Bispecific caninized antibodies and bispecific binding partners for treating atopic dermatitis

Abstract

The present invention provides 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. The present invention further provides a composition comprising a bispecific binding partner comprising the fusion protein and a caninized anti-pruritic antibody or a caninized anti-inflammatory antibody, or a bispecific antibody comprising a caninized anti-pruritic antibody and a caninized anti-inflammatory antibody, and further comprising a fusion protein that binds to canine IL-22.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 092,294, filed October 15, 2020, U.S. Patent Application No. 63 / 092,296, filed October 15, 2020, U.S. Patent Application No. 63 / 015,209, filed April 24, 2020, U.S. Patent Application No. 63 / 015,220, filed April 24, 2020, U.S. Patent Application No. 62 / 951,778, filed December 20, 2019, and U.S. Patent Application No. 62 / 951,793, filed December 20, 2019, the contents of all of which are incorporated herein by reference in their entireties.

[0002] The present invention relates to a composition for treating atopic dermatitis in dogs, comprising a fusion protein that binds to canine IL-22, a caninized anti-pruritic antibody, and a caninized anti-inflammatory antibody. The present invention further relates to a bispecific antibody comprising the fusion protein and a caninized anti-pruritic antibody or a caninized anti-inflammatory antibody, or a bispecific antibody comprising a caninized anti-pruritic antibody and a caninized anti-inflammatory antibody, and further comprising a fusion protein that binds to canine IL-22. [Background technology]

[0003] The immune system comprises a network of resident and recirculating specialized cells that function cooperatively to protect the host from infectious diseases and cancer. The immune system's ability to perform this function depends largely on the biological activity of a group of proteins secreted by white blood cells and collectively known as interleukins. Three well-studied interleukins have been identified: interleukin-4 (IL-4), interleukin-31 (IL-31), and interleukin-22 (IL-22). IL-4, IL-31, and IL-22 are key cytokines for generating the immune responses required for protection against extracellular pathogens (e.g., tissue- or intraluminal parasites). However, these cytokines also play a role in the pathogenesis of allergic diseases in humans and animals, including atopic dermatitis.

[0004] Atopic dermatitis (AD) is a recurrent, pruritic, and chronic inflammatory skin disease characterized by immune system dysregulation and epidermal barrier abnormalities in humans. The pathological and immunological characteristics of atopic dermatitis have been the subject of extensive investigation (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 condition 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 characterized by skin infiltration by various immune cells and CD4, including a predominance of IL-4, IL-13, and IL-31. + It shows striking similarities to the pathogenesis of human atopic dermatitis, including a Th2-polarized cytokine environment. Furthermore, IL-22 is involved in the excessive epidermal proliferation that leads to the epidermal thickening characteristic of atopic dermatitis.

[0005] For example, antibodies against canine IL-31 have been shown to have a significant effect on pruritus associated with atopic dermatitis in dogs [U.S. Patent No. 8,790,651; U.S. Patent No. 10,093,731]. Furthermore, antibodies against human IL-31 receptor alpha (IL-31RA) 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)]. Therefore, blocking IL-31 binding to its receptor IL-31RA reduces pruritus associated with atopic dermatitis.

[0006] Monoclonal antibodies raised against human interleukin-4 receptor α have been developed, and some of these antibodies have been extensively tested for their therapeutic efficacy in treating atopic dermatitis in humans (see, e.g., U.S. Patent Application Publication No. 2015 / 0017176). More recently, the canine IL-4R of canine IL-4 has been shown to inhibit the canine IL-4 receptor α. α Canine interleukin-4 receptor alpha (canine IL-4Rα, canine IL-4R) blocks binding to α , cIL-4Rα or cIL-4R α A caninized antibody against canine IL-4R has also been disclosed (U.S. Patent Application Publication No. 2018 / 0346580, the entire contents of which are incorporated herein by reference). Since type II IL-4 receptor consists of the IL-4 receptor α chain and the IL-13 receptor α1 chain, canine IL-4R can block the binding of both canine IL-4 and canine IL-13 to type II canine IL-4 receptor. α Antibodies against α-glucan have been obtained that help block inflammation associated with atopic dermatitis [US Patent Application Publication No. 2018 / 0346580].

[0007] Interleukin-22 (IL-22) belongs to the IL-10 cytokine family. IL-22 specifically binds to and signals through a receptor complex consisting of a heterodimeric complex of IL-10R2 (also known as IL-10Rβ) and the interleukin-22 receptor (IL-22R) [see Lee et al., Pharmacology Research & Perspectives, Pages 1-13 (2018: e00434)]. The interleukin-22 receptor is also known as interleukin-22R, α1; 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 during wound healing, and its deficiency can promote tumorigenesis [Huber et al., Nature 491:259-263(2012)].

[0008] Interleukin-22 binding protein (IL-22BP), also known as IL-22RA2, CRF2-10, and CRF2-X, is important for limiting epidermal thickening, a hallmark of atopic dermatitis, by binding to IL-22, thereby blocking the interaction of IL-22 with IL-22R and thus the signaling pathway leading to epithelial 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 regulates the pro-inflammatory effects of IL-22 (e.g., neutrophil infiltration) but not those of IL-10 [Weber, et al., Infect. Immun. 75:1690-1697(2007)]. Thus, IL-22BP acts as an antiproliferative agent by blocking the interaction of IL-22 with IL-22R.

[0009] Furthermore, a biosynthetic binding partner for IL-22, a fusion protein containing the extracellular domain of human IL-22RA1 [AAH29273.1; Pro-18-Thr228] fused to the N-terminus of the Fc region of mouse IgG2a [commercially available 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 [Creative BioMart data sheet].

[0010] Pharmaceuticals that have been proven and / or are expected to aid in the treatment of atopic dermatitis include Janus kinase (JAK) inhibitors (see, e.g., U.S. Pat. Nos. 8,133,899; 8,987,283; WO 2018 / 108969), spleen tyrosine kinase (SYK) inhibitors (see, e.g., U.S. Pat. No. 8,759,366), and antagonists to chemoattractant receptor-homologous molecules expressed on TH2 cells (see, e.g., U.S. Pat. Nos. 7,696,222, 8,546,422, 8,637,541, and 8,546,422).

[0011] However, despite recent successes in treating atopic dermatitis, none of the current treatments in use provide significant effects on skin inflammation and rapid onset of antipruritic effects, while also improving skin barrier function. Therefore, there is a need to design better treatments that can simultaneously address these three symptoms of atopic dermatitis.

[0012] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application. [Prior art documents] [Patent documents]

[0013]

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Non-licensed literature

[0014] [Non-licensed document 1] Rahman et al.,Inflammation & Allergy-drug target 10:486-496(2011) [Non-licensed document 2] Harskamp et al., Seminar in Cutaneous Medicine and Surgery 32:132-139(2013) [Non-licensed document 3] Nuttall et al., Veterinary Records 172(8):201-207(2013)

Non-licensed Document 4

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

[0016] Thus, the present invention includes a composition for treating atopic dermatitis in dogs, comprising a fusion protein that binds to canine interleukin IL-22 (IL-22), a caninized anti-pruritic antibody, and a caninized anti-inflammatory antibody. In certain embodiments, the fusion protein that binds to canine IL-22 is a canine IL-22RA1-Fc fusion protein. In a related embodiment, the fusion protein that binds to canine IL-22 is a canine IL-22RA2-Fc fusion protein.

[0017] In certain embodiments, the composition comprises a caninized anti-inflammatory antibody and a bispecific binding partner comprising a canine IL-22RA1-Fc fusion protein and a caninized anti-pruritic antibody monomer. In certain embodiments, the caninized anti-pruritic antibody is a caninized interleukin-31 (cIL-31) antibody. In certain 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 certain embodiments, the light chain of the caninized IL-31 antibody has been modified to comprise heavy chain constant region 1 (CH1) from the heavy chain of the caninized IL-31 antibody in place of the constant light domain (CL), and the heavy chain of the caninized IL-31 antibody has been modified to comprise the constant light domain (CL) from the light chain of the caninized IL-31 antibody in place of the CH1. In certain 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 certain 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 a related embodiment, the composition comprises a caninized anti-inflammatory antibody and a bispecific binding partner comprising a canine IL-22RA2-Fc fusion protein and a caninized anti-pruritic antibody monomer. In certain embodiments, the caninized anti-pruritic antibody is a caninized interleukin-31 (cIL-31) antibody. In certain 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 certain embodiments, the light chain of the caninized IL-31 antibody has been modified to comprise heavy chain constant region 1 (CH1) from the heavy chain of the caninized IL-31 antibody in place of the constant light domain (CL), and the heavy chain of the caninized IL-31 antibody has been modified to comprise the constant light domain (CL) from the light chain of the caninized IL-31 antibody in place of the CH1. In certain 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 certain 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 caninized anti-pruritic antibody monomer comprising a heavy chain and a light chain and a caninized anti-inflammatory antibody monomer 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-4Rα 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-4Rα antibody. In particular embodiments, 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. In a related embodiment, 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. In certain embodiments, the light chain of the caninized IL-4Rα antibody has been modified to include the heavy chain constant region 1 (CH1) from the heavy chain of the caninized IL-4Rα antibody in place of the constant light domain (CL), and the heavy chain of the caninized IL-4Rα antibody has been modified to include the constant light domain (CL) from the light chain of the caninized IL-4Rα antibody in place of the CH1. In certain embodiments, the caninized anti-pruritic antibody of the bispecific antibody is a monomeric 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 an even more specific embodiment, the canine IL-22RA1-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 2.

[0020] In a related embodiment, the composition comprises a canine IL-22RA2-Fc fusion protein and a bispecific antibody comprising a caninized anti-pruritic antibody monomer comprising a heavy chain and a light chain and a caninized anti-inflammatory antibody monomer 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-4Rα 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-4Rα antibody. In particular embodiments, 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. In a related embodiment, 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. In certain embodiments, the light chain of the caninized IL-4Rα antibody has been modified to include the heavy chain constant region 1 (CH1) from the heavy chain of the caninized IL-4Rα antibody in place of the constant light domain (CL), and the heavy chain of the caninized IL-4Rα antibody has been modified to include the constant light domain (CL) from the light chain of the caninized IL-4Rα antibody in place of the CH1. In certain embodiments, the caninized anti-pruritic antibody of the bispecific antibody is a monomeric 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 an even more specific embodiment, the canine IL-22RA2-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 3.

[0021] The present invention further provides bispecific binding partners of the present invention.

[0022] In certain embodiments, the bispecific binding partner comprises a canine IL-22RA1-Fc fusion protein and a caninized anti-pruritic antibody monomer. In particular such embodiments, 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 certain embodiments, the light chain of the caninized IL-31 antibody has been modified to comprise heavy chain constant region 1 (CH1) from the heavy chain of the caninized IL-31 antibody in place of the constant light domain (CL), and the heavy chain of the caninized IL-31 antibody has been modified to comprise the constant light domain (CL) from the light chain of the caninized IL-31 antibody in place of the CH1. In a more specific embodiment, the canine IL-22RA1-Fc fusion protein comprises the amino acid sequence of SEQ ID NO:16.

[0023] In a related embodiment, the bispecific binding partner comprises a canine IL-22RA2-Fc fusion protein and a caninized anti-pruritic antibody monomer. In particular embodiments of this type, the caninized anti-pruritic antibody is a caninized interleukin-31 (cIL-31) antibody. In a more particular embodiment, 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 particular embodiments, the light chain of the caninized IL-31 antibody has been modified to comprise heavy chain constant region 1 (CH1) from the heavy chain of the caninized IL-31 antibody in place of the constant light domain (CL), and the heavy chain of the caninized IL-31 antibody has been modified to comprise the constant light domain (CL) from the light chain of the caninized IL-31 antibody in place of the CH1. In a more specific embodiment, the canine IL-22RA2-Fc fusion protein comprises the amino acid sequence of SEQ ID NO:17.

[0024] In yet other embodiments, compositions comprising the antibodies and / or bispecific antibodies and / or bispecific binding partners and / or fusion proteins of the present invention further comprise one or more additional therapeutic components. In one such embodiment, the therapeutic component is a Janus kinase (JAK) inhibitor. In a particular embodiment of this type, the JAK inhibitor is oclacitinib and its pharmaceutically acceptable salts. In another embodiment, the JAK inhibitor is 1-[(3R,4S)-4-cyanotetrahydropyran-3-yl]-3-[(2-fluoro-6-methoxy-4-pyridyl)amino]pyrazole-4-carboxamide and its pharmaceutically acceptable salts. In another embodiment, the therapeutic component is a spleen tyrosine kinase (SYK) inhibitor. In certain such embodiments, 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 yet another embodiment, the therapeutic component is an antagonist of a chemoattractant receptor homologous molecule expressed on TH2 cells.

[0025] The present invention further provides a method of treating atopic dermatitis, comprising administering one of the compositions described above to a dog with atopic dermatitis.

[0026] In yet another aspect, the present invention provides a method for producing canine interleukin-4 receptor alpha (IL-4R α In one such embodiment, an isolated mammalian antibody or antigen-binding fragment thereof specifically binds 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: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.

[0027] In another embodiment, 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 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.

[0028] In certain embodiments, the isolated mammalian antibody or antigen-binding fragment thereof is a canine IL-4R α binds to canine IL-4R α In a more particular embodiment, the isolated mammalian antibody blocks the binding of IL-4R to canine interleukin-4. α or the antigen-binding fragment thereof is a caninized antibody or caninized antigen-binding fragment thereof.

[0029] In certain embodiments, 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 that 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 that antibody. In yet 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 that 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 that antibody.

[0030] These and other aspects of the present invention will be better understood by reference to the following brief description and detailed description of the drawings. [Brief explanation of the drawings]

[0031] [Figure 1] Figure 1 is a graph showing the binding of canine IL-22 to a canine IL-22RA1-Fc fusion protein. The canine IL-22RA1-Fc fusion protein used is a fusion protein of (i) the extracellular domain of canine IL-22RA1 with (ii) canine IgG-B Fc (canine IL-22RA1-FcB). The canine IL-22RA1-FcB fusion protein was tested for its ability to bind to canine IL-22, and the results show that the canine IL-22RA1-FcB fusion protein binds to canine IL-22 specifically and in a dose-dependent manner. [Figure 2]Figure 2 is a graph showing the binding of canine IL-22 to canine IL-22BP (IL-22RA2). The canine IL-22RA2-Fc fusion protein used is a fusion protein of canine (i) IL-22BP (IL-22RA2) with (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 show that the canine IL-22BP-FcB fusion protein binds to canine IL-22 specifically and in a dose-dependent manner. [Figure 3] Figure 3 is a graph showing the inhibition of IL-4-mediated STAT-6 phosphorylation by IL-4Rα (IL-4Rα) antibodies. Three different caninized monoclonal anti-canine IL-4Rα antibodies, designated c4H3, c146E2-H3L3, and c152H11-H3L3, were evaluated for their ability to inhibit αSTAT-6 phosphorylation. The data show that all three antibodies result in dose-dependent inhibition of STAT-6 phosphorylation in the presence of IL-4. The IL-4 control in the absence of IL-4Rα (IL-4Rα) antibodies is shown in the upper left portion of the graph. [Figure 4] Figure 4 is a graph showing the inhibition of IL-13-mediated STAT-6 phosphorylation by IL-4Rα antibodies. Three different caninized monoclonal anti-canine IL-4Rα antibodies, designated c4H3, c146E2-H3L3, and c152H11-H3L3, were evaluated for their ability to inhibit STAT-6 phosphorylation. The data show that all three antibodies result in dose-dependent inhibition of STAT-6 phosphorylation in the presence of IL-13. The IL-13 control in the absence of IL-4Rα (IL-4Rα) antibody is shown in the upper left portion of the graph. [Figure 5] Figure 5 is a graph showing the binding of IL-31 to the extracellular domain of IL-31R (IL-31RA). The extracellular domain (ECD) of canine IL-31RA was tested for its ability to bind to canine IL-31. The results show 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 OF THE INVENTION

[0032] In response to the need for better treatments for atopic dermatitis, the present invention provides formulations and methodologies 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 provide rapid onset of anti-pruritic effects with significant effects on skin inflammation and improved skin barrier function.

[0033] Abbreviation The following abbreviations are used throughout the detailed description and examples of the present invention: ADCC antibody-dependent cytotoxicity CDC Complement-dependent cytotoxicity CDR Complementarity determining region in an immunoglobulin variable region, defined using the Kabat numbering system CHO Chinese hamster ovary EC50 Concentration that produces 50% efficacy or binding ELISA enzyme-linked immunosorbent assay FR Antibody framework region: immunoglobulin variable region excluding the CDR regions. HRP horseradish peroxidase IFN Interferon IC50: Concentration that produces 50% inhibition IgG immunoglobulin G The immunoglobulin alignment and numbering system pioneered by Elvin A. Kabat [Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)] mAb Monoclonal antibody (also Mab or MAb) MES 2-(N-morpholino)ethanesulfonic acid MOA Mechanism of Action NHS Normal Human Serum PCR polymerase chain reaction PK Pharmacokinetics SEB Staphylococcal enterotoxin B TT tetanus toxoid VH immunoglobulin heavy chain variable region VL immunoglobulin light chain variable region VK immunoglobulin kappa light chain variable region

[0034] definition So that the present invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0035] As used in this specification, including the appended claims, the singular forms of words such as "a," "an," and "the" include their corresponding plural references unless the context clearly dictates otherwise.

[0036] "Activation," when applied to a cell or receptor, refers to the activation or treatment of the cell or receptor by a ligand, unless the context or explicitly indicates otherwise. "Ligand" encompasses natural and synthetic ligands, e.g., cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies. "Ligand" also encompasses small molecules, e.g., peptide mimetics of cytokines and peptide mimetics of antibodies. "Activation" can refer to cell activation regulated by internal mechanisms as well as by external or environmental factors.

[0037] The "activity" of a molecule can describe or refer to the molecule's binding to a ligand or receptor, catalytic activity; ability to stimulate gene expression or cell signaling, differentiation or maturation; antigenic activity, modulation of the activity of other molecules, etc. The "activity" of a molecule can also refer to activity in modulating or maintaining cell-cell interactions, e.g., adhesion, or activity in maintaining cellular structure, e.g., cell membrane or cytoskeleton. "Activity" can also refer to specific activity, e.g., [catalytic activity] / [mg protein], or [immune activity] / [mg protein], concentration in a biological compartment, etc. "Activity" can refer to modulation of components of the innate or adaptive immune system.

[0038] "Administration" and "treatment," as applied to an animal, e.g., a canine subject, a cell, a tissue, an organ, or a biological fluid, refer to the contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition with the animal, e.g., a canine subject, a cell, a tissue, an organ, or a biological fluid. Treatment of a cell encompasses contact of a reagent with the cell, as well as contact of a reagent with a fluid in contact with the cell.

[0039] "Administration" and "treatment" also refer to in vitro and ex vivo treatments, e.g., of a cell, with 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 dog, cat, or human), most preferably a dog.

[0040] "Treat" or "treating" means administering a therapeutic agent, such as a composition containing any of the antibodies and / or bispecific antibodies and / or fusion proteins of the invention, internally or externally, to a canine subject or patient who has or is suspected of having one or more symptoms for which the therapeutic agent has therapeutic activity.

[0041] Typically, a therapeutic agent is administered in an amount effective to alleviate and / or ameliorate one or more disease / condition symptoms in the treated subject or population by inducing regression or inhibiting progression of such symptom(s) to any clinically measurable extent. The amount of a therapeutic agent that is effective in alleviating any particular disease / condition symptom (also referred to as a "therapeutically effective amount") may vary depending on factors such as the condition, age, and weight of the patient (e.g., dog), as well as the ability of the pharmaceutical composition to induce a desired response in the subject. Whether a disease / condition symptom has been alleviated or improved can be assessed by any clinical measurement commonly used by veterinarians or other skilled medical professionals to assess the severity or progression of the symptom. An embodiment of the invention (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the target disease / condition symptom(s) in every subject, but should alleviate the target disease / condition symptom(s) in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-squared test, Mann-Whitney U test, Kruskal-Wallis test (H test), Joncke-Terpstra test, and Wilcoxon test.

[0042] "Treatment," when applied to a human, veterinary subject (e.g., a dog), or research subject, refers to therapeutic treatment as well as research and diagnostic uses. When applied to a human, veterinary subject (e.g., a dog), or research subject, or to a cell, tissue, or organ, "treatment" encompasses contacting an antibody and / or bispecific antibody and / or fusion protein of the invention with, for example, a dog or other animal subject, cell, tissue, physiological compartment, or physiological fluid.

[0043] As used herein, the term "dog" includes all domestic dogs, Canis lupus familiaris or Canis familiaris, unless otherwise specified.

[0044] As used herein, the term "cat" refers to any member of the Felidae family, including wild, zoo, and domestic members, including domestic cats, purebred and / or mixed breed pet cats, show cats, laboratory cats, cloned cats, and wild or feral cats.

[0045] As used herein, the term "canine framework" refers to the amino acid sequences of the heavy and light chains of a canine antibody, excluding the hypervariable region residues defined herein as CDR residues. For caninized antibodies, in most embodiments, the amino acid sequences of the native canine CDRs are replaced in both chains with the corresponding foreign CDRs (e.g., from a mouse or rat antibody). The heavy and / or light chains of the canine antibody may contain some foreign non-CDR residues to preserve the conformation of the foreign CDRs in the canine antibody and / or to modify Fc function, for example, as exemplified below and / or as disclosed in U.S. Pat. No. 10,106,607, the entire contents of which are incorporated herein by reference.

[0046] Canine antibodies (also called immunoglobulin G or IgG) are large tetrameric proteins of approximately 150 Kd. Each IgG protein is composed of two identical light chains of approximately 25 Kd each and two identical heavy chains of approximately 50 Kd each. There are four known IgG heavy chain subclasses of canine IgG, designated IgGA, IgGB, IgGC, and IgGD. Two types of light chains exist: kappa and lambda. Each kappa or lambda light chain consists of one variable domain (VL) and one constant domain (CL). Each of the two heavy chains consists of one variable domain (VH) and three constant domains called 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 CH-3). The CH1 domain is connected to the CH2 domain via an amino acid sequence called the "hinge" or "hinge region." In the present invention, the amino acid sequences for 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, called IgG1, IgG2, IgG3, and IgG4. The IgG subclass is primarily determined by the sequence of the hinge region, which differs among the four IgG subclasses. The two heavy chains are linked to each other by disulfide bonds, and each heavy chain is also linked to one of the light chains via a disulfide bond.

[0047] Digestion of IgG antibodies with the enzyme papain cleaves the antibody molecule at the hinge region, resulting in the formation of three fragments. Two of these fragments are identical and consist of the light chain together with the VH and CH1 domains of the heavy chain. These fragments, called "Fab" fragments, contain the antigen-binding site of the antibody. The Fab fragment is the VL-CL chain attached to the VH-CH1 chain by a disulfide bridge. The third fragment resulting from papain digestion is called "Fc" and contains the remainder of the two heavy chains held together by disulfide bonds. Thus, Fc contains a dimer consisting of the CH2 and CH3 domains of each of the two heavy chains. While Fab allows the antibody to bind to its cognate epitope, Fc allows 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" is composed of one light chain (VL and CL domains) and the CH1 and variable region (VH) of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0048] The "fragment crystallizable region," abbreviated as "Fc," corresponds to the CH3-CH2 portion of an antibody that interacts with a cell surface receptor called the Fc receptor. As used herein, particularly with respect to being part of a fusion protein with canine IL-22RA1 or canine IL-22RA2, "Fc" refers to the monomeric CH3-CH2 portion of an antibody. The canine fragment crystallizable region (cFc) of each of the four canine IgGs was first described by Tang et al. [Vet. Immunol. Immunopathol. 80:259-270 (2001); see also Bergeron et al., Vet. Immunol. Immunopathol. 157:31-41 (2014)].

[0049] As used herein, canine Fc "IgG-Bm" refers to canine IgG-B Fc containing two amino acid residue substitutions, D31A and N63A, in the amino acid sequence of SEQ ID NO: 14 of IgG-B (see below). 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 both replaced by alanine residues (A) in IgG-Bm. These two amino acid residue substitutions serve to significantly attenuate the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of naturally occurring canine IgG-B (see U.S. Pat. No. 10,106,607, the entire contents of which are incorporated herein by reference). Additional amino acid substitutions to IgG-Bm are also contemplated, similar to those that can be made in IgG-B, and may include amino acid substitutions that promote heterodimer formation in bispecific antibodies and / or bispecific binding partners, as described below.

[0050] Interleukin-22 (IL-22) specifically binds to a receptor complex consisting of a heterodimeric complex of interleukin-22 receptor and IL-10R2, and transmits signals through the receptor complex. The interleukin-22 receptor is also known as interleukin-22Rα1 (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. Canine IL-22RA1-Fc fusion proteins can be produced through genetic engineering from chemically synthesized nucleic acids encoding the IL-22RA1 extracellular domain fused to the N-terminus of canine IgG Fc.

[0051] Interleukin-22 binding protein (also known as IL-22RA2 or IL-22BP) acts as an antiproliferative agent by blocking the interaction of IL-22 with its receptor (see above). As used herein, "canine IL-22RA2-Fc fusion protein" includes a canine interleukin-22 binding protein fused to canine IgG Fc. Canine IL-22RA2-Fc fusion protein can be produced through genetic engineering from a chemically synthesized nucleic acid encoding canine IL-22RA2 fused to the N-terminus of canine IgG Fc.

[0052] As used herein, "substitution of an amino acid residue" in the amino acid sequence of an antibody with another amino acid residue is equivalent to, for example, "replacing an amino acid residue" with another amino acid residue, and indicates that a 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., an alanine at a particular position in the amino acid sequence can be intentionally replaced with a serine, for example, by recombinant DNA technology. Alternatively, a particular amino acid residue or series of amino acid residues in 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 an antibody produced by a cell to bind to a given region on its antigen, e.g., a region containing an epitope or portion thereof, and / or such that the antibody contains a particular CDR that retains the same canonical structure as the CDR that the particular CDR replaces. Such substitutions / replacements can result in "variant" CDRs and / or mutant antibodies.

[0053] As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological activity. Antibodies can be monomeric, dimeric, or larger multimeric. Thus, the term "antibody" 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 "parent antibody" is an antibody obtained by exposing the immune system to an antigen prior to modification of the antibody for its intended use, such as caninizing an antibody for use as a canine therapeutic antibody.

[0054] As used herein, an antibody and / or bispecific antibody and / or fusion protein and / or bispecific binding partner of the invention that, for example, "blocks" or "is blocking" or "blocking the binding of" a canine receptor to its binding partner (ligand) is an antibody and / or bispecific antibody and / or fusion protein and / or bispecific binding partner that blocks (partially or fully) the binding of the canine receptor to its canine ligand, and vice versa, as determined in a standard binding assay (e.g., BIACore®, ELISA or flow cytometry).

[0055] A "bivalent antibody" comprises two antigen-binding sites. In some instances, the two binding sites have the same antigen specificity.

[0056] As used herein, the terms "bispecific binding partner" and "bispecific binding protein" are used interchangeably and refer to an engineered protein molecule capable of simultaneously targeting two different antigens, formed by the association of the monomeric heavy and light chains of a monoclonal antibody with a fusion protein comprising a canine IgG Fc (or a modified canine IgG Fc, e.g., IgG-Bm, as defined below) genetically fused to another protein moiety that binds to the target protein. When a 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 against canine IL-31 that is linked to a fusion protein comprising the extracellular domain of canine IL-22RA1 genetically fused to canine IgG Fc, as exemplified herein; in this case, the bispecific binding partner can simultaneously target canine IL-31 and canine IL-22.

[0057] As used herein, a "bispecific antibody" is an artificial protein that can simultaneously target two different antigens. One preferred type of bispecific antibody is an IgG-like antibody composed of four different polypeptide chains. Thus, a bispecific antibody may be a heterodimer containing two monomers, each containing a heavy chain and a light chain. The first monomer may be from an antibody directed against one specific antigen, while the second monomer may be from an antibody directed against a different antigen. For example, a particular bispecific antibody of the present invention may be from an antibody directed against IL-31RA and IL-4R. α Such antibodies can simultaneously target IL-4R α It is formed by the association of one heavy chain and one light chain with specificity for IL-31RA with a heavy chain and a light chain with specificity for IL-4R. α Promotes the association of the heavy and light chains of the antibody with each other and the association of the heavy and light chains of the IL-31RA antibody with each other, while at the same time inhibiting the IL-4R α Another IL-4R heavy chain αIL-4R heavy chain, rather than the association of the IL-31RA heavy chain with another IL-31RA heavy chain, or the association of the IL-31RA heavy chain with another IL-31RA heavy chain. α Each of the heavy and light chains can be modified with specific mutations in their amino acid sequences to promote association of the heavy chain from the antibody with the heavy chain of the IL-31RA antibody.

[0058] Within the context of both bispecific antibodies and bispecific binding partners, a "monomer" of an antibody consists of one heavy chain and one light chain of that antibody.

[0059] As used herein, "artificial protein" and "artificial protein molecule" are used interchangeably to refer to a protein (or multimer of proteins, such as dimers, heterodimers, tetramers and heterotetramers) that does not occur in nature, such as an artificial fusion protein or a heterodimer of monomers derived from two different antibodies.

[0060] Typically, an antibody or antigen-binding fragment of the present invention retains at least 10% of its canine antigen-binding activity (compared to the parent antibody), when that activity is expressed on a molar basis. Preferably, an antibody or antigen-binding fragment of the present invention retains 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 antibody or antigen-binding fragment of the present invention may include conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "functionally conservative variants" of the antibody) that do not substantially alter its biological activity.

[0061] "Isolated antibody" refers to a purified state, 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 cell debris and growth medium. In general, the term "isolated" is not intended to refer to the complete absence of such materials, or the absence of water, buffers, or salts, unless present in amounts that would substantially interfere with experimental or therapeutic uses of the binding compounds described herein.

[0062] As used herein, a "chimeric antibody" is an antibody having variable domains derived from a first antibody and constant domains derived from a second antibody, the first and second antibodies being derived from different species. [U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)]. Typically, the variable domains are derived from an antibody derived from a laboratory animal such as a rodent (the "parent antibody"), and the constant domain sequences are derived from an animal subject antibody, e.g., human or canine, so that the resulting chimeric antibody is less likely to provoke an adverse immune response in a human or canine subject, respectively, than the parent (e.g., rodent) antibody.

[0063] As used herein, the term "caninized antibody" refers to a form of antibody that contains sequences from both canine and non-canine (e.g., mouse or rat) antibodies. Generally, caninized antibodies comprise at least one or more, typically substantially all of two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-canine immunoglobulin (e.g., including six CDRs as exemplified below), and all or substantially all of the framework (FR) regions (and typically all or substantially all of the remaining framework) are those of a canine immunoglobulin sequence. As exemplified herein, caninized antibodies comprise both three heavy chain CDRs and three light chain CDRs from a mouse or rat anti-canine antigen antibody, together with a canine or modified canine frame. For example, the modified canine frame contains one or more amino acid changes, as exemplified herein, that further optimize the effectiveness of the caninized antibody to increase binding of the caninized antibody to its canine antigen and / or its ability to block binding of the canine antigen to its natural binding partner.

[0064] The variable regions of each light / heavy chain pair form the antibody binding site. Thus, an intact antibody generally has two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites are generally the same.

[0065] Typically, both heavy and light chain variable domains contain three hypervariable regions, also called complementarity-determining regions (CDRs), located within relatively conserved framework regions (FRs). The CDRs are usually aligned with the framework regions, enabling binding to specific epitopes. Generally, from N- to C-terminus, both light and heavy chain variable domains contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain is generally based on the information in "Sequences of Proteins of Immunological Interest," Kabat, et al., National Institutes of Health, Bethesda, Md., 500-505. th 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)].

[0066] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are involved in antigen binding. The hypervariable region comprises amino acid residues from the "complementarity determining region" or "CDR" (i.e., CDRL1 (or LCDR1), CDRL2 (or LCDR2), and CDRL3 (or LCDR3) in the light chain variable domain and CDRH1 (or HCDR1), CDRH2 (or HCDR2), and CDRH3 (or HCDR3) in the heavy chain variable domain). (See Kabat et al., "Sequences of Proteins of Immunological Interest," 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991), which defines the CDR region of an antibody by sequence; see also Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987), which defines the CDR region of an antibody by structure).

[0067] As used herein, the terms "framework" or "FR" residues refer to variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0068] There are four known IgG heavy chain subtypes of canine IgG, termed IgG-A, IgG-B, IgG-C, and IgG-D. The two known light chain subtypes are termed λ and κ. In certain embodiments of the present invention, aside from binding and activating canine immune cells, canine or caninized antibodies of the present invention directed against their antigens optimally possess the following two attributes: 1. Lack of effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and 2. Easily purified on a large scale using industry standard techniques such as those based on Protein A chromatography.

[0069] None of the naturally occurring canine IgG isotypes meets 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 exhibits ADCC activity. Furthermore, neither IgG-C nor IgG-D can be purified on a Protein A column, yet IgG-D does not exhibit ADCC activity. (IgG-C does have significant ADCC activity.) One way the present invention addresses these problems is by providing modified canine IgG-B antibodies, and / or bispecific antibodies, and / or bispecific binding partners, and / or fusion proteins of the present invention specific for the antigens of the present invention that lack effector functions such as ADCC and can be easily purified using industry-standard Protein A chromatography.

[0070] In an alternative embodiment of the invention, the canine IgG-B or IgG-C antibodies, and / or bispecific antibodies, and / or bispecific binding partners, and / or fusion proteins of the invention specific for the antigens of the invention have not been intentionally modified to eliminate / substantially reduce effector function, such as ADCC, and thus retain ADCC-like effector function.

[0071] As used herein, the term "anti-pruritic agent" is used interchangeably with "antipruritic agent" and is a compound, macromolecule and / or formulation that tends to inhibit, relieve, and / or prevent itch. Anti-pruritic agents are colloquially referred to as antipruritics.

[0072] As used herein, the term "anti-pruritic antibody" is used interchangeably with "antipruritic antibody" and refers to an antibody that can act as an anti-pruritic agent in animals, including mammals such as humans, dogs, and / or cats, particularly with respect to atopic dermatitis. In certain embodiments, the anti-pruritic antibody binds to a specific protein in the IL-31 signaling pathway, such as IL-31 or its receptor IL-31RA. Binding of the anti-pruritic antibody to its corresponding antigen (e.g., IL-31 or IL-31RA) inhibits, for example, the binding of IL-31 to IL-31RA and disrupts and / or prevents successful signaling of this pathway, thereby inhibiting, alleviating, and / or preventing itch that would otherwise be caused by the IL-31 signaling pathway.

[0073] As used herein, an "anti-inflammatory agent" is a compound, macromolecule and / or preparation that reduces inflammation by blocking the interaction of certain substances in the body that cause inflammation.

[0074] As used herein, an "anti-inflammatory antibody" is an antibody that can act as an anti-inflammatory agent in animals, including mammals such as humans, dogs, and / or cats, particularly with respect to atopic dermatitis. In certain embodiments, the anti-inflammatory antibody is an antibody that inhibits IL-4 or the receptor IL-4R. α Anti-inflammatory antibodies bind to specific proteins in the IL-4 / IL-13 signaling pathway, such as IL-4 or IL-4R. α ) is bound to, for example, IL-4R α The present invention inhibits the binding of α-glucan to β-glucan, disrupting and / or preventing signaling of this pathway, thereby disrupting or preventing the chronic inflammation associated with atopic dermatitis.

[0075] As used herein, "antiproliferative agents" refer to compounds, macromolecules, and / or preparations that counteract the induction of epithelial cell proliferation, particularly the induction of keratinocyte cell proliferation, particularly in relation to atopic dermatitis. Interleukin-22 binding protein (IL-22BP) is an example of a naturally occurring antiproliferative agent. Canine IL-22RA2-Fc fusion protein is an example of an artificial canine antiproliferative agent. Canine IL-22RA1-Fc fusion protein is another example of an artificial canine antiproliferative agent.

[0076] As used herein, an "anti-proliferative antibody" refers to an antibody that can act as an anti-proliferative agent in animals, including mammals such as humans, dogs, and / or cats, particularly with respect to atopic dermatitis. In certain embodiments, the anti-proliferative antibody binds to a specific protein in the IL-22 signaling pathway, such as IL-22 or the IL-22 receptor (IL-22R). The binding of the 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, disrupting and / or preventing signal transduction in this pathway, thereby disrupting or preventing keratinocyte cell proliferation associated with atopic dermatitis.

[0077] "Homology" refers to the sequence similarity between two polynucleotide sequences or two polypeptide sequences when they are optimally aligned. If a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if a position in each of the two DNA molecules is occupied by adenine, the molecules are homologous at that position. The percentage of 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 out of 10 positions in the two sequences are identical or homologous when the sequences are optimally aligned, the two sequences are 60% homologous. Generally, comparison is performed when the two sequences are aligned to give the maximum percent homology.

[0078] An "isolated nucleic acid molecule" refers to DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or some combination thereof, wherein the isolated polynucleotide is unaccompanied by all or part of polynucleotides with which it is found in nature, or wherein the isolated nucleic acid molecule is linked to polynucleotides with which it is not naturally linked. It is understood that, in this disclosure, a "nucleic acid molecule comprising" a particular nucleotide sequence does not encompass intact chromosomes. An isolated nucleic acid molecule "comprising" a specified nucleic acid sequence may, in addition to the specified sequence, include coding sequences for up to 10 or up to 20 or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequences that control expression of the coding region of the described nucleic acid sequence, and / or may include vector sequences.

[0079] The term "control sequence" refers to a DNA sequence necessary for the expression of a functionally linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, promoters, and may include operator sequences and ribosome binding sites. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.

[0080] 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 secretory 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 promote translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0081] As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the terms "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of introductions. It is also understood that not all progeny will have precisely identical DNA content due to intentional or unintentional mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where a different designation is intended, it will be clear from the context.

[0082] The present invention provides isolated caninized antibodies and / or bispecific antibodies and / or bispecific binding partners and / or fusion proteins of the present invention, and methods for using the antibodies and / or bispecific antibodies and / or bispecific binding partners and / or these fusion proteins in the treatment of conditions, such as atopic dermatitis in dogs. In dogs, four IgG heavy chains exist, designated A, B, C, and D. These heavy chains represent four distinct 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 via an amino acid sequence called the "hinge" or "hinge region."

[0083] 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 for these heavy chains are also available from the GenBank database. For example, the amino acid sequence of the IgGA heavy chain has accession number AAL35301.1, the IgGB has accession number AAL35302.1, the IgGC has accession number AAL35303.1, and the IgGD has accession number AAL35304.1. Canine antibodies also contain 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 kappa light chain amino acid sequence has accession number ABY57289.1, and the lambda light chain has accession number ABY55569.1.

[0084] Caninized mouse or rat anti-canine antibodies that bind to 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 invention comprising canine IgG-A, IgG-B, IgG-C, and IgG-D heavy chains and / or canine Kappa or Lambda light chains, together with mouse or rat anti-canine antigen CDRs. Accordingly, the invention provides isolated caninized mouse or rat anti-canine antibodies and / or bispecific antibodies and / or bispecific binding partners and / or fusion proteins of the invention that bind to their corresponding canine antigens and block binding of the canine antigens to their natural binding partners.

[0085] Accordingly, the invention further provides caninized mouse or rat antibodies and methods of using the antibodies, and / or bispecific antibodies, and / or bispecific binding partners, and / or fusion proteins of the invention in the treatment of conditions, such as the treatment of atopic dermatitis in dogs.

[0086] The present invention further provides full-length canine heavy chains that can be matched with corresponding light chains to create caninized antibodies. Accordingly, the present invention further provides caninized mouse or rat anti-canine antigen antibodies (including isolated caninized mouse or rat anti-canine antibodies) and / or bispecific antibodies and / or bispecific binding partners comprising the caninized antibodies and / or fusion proteins of the invention, as well as methods of using the antibodies and / or bispecific antibodies and / or bispecific binding partners and / or fusion proteins of the invention in the treatment of conditions, such as the treatment of atopic dermatitis in dogs.

[0087] The present invention also provides antibodies, bispecific antibodies, bispecific binding partners, and / or fusion proteins of the present invention comprising a canine fragment crystallizable region (cFc region), wherein the cFc has been genetically modified to enhance, reduce, or eliminate one or more effector functions. In one embodiment of the present invention, the genetically modified cFc reduces or eliminates one or more effector functions. In another embodiment of the present invention, the genetically modified cFc region 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 certain embodiments, the effector function is enhanced, reduced, or eliminated antibody-dependent cellular cytotoxicity (ADCC). In another embodiment, the effector function is enhanced, reduced, or eliminated complement-dependent cytotoxicity (CDC). In yet another embodiment, the cFc region is genetically modified to enhance, reduce, or eliminate both ADCC and CDC.

[0088] To generate mutant forms of canine IgG lacking effector functions, a number of mutant canine IgGB heavy chains were generated. These mutant forms can contain one or more of the following single or combined substitutions in the Fc portion of the heavy chain amino acid sequence: P4A, D31A, N63A, G64P, T65A, A93G, and P95A. The mutant heavy chains (i.e., containing such amino acid substitutions) were cloned into expression plasmids and transfected into HEK293 cells together with a plasmid containing a gene encoding the light chain. To evaluate their potential for mediating immune effector functions, the Fc γ Intact antibodies expressed and purified from HEK293 cells were evaluated for binding to RI and C1q (see U.S. Pat. No. 10,106,607, the entire contents of which are incorporated herein by reference).

[0089] The present invention also provides a modified canine IgG-D that comprises, in place of its native IgG-D hinge region, a hinge region from: IgG-A: FNECRCTDTPPCPVPEP SEQ ID NO: 23 IgG-B:PKRENGRVPRPPDCPKCPAPEM SEQ ID NO:24; or IgG-C:AKECECKCNCNNCPCPGCGL SEQ ID NO:25.

[0090] Alternatively, the IgG-D hinge region can be genetically modified by substituting serine residues with proline residues, i.e., PKESTCKCI. P PCPVPES, SEQ ID NO: 26 (the proline residue (P) is underlined and shown in bold, replacing the naturally occurring serine residue). Such modifications can result in a canine IgG-D lacking Fab arm exchange. 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 mutants, a nucleic acid encoding the amino acid sequence of canine IgG-D can be modified to encode the modified IgGD. The modified nucleic acid sequence is then cloned into an expression plasmid for protein expression.

[0091] Sequence identity refers to the degree to which the amino acids of two polypeptides are the same at equivalent positions when the two sequences are optimally aligned.As used herein, if the amino acid residues of both sequences are identical, one amino acid sequence is 100% "identical" to a second amino acid sequence.Thus, if 50% of the amino acid residues of the two amino acid sequences are identical, the amino acid sequence is 50% "identical" to the second amino acid sequence.Sequence comparison is performed over a contiguous block of amino acid residues contained by a given protein, for example, a protein or polypeptide portion being compared.In certain embodiments, selected deletions or insertions that would otherwise change the correspondence between two amino acid sequences are taken into account.

[0092] Sequence similarity includes identical residues and non-identical biochemically related amino acids that share similar properties and may be interchangeable.

[0093] "Conservatively modified variants" or "conservative substitutions" refer to the substitution of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.), such that changes can frequently be made without altering the biological activity of the protein. Those skilled in the art generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al., Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.; 1987)). Furthermore, substitution of structurally or functionally similar amino acids is less likely to destroy biological activity. Exemplary conservative substitutions are shown in Table A immediately below.

[0094] [Table 1]

[0095] Function-conservative variants of the antibodies of the present invention are also contemplated by the present invention. As used herein, the term "function-conservative variant" refers to an antibody or fragment in which one or more amino acid residues have been altered without altering desired properties such as antigen affinity and / or specificity. Such variants include, but are not limited to, replacing an amino acid with an amino acid having similar properties, such as the conservative amino acid substitutions in Table A above.

[0096] nucleic acid The present invention further includes nucleic acids encoding the antibodies and / or bispecific antibodies and / or bispecific binding partners and / or fusion proteins of the invention (see, eg, the Examples below).

[0097] Also included in the present invention are 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 the comparison is performed using a BLAST algorithm with the algorithm parameters selected to give the largest match between the respective sequences over the entire length of the respective reference sequences. The present invention further provides and is included in the present invention 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 the comparison is performed using a BLAST algorithm with the algorithm parameters selected to give the largest match between the respective sequences over the entire length of the respective reference sequences.

[0098] As used herein, the percent identity of nucleotide and amino acid sequences can be determined using the C, MacVector (MacVector, Inc. Cary, NC 27519), Vector NTI (Informax, Inc. MD), Oxford Molecular Group PLC (1996) and Clustal W algorithms with default alignment and identity parameters. These commercially available programs can also be used to determine sequence similarity using the same or similar default parameters. Alternatively, for example, Advanced Blast search can be performed under default filter conditions using the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program with default parameters.

[0099] The following references relate to the BLAST algorithm, which is often 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. Met al.,Comput.Appl.Biosci.10:67-70(1994);ALIGNMENT SCORING SYSTEMS:Dayhoff,MO,et al.,“A model of evolutionary change in proteins.”in Atlas of Protein Sequence and Structure, vol. 5, sup. Structure, vol. 5, suppl. 3.” (1978), MODayhoff (ed.), pp. 353-358 (1978), Natl. Biomed. Res. Found., Washington, DC; Altschul, SF, J. Mol. Biol. al.,Methods 3:66-70(1991);Henikoff,S.,et al.,Proc.Natl.Acad.Sci.USA 89:10915-10919(1992);Altschul,SF,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);および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)。.

[0100] The antibodies and / or bispecific antibodies, and / or bispecific binding partners, and / or fusion proteins of the present invention can be recombinantly produced by methods known in the art. Mammalian cell lines available as hosts for expressing 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). These include, among others, 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., HepG2), A549 cells, 3T3 cells, HEK-293 cells, and numerous other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse, 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 an antigen-binding portion or fragment thereof, a light chain and / or an 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 expression of the antibody in the host cell, or more preferably, secretion of the antibody into the culture medium in which the host cell is grown.

[0101] The antibody can be recovered from the culture medium using standard protein purification methods. Furthermore, expression of the antibody of the present invention (or other moieties thereof) from the production cell line can be enhanced using several known techniques. For example, the glutamine synthetase gene expression system (GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or in part in connection with European Patent Nos. 0216846, 0256055, and 0323997, as well as European Patent Application No. 89303964.4.

[0102] Generally, glycoproteins produced in a particular cell line or transgenic animal have a glycosylation pattern characteristic of the glycoprotein produced in that cell line or transgenic animal. Thus, the specific glycosylation pattern of an antibody depends 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 present invention, regardless of the glycosylation pattern the antibody may have. Similarly, in certain embodiments, antibodies with a glycosylation pattern containing only nonfucosylated N-glycans may be advantageous, as these antibodies have typically been shown to exhibit stronger efficacy both 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).

[0103] Antibody and fusion protein engineering In the fusion proteins of the present invention, a heavy chain constant region, e.g., a canine constant region such as IgGA, IgGB, IgGC, or IgGD, or a variant thereof, is conjugated to a protein that binds to IL-22. Thus, a protein that binds to IL-22 can be conjugated to an antibody monomer to form a bispecific binding partner. Such antibodies or antigen-binding fragments also contain a light chain constant region, e.g., a canine light chain constant region such as 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 derived from IgG-B or a modified cFc, such as IgG-Bm, as used herein (see U.S. Pat. No. 10,106,607, incorporated herein by reference in its entirety), and the canine light chain constant region can be derived from kappa.

[0104] The antibodies, and / or bispecific antibodies, and / or bispecific binding partners of the invention can be engineered to contain modifications to the canine framework and / or canine frame residues within the variable domains of the parent (i.e., mouse or rat) monoclonal antibody, e.g., to improve the properties of the antibody.

[0105] Pharmaceutical Compositions and Administration To prepare pharmaceutical or sterile compositions comprising the antibodies and / or bispecific antibodies and / or bispecific binding partners and / or fusion proteins of the invention, the antibodies and / or bispecific antibodies and / or bispecific binding partners and / or fusion proteins can be mixed with a pharmaceutically acceptable carrier or excipient. [See, e.g., Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984)].

[0106] Formulations of therapeutic and diagnostic agents can be prepared, for example, by mixing with acceptable carriers, excipients, or stabilizers in the form of lyophilized powders, slurries, aqueous solutions, or suspensions [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: Tablets, Marcel Dekker, NY]. Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY]. In one embodiment, the antibodies and / or bispecific antibodies, and / or bispecific binding partners and / or fusion proteins of the invention are diluted to an appropriate concentration in sodium acetate solution, pH 5-6, with NaCl or sucrose added for isotonicity. Additional agents, such as polysorbate 20 or polysorbate 80, may be added to enhance stability.

[0107] The toxicity and therapeutic efficacy of an antibody composition administered alone or in combination with another agent can be determined, for example, by the LD 50 (a dose lethal to 50% of the population) and ED 50The dose that is therapeutically effective in 50% of the population can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects is known as the therapeutic index (LD 50 / ED 50 ) In certain aspects, antibodies that exhibit a high therapeutic index are desirable. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosages for use in dogs. The dosage of such compounds is preferably such that the ED 50 The dosage may vary within this range depending upon the dosage form used and the route of administration.

[0108] The mode of administration can vary. Suitable routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intracerebroventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal, or intraarterial. In certain embodiments, the antibodies and / or bispecific antibodies and / or bispecific binding partners and / or fusion proteins of the invention can be administered by an invasive route such as injection. In further embodiments of the invention, the antibodies and / or bispecific antibodies and / or bispecific binding partners and / or fusion proteins of the invention, or pharmaceutical compositions thereof, are administered intravenously, subcutaneously, intramuscularly, intraarterially, or by inhalation or aerosol delivery. Administration by non-invasive routes (e.g., oral; e.g., pill, capsule, or tablet) is also within the scope of the invention.

[0109] Composition can be administered using medical equipment known in the art.For example, pharmaceutical compositions of the present invention can be administered by injection with hypodermic needle, including for example pre-filled syringe or auto-injector.Pharmaceutical compositions disclosed herein can also be administered using needleless hypodermic injection device, such as the device disclosed in United States Patent No. 6,620,135; United States Patent No. 6,096,002; United States Patent No. 5,399,163; United States Patent No. 5,383,851; United States Patent No. 5,312,335; United States Patent No. 5,064,413; United States Patent No. 4,941,880; United States Patent No. 4,790,824 or United States Patent No. 4,596,556.

[0110] The pharmaceutical compositions disclosed herein can also be administered by infusion.The well-known implant and module forms for administering pharmaceutical compositions include: United States Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at controlled speed; United States Patent No. 4,447,233, which discloses an infusion pump for delivering drugs at precise infusion rates; United States Patent No. 4,447,224, which discloses an implantable variable flow rate infusion device for continuous drug delivery; United States Patent No. 4,439,196, which discloses an osmotic drug delivery system with multi-chamber compartments.Many other such implants, delivery systems and modules are well known to those skilled in the art.

[0111] Alternatively, antibodies, and / or bispecific antibodies, and / or bispecific binding partners, and / or fusion proteins of the invention may be administered locally rather than systemically, often in a depot or sustained release formulation.

[0112] 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 target cells in the biological matrix. Preferably, the administration regimen delivers enough therapeutic antibody, and / or bispecific antibody, and / or bispecific binding partner, and / or fusion protein to result in improvement of the target disease / symptom state while simultaneously minimizing undesirable side effects. Thus, the amount of biologic delivered will depend, 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 condition being treated.Guidance on selecting appropriate doses of therapeutic antibodies is available [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)].

[0113] Determination of appropriate dosages will be made by a veterinarian, for example, using parameters or factors known or suspected in the art to affect treatment. Generally, dosages will begin somewhat less than the optimum dose and then be increased by small increments until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include symptomatic diagnostic measures.

[0114] 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 to 7 times per week, weekly, biweekly, monthly, bimonthly, quarterly, semi-annually, yearly, etc. Doses can be provided, for example, intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, intraspinally, or by inhalation. The total weekly dose is generally at least 0.05 μg / kg body weight, more usually at least 0.2 μg / kg, 0.5 μg / kg, 1 μg / kg, 10 μg / kg, 100 μg / kg, 0.25 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 5.0 mg / kg, 10 mg / kg, 25 mg / kg, 50 mg / kg, or more [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)]. Dosages can also be provided to achieve a predetermined target concentration of the antibody, and / or bispecific antibody, and / or bispecific binding partner, and / or fusion protein of the invention in the serum of the dog, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg / ml or more. In other embodiments, the antibody, and / or bispecific antibody, and / or bispecific binding partner, and / or fusion protein of the invention is administered subcutaneously or intravenously at 10, 20, 50, 80, 100, 200, 500, 1000, or 2500 mg / subject weekly, biweekly, "every four weeks," monthly, bimonthly, or quarterly.

[0115] As used herein, "inhibiting" or "treating" or "treatment" includes delaying the onset of symptoms associated with a disorder and / or reducing the severity of symptoms of such a disorder. These terms further include ameliorating existing uncontrolled or undesirable symptoms, preventing further symptoms, and ameliorating or preventing the underlying cause of such symptoms. Thus, these terms refer to a beneficial result being imparted to a vertebrate subject (e.g., a dog) having a disorder, condition, and / or symptom, or having a potential for developing such a disorder, disease, or symptom.

[0116] As used herein, the terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of an antibody, and / or bispecific antibody, and / or bispecific binding partner, and / or fusion protein of the invention that, when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, e.g., a dog, is effective in causing a measurable improvement in one or more symptoms of a disease or condition or the progression of such a disease or condition. A therapeutically effective dose further refers to an amount of an antibody, and / or bispecific antibody, and / or bispecific binding partner, and / or fusion protein sufficient to cause at least a partial improvement of a symptom, e.g., treatment, cure, prevention, or amelioration of an associated medical condition, or an increased rate of treatment, cure, prevention, or amelioration of such a condition. When applied to a combination, a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent will produce an improvement of at least 10%; usually at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably at least 50% in a diagnostic measure or parameter. An effective amount can also result in an improvement in a subjective scale when a subjective scale is used to assess the severity of the condition.

[0117] Other combination therapies Compositions comprising the antibodies and / or bispecific antibodies and / or bispecific binding partners and / or fusion proteins of the invention can comprise one or more additional therapeutic components. One such family of therapeutic components is Janus kinase (JAK) inhibitors. In particular embodiments of this type, the JAK inhibitor comprises the following chemical formula, and pharmaceutically acceptable salts thereof: [ka] In the formula, R 1 C optionally substituted with hydroxy 1~4 [U.S. Patent No. 8,133,899; U.S. Patent No. 8,987,283]. More specifically, the JAK inhibitor is oclacitinib, and even more specifically, oclacitinib maleate. Another JAK inhibitor that preferentially inhibits JAK1 compared to JAK3 is: [ka] and pharmaceutically acceptable salts thereof [see WO 2018 / 108969].

[0118] Another therapeutic component that can be added to the compositions of the present invention 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, for example, U.S. Patent No. 8,759,366).

[0119] Additionally, additional therapeutic ingredients that can be added to the compositions of the present invention include: [ka] and pharmaceutically acceptable salts thereof (see also U.S. Pat. Nos. 7,696,222, 8,546,422, 8,637,541, WO 2010 / 099039; WO 2010 / 031183; and U.S. Pat. No. 8,546,422).

[0120] These additional therapeutic components may be administered to the canine subject before, together with, or after administration of a composition comprising an antibody, and / or bispecific antibody, and / or bispecific binding partner, and / or fusion protein of the invention.

[0121] [Example] General Materials and Methods: In all of the following examples, recombinant proteins were obtained by providing the amino acid sequence of a selected protein to a commercial manufacturer (ATUM, Newark, California), who then selected an appropriate nucleotide sequence encoding this amino acid sequence. Nucleotide sequences 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 either HEK-293 or CHO cells to express the recombinant protein, which was then isolated by conventional methods.

[0122] [Example 1] Canine IL-22 and IL-22-binding proteins A nucleic acid encoding the extracellular domain (ECD) of canine IL-22RA1 was linked to a nucleic acid encoding IgG-B Fc to form a nucleic acid encoding a canine IL-22RA1-Fc fusion protein by chemical synthesis. Similarly, a nucleic acid encoding a canine IL-22 binding protein (cIL-22BP; also known as cIL-22 receptor α2 and cIL-22RA2) was linked to a nucleic acid encoding canine IgG-B Fc to form a nucleic acid encoding a canine IL-22BP-Fc fusion protein by chemical synthesis. These nucleic acids were then individually cloned into expression plasmids suitable for producing the corresponding proteins in eukaryotic cells such as HEK-293 cells or CHO cells. Both the canine IL-22RA1-Fc fusion protein and the canine IL-22BP-Fc fusion protein bind to canine IL-22 and subsequently block the binding of canine IL-22 to full-length membrane-bound canine IL-22RA1.

[0123] Binding of canine IL-22RA1 and canine IL-22BP (IL-RA2) to canine IL-22 material: 1. Coating antigen: cIL-22-His (canine IL-22 with a His tag) 2. cIL-22BP-Fc fusion protein (canine IL-22BP-Fc fusion protein) 3. cIL-22RA1-Fc fusion protein (canine IL-22RA1-Fc fusion protein) 4. cPD-L1-Fc, 6.8 mg / mL (canine PD-L1-IgG-B Fc fusion protein, used as a control). 5. Horseradish peroxidase (HRP) conjugated rabbit anti-dog IgG (Fc), Sigma catalog number SAB3700109, 1.5 mg / mL 6. 3,3',5,5'-Tetramethylbenzidine (TMB) peroxidase substrate, SeraCare KPL 5120-0048; 7. Peroxidase Substrate Solution B, SeraCare KPL 5120-0037 8. Phosphate-buffered saline (PBS) 9. Phosphate Buffered Saline with TWEEN® (PBST) National Veterinary Services Laboratories (NVSL) 10. 1.5M Phosphate.

[0124] method: 1. Dilute cIL-22-His to 1 μg / mL in PBS and add 100 μL / well to the ELISA plate. Incubate overnight at 2-7°C. 2. Using an automated plate washer, wash the coated plate(s) three times with 200 μL / well / wash of NVSL-PBST. 3. Block the ELISA plate with 200 μL of 5% non-fat dry milk (NFDM) in NVSL-PBST, seal, and incubate at 37 °C for at least 60 minutes. 4. Using an automated plate washer, wash the blocked ELISA plate three times with 200 μL / well / wash of NVSL-PBST. 5. Dilute cIL-22BP-Fc fusion protein, cIL-22RA1-Fc fusion protein and cPD-L1-Fc fusion protein to 10μg / mL and serially dilute 3-fold across the dilution plate. Transfer 50μL of diluted test sample to the ELISA plate, seal and incubate with rotation at 37℃ for 60 minutes. 6. Using an automated plate washer, wash the plate 3 times with 200 μL / well / wash of NVSL-PBST. 7. Add 100 μl / well of 1:2500 diluted HRP-conjugated anti-dog IgG(Fc), seal and incubate at 37° C. for 60 minutes. 8. Mix equal volumes of TMB peroxidase substrate and TMB peroxidase substrate solution B and add 100 μl / well. Incubate at 37°C for 20 minutes. 9. Stop with 100 μl / well of 1.5 M phosphoric acid. 10. Measure A450 to A540 using a spectrophotometer.

[0125] cIL-22-HIS: [SEQ ID NO: 1] LPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVNMGERCYLMKEVLNFTLEEVLLPQSDRFQPYMQEVVPFLARLSNKLSQCHIENDDQHIQRNVQKLKDTVQKLGENGEIKAIGELDLLFMALRNACVHHHHHH

[0126] cIL-22RA1-FcB: [SEQ ID NO: 2] AEDTSDLLQYVKFQSSNFENILTWDSGLESAPDVVYSVEYKTYGKKEWLAKEGCQRITRKSCNLTTETGNHTEHYYARVTAVSAGGRSATKMTDRFSSMQQTTIKPPDVTCIPK VRSIQMIVHPTSTPIHAEDGHRLTLEDIFQDLFYRLELQVNHTYQMHLGGKQRDYEFIGLSPDTEFLGTITISVPNFFKESAPYVCRVKTLPDRTWTGGGGSGGGGSPKRENGR VPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIE RTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

[0127] cIL-22RA2-FcB: [SEQ ID NO: 3] TQSAYESLKPQRVHFQSRNFHNILHWQPGRACTSNGSVYFVQYKMYGQRQWKNKEDCWGILECFCDLTNETSDIQEPYYGRVRTTSAGIHSGWTMTQRFTPWWETKIDPPIINI TQVNGSLLVILHAPSLPYRDQKGKNVSIENYYELLYRVFIINNSLEKEQKVYEGAHRIVEIGALAPHTGYCVVAEMYQPMLDRRSPRSEERCMELPGGGGSGGGGSPKRENGRV PRPPDCPKCPOPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIER TISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

[0128] [Example 2] Anti-IL-4 receptor α antibody Anti-canine IL-4 receptor α antibodies useful in the present invention are exemplified by antibody c152H11VL3-cCLk-s / c152H11VH3-cIgG-Bm and antibody c146E2VL3-cCLk-s / c146E2VH3-cIgG-Bm. The sets of six CDRs (three individual light chain (LC) and three heavy chain (HC) sequences) for these two antibodies are shown in Table 1A (nucleic acid sequences) and Table 1B (amino acid sequences), respectively. The amino acid sequences of the full-length LC and HC of these caninized antibodies are provided in Tables 1A and 1B, respectively.

[0129] IL-4Rα antibody CDR Nucleic acid and amino acid sequences

[0130] [Table 2]

[0131] [Table 3]

[0132] c152H11VL3-cCLk-s (light chain): [SEQ ID NO: 4] EIVMTQSPASLSLSQEEKVTITCKASQNVGTNVAWYQQKPGQAPKLLIYSASYRYSGLPDRFSGSGSGTDFSFTISSLEPEDVAEFFCQQYNSYPYTFGQGTKLEIKR NDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD

[0133] c152H11VH3-cIgG-Bm (heavy chain): [SEQ ID NO: 5] EVQLVESGGDLVKPGGSLRLSCAASGFTFSSYGMSWVRQAPDKRLQWVATISRGGDYTYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCARGTLNNRGFASWGQGT LVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVP RPPDCPKCPOPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIER TISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

[0134] c146E2VL3-cCLk-s (light chain): [SEQ ID NO: 6] DIVLTQTPLSLSVSPGETASIYCRASESVDSYGNSFLNWYQQKPGQPPKLLIYRASNLASEIPDRFSGSGSRTEFTLKISRVEADDAGVYYCQQNYENPRTFGQGTKLEI KRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD

[0135] c146E2VH3-cIgG-Bm (heavy chain): [SEQ ID NO: 7] EVQLVQSGAEVKKPGASVKVSCKASGYTFARYWMHWMKQAPGAGLDWIGMIHPDSGNINYNERFKTKATLTVDKSTSTAYMELSSLRAGDIAVYYCARQLRNAMDYWGQGTL VTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPR PPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERT ISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

[0136] Antibodies against canine IL-4 receptor alpha were tested for their ability to inhibit STA-6 phosphorylation in DH82 cells as follows: material 1. Actively growing DH82 cells 2. DH82 Cell Growth Media (ATCC® 302003™, Eagle's Minimum Essential Medium supplemented with heat-inactivated fetal bovine serum to a final concentration of 15% w / v) 3. AlphaLISA p-STAT6 (Tyr641) Assay Kit: Perkin Elmer Catalog: ALSU-PST6-A-HV 4. Recombinant canine IL-4: R&D Systems, Catalog: 752-CL / CF 5. Recombinant canine IL-13: R&D Systems, Catalog: 5894-CL / CF 6. Perkin Elmer Envision a. Caninized anti-canine IL-4R α Monoclonal antibodies b.c146E2-H3L3 c.c152H11-H3L3 d.c4H3

[0137] method 1. Two tissue culture plates, 8 x 10 cells per well 4 DH82 cells (4 × 10 5 The cells were seeded in 200 μL at a density of 100 cells / mL and incubated overnight at 37°C. 2. Test antibodies were pre-diluted to 500 μg / mL and then serially diluted 3-fold in DH82 Cell Growth Media. The medium was removed from the cell culture plates and 50 μL / well of the serially diluted test sample was transferred to each plate. 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 a second plate. The plates were incubated at 37°C for 15 minutes. 4. Remove the medium from the plate and add 100 μL / well of freshly prepared 1× lysis buffer from the AlphaLISA p-STAT-6 Assay Kit to the plate. The plate was agitated at 350 rpm on a plate shaker at room temperature for 10 minutes. 5. Prepare Acceptor Mix from the AlphaLISA p-STAT6 Assay Kit and add 15 μL / well to 30 μL of cell lysate in 96-well ½ area plates. The plates were sealed and agitated at 350 rpm for 2 minutes, then incubated at room temperature for 2 hours. 6. Under dimmed laboratory lighting, prepare the Donor Mix from the AlphaLISA p-STAT6 Assay kit and add 15 μL / well to each plate. The plates were sealed, covered with foil, and agitated at 350 rpm for 2 minutes, then incubated at room temperature for 2 hours. 7. Plates were read using the AlphaScreen setting on a Perkin Elmer EnVison.

[0138] [Example 3] IL-31 receptor alpha Nucleotide sequence 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 production of the corresponding protein in eukaryotic cells, such as HEK-293 or CHO cells.

[0139] Canine IL-31RA ECD-10His: [SEQ ID NO: 8]

[0140] Expression and purification of IL-31 receptor αECD A plasmid containing the nucleotide sequence of SEQ ID NO:8 was transfected into HEK-293 or CHO cells using electroporation via a MaxCyte instrument according to the manufacturer's recommendations. Several days after transfection, the supernatants of the transfected cells and non-transfected controls were harvested and centrifuged to remove cellular debris. Histidine-tagged IL-31RA was purified from the cell culture medium by passing the clarified and collected fluid from the transfected cells through a nickel column according to the manufacturer's recommendations. The purified protein was quantified by measuring the absorbance of ultraviolet light at 280 nm.

[0141] Canine IL-31RA ECD-10His: [SEQ ID NO: 9] VLPAKPENISCIFYYEENFTCTWSPEKEASYTWYKVKRTYSYGYKSDICSTDNSTRGNHASCSFLPPTITNPDNYTIQVEAQNADGIMKSDITYWNLDAIMKIEPPEIFSVKSVLGIKRMLQIKWI RPVLAPHSSTLKYTLRFRTINSAYWMEVNFTKEDIDRDETYNLTELQAFTEYVMTLRCAPAESMFWSGWSQEKVGTTEEEAPYGLDLWRVLKPAMVDGRRPVQLMWKKATGAPVLEKALGYNIWYFP ENNTNLTETVNTTNQTHELYLGGKTYWVYVVSYNSLGESPVATLRIPALNEKTFQCIEAMQACLTQDQLVVEWQSSAPEVDTWMVEWFPDVDSEPSSFSWESVSQARNWTIQKDELKPLWCYNISVY PVLRDRVGQPYSTQAYVQEGIPSAGPVTQADSIGVKTVTITWKEIPKSKRNGFIKNYTIFYQAEDGKEFSKTVNSNILQYRLESLTRRTSYSLQVMASTNAGGTNGTKINFKTLSISHHHHHHHHHH

[0142] Binding of canine IL-31RA to biotinylated canine IL-31: 1. Coat immunoplate(s) with IL-31RA protein by diluting to 10 μg / mL in PBS. Add 100 μL / well. Incubate plate(s) overnight at 2-7°C. 2. Wash the plate three times with 275 μL / well of PBST. 3. Block the plate with 200 μL / well of blocking buffer (1% Dry Milk in PBST) for 30-45 minutes at 36±2°C with gentle shaking (120±20 RPM). 4. Wash plate 3 times with 275 μL / well of PBST. 5. Dilute biotinylated IL-31 to 10ug / mL in 1% NFDM in PBST. 6. Dilute biotinylated IL-31 (10 μg / mL) 3-fold in 1% NFDM in PBST and transfer 100 μL / well to the immunoplate(s). Incubate with gentle shaking (120 ± 20 RPM) at 36 ± 2°C for 30-45 minutes. 7. Wash plate 3 times with 275 μL / well PBST. 8. Dilute HRP-Streptavidin in 1% NFDM in PBST to a final dilution of 1:1000. 9. Add 100 μL / well of HRP-streptavidin to the immunoplate(s) and incubate with gentle shaking (120 ± 20 RPM) at 36 ± 2°C for 30-45 minutes. Wash the plate three times with 10.275 μL / well of PBST. 11. Combine equal volumes of pre-warmed TMB 2-Component Substrate immediately before use. 12. Add 100 μL / well of prepared TMB substrate to the immunoplate(s) and incubate in the dark with gentle shaking (120 ± 20 RPM) at 36 ± 2°C for 10-15 minutes. 13. Stop the reaction by adding 100 μL / well of 1 M H3PO4. 14. Using a microplate reader, read the plate at a wavelength of 450 nm, with a reference wavelength of 540 nm.

[0143] Monoclonal antibody against canine IL-31 receptor α Monoclonal antibodies against canine IL-31RA were generated by multiple immunizations of two Lewis rats with canine IL-31RA ECD (using 10 μg or 25 μg of antigen per rat) over a 3-4 week period. After immunization, serum was collected from each rat and tested against canine IL-31RA by ELISA. Lymph node cells from the rat 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 the growing hybridomas were screened by ELISA on plates coated with IL-31RA ECD protein using the protocol described below. Approximately 263 clones that showed potential binding to IL-31RA were selected in this ELISA, with the majority of clones having an OD of >1.

[0144] ELISA procedure: 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. 2. Wash the plate three times with PBST (PBS + 0.05% Tween 20). 3. Block plates with blocking buffer (PBS with 5% FBS), 25 ul / well, for 30 minutes at room temperature. 4.25 ul / well of hybridoma supernatant is transferred to a 96-well plate and incubated at room temperature for 60 minutes. 5. Wash the plate 3 times with PBST. 6. Add 25 ul / well of anti-rat HRP diluted 1:4000 in blocking buffer to the plate and incubate at room temperature for 60 minutes. 7. Wash the plate 5 times with PBST. 8. Add TMB-based reagent to the plate for a 2-3 min colorimetric reaction. 9. Stop the reaction with 0.16 M sulfuric acid. 10. Read the plate with a plate reader.

[0145] Blocking activity of anti-IL-31 receptor α antibody The ability of anti-canine IL-31RA hybridoma supernatants to block IL-31 binding to IL-31RA was assessed in the following blocking ELISA. Of the 263 clones that showed binding to IL-31RA, approximately 24 clones showed potential blocking of IL-31 binding to IL-31RA. 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. 2. Wash the plate three times with PBST (PBS + 0.05% Tween 20). 3. Block plates with blocking buffer (PBS with 5% FBS), 25 ul / well, for 30 minutes at room temperature. 4.25 ul / well of hybridoma supernatant is transferred to a 96-well plate and incubated at room temperature for 60 minutes. 5. Wash the plate 3 times with PBST. Transfer 6.25 μL / well of biotinylated IL-31 (0.5 μg / mL in blocking buffer) and incubate for 60 minutes at room temperature. 7. Wash the plate 3 times with PBST. 8. Add 25 μl / well of 1:5000 diluted Streptavidin-HRP in Blocking Buffer to the plate and incubate at room temperature for 60 minutes. Wash the plate 5 times with PBST. Add TMB-based reagent to the plate for a 2-3 minute colorimetric reaction. · Stop the reaction with 0.16M sulfuric acid. Read the plate with a plate reader.

[0146] Biological activity of anti-IL-31RA antibodies: The ability of anti-IL-31RA antibodies to inhibit STAT-3 activation was evaluated using a Baf3 cell line transfected with the nucleotide sequences of the full-length canine IL-31RA chain and the full-length canine oncostatin M receptor (OSMR) chain. This 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 expression of the corresponding proteins in baf3 cells.

[0147] Biological activity of anti-IL-31RA antibody: Stat-3 inhibition in Baf3 cells The ability of anti-IL-31 receptor alpha antibodies to inhibit activation of STAT-3 in Baf3 cells is assessed as follows. 1.5×10 5 Prepare a cell suspension with a viable cell density of 1 x 10 cells / mL and add 200 µL to each well of a 96-well tissue culture plate (1 x 10). 5 cells / well). Incubate the plate in a humidified 37°C incubator for 22-24 hours. 2. Remove the medium from the plate and add 200 μL of Eagle's Minimum Essential Medium (EMEM) to each well of the cell plate. Return the plate to a 37°C incubator for 2 hours. 3. Remove medium from cell plates and add 50 μL / well of supernatant from anti-IL-31RA hybridoma grown in PBS. Incubate plates at 37° C. for 1 hour. Add 50 μL / well of 80 ng / mL cIL-31 diluted in PBS and incubate plates at 37° C. for 5 minutes. 4. Remove the medium from the plate and add 100 μL / well of freshly prepared 1× lysis buffer to all wells. Agitate the plate on a plate shaker at 350 rpm at room temperature for 10 minutes. At this point, the cell lysate can be stored frozen at -20°C. 5. pSTAT-3 AlphaLISA: - Prepare reagents from the AlphaLISA® SureFire® Ultra™ p-STAT3 Assay Kit according to kit instructions. - Transfer 30 μL of cell lysate to a 96-well half-area plate and add 15 μL / well of Acceptor Mix to the cell lysate. - Seal the plate and incubate at room temperature for 1 hour. Add 15 μL / well of Donor Mix to the cell plate. Seal the plate, cover with foil, and incubate at room temperature for 1 hour or overnight. Note: Donor Mix is light-sensitive. -Read plates on a Perkin Elmer EnVison using Alpha Screen Assay settings.

[0148] [Example 4] Antibody against canine IL-31 Antibodies that may be useful in the present invention are those described in U.S. Patent Nos. 9,206,253 and 10,150,810. Preferably, these antibodies have the following light and heavy chain sequences: Caninized heavy chain sequences from mouse antibody clone M14 and canine IgG-B: [SEQ ID NO: 10] EVQLVESGPSLVKPGGSLRLTCSVTGDSITSGYWNWIRKFPGNKLEYMGYISYSGITDYNPSLKSRITISRDTSKNQYYLQLNSVTTEDTATYYCARYGNYGYAMDYWGQGT LVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVP RPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVWDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRWSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTI SKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGK

[0149] Caninized light chain sequence from mouse antibody clone M14 and canine light chain constant region: [SEQ ID NO: 11] DIVMTQSPASLSVSLGQRATISCRASESVDTYGNSFMHWYQQKPGQSPKLLIYRASNLESGIPARFGGSGSGTDFTLTIDPVQADDVATYYCQQSYEDPWTFGGGTKLEI KRNDAQPAVYLFQPSPDQLHTGSASWCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD

[0150] Zoe-LC: caninized light chain sequence: [SEQ ID NO: 12] EIVMTQSPASLSLSQEEKVTITCKASQSVSFAGTGLMHWYQQKPGQAPKLLIYRASNLEAGVPSRFSGSGSGTDFSFTISSLEPEDVAVYYCQQSREYPWTFGQGTKL EIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSEC

[0151] Zoe-HC: caninized heavy chain sequence: [SEQ ID NO: 13] EVQLVESGGDLVKPGGSLRLSCVASGFTFSNYGMSWVRQAPGKGLQWVATISYGGSYTYYPDNIKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCVRGYGYDTMDYWGQGTL VTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPR PPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERT ISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

[0152] [Example 5] Caninized antibody against IL-31RA IL-31 receptor αc10A12VH1-cIgGBm [SEQ ID NO: 51] EVQLVESGGDLVKPGGSLRLSCVASGFTFSNYYMAWVRQAPGKGLQWVASISTGGGNTYYRDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCAKHGTLYFDYWGQGTLV TVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRP PDCPKCPPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTI SKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGK

[0153] IL-31 receptor alpha c10A12VL5-cCL [SEQ ID NO: 52] QPVLTQPPSLSASLGTTARLTCERSSGDIGDSYVSWYQQKPGSPPRDLLYVDDQRPSGVSKSFSGSKDTSANAGLLLISGLQPEDEADYYCQSYDSNIDGPVFGGGTHL TVLGQPKASPSVTLFPPSSEELGANKATLVCLISDFYPSGVTVAWKADGSPVTQGVETTKPSKQSNNKYAASSYLSLTPDKWKSHSSFSCLVTHEGSTVEKKVAPAECS

[0154] [Example 6] Bispecific Binding Partners and Bispecific Antibodies The bispecific binding partners of the present invention are artificial protein molecules formed by the association of one heavy chain (HC) and one light chain (LC) of a monoclonal antibody with a fusion protein composed 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 into the light or heavy chain of the anti-IL-31 antibody and the Fc portion of the fusion protein to favor heterodimer formation over homodimer formation. This means, for example, that the heavy chain of the canine IL-31 antibody is paired with the Fc of the canine IL-22RA1-Fc fusion protein rather than with the second heavy chain of the canine IL-31 antibody.

[0155] 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 provided by the association of proteins corresponding to three amino acid sequences: (i) the caninized M14 light chain, the caninized M14 heavy chain, and the canine IL-22RA1-Fc fusion protein, or (ii) the caninized M14 light chain, the caninized M14 heavy chain, and the canine IL-22RA2-Fc fusion protein. Another example is provided by the association of proteins corresponding to three amino acid sequences: (a) the Zoe-LC light chain, the Zoe-HC heavy chain, and the canine IL-22RA1-Fc fusion protein, or (b) the Zoe-LC light chain, the Zoe-HC heavy chain, and the canine IL-22RA2-Fc fusion protein.

[0156] Bispecific antibodies are artificial protein molecules that can simultaneously target two different antigens. One preferred type of bispecific antibody is an IgG-like antibody that consists of four different polypeptide chains. Examples of bispecific antibodies of the present invention include those targeting IL-31RA and IL-4R. α Such antibodies include antibody molecules that can simultaneously target IL-4R and IL-5R. αThe heavy and light chains are formed by the association of one HC and one LC chain with specificity for IL-4R with one HC and one LC chain with specificity for IL-31RA. α The present invention promotes the association of the HC and LC of the antibody with each other, and the association of the HC and LC of the IL-31RA antibody with each other, and at the same time, promotes the association of each HC with IL-4R more than with itself. α The HCs from the antibodies are modified with specific substitutions / mutations in their amino acid sequences to promote association with the HC of the IL-31RA antibody. A list of amino acid substitutions in antibody HCs to promote HC heterodimer formation is listed in Table 2A above.

[0157] 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, provided below.

[0158] [Table 4] TIFF0007719077000008.tif22157

[0159] Canine IgG-Bm differs from naturally occurring canine IgG-B by containing two amino acid residue substitutions, namely, D31A and N63A in the amino acid sequence of IgG-B of SEQ ID NO: 14, 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 by alanine residues (A). The positions of these residues in the amino acid sequence are shown in bold and underlined. These two amino acid residue substitutions serve to significantly attenuate the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of naturally occurring canine IgG-B (see U.S. Pat. No. 10,106,607, the entire contents of which are incorporated herein by reference).

[0160] Additionally, potential embodiments or combinations of specific amino acid substitutions in the Fc portion of a canine IL-31 antibody or a canine IL-22RA1-Fc fusion protein or a canine IL-22RA2-Fc fusion that can be used to promote heterodimer formation are provided in Table 2A below. These substitutions favor a knobs-into-holes approach for heavy chain heterodimerization, or favor electrostatic attraction between different heavy chains to enable heterodimerization. [For a comprehensive discussion of these amino acid substitutions, see Moore et al., Methods, 154:38-50 (2019) and Brinkmann & Kontermann, MABS, 9:182-212 (2017)]. Within the context of the amino acid substitutions listed in Table 2A below, in some embodiments, chain 1 refers to the canine IL-31 heavy chain (HC) and chain 2 refers to the canine IL-22RA1-Fc fusion protein or the canine IL-22RA2-Fc fusion protein. In other embodiments, chain 1 refers to the canine IL-22RA1-Fc fusion protein or the canine IL-22RA2-Fc fusion protein and chain 2 refers to the canine IL-31 heavy chain (HC). Additionally, in some embodiments, chain 1 refers to the anti-IL-31RA heavy chain (HC) and chain 2 refers to the anti-IL-4R α In other embodiments, chain 1 refers to anti-IL-4R α In yet other embodiments, chain 1 refers to IL-22HC and chain 2 refers to anti-IL-31HC. In yet other embodiments, chain 1 refers to anti-IL-31HC and chain 2 refers to IL-22HC.

[0161] Furthermore, a variable light (VL) domain can be swapped with a variable heavy (VH) domain in a domain swap, i.e., one of the various domains can be swapped for another within the Fab fragment of an antibody. Light chains (such as those listed above) that are candidates for inclusion in a bispecific antibody can be adapted for this purpose by, for example, swapping the position of the constant light (CL) domain with the position of the CH1 domain in one of the antibody pairs, resulting in a VL-CH1 and VH-CL1 antibody domain configuration, without changing the light chain in the other antibody pair of the bispecific antibody. Other options are also possible, such as swapping a CL-VL domain with a CH1-VH domain or swapping the VL and VH domains in one antibody pair. Table 2B provides examples of (i) the amino acid sequence at the VH-CL domain junction and (ii) the amino acid sequence at the VL-CH1 domain junction after such domain swapping. In another example, the entire Fab fragment can be swapped, such that the VH-CH1 domain is swapped with the VL-CL domain.

[0162] Therefore, to optimally provide IgG-like bispecific antibodies, two types of modifications can be made: a) modifying the Fc of the antibody to promote heterodimerization over homodimerization of the heavy chains, and b) modifying the light chains to promote the association of each of the two light chains with its cognate heavy chain. The amino acid substitutions listed in Table 2 can be used with chain 1 and chain 2 to modify the Fc. To make the light chain suitable for inclusion in a bispecific antibody, a method called cross-mab can be used [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 cross-mab technology, a light chain (such as a light chain of the invention) that is a candidate for inclusion in a bispecific antibody can be made suitable for this purpose, for example, by incorporating the light chain into an antibody domain arrangement in which the position of the CL domain is swapped for 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 unchanged. As mentioned above, other options are also possible, such as swapping the CL-VL domain with the CH1-VH domain, or swapping the VL and VH domains in one of the antibody pairs.

[0163] [Table 5]

[0164] [Table 6]

[0165] Caninized M14 LC amino acid: [SEQ ID NO: 11] DIVMTQSPASLSVSLGQRATISCRASESVDTYGNSFMHWYQQKPGQSPKLLIYRASNLESGIPARFGGSGSGTDFTLTIDPVQADDVATYYCQQSYEDPWTFGGGTKLEI KRNDAQPAVYLFQPSPDQLHTGSASWCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD

[0166] Caninized M14 HC amino acid: [SEQ ID NO: 15] This amino acid sequence is identical to SEQ ID NO: 10 except for the bolded and underlined amino acids. EVQLVESGPSLVKPGGSLRLTCSVTGDSITSGYWNWIRKFPGNKLEYMGYISYSGITDYNPSLKSRITISRDTSKNQYYLQLNSVTTEDTATYYCARYGNYGYAMDYWGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQS SGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVWDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRWSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPP C REELSKNTVSL W CLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGK

[0167] cIL-22RA1-FcB with amino acid substitutions to promote heterologous: [SEQ ID NO: 16] AEDTSDLLQYVKFQSSNFENILTWDSGLESAPDVVYSVEYKTYGKKEWLAKEGCQRITRKSCNLTTETGNHTEHYYARVTAVSAGGRSATKMTDRFSSMQQTTIKPPDVTCIPKVRSIQMIVHPTSTPIHAEDGHRLTLEDIFQDLFYRLELQVNHTYQMHLGGKQRDYEFIGLSPDT EFLGTITISVPNFFKESAPYVCRVKTLPDRTWTGGGGSGGGGSPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSV C VLPPSREELSKNTVSL S C A IKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFL V SKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

[0168] cIL-22RA2-FcB with mutations to promote heterozygosity: [SEQ ID NO: 17] TQSAYESLKPQRVHFQSRNFHNILHWQPGRACTSNGSVYFVQYKMYGQRQWKNKEDCWGILECFCDLTNETSDIQEPYYGRVRTTSAGIHSGWTMTQRFTPWWETKIDPPIINITQVNGSLLVILHAPSLPYRDQKGKNVSIENYYELLYRVFIINNSLEKEQKVYEGAHRIVEIGAL APHTGYCVVAEMYQPMLDRRSPRSEERCMELPGGGSGGGGSPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSV C VLPPSREELSKNTVSL S CA IKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFL V SKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

[0169] Zoe-HC: caninized heavy chain sequence: [SEQ ID NO: 18] EVQLVESGGDLVKPGGSLRLSCVASGFTFSNYGMSWVRQAPGKGLQWVATISYGGSYTYYPDNIKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCVRGYGYDTMDYWGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSS GLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPP C REELSKNTVSL W CLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

[0170] IL-4R for bispecific antibody formation α An example of modifying the heavy chain of an antibody.

[0171] IL-4 receptor alpha c152H11VL3-cCLk-s (light chain): [SEQ ID NO: 19] EIVMTQSPASLSLSQEEKVTITCKASQNVGTNVAWYQQKPGQAPKLLIYSASYRYSGLPDRFSGSGSGTDFSFTISSLEPEDVAEFFCQQYNSYPYTFGQGTKLEIKR NDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD

[0172] Modified IL-4 receptor alpha c152H11VH3-cIgG-Bm (heavy chain): [SEQ ID NO: 20] EVQLVESGGDLVKPGGSLRLSCAASGFTFSSYGMSWVRQAPDKRLQWVATISRGGDYTYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCARGTLNNRGFASWGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVL QSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSV C VLPPSREELSKNTVSL S C A IKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFL V SKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

[0173] IL-4 receptor alpha c146E2VL3-cCLk-s (light chain): [SEQ ID NO: 21] DIVLTQTPLSLSVSPGETASIYCRASESVDSYGNSFLNWYQQKPGQPPKLLIYRASNLASEIPDRFSGSGSRTEFTLKISRVEADDAGVYYCQQNYENPRTFGQGTKLEI KRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD

[0174] Modified IL-4 receptor αc146E2VH3-cIgG-Bm: (heavy chain): [SEQ ID NO: 22] EVQLVQSGAEVKKPGASVKVSCKASGYTFARYWMHWMKQAPGAGLDWIGMIHPDSGNINYNERFKTKATLTVDKSTSTAYMELSSLRAGDIAVYYCARQLRNAMDYWGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQ SSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSV C VLPPSREELSKNTVSL S C A IKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFL V SKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

[0175] Engineered IL-31 receptor alpha caninized heavy chain 10A12VH1-cIgGBm [SEQ ID NO: 53] EVQLVESGGDLVKPGGSLRLSCVASGFTFSNYYMAWVRQAPGKGLQWVASISTGGGNTYYRDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCAKHGTLYFDYWGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSG LYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPP C REELSKNTVSL W CLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGK

[0176] [Table 7]

[0177] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be included within the scope of the appended claims.

Claims

1. A composition for treating atopic dermatitis in dogs, comprising: wherein the composition comprises: (a) a bispecific binding partner comprising (i) a fusion protein that binds canine IL-22 and (ii) a monomer of a caninized anti-pruritic antibody; or (b) a bispecific binding partner comprising (i) a fusion protein that binds canine IL-22 and (ii) a monomer of a caninized anti-inflammatory antibody; wherein the fusion protein that binds 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, the caninized anti-pruritic antibody is a caninized interleukin-31 (IL-31) antibody or a caninized interleukin-31 receptor alpha (IL-31RA) antibody, and the caninized anti-inflammatory antibody is a caninized IL-4Rα antibody; composition.

2. the canine IL-22RA1-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 16; 2. The composition of claim 1, wherein the IL-22RA2-canine Fc fusion protein comprises the amino acid sequence of SEQ ID NO:

17.

3. The caninized IL-31 antibody is 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; or 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; The composition of claim 1.

4. the caninized IL-31 antibody comprises a heavy chain and a light chain, wherein the light chain comprises a heavy chain constant domain 1 (CH1) instead of a light chain constant domain (CL), and wherein: The composition of claim 1, wherein the heavy chain of the caninized IL-31 antibody comprises the CL in place of the CH1.

5. The caninized IL-4Rα antibody is 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, or 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; The composition of claim 1.

6. The caninized IL-4Rα antibody is 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, or 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; The composition of claim 1.

7. The caninized IL-4Rα antibody comprises a heavy chain and a light chain, wherein the light chain comprises a heavy chain constant domain 1 (CH1) instead of a light chain constant domain (CL), and wherein: The composition of claim 1 , wherein the heavy chain comprises the CL in place of the CH1.

8. The composition of claim 1, 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.

9. 2. The composition of claim 1, wherein the canine IL-22RA1-Fc fusion protein comprises the amino acid sequence of SEQ ID NO:2, or the canine IL-22RA2-Fc fusion protein comprises the amino acid sequence of SEQ ID NO:

3.

10. The composition of any one of claims 1 to 9, 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 chemoattractant receptor homologous molecule expressed on TH2 cells.

11. The JAK inhibitor 【Chemical 1】 In the formula, R １ is C optionally substituted by hydroxy １～４ alkyl, and pharmaceutically acceptable salts thereof, and 【Chemistry 2】 and pharmaceutically acceptable salts thereof, The composition of claim 10 selected from the group consisting of:

12. A method for treating atopic dermatitis, comprising administering the composition according to any one of claims 1 to 11 to a dog having atopic dermatitis.

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