), wherein the cFc is genetically modified to enhance, reduce or eliminate one or more effector functions. In one aspect of the present application, the genetically modified cFc reduces or eliminates one or more effector functions. In another aspect of the present application, the genetically modified cFc enhances one or more effector functions. In certain embodiments, the genetically modified cFc region is a genetically modified canine IgGB Fc region. In another such embodiment, the genetically modified cFc region is a genetically modified canine IgGC Fc region. In a specific embodiment, the effector function that is enhanced, reduced or eliminated is antibody-dependent cellular cytotoxicity (ADCC). In another embodiment, the effector function that is enhanced, reduced or eliminated is complement-dependent cytotoxicity (CDC). In yet another embodiment, the cFc region is genetically modified to increase, reduce or eliminate both ADCC and CDC.

[0117] To generate variants of canine IgG that lack effector functions, a number of mutated canine IgGB heavy chains were generated. These variants can include one or more of the following single or combinations of substitutions in the Fc portion of the heavy chain amino acid sequence: P4A, D31A, N63A, G64P, T65A, A93G, and P95A. The variant heavy chains (i.e., comprising such amino acid substitutions) were cloned into expression plasmids and transfected into HEK 293 cells along with plasmids comprising genes encoding light chains. The intact antibodies expressed and purified from HEK 293 cells were evaluated for binding to Fc γ RI and Clq to assess their potential to mediate immune effector functions. [See US 10,106,607 B2, the contents of which are incorporated by reference in their entirety.]

[0118] The present application also provides modified canine IgG-Ds, which comprise, in place of their native IgG-D hinge regions, hinge regions from:

[0119] IgG-A: FNECRCTDTPPCPVPEP SEQ ID NO: 23

[0120] IgG-B: PKRENGRVPRPPDCPKCPAPEM SEQ ID NO: 24; or

[0121] IgG-C: AKECECKCNCNNCPCPGCGL SEQ ID NO: 25.

[0122] Alternatively, the IgG-D hinge region can be genetically modified by replacing a serine residue with a proline residue, i.e., SEQ ID NO: 26 (proline residues (P) substituted for naturally occurring serine residues are underlined and in bold). Such modifications can result in a canine IgG-D that lacks Fab arm exchange. The modified canine IgG-D can be constructed using standard methods of recombinant DNA technology [e.g., Maniatis et al., Molecular Cloning, A Laboratory Manual (1982)]. To construct these variants, the nucleic acid encoding the amino acid sequence of canine IgG-D can be modified so that it encodes the modified IgG-D. The modified nucleic acid sequence is then cloned into an expression plasmid for protein expression.

[0123] Sequence identity refers to the extent to which the amino acids of two polypeptides are identical at equivalent positions when the two sequences are optimally aligned. As used herein, an amino acid sequence is 100% "identical" to a second amino acid sequence when the amino acid residues of the two sequences are the same at each corresponding position. Thus, an amino acid sequence is 50% "identical" to a second amino acid sequence when 50% of the amino acid residues of the two sequences are the same. Sequence comparisons are made over a segment of the given protein (e.g., a portion of the protein or polypeptide being compared) comprising contiguous amino acid residues. In certain embodiments, selected deletions or insertions are taken into account in order to otherwise alter the correspondence between two amino acid sequences.

[0124] Sequence similarity includes identical residues and non-identical, biochemically related amino acids. Biochemically related amino acids with similar properties are interchangeable.

[0125] A "conservatively modified variant" or "conservative substitution" refers to the substitution of one amino acid for another amino acid in a protein with similar properties (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity, etc.) such that the change can generally be made without altering the biological activity of the protein. In general, those of skill in the art recognize that a single amino acid substitution in a non-essential region of a polypeptide will not significantly alter biological activity [see, e.g., Watson et al., Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.; 1987)]. Moreover, substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity. Exemplary conservative substitutions are listed in Table A, immediately below.

[0126] Table A

[0127] Exemplary conservative amino acid substitutions

[0128] Original Residues Conservative Substitutions Ala (A) Gly; Ser Arg (R) Lys; His Asn (N) Gln; His Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn Glu (E) Asp; Gln Gly (G) Ala His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg; His Met (M) Leu; Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala; Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu

[0129] The present application also encompasses function-conservative variants of the antibodies of the present application. As used herein, "function-conservative variants" refer to antibodies or fragments in which one or more amino acid residues have been altered without altering the desired properties (e.g., antigen affinity and / or specificity). Such variants include, but are not limited to, substitution of an amino acid with one having similar properties, e.g., the conservative amino acid substitutions of Table A above.

[0130] Nucleic acids

[0131] The present application also includes nucleic acids encoding the antibodies and / or bispecific antibodies, and / or bispecific binding partners, and / or fusion proteins of the present application (see, e.g., the Examples below).

[0132] The present application also includes nucleic acids encoding immunoglobulin polypeptides comprising an amino acid sequence that is at least about 70% identical, preferably at least about 80% identical, more preferably at least about 90% identical, and most preferably at least about 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to the amino acid sequence of a caninized antibody provided herein when compared using the BLAST algorithm, wherein the parameters of the algorithm are selected to give the maximum match over the entire length of the corresponding sequence in the corresponding reference sequence. The present application further provides nucleic acids encoding immunoglobulin polypeptides comprising an amino acid sequence that is at least about 70% similar, preferably at least about 80% similar, more preferably at least about 90% similar, and most preferably at least about 95% similar (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to any of the reference amino acid sequences when compared using the BLAST algorithm, wherein the parameters of the algorithm are selected to give the maximum match over the entire length of the corresponding sequence in the corresponding reference sequence, are also encompassed by the present application.

[0133] As used herein, percent identity of nucleotide and amino acid sequences can be determined using C, MacVector (MacVector, Inc. Cary, NC 27519), Vector NTI (Informax, Inc. MD), Oxford Molecular Group PLC (1996), and the Clustal W algorithm with the alignment default parameters and default parameters for identity. These commercially available programs can also be used to determine sequence similarity using the same or similar default parameters. Alternatively, high advanced Blast searches under default filter conditions can be used, e.g., the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program using default parameters.

[0134] The following references relate to the BLAST algorithm commonly used for sequence analysis: BLAST ALGORITHMS: Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990); Gish, W. et al., Nature Genet. 3:266-272 (1993); Madden, T. L. et al., Meth. Enzymol. 266:131-141 (1996); Altschul, S. F. et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang, J. et al., Genome Res. 7:649-656 (1997); Wootton, J. C. et al., Comput. Chem. 17:149-163 (1993); Hancock, J. M. et al., Comput. Appl. Biosci. 10:67-70 (1994); ALIGNMENT SCORING SYSTEMS: Dayhoff, M. O. et al., "A model of evolutionary change in proteins." in Atlas of Protein Sequence and Structure, vol. 5, suppl. 3. M. O. Dayhoff (ed.), pp. 345-352, (1978) Natl. Biomed. Res. Found., Washington, DC; Schwartz, R. M. et al., "Matrices for detecting distant relationships." in Atlas of Protein Sequence and Structure, vol. 5, suppl. 3. (1978), M. O. Dayhoff (ed.), pp. 353-358 (1978), Natl. Biomed. Res. Found., Washington, DC; Altschul, S. F., J. Mol. Biol. 219:555-565 (1991); States, D. J. et al., Methods 3:66-70 (1991); Henikoff, S. et al., Proc. Natl. Acad. Sci. USA 89:10915-10919 (1992); Altschul, S. F. et al., J. Mol. Evol. 36:290-300 (1993); ALIGNMENT STATISTICS: Karlin, S. et al., Proc. Natl. Acad. Sci.USA 87:2264-2268 (1990); Karlin, S. et al., Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); Dembo, A. et al., Ann. Prob. 22:2022-2039 (1994); and Altschul, S. F. "Evaluating the statistical significance of multiple distinct local alignments." in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), pp. 1-14, Plenum, New York (1997).

[0135] The antibodies and / or bispecific antibodies and / or bispecific binding partners and / or fusion proteins of the present application can be produced recombinantly by methods known in the art. Mammalian cell lines useful as hosts for the expression of the antibodies or fragments disclosed herein are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). In particular, these cell lines include Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells, and many others. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, equine, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels. Other cell lines that can be used are insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells, and fungal cells. When a recombinant expression vector encoding a heavy chain or antigen binding portion thereof or fragment thereof, a light chain and / or antigen binding fragment thereof is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow for expression of the antibody in the host cell, or more, preferably, for the antibody to be secreted into the culture medium in which the host cells are grown.

[0136] Antibodies can be recovered from the culture medium using standard protein purification methods. Furthermore, a number of known techniques can be used to enhance expression of the antibodies of the present application (or other portions therein). For example, the glutamine synthetase gene expression system (GS system) is a commonly used method to enhance expression under certain conditions. The GS system is discussed in whole or in part in connection with European Patent Nos. 0 216 846, 0 256 055, and 0 323 997, and European Patent Application No. 89303964.4.

[0137] In general, a glycoprotein produced in a particular cell line or transgenic animal will have a glycosylation pattern that is characteristic of glycoproteins produced in the cell line or transgenic animal. Thus, the particular glycosylation pattern of an antibody will depend on the particular cell line or transgenic animal used to produce the antibody. However, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein constitute the invention, regardless of the glycosylation pattern the antibodies can have. Similarly, in certain embodiments, antibodies having a glycosylation pattern comprising only non-fucosylated N-glycans can be advantageous, as these antibodies have been shown to generally exhibit stronger efficacy in vitro and in vivo than their fucosylated counterparts [see, e.g., Shinkawa et al., J. Biol. Chem. 278:3466-3473 (2003); U.S. Patent Nos. 6,946,292 and 7,214,775].

[0138] Antibody and fusion protein engineering

[0139] For the fusion proteins of the invention, a heavy chain constant region, e.g., a canine constant region, e.g., IgGA, IgGB, IgGC, and IgGD, or a variant thereof is joined to a protein that binds IL-22. As such, the protein that binds IL-22 can be bound to an antibody monomer or form a bispecific binding partner. Such antibodies or antigen binding fragments further comprise a light chain constant region, e.g., a canine light chain constant region, e.g., a lambda or kappa canine light chain constant region or a variant thereof. By way of example and not limitation, the canine heavy chain constant region can be from IgG B or a modified cFc, e.g., IgG-Bm as used herein [see US 10,106,607 B2, incorporated by reference in its entirety herein], and the canine light chain constant region can be from kappa.

[0140] The antibodies and / or bispecific antibodies and / or bispecific binding partners of the invention can be engineered to include modifications to the canine framework and / or canine framework residues within the parental (i.e., mouse or rat) monoclonal antibody variable domains, e.g., to improve the properties of the antibody.

[0141] Pharmaceutical compositions and administration

[0142] To prepare a pharmaceutical or sterile composition comprising an antibody, and / or bispecific antibody, and / or bispecific binding partner, and / or fusion protein of the present application, the antibody and / or bispecific antibody, and / or bispecific binding partner, and / or fusion protein can be admixed with a pharmaceutically acceptable carrier or excipient. [See, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984)].

[0143] Formulations of therapeutic and diagnostic agents can be prepared by mixing with acceptable carriers, excipients, or stabilizers in the form of, e.g., a lyophilized powder, a syrup, a solution, or a suspension [see, e.g., Hardman et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams and Wilkins, New York, NY; Avis et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY]. In one embodiment, the antibody and / or bispecific antibody, and / or bispecific binding partner, and / or fusion protein of the present application is diluted to the appropriate concentration in a sodium acetate solution at pH 5-6, and NaCl or sucrose is added to obtain tonicity. Additional agents, such as polysorbate 20 or polysorbate 80, can be added to enhance stability.

[0144] Toxicity and therapeutic efficacy of the antibody compositions alone or in combination with another pharmaceutical agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index (LD50 / ED50). In certain aspects, antibodies exhibiting a high therapeutic index are desired. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in canids. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. 50 (therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index (LD50 / ED50). In certain aspects, antibodies exhibiting a high therapeutic index are desired. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in canids. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. 50 50 50 )· In certain aspects, antibodies exhibiting a high therapeutic index are desired. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in canids. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. 50

[0145] The mode of administration can vary. Suitable routes of administration include oral, rectal, transmucosal, enteral, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal, or intraarterial. In particular embodiments, the antibodies and / or bispecific antibodies of the application, and / or bispecific binding partners, and / or fusion proteins can be administered by an invasive route, e.g., by injection. In further embodiments of the application, the antibodies and / or bispecific antibodies of the application, and / or bispecific binding partners, and / or fusion proteins, or pharmaceutical compositions thereof, are administered intravenously, subcutaneously, intramuscularly, intraarterially, or by inhalation, aerosol delivery. Administration by non-invasive routes, e.g., oral; e.g., in a pill, capsule, or tablet, is also within the scope of the application.

[0146] The compositions can be administered with medical devices known in the art. For example, the pharmaceutical compositions of the application can be administered by injection with a hypodermic needle, including, e.g., a pre-filled syringe or an auto-injector. The pharmaceutical compositions disclosed herein can also be administered with needle-free hypodermic injection devices; e.g., the devices disclosed in U.S. Patent Nos.: 6,620,135; 6,096,002; 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556.

[0147] ​​​The pharmaceutical compositions disclosed herein can also be administered by infusion. Examples of well-known infusion devices and modules for administering pharmaceutical compositions include U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at precise infusion rates; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic pharmaceutical delivery system having multi-chamber compartments. Many other such implantation devices, delivery systems, and modules are known to those skilled in the art.

[0148] Alternatively, the antibodies of the application, and / or bispecific antibodies, and / or bispecific binding partners, and / or fusion proteins can be administered in a local rather than systemic manner, for example, via injection of the antibody or protein at the site to be treated. The antibodies of the application, and / or bispecific antibodies, and / or bispecific binding partners, and / or fusion proteins can be formulated in an acceptable diluent, such as sterile water or sterile saline, and released using a pump, or other device.

[0149] The administration regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic antibody, and / or bispecific antibody, and / or bispecific binding partner, and / or fusion protein, the level of symptoms, the immunogenicity of the therapeutic antibody and / or bispecific antibody, and / or bispecific binding partner, and / or fusion protein, and the accessibility of the target cells in the biological matrix. Preferably, the administration regimen delivers sufficient therapeutic antibody, and / or bispecific antibody, and / or bispecific binding partner, and / or fusion protein to ameliorate the target disease / disorder state while minimizing adverse side effects. Thus, the amount of biologic delivered depends in part on the particular therapeutic antibody, and / or bispecific antibody, and / or bispecific binding partner, and / or fusion protein and the severity of the disorder being treated. Guidance for selecting appropriate dosages of therapeutic antibodies is provided [see, e.g., Wawrzynczak Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK (1996); Kresina (ed.) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY (1991); Bach (ed.) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY (1993); Baert et al. New Engl. J. Med. 348:601-608 (2003); Milgrom et al. New Engl. J. Med. 341 :1966-1973 (1999); Slamon et al. New Engl. J. Med. 344:783-792 (2001); Beniaminovitz et al. New Engl. J. Med. 342:613-619 (2000); Ghosh et al. New Engl. J. Med. 348:24-32 (2003); Lipsky et al. New Engl. J. Med. 343:1594-1602 (2000)].

[0150] Determining appropriate dosages is performed by a veterinarian, e.g., using parameters or factors known in the art or suspected of affecting treatment. Typically, the dosage is started at a lower amount than the optimal dosage, and increased in small increments until the desired or optimal effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms.

[0151] The antibodies, and / or bispecific antibodies, and / or bispecific binding partners, and / or fusion proteins provided herein can be provided by continuous infusion or by doses administered, e.g., daily, 1-7 times per week, weekly, biweekly, monthly, bimonthly, quarterly, semiannually, annually, etc. The doses can be provided, e.g., intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, intraspinally, or by inhalation. The total weekly dose is typically at least 0.05 pg / kg body weight, more typically at least 0.2 pg / kg, 0.5 pg / kg, 1 pg / kg, 10 pg / kg, 100 pg / kg, 0.25 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 5.0 mg / ml, 10 mg / kg, 25 mg / kg, 50 mg / kg, or more [see, e.g., Yang et al. New Engl. J. Med. 349:427-434 (2003); Herold et al. New Engl. J. Med. 346:1692-1698 (2002); Liu et al. J. Neurol. Neurosurg. Psych. 67:451-456 (1999); Portielji et al. Cancer Immunol. Immunother. 52:133-144 (2003)]. Doses can also be provided to achieve a predetermined target concentration of the antibodies, and / or bispecific antibodies, and / or bispecific binding partners, and / or fusion proteins of the application in the serum of a canine, e.g., 0.1, 0.3, 1, 3, 10, 30, 100, 300 pg / ml, or more. In other embodiments, the antibodies and / or bispecific antibodies, and / or bispecific binding partners, and / or fusion proteins of the application are administered subcutaneously or intravenously at 10, 20, 50, 80, 100, 200, 500, 1000, or 2500 mg per subject per week, biweekly, “every 4 weeks,” monthly, bimonthly, or quarterly.

[0152] As used herein, “inhibit” or “treat” or “treating” includes delaying the onset and / or reducing the severity of symptoms associated with a disorder. The term also includes ameliorating existing uncontrolled or unwanted symptoms, preventing additional symptoms, and ameliorating or preventing the underlying cause of these symptoms. The terms also include ameliorating existing uncontrolled or unwanted symptoms, preventing additional symptoms, and ameliorating or preventing the underlying cause of these symptoms. Thus, the terms denote a beneficial result to a vertebrate subject (e.g., a canine) that is suffering from a disease, condition, and / or symptom or that has a likelihood of developing such a disease, condition, or symptom.

[0153] As used herein, the terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to the amount of the antibody, and / or bispecific antibody, and / or bispecific binding partner, and / or fusion protein of the present application that, when administered alone or in combination with another therapeutic agent, is effective to elicit a measurable improvement in one or more symptoms of a disease or condition, or the progression of such disease or condition. A therapeutically effective dose further refers to an amount of the antibody, and / or bispecific antibody, and / or bispecific binding partner, and / or fusion protein that is sufficient to result in at least partial amelioration of symptoms, e.g., treatment, cure, prevention, or amelioration of the relevant medical condition, or an increase in the rate of treatment, cure, prevention, or amelioration of such conditions. When applied to combination therapy, a therapeutically effective dose refers to combined amounts of active ingredients that produce the therapeutic effect whether administered jointly, sequentially, or concurrently. An effective amount of the therapeutic agent will produce an improvement in a diagnostic measure or parameter of at least 10%; usually at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably at least 50%. Where subjective parameters are used to assess the severity of illness, an effective amount can also produce an improvement in subjective parameters.

[0154] Other Combination Therapies

[0155] Compositions comprising the antibody, and / or bispecific antibody, and / or bispecific binding partner, and / or fusion protein of the present application can comprise one or more additional therapeutic components. One such family of therapeutic components is Janus Kinase (JAK) inhibitors. In a particular embodiment of this type, the JAK inhibitor comprises the following chemical formula:

[0156]

[0157] wherein R 1 is C 1-4 alkyl optionally substituted with hydroxyl, and pharmaceutically acceptable salts thereof [US 8,133,899; US 8,987,283]. More particularly, the JAK inhibitor is oclacitinib, and even more particularly oclacitinib maleate. An alternative JAK inhibitor that preferentially inhibits JAK1 over JAK3 is 1-[(3R,4S)-4-cyanotetrahydropyran-3-yl]-3-[(2-fluoro-6-methoxy-4-pyridyl)amino]pyrazole-4-carboxamide, which comprises the following chemical formula:

[0158]

[0159] and pharmaceutically acceptable salts thereof [see, WO 2018 / 108969].

[0160] Another therapeutic component that can be added to the compositions of the present application can be a spleen tyrosine kinase (SYK) inhibitor. One such SYK inhibitor is (1S,4R)-4-hydroxy-2,2-dimethyl-4-{5-[3-methyl-5-(4-methyl-pyrimidin-2-ylamino)-phenyl]-1,3-thiazol-2-yl}- cyclohexanecarboxylic acid or a pharmaceutically acceptable salt thereof [see, e.g., US 8,759,366].

[0161] In addition, yet another therapeutic component that can be added to the compositions of the present application is an antagonist of a chemoattractant receptor homolog molecule expressed on TH2 cells, which comprises the following chemical formula:

[0162]

[0163] and pharmaceutically acceptable salts thereof [see also, US 7,696,222; US 8,546,422; US 8,637,541; WO2010 / 099039; WO 2010 / 031183; and US 8,546,422].

[0164] These additional therapeutic components can be administered to the canine subject prior to, in conjunction with, or after administration of a composition comprising the antibodies, and / or bispecific antibodies, and / or bispecific binding partners, and / or fusion proteins of the present application.

[0165] Examples

[0166] General Materials and Methods:

[0167] In all of the following examples, the recombinant proteins were obtained by providing the amino acid sequence of the selected protein to a commercial manufacturer (ATUM, Newark, California), who in turn selected an appropriate nucleotide sequence that encoded the amino acid sequence. The nucleotide sequence can also be obtained from publicly available DNA databases, such as GenBank®. The commercial manufacturer then chemically synthesized the nucleic acid, which was then cloned by ATUM into an expression plasmid (pD2610-v10; available from AUTM) to produce the corresponding recombinant protein. The plasmid was placed into HEK-293 cells or CHO cells to express the recombinant protein, which was then isolated by conventional methods.

[0168] Example 1

[0169] Canine IL-22 and IL-22 Binding Proteins

[0170] Nucleic acid encoding the extracellular domain (ECD) of canine IL-22RA1 was chemically synthesized and linked to nucleic acid encoding canine IgG-B Fc to form nucleic acid encoding a canine IL-22RA1-Fc fusion protein. Similarly, nucleic acid encoding canine IL-22 binding protein (cIL-22BP; also known as cIL-22 receptor alpha 2 and cIL-22RA2) was chemically synthesized and linked to nucleic acid encoding canine IgG-B Fc to form nucleic acid encoding a canine IL-22BP-Fc fusion protein. These nucleic acids were then individually cloned into expression plasmids suitable for production of the corresponding proteins in eukaryotic cells such as HEK-293 or CHO cells. Both canine IL-22RA1-Fc fusion protein and canine IL-22BP-Fc fusion protein bind canine IL-22 and subsequently block binding of canine IL-22 to full-length membrane-bound canine IL-22RA1.

[0171] Binding of canine IL-22RA1 and canine IL-22BP (IL-RA2) to canine IL-22

[0172] Materials:

[0173] 1. Coating antigen: cIL-22-His (canine IL 22 with His tag)

[0174] 2. cIL-22BP-Fc fusion protein (canine IL-22BP-Fc fusion protein)

[0175] 3. cIL 22RA1-Fc fusion protein (canine IL 22RA1-Fc fusion protein)

[0176] 4. cPD-L1-Fc, 6.8 mg / mL. (canine PD-L1-IgG-B Fc fusion protein, used as control).

[0177] 5. Horseradish peroxidase (HRP) conjugated rabbit anti-dog IgG (Fc), Sigma catalog number SAB3700109, 1.5 mg / mL.

[0178] 6. 3,3',5,5'-tetramethylbenzidine (TMB) peroxidase substrate, SeraCare KPL 5120-0048,

[0179] 7. Peroxidase substrate solution B, SeraCare KPL 5120-0037

[0180] 8. Phosphate buffered saline (PBS)

[0181] 9. Phosphate buffered saline with (PBST), National Veterinary Services Laboratories (NVSL)

[0182] 10.1.5 M phosphoric acid.

[0183] Method:

[0184] 1. Dilute cIL-22-His to 1 pg / mL in PBS, then add 100 pL / well to ELISA plate. Incubate overnight at 2-7 °C.

[0185] 2. Wash coated plates 3 times with NVSL-PBST using an automated plate washer, 200 pL / well / wash.

[0186] 3. Block ELISA plates with 200 pL of 5% nonfat dried milk (NFDM) in NVSL-PBST, seal and incubate at 37 °C for at least 60 minutes.

[0187] 4. Wash blocked ELISA plates 3 times with NVSL-PBST using an automated plate washer, 200 pL / well / wash.

[0188] 5. Dilute cIL-22BP-Fc fusion protein, cIL-22RA1-Fc fusion protein, and cPD-L1-Fc fusion protein to 10 pg / mL and serially dilute 3-fold on a dilution plate. Transfer 50 pL of diluted test sample to ELISA plate, seal and incubate at 37 °C for 60 minutes with rotation.

[0189] 6. Wash plates 3 times with NVSL-PBST using an automated plate washer, 200 pL / well / wash.

[0190] 7. Add 100 pL / well of 1 :2500 diluted HRP conjugated anti-dog IgG (Fc), seal and incubate at 37 °C for 60 minutes.

[0191] 8. Mix equal amounts of TMB Peroxidase Substrate and TMB Peroxidase Substrate Solution B, add 100 pL / well. Incubate at 37 °C for 20 minutes.

[0192] 9. Stop with 100 pL / well of 1.5 M phosphoric acid.

[0193] 10. Measure A450-A540 on a spectrophotometer.

[0194] cIL-22-HIS: [SEQ ID NO: 1]

[0195] LPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVNMGERCYLMKEVLNFTLEEVLLPQSDRFQPYMQEVVPFLARLSNKLSQCHIENDDQHIQRNVQKLKDTVQKLGENGEIKAIGELDLLFMALRNACVHHHHHH

[0196] cIL-22RA1-FcB: [SEQ ID NO: 2]

[0197] AEDTSDLLQYVKFQSSNFENILTWDSGLESAPDVVYSVEYKTYGKKEWLAKEGCQRITRKSCNLTTETGNHTEHYYARVTAVSAGGRSATKMTDRFSSMQQTTIKPPDVTCIPKVRSIQMIVHPTSTPIHAEDGHRLTLEDIFQDLFYRLELQVNHTYQMHLGGKQRDYEFIGLSPDTEFLGTITISVPNFFKESAPYVCRVKTLPDRTWTGGGGSGGGGSPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

[0198] cIL-22RA2-FcB: [SEQ ID NO: 3]

[0199] TQSAYESLKPQRVHFQSRNFHNILHWQPGRACTSNGSVYFVQYKMYGQRQWKNKED CWGILECFCDLTNETSDIQEPYYGRVRTTSAGIHSGWTMTQRFTPWWETKIDPPIIN ITQVNGSLLVILHAPSLPYRDQKGKNVSIENYYELLYRVFIINNSLEKEQKVYEGA HRIVEIGALAPHTGYCVVAEMYQPMLDRRSPRSEERCMELPGGGGSGGGGSPKRE NGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPE VQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNK ALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQS NGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHY TQESLSHSPG

[0200] Example 2

[0201] Anti-IL-4 Receptor Alpha Antibodies

[0202] Anti-canine IL-4 receptor alpha antibodies useful in the present application are exemplified by antibody c152H11 VL3-cCLk-s / c152H11 VH3-cIgG Bm and antibody c146E2 VL3-cCLk-s / c146E2 VH3-cIgG Bm. The sets of six (6) CDRs for these two antibodies (three separate light chain (LC) and three heavy chain (HC) sequences) are provided in Table 1A (nucleic acid sequences) and Table 1B (amino acid sequences) below. The amino acid sequences of the full-length LC and HC of these caninized antibodies are provided in Table 1A and 1B below.

[0203] IL-4R alpha Antibody CDRs

[0204] Nucleic Acid and Amino Acid Sequences

[0205] Table 1A

[0206]

[0207] Table 1B

[0208]

[0209] c152H11 VL3-cCLk-s (light chain): [SEQ ID NO: 4]

[0210] EIVMTQSPASLSLSQEEKVTITCKASQNVGTNVAWYQQKPGQAPKLLIYSASYRYSGLPDRFSGSGSGTDFSFTISSLEPEDVAEFFCQQYNSYPYTFGQGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD

[0211] c152H11 VH3-cIgG-Bm (heavy chain): [SEQ ID NO: 5]

[0212] EVQLVESGGDLVKPGGSLRLSCAASGFTFSSYGMSWVRQAPDKRLQWVATISRGGDYTYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCARGTLNNRGFASWGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

[0213] c146E2 VL3-cCLk-s (light chain): [SEQ ID NO: 6]

[0214] DIVLTQTPLSLSVSPGETASIYCRASESVDSYGNSFLNWYQQKPGQPPKLLIYRASNLASEIPDRFSGSGSRTEFTLKISRVEADDAGVYYCQQNYENPRTFGQGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD

[0215] c146E2VH3-cIgG-Bm (heavy chain): [SEQ ID NO: 7]

[0216] EVQLVQSGAEVKKPGASVKVSCKASGYTFARYWMHWMKQAPGAGLDWIGMIHPDSGNINYNERFKTKATLTVDKSTSTAYMELSSLRAGDIAVYYCARQLRNAMDYWGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

[0217] The ability of antibodies against canine IL-4 receptor alpha to inhibit STA-6 phosphorylation in DH82 cells was tested as follows:

[0218] Materials

[0219] 1. Actively growing DH82 cells

[0220] 2. DH82 cell growth medium (DMEM, 10% FBS, 2 mM L-glutamine, 1% Penicillin-Streptomycin) 302003 TMEagle's Minimum Essential Medium, supplemented with heat-inactivated fetal bovine serum to a final concentration of 15% w / v)

[0221] 3. AlphaLISA p-STAT6 (Tyr641) Assay Kit: Perkin Elmer Cat# ALSU-PST6-A-HV

[0222] 4. Recombinant canine IL-4: R&D Systems, Cat# 752-CL / CF

[0223] 5. Recombinant canine IL-13: R&D Systems, Cat# 5894-CL / CF

[0224] 6. Perkin Elmer Envision

[0225] a. Caninized Anti-Canine IL-4R α Monoclonal Antibodies

[0226] b. c146E2-H3L3

[0227] c. c152H11-H3L3

[0228] d. c4H3

[0229] Methods

[0230] 1. Two tissue culture plates were seeded with 8 x 10 4 DH82 cells per well (200 μL at a density of 4 x 10 5 cells / mL) and incubated at 37°C overnight.

[0231] 2. Test antibodies were pre-diluted to 500 μg / mL and then serially 3-fold diluted in DH82 cell growth media. The media was removed from the cell culture plates and 50 μL / well of the serially diluted test samples were transferred to each plate.

[0232] 3. Canine IL-4 was diluted to 5 ng / mL in DH82 cell growth media and 50 μL was added to each well of one plate. Canine IL-13 was diluted to 10 ng / mL in DH82 cell growth media and 50 μL was added to each well of the second plate. The plates were incubated at 37°C for 15 minutes.

[0233] 4. The media was removed from the plates and 100 μL per well of freshly prepared lx lysis buffer from the AlphaLISA p-STAT-6 assay kit was added to the plates. The plates were agitated at 350 rpm on a plate shaker at room temperature for 10 minutes.

[0234] 5. The Acceptor Mix was prepared from the AlphaLISA p-STAT6 assay kit and 15 μΐ, per well was added to 30 μΐ, of cell lysate in a 96-well ½ Area Plates. The plates were sealed, agitated at 350 rpm for 2 minutes, and then incubated at room temperature for 2 hours.

[0235] 6. The Donor Mix was prepared from the AlphaLISA p-STAT6 assay kit under soft laboratory lighting and 15 μΐ, per well was added to each plate. The plates were sealed, covered with foil, agitated at 350 rpm for 2 minutes, and then incubated at room temperature for 2 hours.

[0236] 7. The plates were read on a Perkin Elmer EnVison using AlphaScreen settings.

[0237] Example 3

[0238] IL-31 Receptor Alpha

[0239] Nucleotide Sequence

[0240] The nucleotide sequence of SEQ ID NO: 8 encodes the extracellular domain of canine IL-31 Receptor Alpha (IL-31RA) fused to a HIS tag. The canine IL-31RA ECD HIS-tagged protein comprises the amino acid sequence of SEQ ID NO: 9. The nucleotide sequence was prepared by chemical synthesis and then cloned into an expression plasmid suitable for producing the corresponding protein in eukaryotic cells such as HEK-293 or CHO cells. Canine IL-31RA ECD-10His: [SEQ ID NO: 8]

[0241]

[0242] Expression and purification of IL-31 receptor alpha ECD

[0243] Plasmids containing the nucleotide sequence of SEQ ID NO: 8 were transfected into HEK-293 or CHO cells by MaxCyte instruments using electroporation according to the manufacturer's recommendations. A few days after transfection, the supernatant of transfected cells and untransfected controls was harvested and centrifuged to remove cell debris. The clarified harvest from transfected cells was passed over a nickel column to purify the IL-31RA with a histidine tag from the cell culture fluid according to the manufacturer's recommendations. Purified protein was quantified by measuring their absorbance of ultraviolet light at 280 nm.

[0244] Canine IL-31RA ECD-10His: [SEQ ID NO: 9]

[0245] VLPAKPENISCIFYYEENFTCTWSPEKEASYTWYKVKRTYSYGYKSDICSTDNSTRGNHASCSFLPPTITNPDNYTIQVEAQNADGIMKSDITYWNLDAIMKIEPPEIFSVKSVLGIKRMLQIKWIRPVLAPHSSTLKYTLRFRTINSAYWMEVNFTKEDIDRDETYNLTELQAFTEYVMTLRCAPAESMFWSGWSQEKVGTTEEEAPYGLDLWRVLKPAMVDGRRPVQLMWKKATGAPVLEKALGYNIWYFPENNTNLTETVNTTNQTHELYLGGKTYWVYVVSYNSLGESPVATLRIPALNEKTFQCIEAMQACLTQDQLVVEWQSSAPEVDTWMVEWFPDVDSEPSSFSWESVSQARNWTIQKDELKPLWCYNISVYPVLRDRVGQPYSTQAYVQEGIPSAGPVTQADSIGVKTVTITWKEIPKSKRNGFIKNYTIFYQAEDGKEFSKTVNSNILQYRLESLTRRTSYSLQVMASTNAGGTNGTKINFKTLSISHHHHHHHHHH

[0246] Binding of canine IL-31RA to biotinylated canine IL-31:

[0247] 1. Coat immunoplates with IL-31RA protein at 10 μg / mL diluted in PBS. Add 100 μL / well. Incubate plates overnight at 2-7°C.

[0248] 2. Wash plate 3 times with 275 μL / well of PBST.

[0249] 3. Block plate with 200 μL / well of blocking buffer (1% NFDM in PBST) for 30-45 minutes at 36 ± 2°C with gentle shaking (120 ± 20 RPM).

[0250] 4. Wash plate 3 times with 275 μL / well of PBST.

[0251] 5. Dilute biotinylated IL-31 to 10 ug / mL in 1% NFDM in PBST.

[0252] 6. Dilute biotinylated IL-31 (10 ug / mL) 3-fold in 1% NFDM in PBST and transfer 100 μL / well to the immuno plate. Incubate for 30-45 minutes at 36 ± 2°C with gentle shaking (120 ± 20 RPM).

[0253] 7. Wash plate 3 times with 275 μL / well of PBST.

[0254] 8. Dilute HRP-Streptavidin to a final dilution of 1:1000 in 1% NFDM in PBST.

[0255] 9. Add 100 μL / well of HRP-Streptavidin to the immuno plate and incubate for 30-45 minutes at 36 ± 2°C with gentle shaking (120 ± 20 RPM).

[0256] 10. Wash plate 3 times with 275 μL / well of PBST.

[0257] 11. Mix equal amounts of pre-warmed TMB 2-component substrate immediately before use.

[0258] 12. Add 100 μL / well of prepared TMB substrate to the immuno plate and incubate for 10 to 15 minutes at 36 ± 2°C in the dark with gentle shaking (120 ± 20 RPM).

[0259] 13. Stop the reaction by adding 100 μL / well of 1 M H3PO4.

[0260] 14. Read the plate using a microplate reader at a wavelength of 450 nm with a reference wavelength of 540 nm.

[0261] Monoclonal antibodies against canine IL-31 receptor alpha

[0262] Monoclonal antibodies to canine IL-31RA were generated by multiple immunizations of 2 Lewis rats with canine IL-31RA ECD (10 μg or 25 μg antigen per rat per use) over a period of 3 to 4 weeks. After immunization, serum was collected from each rat and tested against canine IL-31RA by ELISA. The rat lymph node cells with the highest IL-31RA ECD reactivity were fused with the myeloma SP2 / 0 cell line to generate hybridomas. Approximately 10 days after fusion, supernatants from growing hybridomas were screened by ELISA on IL-31RA ECD protein coated plates using the following protocol. Approximately 263 clones were selected in this ELISA that showed potential binding to IL-31RA with an OD > 1 for most of the clones.

[0263] Procedure for ELISA:

[0264] 1. Coat 96 well half area plates with IL-31RA (1 μg / mL in PBS buffer), 25 μL / well. Incubate plates at 4°C overnight.

[0265] 2. Wash plates 3 times with PBST (PBS + 0.05% Tween 20)

[0266] 3. Block plates with blocking buffer (PBS with 5% FBS), 25 ul / well, for 30 minutes at room temperature.

[0267] 4. Transfer 25 ul / well of hybridoma supernatant to 96 well plates and incubate for 60 minutes at room temperature.

[0268] 5. Wash plates 3 times with PBST.

[0269] 6. Add 25 ul / well of anti-rat HRP (diluted 1 :4000 in blocking buffer) to plates and incubate for 60 minutes at room temperature.

[0270] 7. Wash plates 5 times with PBST.

[0271] 8. Add TMB based reagent to plates for colorimetric reaction for 2-3 minutes.

[0272] 9. Stop reaction with 0.16 M sulfuric acid.

[0273] 10. Read plates with plate reader.

[0274] Blocking activity of anti-IL-31 receptor alpha antibodies

[0275] The ability of anti-canine IL-31RA hybridoma supernatants to block IL-31 binding to IL-31RA was assessed in a blocking ELISA described below. Of the 263 clones shown to bind to IL-31RA, approximately 24 clones showed potential for blocking IL-31 binding to IL-31RA.

[0276] 1. Coat 96 well half area plates with IL-31RA (1 μg / mL in PBS buffer), 25 μL / well. Incubate plates at 4°C overnight.

[0277] 2. Wash plates 3 times with PBST (PBS + 0.05% Tween 20)

[0278] 3. Block plates with blocking buffer (PBS with 5% FBS), 25 ul / well, for 30 minutes at room temperature.

[0279] 4. Transfer 25 ul / well of hybridoma supernatant to 96 well plates and incubate for 60 minutes at room temperature.

[0280] 5. Wash plates 3 times with PBST.

[0281] 6. Transfer 25 μL / well of biotinylated IL-31 (0.5 μg / mL in blocking buffer) and incubate for 60 minutes at room temperature.

[0282] 7. Wash plates 3 times with PBST.

[0283] 8. Add 25 μl / well of streptavidin-HRP (1 :5000 dilution in blocking buffer) to plates and incubate for 60 minutes at room temperature.

[0284] • Wash plates five (5) times with PBST.

[0285] • Add TMB substrate to plates for colorimetric reaction for 2-3 minutes.

[0286] • Stop reaction with 0.16 M sulfuric acid.

[0287] • Read plates by plate reader.

[0288] Biological activity of anti-IL-31RA antibodies:

[0289] A Baf3 cell line transfected with nucleotide sequences of the full length canine IL-31RA chain and the full length canine Oncostatin M receptor (OSMR) chain was used to assess the ability of anti-IL-31RA antibodies to inhibit STAT-3 activation. The cell line was also transfected with a luciferase reporter gene. The nucleotide sequences of these receptors were prepared by chemical synthesis and then cloned into expression plasmids suitable for expressing the corresponding proteins in baf3 cells.

[0290] Biological activity of anti-IL-31RA antibodies: Stat-3 inhibition in Baf3 cells

[0291] The ability of anti-IL-31 receptor alpha antibodies to inhibit STAT-3 activation in Baf3 cells was assessed as follows:

[0292] 1. Prepare a cell suspension with a viable cell density of 5 x 10 5 cells / mL and add 200 μL (1 x 10 5 cells / well) to each well of a 96-well tissue culture plate. Incubate the plate in a humidified 37°C incubator for 22-24 hours.

[0293] 2. Remove the media from the plate and add 200 μL Eagle's Minimum Essential Media (EMEM) to each well of the cell plate. Place the plate back in the 37°C incubator for 2 hours.

[0294] 3. Remove the media from the cell plate and add 50 μL / well of supernatant from anti-IL-31RA hybridomas grown in PBS. Incubate the plate at 37°C for 1 hour. Add 50 μL / well of 80 ng / mL cIL-31 diluted in PBS and incubate the plate at 37°C for 5 minutes.

[0295] 4. Remove the media from the plate and add 100 μL / well of freshly prepared 1 x lysis buffer to all wells. Shake the plate on a plate shaker at room temperature at 350 rpm for 10 minutes.

[0296] At this point, the cell lysate can be stored frozen at -20°C.

[0297] 5. pSTAT-3 AlphaLISA:

[0298] - Prepare reagents from the Ultra TM p-STAT3 Detection Kit according to the kit instructions.

[0299] - Transfer 30 μL of cell lysate to a 96-well half-area plate and add 15 μL / well Acceptor Mix to the cell lysate. Seal the plate and incubate at room temperature for 1 hour.

[0300] - Add 15 μL / well of Donor Mix to the cell plate. Seal the plate, cover with foil and incubate at room temperature for one hour or overnight. Note - donor mix is light sensitive.

[0301] - Read the plate using the Alpha Screen Assay setup on a Perkin Elmer EnVison.

[0302] Example 4

[0303] Antibodies to canine IL-31

[0304] Antibodies useful in the present application are those described in US 9,206,253 B2 and US 10,150,810 B2. Preferably, these antibodies have the following light and heavy chain sequences:

[0305] Canine heavy chain sequence from mouse antibody clone M14 and canine IgG-B:

[0306] [SEQ ID NO: 10]

[0307] EVQLVESGPSLVKPGGSLRLTCSVTGDSITSGYWNWIRKFPGNKLEYMGYISYSGITDYNPSLKSRITISRDTSKNQYYLQLNSVTTEDTATYYCARYGNYGYAMDYWGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVWDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRWSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGK

[0308] Caninized light chain sequence from mouse antibody clone M14 and canine light chain constant region: [SEQ ID NO: 11]

[0309] DIVMTQSPASLSVSLGQRATISCRASESVDTYGNSFMHWYQQKPGQSPKLLIYRASNLESGIPARFGGSGSGTDFTLTIDPVQADDVATYYCQQSYEDPWTFGGGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASWCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD

[0310] Zoe-LC: Caninized light chain sequence: [SEQ ID NO: 12]

[0311] EIVMTQSPASLSLSQEEKVTITCKASQSVSFAGTGLMHWYQQKPGQAPKLLIYRASNLEAGVPSRFSGSGSGTDFSFTISSLEPEDVAVYYCQQSREYPWTFGQGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSEC

[0312] Zoe-HC: Caninized heavy chain sequence: [SEQ ID NO: 13]

[0313] EVQLVESGGDLVKPGGSLRLSCVASGFTFSNYGMSWVRQAPGKGLQWVATISYGGSYTYYPDNIKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCVRGYGYDTMDYWGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

[0314] Example 5

[0315] Caninized antibodies to IL-31RA

[0316] IL-31 Receptor Alpha C10A12 VH1 -CIGGBM [SEQ ID NO: 51]

[0317] EVQLVESGGDLVKPGGSLRLSCVASGFTFSNYYMAWVRQAPGKGLQWVASISTGGGNTYYRDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCAKHGTLYFDYWGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGK

[0318] IL-31 receptor alpha C10A12 VL5-CCL [SEQ ID NO: 52]

[0319] QPVLTQPPSLSASLGTTARLTCERSSGDIGDSYVSWYQQKPGSPPRDLLYVDDQRPSGVSKSFSGSKDTSANAGLLLISGLQPEDEADYYCQSYDSNIDGPVFGGGTHLTVLGQPKASPSVTLFPPSSEELGANKATLVCLISDFYPSGVTVAWKADGSPVTQGVETTKPSKQSNNKYAASSYLSLTPDKWKSHSSFSCLVTHEGSTVEKKVAPAECS

[0320] Example 6

[0321] Bispecific binding partners and bispecific antibodies

[0322] The bispecific binding partner of the present invention is an artificial protein molecule formed by the binding of one heavy chain (HC) and one light chain (LC) of a monoclonal antibody to a fusion protein consisting of an IgG Fc (e.g., IgG-B) fused to another protein moiety. Bispecific binding partners comprising a canine IL-22RA1-Fc fusion protein or a canine IL-22RA2-Fc fusion protein and an anti-canine IL-31 antibody can be constructed by introducing specific mutations in the light chain or heavy chain of the anti-IL-31 and in the Fc moiety of the fusion protein so as to favor the formation of heterodimers over homodimers. This means, for example, forcing the heavy chain of the canine IL-31 antibody to pair with the Fc of the canine IL-22RA1-Fc fusion protein rather than with the second heavy chain of the canine IL-31 antibody.

[0323] An example of an IL-31 antibody / IL-22RA1 Fc fusion protein bispecific binding partner or an IL-31 antibody / IL-22RA2-Fc fusion protein bispecific binding partner is given by the association of proteins representing three (3) amino acid sequences: (i) the light chain of caninized M14, the heavy chain of caninized M14 and canine IL-22RA1-Fc fusion protein or (ii) the light chain of caninized M14, the heavy chain of caninized M14 and canine IL-22RA2-Fc fusion protein. Another example is given by the association of proteins representing three (3) amino acid sequences: (a) the light chain of Zoe-LC, the heavy chain of Zoe-HC and canine IL-22RA1-Fc fusion protein or (b) the light chain of Zoe-LC, the heavy chain of Zoe-HC and canine IL-22RA2-Fc fusion protein.

[0324] Bispecific antibodies are artificial protein molecules that can target two different antigens simultaneously. One preferred type of bispecific antibody is an IgG-like antibody composed of four different polypeptide chains. Examples of bispecific antibodies of the present invention include antibody molecules that can target IL-31RA and IL-4R α simultaneously. Such antibodies are formed by the association of one HC and one LC chain having specificity for IL-4R α with one HC and one LC having specificity for IL-31RA. Each heavy and light chain is modified with specific substitutions / mutations of its amino acid sequence to favor the association of the HC and LC of the IL-4R α antibody with the HC and LC of the IL-31RA antibody with each other and simultaneously favoring the association of the HC from the IL-4R α antibody with the HC of the IL-31RA antibody rather than each HC with itself. A list of amino acid substitutions in the antibody HC that favor the formation of HC heterodimers is presented in Table 2A above.

[0325] Canine IgG-B Fc was first defined by Tang et al. [Vet Immunology & Immunopathology, 80:259-270 (2001)] as comprising the amino acid sequence of SEQ ID NO: 14 as set forth below.

[0326]

[0327] Canine IgG-Bm differs from naturally occurring canine IgG-B in that it comprises two (2) amino acid residue substitutions, D31A and N63A, in the amino acid sequence of SEQ ID NO: 14 of IgG B, i.e., the aspartic acid residue (D) at position 31 and the asparagine residue (N) at position 63 of SEQ ID NO: 14 are replaced with an alanine residue (A). The positions of these residues in the amino acid sequence are indicated in bold and underlined. These two amino acid residue substitutions serve to significantly reduce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of naturally occurring canine IgG-B [see US 10,106,607 B2, the contents of which are incorporated by reference in their entirety].

[0328] In addition, potential ways or combinations of specific amino acid substitutions in the Fc or Fc portion of a canine IL-31 antibody or canine IL-22RA1-Fc fusion protein or canine IL-22RA2-Fc fusion protein can be used to facilitate heterodimer formation are provided in Table 2A below. These substitutions favor a knob-in-hole approach to heavy chain heterodimerization, or favor electrostatic attraction between different heavy chains that also allows for heterodimerization [for a thorough discussion of these amino acid substitutions see Moore et al., Methods, 154:38-50 (2019) and Brinkmann & Kontermann, MABS, 9:182-212 (2017)]. In the case of the amino acid substitutions listed in Table 2A below, in some embodiments, chain 1 refers to a canine IL-31 heavy chain (HC) and chain 2 refers to a canine IL-22RA1-Fc fusion protein or canine IL-22RA2-Fc fusion protein. In other embodiments, chain 1 refers to a canine IL-22RA1-Fc fusion protein or canine IL-22RA2-Fc fusion protein and chain 2 refers to a canine IL-31 heavy chain (HC). In addition, in some embodiments, chain 1 refers to an anti-IL-31RA heavy chain (HC) and chain 2 refers to an anti-IL-4R α HC. In other embodiments, chain 1 refers to an anti-IL-4R α HC, chain 2 refers to an anti-IL-31RA HC. In yet other embodiments, chain 1 refers to an IL-22 HC and chain 2 refers to an anti-IL-31 HC. In other embodiments, chain 1 refers to an anti-IL-31 HC and chain 2 refers to an IL-22 HC.

[0329] Furthermore, in a domain swap, the variable light (VL) domain can be swapped with the variable heavy (VH) domain, i.e., the exchange of one of the various domains within a fab fragment of an antibody with another domain. Light chains that are candidates for inclusion in a bispecific antibody (such as those listed above) can be made suitable for this purpose by incorporating them into, for example, an antibody domain arrangement that swaps the position of the light chain constant (CL) domain with the position of the CH1 domain in one of the antibody pairs, thereby creating a VL-CH1 and a VH-CL1, while leaving the light chain in the other antibody pair of the bispecific unaltered. Other options are possible as well, such as a swap of the CL-VL domain with the CH1-VH domain or a swap of the VL and VH domains in one of the antibody pairs. Table 2B provides the following examples: (i) the amino acid sequence at the junction of the VH-CL domain and (ii) the amino acid sequence at the junction of the VL-CH1 domain after such a domain swap. In another case, the entire fab fragment can be swapped such that the VH-CH1 domain is swapped for a VL-CL domain.

[0330] Accordingly, to optimally provide an IgG-like bispecific antibody, two types of modifications can be made: a) modification of the Fc of the antibody to favor heterodimerization of the heavy chains over homodimerization, and b) modification of the light chains to favor binding of each of the two light chains to its cognate heavy chain. The amino acid substitutions described in Table 2 can be used in the context of chain 1 and chain 2 to modify the Fc. To make light chains suitable for inclusion in a bispecific antibody, the method known as cross-mab can be employed [Klein et al., Methods: 154:21-23 (2019); Klein et al., MABS, 8:1010-1020 (2016); and Schaefer et al., Proc. Nat’l Acad. Sci. 108:11187-11192 (2011)]. Using the cross-mab technology, light chains that are candidates for inclusion in a bispecific antibody (such as the light chains of the present invention) can be made suitable for this purpose by incorporating them into, for example, an antibody domain arrangement that swaps the position of the light chain constant (CL) domain with the position of the CH1 domain in one of the antibody pairs, while leaving the light chain in the other antibody pair of the bispecific unaltered. Other options are possible as well, such as a swap of the CL-VL domain with the CH1-VH domain or a swap of the VL and VH domains in one of the antibody pairs, as described above.

[0331] Table 2A

[0332] Amino acid substitutions for canine IGG-B FC (SEQ ID NO: 14)

[0333] Numbering Chain 1 Chain 2 1 T132W T132S / L134A / Y175V 2 T132W / S120C T132S / L134A / Y175V / Y115C 3 K177D / R160D D167K / E122K 4 S130H / F173A Y115T / T162F 5 F173L K177R 6 T132L / R160L / T162W L117Y / F173A / Y175V 7 K126E / K177W S113R / D167V / F173T 8 K126E / K177W / Y115C S113R / D167V / F173T / S120C 9 K136E / K177W E123N / D167V / F173T 10 K126D / D167M / Y175A Q111R / S113R / T132V / K177V 11 Y115S / K136Y / T132M / K177V E122G / E123D / S130Q / Y175A 12 L117D / L134E L117K / T132K 13 L134D / K136S E122Q / S130K

[0334] Table 2B

[0335] Kappa and lambda crossover sequences

[0336] Domain swapping of CL and CH1

[0337]

[0338]

[0339] FW4 (FR4) of the VH or VL variable region, font slightly enlarged; a stretch of an "elbow" sequence that can contain a pair of added amino acids (in bold); the entire "elbow" sequence is underlined; the N-terminus of the CH1 or CL region is indicated in italics [see, Klein et al., MABS 8:1010-1020 (2016) and Schaefer, et al., Proc. Nat'l. Acad. Sci., 108:11187-11192 (2011)].

[0340] LC amino acid of caninized M14: [SEQ ID NO: 11]

[0341] DIVMTQSPASLSVSLGQRATISCRASESVDTYGNSFMHWYQQKPGQSPKLLIYRASNLESGIPARFGGSGSGTDFTLTIDPVQADDVATYYCQQSYEDPWTFGGGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASWCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD

[0342] HC amino acid of caninized M14: [SEQ ID NO: 15] This amino acid sequence is identical to SEQ ID NO: 10, except for the amino acids in bold and underlined:

[0343]

[0344] cIL-22RA1-FcB with amino acid substitutions to promote heterodimerization: [SEQ ID NO: 16]

[0345]

[0346] cIL-22RA2-FcB with mutations to promote heterodimerization: [SEQ ID NO: 17]

[0347]

[0348]

[0349] Zoe-HC: Caninized heavy chain sequence: [SEQ ID NO: 18]

[0350]

[0351] Modified IL-4R α Examples of heavy chains of antibodies for use in the formation of bispecific antibodies.

[0352] IL-4 Receptor alpha c152H11 VL3-cCLk-s (light chain): [SEQ ID NO: 19]

[0353] EIVMTQSPASLSLSQEEKVTITCKASQNVGTNVAWYQQKPGQAPKLLIYSASYRYSGLPDRFSGSGSGTDFSFTISSLEPEDVAEFFCQQYNSYPYTFGQGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD

[0354] Modified IL-4 Receptor alpha c152H11 VH3-cIgG-Bm (heavy chain): [SEQ ID NO: 20]

[0355]

[0356] IL-4 Receptor alpha c146E2 VL3-cCLk-s (light chain): [SEQ ID NO: 21]

[0357] DIVLTQTPLSLSVSPGETASIYCRASESVDSYGNSFLNWYQQKPGQPPKLLIYRASNLASEIPDRFSGSGSRTEFTLKISRVEADDAGVYYCQQNYENPRTFGQGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD

[0358] Modified IL-4 Receptor Alpha c146E2 VH3-cIgG-Bm: (Heavy Chain): [SEQ ID NO: 22]

[0359]

[0360]

[0361] Modified IL-31 Receptor Alpha Caninized Heavy Chain 10A12 VH1-CIGG BM [SEQ ID NO: 53]

[0362]

[0363] Table 3

[0364] Table of Sequences (See also Tables 1A and IB above)

[0365]

[0366]

[0367] The scope of the application is not intended to be limited to the particular embodiments described herein. Indeed, a variety of modifications to the application can be made without departing from the spirit of the application. Such modifications are intended to fall within the scope of the following claims.

Claims

1. A composition for treating canine atopic dermatitis, comprising a fusion protein that binds to canine IL-22, a caninized anti-pruritic antibody, and a caninized anti-inflammatory antibody; wherein the fusion protein that binds to canine IL-22 is selected from the group consisting of a canine IL-22RA1-Fc fusion protein and a canine IL-22RA2-Fc fusion protein.

2. The composition of claim 1, wherein the fusion protein is the canine IL-22RA1-Fc fusion protein.

3. The composition of claim 2, wherein the canine IL-22RA1-Fc fusion protein comprises the amino acid sequence of SEQ ID NO:

16.

4. The composition of claim 1, wherein the fusion protein is the canine IL-22RA2-Fc fusion protein.

5. The composition of claim 4, wherein the IL-22RA2-canine Fc fusion protein comprises the amino acid sequence of SEQ ID NO:

17.

6. The composition of claims 1-5, wherein the bispecific binding partner comprises the fusion protein and a monomer of the caninized anti-pruritic antibody comprising a heavy chain and a light chain.

7. The composition of claim 6, wherein the caninized anti-pruritic antibody is a caninized interleukin 31 (IL-31) antibody.

8. The composition of claim 7, wherein the caninized IL-31 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 12 and a heavy chain comprising the amino acid sequence of SEQ ID NO:

18. 9 . The composition of claim 7 , wherein the caninized IL-31 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 11 and a heavy chain comprising the amino acid sequence of SEQ ID NO:

15.

10. The composition according to any one of claims 6 to 9, wherein the light chain of the caninized IL-31 antibody is modified to comprise the heavy chain constant region 1 (CH1) from the heavy chain of the caninized IL-31 antibody instead of the constant light chain domain (CL); and wherein the heavy chain of the caninized IL-31 antibody is modified to comprise the constant light chain domain (CL) from the light chain of the caninized IL-31 antibody instead of the CH1.

11. The composition of claims 1-10, wherein the caninized anti-inflammatory antibody is a caninized IL-4Rα antibody. 12 . The composition of claim 11 , wherein the caninized IL-4Rα antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 4 and a heavy chain comprising the amino acid sequence of SEQ ID NO:

5. 13 . The composition of claim 11 , wherein the caninized IL-4Rα antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 6 and a heavy chain comprising the amino acid sequence of SEQ ID NO:

7.

14. The composition of any one of claims 1, 2 or 4, wherein the bispecific antibody comprises a monomer comprising the heavy chain and the light chain of the caninized anti-pruritic antibody and a monomer comprising the heavy chain and the light chain of the caninized anti-inflammatory antibody.

15. The composition of claim 14, wherein the caninized anti-inflammatory antibody is a caninized interleukin-4 receptor alpha (IL-4Rα) antibody. The composition of claim 15 , wherein the caninized IL-4Rα antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 19 and a heavy chain comprising the amino acid sequence of SEQ ID NO:

20. The composition of claim 15 , wherein the caninized IL-4Rα antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 21 and a heavy chain comprising the amino acid sequence of SEQ ID NO:

22.

18. The composition of any one of claims 15-17, wherein the light chain of the caninized IL-4Rα antibody is modified to comprise the heavy chain constant region 1 (CH1) from the heavy chain of the caninized IL-4Rα antibody in place of the constant light chain domain (CL); and wherein the heavy chain of the caninized IL-4Rα antibody is modified to comprise the constant light chain domain (CL) from the light chain of the caninized IL-4Rα antibody in place of the CH1.

19. The composition of any one of claims 14-18, wherein the caninized anti-pruritic antibody is a caninized interleukin 31 receptor alpha (IL-31RA) antibody.

20. The composition of claim 19, wherein the caninized IL-31RA antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 52 and a heavy chain comprising the amino acid sequence of SEQ ID NO:

53.

21. The composition of any one of claims 14-20, wherein the fusion protein is the canine IL-22RA1-Fc fusion protein.

22. The composition of claim 21, wherein the canine IL-22RA1-Fc fusion protein comprises the amino acid sequence of SEQ ID NO:

2.

23. The composition of any one of claims 14-20, wherein the fusion protein is the canine IL-22RA2-Fc fusion protein.

24. The composition of claim 23, wherein the IL-22RA2-canine Fc fusion protein comprises the amino acid sequence of SEQ ID NO:

3.

25. The composition of claims 1-24, further comprising one or more additional components selected from the group consisting of a Janus kinase (JAK) inhibitor, a spleen tyrosine kinase (SYK) inhibitor, or an antagonist of a chemotactic receptor-homologous molecule expressed on TH2 cells.

26. The composition of claim 25, wherein the JAK inhibitor is selected from the group consisting of: where R 1 is C optionally substituted by hydroxy 1-4 Alkyl, and pharmaceutically acceptable salts thereof; and and pharmaceutically acceptable salts thereof.

27. A method of treating atopic dermatitis comprising administering the composition of claims 1-26 to a canine suffering from atopic dermatitis.

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