Canine antibodies against canine interleukin-31 receptor alpha

Abstract

The present invention provides a caninized rat antibody against canine IL-31 receptor alpha, which has high binding affinity to canine IL-31 receptor alpha and is capable of blocking the binding of canine IL-31 to canine IL-31 receptor alpha. The present invention further provides use of the antibody for the treatment of atopic dermatitis in dogs.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority under 119(e) of the United States Patent Act to U.S. Provisional Patent Application No. 63 / 092,294, filed on 15 October 2020, U.S. Provisional Patent Application No. 63 / 092,296, filed on 15 October 2020, U.S. Provisional Patent Application No. 63 / 127,184, filed on 18 December 2020, U.S. Provisional Patent Application No. 63 / 235,258, filed on 20 August 2021, and U.S. Provisional Patent Application No. 63 / 235,257, filed on 20 August 2021, the entire contents of which are incorporated herein by reference.

[0002] Sequence List This application includes a sequence listing, which is filed electronically in ASCII format and is incorporated herein by reference in its entirety. The above ASCII copy, created on October 1, 2021, is named 25071-WO-PCT_SL.txt and has a size of 103,453 bytes.

[0003] The present invention relates to an antibody against the canine IL-31 receptor alpha that has a high binding affinity to the canine IL-31 receptor alpha and can block the binding of canine IL-31 to the canine IL-31 receptor alpha. The present invention also relates to the use of the antibody of the present invention for the treatment of canine atopic dermatitis. [Background technology]

[0004] The immune system consists of a network of resident and recirculating specialized cells that work together 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 leukocytes and collectively called interleukins. Among the well-studied interleukins are four important molecules identified as interleukin-31 (IL-31), interleukin-4 (IL-4), interleukin-13 (IL-13), and interleukin-22 (IL-22). While IL-4, IL-13, IL-22, and IL-31 are crucial cytokines for generating the immune response necessary for protection against extracellular pathogens (e.g., parasites present in tissues or tubules), these cytokines are also involved in the pathogenesis of allergic diseases in humans and animals, including atopic dermatitis.

[0005] Atopic dermatitis (AD) is a recurrent, pruritic, and chronic inflammatory skin disease characterized in humans by immune system dysregulation and epidermal barrier abnormalities. The pathological and immunological attributes of atopic dermatitis have been the subject of extensive research [seen 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 etiology of atopic dermatitis in dogs and cats [seen in Nuttall et al., Veterinary Records 172(8):201-207(2013)] shows remarkable similarities to the etiology of human atopic dermatitis, including skin infiltration by various immune cells, as well as CD4, which is predominantly IL-31, IL-4, and IL-13. +This includes a Th2-polarizing cytokine environment. Furthermore, IL-22 is involved in the excessive epithelial proliferation that leads to the epidermal hyperplasia characteristic of atopic dermatitis.

[0006] For example, antibodies against canine IL-31 have been shown to be effective against pruritus associated with atopic dermatitis in dogs [U.S. Patent No. 8,790,651; U.S. Patent No. 10,093,731]. In addition, antibodies against human IL-31 receptor alpha (IL-31RA) have been tested and found to be effective against pruritus associated with atopic dermatitis in humans [Ruzicka, et al., New England Journal of Medicine, 376(9), 826-835 (2017)].

[0007] IL-4 and IL-13 are CD4 + IL-4 and IL-13 are closely related proteins that can be secreted by many cell types, including Th2 cells, natural killer T cells (NKTs), macrophages, mast cells, and basophils. They exhibit many overlapping functions and are important for the development of the T cell-dependent humoral immune response. IL-4 is known to bind with high affinity to two receptors, namely type I and type II IL-4 receptors. Human IL-4 receptor alpha (IL-4 R α Monoclonal antibodies against IL-4 have been developed, and some of these antibodies have been extensively tested in humans for their therapeutic effects in treating atopic dermatitis [see, for example, U.S. Patent No. 2015 / 0017176]. More recently, canine IL-4 R α Canine IL-4 R blocks the binding of canine IL-4 to [the target of the drug]. αCanine antibodies against canine IL-4 are also disclosed [U.S. Patent No. 2018 / 0346580, which is incorporated herein in its entirety by reference]. Since the type II IL-4 receptor consists of the IL-4 receptor α chain and the IL-13 receptor α1 chain, it is possible to block both canine IL-4 and canine IL-13 from binding to the type II canine IL-4 receptor, thereby helping to block inflammation associated with atopic dermatitis, canine IL-4 R α Antibodies against it have been obtained [U.S. Patent No. 2018 / 0346580].

[0008] Interleukin-22 (IL-22), also known as IL-10-related T cell-derived inducer (IL-TIF), belongs to the IL-10 cytokine family. In humans, IL-22 is produced by normal T cells in response to anti-CD3 stimulation. Furthermore, lipopolysaccharide injection induces IL-22 expression in various organs in mice, suggesting that IL-22 may be involved in inflammatory responses. IL-22 specifically binds to a receptor complex consisting of a heterodimer complex of IL-10R2 (also known as IL-10R beta) and the interleukin-22 receptor (IL-22R), and signals through this complex [see Lee et al., Pharmacology Research & Perspectives, Pages 1-13 (2018:e00434)]. The interleukin-22 receptor is also known as interleukin-22R, alpha-1; IL-22RA1; IL-22R1; zcytor11; and CRF2-9 [Xu et al., Proc. Nat. Acad. Sci. 98(17) 9511-9516 (2001); Gelebart and Lai, Atlas of Genetics and Cytogenetics 14(12): 1106-1110 (2010)]. IL-22 induces epithelial cell proliferation during wound healing, and its deficiency can enable uncontrolled proliferation and enhance tumorigenesis [Huber et al., Nature 491:259-263 (2012)]. IL-22 has been shown to activate STAT-1 and STAT-3 in some hepatocellular carcinoma cell lines, upregulating the production of acute-phase proteins. Antibodies against interleukin-22 and IL-22R act as antiproliferative agents by blocking the interaction between IL-22 and IL-22R, thereby blocking the associated signaling pathways that lead to epithelial proliferation.

[0009] Medicines that have been proven to be adjunctive to the treatment of atopic dermatitis, and / or have been shown to be promising in doing so, include Janus kinase (JAK) inhibitors [see, e.g., U.S. Patent Nos. 8,133,899, 8,987,283, and International Publication No. 2018 / 108969], spleen tyrosine kinase (SYK) inhibitors [see, e.g., U.S. Patent No. 8,759,366], and antagonists to chemoattractant receptor homologs expressed on TH2 cells [see, e.g., U.S. Patents Nos. 7,696,222, 8,546,422, 8,637,541, and 8,546,422].

[0010] However, despite some success in treating atopic dermatitis, there is still a need to design alternative and / or better therapies that can address one or more of the symptoms of atopic dermatitis in dogs.

[0011] No reference made herein shall be construed as an acknowledgment that such reference is available as “prior art” to this application. [Prior art documents] [Patent Documents]

[0012] [Patent Document 1] U.S. Patent No. 8,790,651 [Patent Document 2] U.S. Patent No. 10,093,731 [Patent Document 3] U.S. Patent No. 2015 / 0017176 [Patent Document 4] U.S. Patent No. 2018 / 0346580 [Patent Document 5] U.S. Patent No. 8,133,899 [Patent Document 6] U.S. Patent No. 8,987,283 [Patent Document 7] International Publication No. 2018 / 108969

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[0014] The present invention provides novel mammalian antibodies, including canine antibodies, against canine IL-31 receptor alpha (IL-31RA). In certain embodiments, the mammalian antibody against canine IL-31 receptor alpha (cIL-31RA) is an isolated antibody. In preferred embodiments, the mammalian antibody or its antigen-binding fragment binds to canine IL-31RA. In even more specific embodiments, the mammalian antibody or antigen-binding fragment also blocks the binding of canine IL-31RA to canine interleukin-31. In certain embodiments, the antibody is a rat antibody against canine IL-31RA. In even more specific embodiments, the mammalian antibody is a canine rat antibody against canine IL-31RA.

[0015] Accordingly, the present invention provides a mammalian antibody or its antigen-binding fragment comprising a heavy chain that binds to canine IL-31RA and includes a set of three heavy chain complementarity-determining regions (CDRs): CDR heavy 1 (HCDR1), CDR heavy 2 (HCDR2), and CDR heavy 3 (HCDR3), and a set of three light chain CDRs: CDR light 1 (LCDR1), CDR light 2 (LCDR2), and CDR light 3 (LCDR3).

[0016] In certain embodiments, the mammalian antibody or antigen-binding fragment comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 13, HCDR2 containing the amino acid sequence of SEQ ID NO: 14, HCDR3 containing the amino acid sequence of SEQ ID NO: 15, and further comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 16, LCDR2 containing the amino acid sequence of SEQ ID NO: 17, and LCDR3 containing the amino acid sequence of SEQ ID NO: 18. In specific embodiments, when the antibody is bound to canine IL-31RA, it binds to an epitope composed of the amino acids of SEQ ID NO: 102 or SEQ ID NO: 103, or to both SEQ ID NO: 102 and SEQ ID NO: 103. In related embodiments, when the antibody is bound to canine IL-31RA, it binds to at least one amino acid residue, preferably 1 to 3 amino acid residues, more preferably 2 to 5 amino acid residues, and / or even more preferably 3 to 8 amino acid residues or more, in the amino acid sequences of SEQ ID NO: 102 or SEQ ID NO: 103, or both SEQ ID NO: 102 and SEQ ID NO: 103.

[0017] In other embodiments, the mammalian antibody or antigen-binding fragment comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 19, HCDR2 containing the amino acid sequence of SEQ ID NO: 20, HCDR3 containing the amino acid sequence of SEQ ID NO: 21, and further comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 22, LCDR2 containing the amino acid sequence of SEQ ID NO: 23, and LCDR3 containing the amino acid sequence of SEQ ID NO: 24.

[0018] In further embodiments, the mammalian antibody or antigen-binding fragment comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 25, HCDR2 containing the amino acid sequence of SEQ ID NO: 26, HCDR3 containing the amino acid sequence of SEQ ID NO: 27, and further comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 28, LCDR2 containing the amino acid sequence of SEQ ID NO: 29, and LCDR3 containing the amino acid sequence of SEQ ID NO: 30. In specific embodiments, when the antibody is bound to canine IL-31RA, it binds to an epitope composed of the amino acid sequence of SEQ ID NO: 101. In related embodiments, when the antibody is bound to canine IL-31RA, it binds to at least one amino acid residue, preferably 1 to 3 amino acid residues, more preferably 2 to 5 amino acid residues, and / or even more preferably 3 to 8 amino acid residues or more within the amino acid sequence of SEQ ID NO: 101.

[0019] In specific embodiments, the mammalian antibody against canine IL-31RA is a rat antibody. In particular embodiments, the mammalian antibody against canine IL-31RA is a canine rat antibody. In one particular embodiment, the canine antibody contains a heavy chain containing IgG-D cFc, but the naturally occurring IgG-D hinge region is replaced by a hinge region containing the amino acid sequence of SEQ ID NO: 79. In other embodiments, the canine antibody contains a heavy chain containing IgG-D cFc, but the naturally occurring IgG-D hinge region is replaced by a hinge region containing the amino acid sequence of SEQ ID NO: 80. In yet another embodiment, the canine antibody contains a heavy chain containing IgG-D cFc, but the naturally occurring IgG-D hinge region is replaced by a hinge region containing the amino acid sequence of SEQ ID NO: 81. In yet another embodiment, the canine antibody contains a heavy chain containing IgG-D cFc, but the naturally occurring IgG-D hinge region is replaced by a hinge region containing the amino acid sequence of SEQ ID NO: 82.

[0020] In a particular embodiment, the canine antibody comprises a heavy chain containing modified canine IgG-B (IgG-Bm) having the amino acid sequence of SEQ ID NO: 78. In an alternative embodiment, the canine antibody comprises a heavy chain containing unmodified canine IgG-B having the amino acid sequence of SEQ ID NO: 77.

[0021] In certain embodiments of the composition, the canine antibody against canine IL-31RA (cIL-31RA) comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 88 or SEQ ID NO: 89 and a light chain containing the amino acid sequence of SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92 or SEQ ID NO: 93. In specific embodiments, when the antibody binds to canine IL-31RA, it binds to an epitope composed of the amino acid sequence of SEQ ID NO: 101. In related embodiments, when the antibody binds to canine IL-31RA, it binds to at least one amino acid residue, preferably 1 to 3 amino acid residues, more preferably 2 to 5 amino acid residues, and / or even more preferably 3 to 8 amino acid residues or more within the amino acid sequence of SEQ ID NO: 101. The present invention further provides antigen-binding fragments of these canine antibodies.

[0022] In certain embodiments, the canine antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 90 and a heavy chain containing the amino acid sequence of SEQ ID NO: 88. In other embodiments, the canine antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 91 and a heavy chain containing the amino acid sequence of SEQ ID NO: 88. In yet another embodiment, the canine antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 92 and a heavy chain containing the amino acid sequence of SEQ ID NO: 88. In yet another embodiment, the canine antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 93 and a heavy chain containing the amino acid sequence of SEQ ID NO: 88. In yet another embodiment, the canine antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 90 and a heavy chain containing the amino acid sequence of SEQ ID NO: 89. In yet another embodiment, the canine antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 91 and a heavy chain containing the amino acid sequence of SEQ ID NO: 89. In yet another embodiment, the canine antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 92 and a heavy chain containing the amino acid sequence of SEQ ID NO: 89. In yet another embodiment, the canine antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 93 and a heavy chain containing the amino acid sequence of SEQ ID NO: 89. In a specific embodiment, when the antibody is bound to canine IL-31RA, it binds to at least one amino acid residue, preferably 1 to 3 amino acid residues, more preferably 2 to 5 amino acid residues, and / or even more preferably 3 to 8 amino acid residues or more, within the amino acid sequence of SEQ ID NO: 101.

[0023] In certain embodiments of the composition, the canine antibody against cIL-31RA comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 100, and a light chain containing the amino acid sequence of SEQ ID NO: 97, SEQ ID NO: 98, or SEQ ID NO: 99. In specific embodiments, when the antibody binds to canine IL-31RA, it binds to an epitope composed of the amino acids of SEQ ID NO: 102 or SEQ ID NO: 103, or to both SEQ ID NO: 102 and SEQ ID NO: 103. In related embodiments, when the antibody binds to canine IL-31RA, it binds to at least one amino acid residue, preferably 1 to 3 amino acid residues, more preferably 2 to 5 amino acid residues, and / or even more preferably 3 to 8 amino acid residues or more, within the amino acid sequences of SEQ ID NO: 102 or SEQ ID NO: 103, or both SEQ ID NO: 102 and SEQ ID NO: 103.

[0024] The present invention further provides antigen-binding fragments of these canine antibodies.

[0025] In other embodiments, the canine antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 97 and a heavy chain containing the amino acid sequence of SEQ ID NO: 94. In yet another embodiment, the canine antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 97 and a heavy chain containing the amino acid sequence of SEQ ID NO: 95. In yet another embodiment, the canine antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 97 and a heavy chain containing the amino acid sequence of SEQ ID NO: 96. In yet another embodiment, the canine antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 97 and a heavy chain containing the amino acid sequence of SEQ ID NO: 100. In a specific embodiment, when the antibody is bound to canine IL-31RA, it binds to an epitope composed of the amino acids of SEQ ID NO: 102 or SEQ ID NO: 103, or to both SEQ ID NO: 102 and SEQ ID NO: 103. In the relevant embodiments, when the antibody is bound to canine IL-31RA, it binds to at least one amino acid residue, preferably 1 to 3 amino acid residues, more preferably 2 to 5 amino acid residues, and / or even more preferably 3 to 8 amino acid residues or more, in the amino acid sequences of SEQ ID NO: 102 or SEQ ID NO: 103, or both SEQ ID NO: 102 and SEQ ID NO: 103.

[0026] In yet another embodiment, the canine antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 98 and a heavy chain containing the amino acid sequence of SEQ ID NO: 94. In yet another embodiment, the canine antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 98 and a heavy chain containing the amino acid sequence of SEQ ID NO: 95. In yet another embodiment, the canine antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 98 and a heavy chain containing the amino acid sequence of SEQ ID NO: 96. In yet another embodiment, the canine antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 98 and a heavy chain containing the amino acid sequence of SEQ ID NO: 100. In a specific embodiment, when the antibody is bound to canine IL-31RA, it binds to an epitope composed of the amino acids of SEQ ID NO: 102 or SEQ ID NO: 103, or to both SEQ ID NO: 102 and SEQ ID NO: 103. In the relevant embodiments, when the antibody is bound to canine IL-31RA, it binds to at least one amino acid residue, preferably 1 to 3 amino acid residues, more preferably 2 to 5 amino acid residues, and / or even more preferably 3 to 8 amino acid residues or more, in the amino acid sequences of SEQ ID NO: 102 or SEQ ID NO: 103, or both SEQ ID NO: 102 and SEQ ID NO: 103.

[0027] In yet another embodiment, the canine antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 99 and a heavy chain containing the amino acid sequence of SEQ ID NO: 94. In yet another embodiment, the canine antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 99 and a heavy chain containing the amino acid sequence of SEQ ID NO: 95. In yet another embodiment, the canine antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 99 and a heavy chain containing the amino acid sequence of SEQ ID NO: 96. In yet another embodiment, the canine antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 99 and a heavy chain containing the amino acid sequence of SEQ ID NO: 100. In a specific embodiment, when the antibody is bound to canine IL-31RA, it binds to at least one amino acid residue, preferably 2 to 5 amino acid residues, and / or more preferably 3 to 8 amino acid residues or more, within the amino acid sequences of SEQ ID NO: 102 or SEQ ID NO: 103, or both SEQ ID NO: 102 and SEQ ID NO: 103.

[0028] The present invention further provides all of the antigen-binding fragments of these canine antibodies.

[0029] The present invention further provides isolated mammalian antibodies or their antigen-binding fragments (including canine antibodies, canine antibodies or their antigen-binding fragments) that bind to canine IL-31RA and, when bound to canine IL-31RA, bind to an epitope consisting of the amino acid sequence of SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, or any combination thereof, wherein the antibodies bind to canine IL-31RA and block the binding of canine IL-31RA to canine IL-31.

[0030] In certain embodiments, isolated mammalian antibodies or their antigen-binding fragments (including canine antibodies, canine antibodies or their antigen-binding fragments)

[0031] The antibody specifically binds to canine IL-31RA, and when bound to canine IL-31RA, it binds to at least one amino acid residue, preferably 1 to 3 amino acid residues, more preferably 2 to 5 amino acid residues, and / or even more preferably 3 to 8 amino acid residues or more, within the amino acid sequence of SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, or SEQ ID NO: 105, or any combination thereof. In certain embodiments, the antibody or its antigen-binding fragment binds to canine IL-31RA and blocks the binding of canine IL-31RA to canine IL-31.

[0032] The present invention also provides nucleic acids comprising isolated nucleic acids encoding a CDR, a heavy chain of a canine antibody or its antigen-binding fragment, and / or a light chain of a canine antibody or its antigen-binding fragment.

[0033] Accordingly, the present invention further provides nucleic acids encoding a set of three heavy chain complementarity-determining regions (CDRs), CDR heavy 1 (HCDR1), CDR heavy 2 (HCDR2), and CDR heavy 3 (HCDR3), of the mammalian antibody or its antigen-binding fragment of the present invention. In a preferred embodiment, the nucleic acid encodes a set of three heavy chain complementarity-determining regions (CDRs), CDR heavy 1 (HCDR1), CDR heavy 2 (HCDR2), and CDR heavy 3 (HCDR3), of the canine antibody or its antigen-binding fragment of the present invention.

[0034] In one particular embodiment of this type, the nucleic acid encodes HCDR1 containing the amino acid sequence of SEQ ID NO: 13, HCDR2 containing the amino acid sequence of SEQ ID NO: 14, and HCDR3 containing the amino acid sequence of SEQ ID NO: 15. In another embodiment of this type, the nucleic acid encodes HCDR1 containing the amino acid sequence of SEQ ID NO: 19, HCDR2 containing the amino acid sequence of SEQ ID NO: 20, and HCDR3 containing the amino acid sequence of SEQ ID NO: 21. In yet another embodiment of this type, the nucleic acid encodes HCDR1 containing the amino acid sequence of SEQ ID NO: 25, HCDR2 containing the amino acid sequence of SEQ ID NO: 26, and HCDR3 containing the amino acid sequence of SEQ ID NO: 27.

[0035] The present invention also provides nucleic acids encoding a set of three light chain complementarity-determining regions (CDRs), CDR light 1 (LCDR1), CDR light 2 (LCDR2), and CDR light 3 (LCDR3), of the mammalian antibody or its antigen-binding fragment. In a more specific embodiment of this type, the nucleic acid encodes LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, LCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 18. In another embodiment of this type, the nucleic acid encodes LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 24. In yet another embodiment of this type, the nucleic acid encodes LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 30.

[0036] The present invention further provides nucleic acids encoding the heavy chain of the mammalian antibody or its antigen-binding fragment. The present invention also provides nucleic acids encoding the light chain of the mammalian antibody or its antigen-binding fragment. Furthermore, the present invention provides an expression vector comprising one or more of the nucleic acids of the present invention, and a host cell comprising such an expression vector.

[0037] The present invention also provides a pharmaceutical composition comprising the canine antibody of the present invention and its antigen-binding fragment, together with a pharmaceutically acceptable carrier and / or diluent. The present invention further provides a method for treating atopic dermatitis, comprising administering one of the aforementioned compositions to a dog having atopic dermatitis. In a particular embodiment, the present invention provides a method for assisting the blockage of pruritus associated with atopic dermatitis, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention to a dog in need thereof.

[0038] These and other aspects of the present invention will be better understood by referring to the brief and detailed description of the drawings below. [Brief explanation of the drawing]

[0039] [Figure 1] The extracellular domain (ECD) of canine IL-31RA was tested for its ability to bind to canine IL-31. The results showed that the canine IL-31RA ECD binds to biotinylated canine IL-31 in a dose-dependent manner. [Figure 2A] Regarding responsiveness to canine IL-31RA, selected rat mAbs were tested against canine IL-31RA. Figure 2A: 4G7 (●), 20B8 (■), 22B4 (▲), 27A10 (▼) and rat IgG2a / kappa (◆) control. [Figure 2B] Regarding responsiveness to canine IL-31RA, selected rat mAbs were tested against canine IL-31RA. Figure 2B: 38B6 (●), 48B1 (■), 49D3 (▲) and rat IgG2a / kappa (◆) control. [Figure 2C] Regarding responsiveness to canine IL-31RA, selected rat mAbs were tested against canine IL-31RA. Figure 2C: 10A12 (■), 44E2 (▼), and rat IgG2a / kappa (◆) control. [Figure 2D] Regarding responsiveness to canine IL-31RA, selected rat mAbs were tested against canine IL-31RA. Figure 2D: 47F3 (●), 51G4 (■), and rat IgG2a / kappa (▼) control. [Figure 2E] Regarding reactivity to canine IL-31RA, selected rat mAbs were tested against canine IL-31RA. The results showed that the selected rat mAbs bound to canine IL-31RA in a dose-dependent manner. All 14 rat monoclonal antibodies tested exhibited strong binding reactivity to canine IL-31RA. Figure 2E: 7D7 (●), 28F12 (■), 53B3 (▲) and rat IgG2a / kappa (X) control and rat IgG2b / kappa (◆) control. [Figure 3]Selected rat mAbs were tested by ELISA for their ability to block the binding of canine IL-31 to canine IL-31RA. The results showed that some of the selected mAbs could block the binding of canine IL-31 to canine IL-31RA in a dose-dependent manner, while others could not. Figure 3 shows the antibodies: 4G7 (●), 28F12 (▲), 44E2 (▼), 48B1 (■), as well as rat IgG2a / kappa (△) controls and rat IgG2b / kappa (∇) controls. [Figure 4] We tested Ba / f3-OI cells expressing the IL-31 receptor complex for IL-31-induced STAT-3 phosphorylation. The results showed that STAT-3 phosphorylation was dose-dependently induced by IL-31 in Ba / f3-OI cells (■), suggesting that (i) the canine IL-31 receptor complex is successfully expressed on the cell surface, (ii) binding of canine IL-31 to the IL-31 receptor can stimulate endogenous STAT3 phosphorylation, and (iii) subsequently initiate downstream signaling pathways. Ba / f3 cells (●) were used as a control. [Figure 5] Inhibition of IL-31-mediated STAT-3 phosphorylation in Ba / f3-OI cells. The results show inhibition of IL-31-mediated STAT-3 phosphorylation by antibodies 4G7 (●) and 44E2 (▲). Medium containing cIL-31 (X) or medium without cIL-31 (+) was used as a control. [Figure 6A] Figure 6A shows the binding of canine anti-canine IL-31RA antibodies containing either a lambda(L) light chain or a kappa(k) light chain as evaluated by ELISA. Chimeric rat / canine: Chimeric 10A12[●], canine 10A12VH1VL5[∇], canine 10A12VH1VL6[■], canine 10A12VH2VL5[▲] and canine 10A12VH2VL6[▼]. [Figure 6B]Figure 6B shows the binding of canine anti-canine IL-31RA antibodies containing either a lambda (L) light chain or a kappa (k) light chain, as evaluated by ELISA. Chimeric rat / canine: Chimeric 28F12 [●], canine 28F12VH1VK3 [■], canine 28F12VH1VK4 [▲], canine 28F12VH2VK2 [▼], canine 28F12VH2VK3 [∇], and canine 28F12VH2VK4 [△]. [Figure 6C] Figure 6C shows the binding of canine anti-canine IL-31RA antibodies containing either a lambda (L) light chain or a kappa (k) light chain, as evaluated by ELISA. Chimeric rat / canine: Chimeric 44E2[●], canine 44E2VH2VK1[■], canine 44E2VH2VK2[▲], canine 44E2VH5VK1[▼], canine 44E2VH5VK2[∇], and canine 44E2VH4VK1[△]. The results indicate that canine anti-canine IL-31RA antibodies bind to canine IL-31RA. [Figure 7A] This graph shows the inhibition of cIL-31-mediated STAT-3 phosphorylation by cIL-31RA antibody. Figure 7A shows the inhibition of cIL-31-mediated STAT-3 phosphorylation by cIL-31RA antibody (c10A12). Chimeric rat / canine: Chimeric 10A12[●] and canine 10A12VH2VL6[■]. [Figure 7B] This graph shows the inhibition of cIL-31-mediated STAT-3 phosphorylation by cIL-31RA antibody. Figure 7B shows the inhibition of cIL-31-mediated STAT-3 phosphorylation by cIL-31RA antibody (c28F12). Chimeric rat / canine: Chimeric 28F12[●], canine 28F12VH2K2[■] and canine 28F12VH2VK3[▲]. [Figure 7C]This graph shows the inhibition of cIL-31-mediated STAT-3 phosphorylation by cIL-31RA antibodies. Three different canine monoclonal anti-canine IL-31RA antibodies, called canine 10A12, canine c28F12, and canine 44E2, were evaluated for their ability to inhibit STAT-3 phosphorylation. Figure 7C shows the inhibition of cIL-31-mediated STAT-3 phosphorylation by the cIL-31RA antibody (c44E2). Chimeric rat / canine: Chimeric c44E2[●], canine 44E2VH2VK1[■], canine 44E2VH2VK2[▲], and canine 44E2VH5VK2[▼]. The data show that all three antibodies induce dose-dependent inhibition of STAT-3 phosphorylation in the presence of IL-31. [Figure 8A] Figure 8A shows the epitopes on canine IL-31RA for antibodies 10A12, 28F12, and 44E2, respectively. Figure 8A shows the amino acid sequences of SEQ ID NOs. 104 and 105, respectively. [Figure 8B] Figure 8B shows the epitopes on canine IL-31RA for antibodies 10A12, 28F12, and 44E2, respectively. [Figure 8C] Figure 8C shows the epitopes on canine IL-31RA for antibodies 10A12, 28F12, and 44E2, respectively. Figure 8C shows the amino acid sequences of SEQ ID NOs. 102 and 103, respectively. [Modes for carrying out the invention]

[0040] In response to the need for better treatments for atopic dermatitis, the present invention provides formulations and methods that can achieve remarkable effects against skin inflammation associated with atopic dermatitis.

[0041] Abbreviation Throughout the detailed description and examples of this invention, the following abbreviations will be used: ADCC antibody-dependent cytotoxicity CDC complement-dependent cell injury Complementarity-determining regions within immunoglobulin variable regions, defined using the CDR Kabat numbering system. EC50 50% concentration that produces an effect or binding ELISA enzyme-linked immunosorbent assay FR Antibody Framework Region: Immunoglobulin Variable Region excluding the CDR Region Concentration that results in 50% inhibition of IC50 IgG (Immunoglobulin G) An immunoglobulin alignment and numbering system developed 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 known as Mab or MAb) The V region is a segment of the IgG chain whose sequence is variable between different antibodies. VH Immunoglobulin Heavy Chain Variable Region VL (Variable Region of Immunoglobulin Lambda Light Chain) VK Immunoglobulin Kappa Light Chain Variable Region

[0042] definition To make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise specifically defined elsewhere in this specification, all other technical and scientific terms used herein have meanings that are generally understood by those skilled in the art to which the present invention pertains.

[0043] As used herein, including in the attached claims, the singular forms of words, such as "a," "an," and "the," include their corresponding plural references unless the context clearly indicates otherwise.

[0044] "Administration" and "treatment" refer to the contact between an exogenous drug, therapeutic agent, diagnostic agent or composition and an animal, such as a canine subject, cells, tissues, organs or bodily fluids, when applied to an animal, such as a canine subject, cells, tissues, organs or bodily fluids. Cellular treatment includes contact between the reagent and the cell, and, if the fluid is in contact with the cell, contact between the reagent and the fluid.

[0045] "Administration" and "processing" also mean, for example, in vitro and ex vivo processing of cells with reagents, diagnostic compounds, conjugate compounds or other cells. The term "subject" refers to any living organism, preferably an animal, more preferably a mammal (e.g., a dog, cat or human), most preferably a dog.

[0046] "Treat" or "treating" means administering, internally or externally, a therapeutic agent, for example, a composition containing any of the antibodies of the present invention, to a canine subject or patient suspected of having one or more symptoms or a condition for which the agent is therapeutically active, for example. Typically, the agent is administered in an amount effective to alleviate and / or improve one or more disease / condition symptoms in the treated subject or population, whether by inducing regression of such (one or more) symptoms or inhibiting the progression of such (one or more) symptoms to any clinically measurable extent. The amount of therapeutic agent effective to alleviate any particular disease / condition symptom (also called the "therapeutic effective dose") may vary depending on factors such as the disease / condition, the age and weight of the patient (e.g., a dog), and the ability of the pharmaceutical composition to induce the desired response in the subject. Whether the disease / condition symptoms have been alleviated or improved can be assessed by any clinical measurement typically used by a veterinarian or other skilled healthcare provider to assess the severity or progression of the symptoms. Embodiments of the present invention (e.g., a treatment method or product) may not be effective in alleviating (one or more) target disease / condition symptoms in any subject, but should alleviate (one or more) target disease / condition symptoms 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), Jonkhiel-Taapstra test, and Wilcoxon test.

[0047] When applied to human, veterinary (e.g., canine) subjects, or research subjects, “treatment” refers to therapeutic procedures, as well as research and diagnostic uses. When applied to human, veterinary (e.g., canine) subjects, or research subjects, or cells, tissues, or organs, “treatment” includes contact between the antibodies of the present invention and, for example, canine or other animal subjects, cells, tissues, physiological compartments, or physiological fluids.

[0048] As used herein, the term "dog" includes, unless otherwise specified, all domestic dogs, Canis lupus familiaris, or Canis familiaris.

[0049] As used herein, the term “cat” refers to any member of the family Felidae. Members of this family include wild, zoo and domestic members, such as domestic cats, purebred and / or hybrid companion cats, cat show cats, laboratory cats, cloned cats, and wild or feral cats.

[0050] As used herein, the term “canine frame” 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. With respect to canine antibodies, in most embodiments, the amino acid sequences of the native canine CDRs are replaced in both chains by the corresponding foreign CDRs (e.g., those derived from mouse or rat antibodies). The heavy and / or light chains of a canine antibody may contain several foreign non-CDR residues to preserve the conformation of the foreign CDRs in the canine antibody and / or to modify the Fc function, for example, as illustrated below and / or as disclosed in U.S. Patent No. 10,106,607, which is incorporated entirely herein by reference.

[0051] The "fragment crystallizable region," abbreviated as "Fc," corresponds to the CH3-CH2 portion of the antibody that interacts with cell surface receptors called Fc receptors. The canine fragment crystallizable regions (cFc) of each of the four canine IgGs were first described by Tang et al. [Vet.Immunol.Immunopathol.80:259-270(2001); also see Bergeron et al., Vet.Immunol.Immunopathol.157:31-41(2014) and U.S. Patent No. 10,106,607].

[0052] As used herein, canine Fc(cFc) "IgG-Bm" is canine IgG-B Fc containing two amino acid residue substitutions D31A and N63A, as in the amino acid sequence of IgG-B SEQ ID NO: 78 (see below), and lacking a c-terminal lysine ("K"). Both the aspartic acid residue (D) at position 31 of SEQ ID NO: 77 and the asparagine residue (N) at position 63 of SEQ ID NO: 77 are substituted with an alanine residue (A) in IgG-Bm. These two amino acid residue substitutions help significantly reduce antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cell-mediated cytotoxicity (CDC) in naturally occurring canine IgG-B [see U.S. Patent No. 10,106,607, the entirety of which is incorporated herein by reference]. Further amino acid substitutions for IgG-Bm are also conceivable, and these may include amino acid substitutions to promote heterodimerization in bispecific antibodies, in parallel with those that may occur within IgG-B.

[0053] The amino acid sequence of IgG-B, sequence number 77, is as follows: [ka]

[0054] The amino acid sequence of IgG-Bm, sequence number 78, is shown below. [ka]

[0055] As used herein, “substitution of an amino acid residue” by another amino acid residue in the amino acid sequence of an antibody is equivalent to, for example, “replacing an amino acid residue” by another amino acid residue, and indicates that a particular amino acid residue at a particular position in the amino acid sequence is replaced (or substituted) by a different amino acid residue. Such substitutions can be specifically designed, i.e., for example, by recombinant DNA technology, alanine can be intentionally replaced by serine at a particular position in the amino acid sequence. Alternatively, a particular amino acid residue or a set of amino acid residues in an antibody may be replaced by 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., an epitope or part thereof, and / or so that the antibody contains a particular CDR that retains the same canonical structure as the substituted CDR. Such substitutions / replacements may result in “mutant” CDRs and / or mutant antibodies.

[0056] As used herein, the term “antibody” refers to any form of antibody exhibiting desired biological activity. Antibodies can be monomers, dimers, or even larger polymers. Therefore, the term “antibody” is used in its broadest sense and is not limited to, but specifically includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), canine antibodies, fully canine antibodies, chimeric antibodies, and camel-derived 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, for example, canine modification of the antibody for use as a canine therapeutic antibody.

[0057] As used herein, for example, an antibody that “blocks” or “blocks” or “blocks binding” to a canine receptor to its binding partner (ligand) is an antibody that (partially or completely) blocks the binding of a canine receptor to its canine ligand, as determined by a standard binding assay (e.g., BIACore®, ELISA, or flow cytometry), and vice versa.

[0058] Typically, the 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 its activity is expressed in molar terms. Preferably, the 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 contain conserved or non-conserved amino acid substitutions that do not substantially alter its biological activity (referred to as “conserved variants” or “function-conserving variants” of the antibody).

[0059] "Isolated antibody" refers to a purified state, and in such context, means that the molecule is substantially free from other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth media. In general, the term "isolated" is not intended to mean the complete absence of such materials, or the absence of water, buffers, or salts, unless they are present in amounts that substantially prevent the experimental or therapeutic use of the conjugated compounds described herein.

[0060] As used herein, an antibody is said to bind specifically to a polypeptide containing a portion of the amino acid sequence of canine IL-31RA if it binds to a polypeptide containing a portion of the amino acid sequence of canine IL-31RA but does not bind to other canine proteins lacking that portion of the sequence. For example, an antibody that specifically binds to a polypeptide containing canine IL-31RA may bind to the FLAG®-tagged form of canine IL-31RA but not to other FLAG®-tagged canine proteins. An antibody, or a conjugate compound derived from the antigen-binding site of an antibody, binds "specifically" to the canine antigen, its variant, or mutain if it has an affinity for that canine antigen, or its variant, or mutain at least 10 times, more preferably at least 20 times, and even more preferably at least 100 times, than its affinity for any other canine antigen tested.

[0061] As used herein, “chimeric antibody” is an antibody having a variable domain derived from a first antibody and a constant domain derived from a second antibody, and the first and second antibodies originate 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 domain is obtained from an antibody derived from an experimental animal such as a rodent ("parent antibody"), and the constant domain sequence is obtained from an animal subject antibody, e.g., human or dog, and as a result the resulting chimeric antibody is less likely to induce an adverse immune response in human or canine subjects than the parent (e.g., rodent) antibody.

[0062] As used herein, the term “canine antibody” refers to a form of antibody containing sequences derived from both canine and non-canine (e.g., rat) antibodies. Generally, a canine antibody comprises at least one, typically two, substantially all variable domains, where all or substantially all of the hypervariable loops correspond to those of a non-canine immunoglobulin (e.g., including six CDRs as illustrated below), and all or substantially all of the framework (FR) region (and typically all or substantially all of the remaining frame) are those of a canine immunoglobulin sequence. As illustrated herein, a canine antibody comprises both three heavy-chain CDRs and three light-chain CDRs derived from a rat anti-canine antigen antibody, along with a canine frame or a modified canine frame. A modified canine frame includes one or more amino acid changes, as illustrated herein, to further optimize the efficacy of the canine antibody, for example, to increase its ability to block its binding to a canine antigen and / or its binding to a natural binding partner of the canine antigen.

[0063] The variable regions of each light / heavy chain pair form antibody binding sites. Therefore, generally, intact antibodies have two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites are generally identical. Typically, both the heavy and light chain variable domains contain three hypervariable regions, also called complementarity-determining regions (CDRs), located within a relatively conserved framework region (FR). The CDRs are usually aligned by the framework region, enabling binding to specific epitopes. Generally, from the N-terminus to the C-terminus, both the light and heavy chain variable domains contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments to each domain are generally found in *Sequences of Proteins of Immunological Interest*, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5 thed.;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)].

[0064] As used herein, the term “hypervariable region” refers to amino acid residues of an antibody that are involved in antigen binding. The hypervariable region includes amino acid residues derived from the “complementarity-determining region” or “CDR” (i.e., LCDR1, LCDR2, and LCDR3 in the light chain variable domain and HCDR1, HCDR2, and 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) for a sequence definition of the antibody CDR region. Also see Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987) for a structural definition of the antibody CDR region.] As used herein, the term “framework” residue or “FR” residue refers to variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0065] There are four known IgG heavy chain subtypes of canine IgG, which are called IgG-A, IgG-B, IgG-C, and IgG-D. Two known light chain subtypes are called lambda and kappa. In specific embodiments of the present invention, in addition to binding to canine IL-31RA, the canine antibody or canine antibody against its antigen of the present invention optimally has the following two attributes: 1. Lack of effector function such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cell-mediated cytotoxicity (CDC), as well as 2. Purification should be easily carried out on a large scale using industry-standard techniques, such as those based on protein A chromatography.

[0066] None of the naturally occurring canine IgG isotypes meet both criteria. For example, IgG-B can be purified using protein A, but it has high levels of ADCC activity. On the other hand, IgG-A binds weakly to protein A, but it also exhibits ADCC activity. Furthermore, IgG-D does not exhibit ADCC activity, and neither IgG-C nor IgG-D can be purified using a protein A column. (IgG-C has considerable ADCC activity.) One way the present invention addresses these problems is by providing a modified canine IgG-B antibody of the present invention that is specific to the antigen of the present invention, lacks effector function such as ADCC, and can be easily purified using industry-standard protein A chromatography.

[0067] As used herein, “antipruritic” refers to a compound, polymer, and / or formulation that tends to inhibit, reduce, and / or prevent itching. Antipruritic is colloquially called an anti-itch medication.

[0068] As used herein, “antipruritic antibody” is an antibody that can act as an antipruritic agent in animals, including mammals such as humans, dogs, and / or cats, particularly with respect to atopic dermatitis. In certain embodiments, the antipruritic antibody binds to specific proteins of the IL-31 signaling pathway, such as IL-31 or its receptor IL-31RA. The binding of the antipruritic antibody to its corresponding antigen (e.g., IL-31 or IL-31RA) inhibits, for example, the binding of IL-31 to IL-31RA, thereby interfering with and / or preventing good signaling in this pathway, and thereby inhibiting, reducing, and / or preventing itching that would normally be caused by the IL-31 signaling pathway.

[0069] As used herein, “homology” refers to the sequence similarity between two polynucleotide sequences or between two polypeptide sequences when they are optimally aligned. Two sequences being compared are homologous at a given position if the same base or amino acid residue occupies the same position in both sequences; for example, if the position in each of two DNA molecules is occupied by adenine. The percentage of homology is the number of homologous positions shared by the two sequences, divided by the total number of positions compared × 100. For example, if six out of ten positions in two sequences coincide or are homologous when the sequences are optimally aligned, the two sequences are 60% homologous. Generally, comparisons are made when the two sequences are aligned to yield the maximum percentage of homology. 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 considered 100% "identical" to the second amino acid sequence.

[0070] Therefore, if 50% of the amino acid residues of two amino acid sequences are identical, the amino acid sequence is 50% "identical" to the second amino acid sequence. Sequence comparison is performed on a contiguous block of amino acid residues contained in a given protein, e.g., the protein being compared, or a portion of a polypeptide. In certain embodiments, selected deletions or insertions that would normally alter the correspondence between two amino acid sequences are taken into consideration. Sequence similarity includes identical residues and biochemically related amino acids that are not identical. Biochemically related amino acids that share similar properties and may be interchangeable.

[0071] A “conservatively modified variant” or “conservative substitution” refers to the substitution of an amino acid within a protein by another amino acid with similar characteristics (e.g., charge, side chain size, hydrophobic / hydrophilicity, back chain conformation, and rigidity) that can be frequently altered without changing the protein’s biological activity. Those skilled in the art generally recognize that single amino acid substitutions within non-essential regions of polypeptides 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, substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity. Exemplary conservative substitutions are shown in Table A below.

[0072] [Table 1]

[0073] Functionally conserved variants of the antibodies of the present invention are also contemplated by the present invention. As used herein, “functionally conserved variant” refers to an antibody or fragment in which one or more amino acid residues are altered without changing desired properties, such as antigen affinity and / or specificity. Such variants include, but are not limited to, amino acid substitutions having similar properties, such as the conservative amino acid substitutions in Table A above.

[0074] "Isolated nucleic acid molecule" means DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or any combination thereof, that is not related to all or part of a polynucleotide, whether isolated polynucleotide found in nature or ligated to a polynucleotide that is not ligated in nature. For the purposes of this disclosure, it should be understood that a "nucleic acid molecule containing" a particular nucleotide sequence does not include an intact chromosome. An isolated nucleic acid molecule "containing" a specified nucleic acid sequence may, in addition to the specified sequence, contain up to 10, or even up to 20, or more other coding sequences of proteins or parts or fragments thereof, or may contain activatably ligated regulatory sequences that control the expression of the coding region of the listed nucleic acid sequence, and / or may contain vector sequences.

[0075] The present invention provides an isolated canine antibody and a method of using the antibody in the treatment of a disease, for example, canine atopic dermatitis. In dogs, there are four IgG heavy chains called A, B, C, and D. These heavy chains represent four different subclasses of canine IgG, called 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 called CH-1, CH-2, and CH-3. The CH-1 domain is connected to the CH-2 domain via an amino acid sequence called a "hinge" or "hinge region".

[0076] The nucleic acid 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 nucleic acid sequences of 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, IgGB has accession number AAL35302.1, IgGC has accession number AAL35303.1, and IgGD has accession number AAL35304.1. The canine antibody also contains two light chains, kappa and lambda. The DNA and amino acid sequences of these light chains are also available from the GenBank database. For example, the amino acid sequence of the kappa light chain has accession number ABY 57289.1, and the lambda light chain has accession number ABY 55569.1.

[0077] In this invention, the amino acid sequences of each of the four canine IgG Fc fragments are based on the identified boundaries of the CH1 and CH2 domains determined by Tang et al (previously cited). Canine rat anti-canine antibodies that bind to canine IL-31RA include, but are not limited to, antibodies of the present invention that contain canine IgG-A heavy chain, canine IgG-B heavy chain, canine IgG-C heavy chain, and canine IgG-D heavy chain and / or canine kappa light chain or canine lambda light chain together with rat anti-canine IL-31RA CDR. Accordingly, the present invention provides canine rat anti-canine antibodies of the present invention, which include isolated canine rat anti-canine antibodies that bind to canine IL-31RA and preferably also block the binding of that canine IL-31RA to canine IL-31.

[0078] Therefore, the present invention further provides canine rat antibodies and methods for using the antibodies in the treatment of disease conditions, such as canine atopic dermatitis.

[0079] The present invention further provides full-length canine heavy chains that can be adapted to the corresponding light chains for the production of canine antibodies. Accordingly, the present invention further provides canine rat anti-canine antigen antibodies (including isolated canine rat anti-canine antibodies) and methods of using the antibodies in the treatment of pathological conditions, for example, in the treatment of canine atopic dermatitis.

[0080] The present invention also provides antibodies comprising a canine fragment crystallizable region (cFc region) in which cFc is genetically modified to increase, decrease, or eliminate one or more effector functions. In one aspect of the present invention, the genetically modified cFc decreases or eliminates one or more effector functions. In another aspect of the present invention, the genetically modified cFc increases one or more effector functions. In a particular embodiment, the genetically modified cFc region is a genetically modified canine IgGB Fc region. In another such embodiment, the genetically modified cFc region is a genetically modified canine IgGC Fc region. In a particular embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC) which is increased, decreased, or eliminated. In another embodiment, the effector function is complement-dependent cell-mediated cytotoxicity (CDC) which is increased, decreased, or eliminated. In yet another embodiment, the cFc region is genetically modified to increase, decrease, or eliminate both ADCC and CDC.

[0081] Numerous mutant canine IgGB heavy chains were generated to produce canine IgG variants lacking effector function. These variants may contain one or more single or combined substitutions in the Fc portion of the heavy chain amino acid sequence, namely P4A, D31A, N63A, G64P, T65A, A93G, and P95A. The mutant heavy chains (i.e., those containing such amino acid substitutions) are cloned into expression plasmids and transfected into HEK 293 cells along with plasmids containing the gene encoding the light chain. Intact antibodies are expressed and purified from HEK 293 cells and then, to evaluate their potential for mediating immunoeffector function, are used to analyze the Fc portion. γ The binding to RI and C1q can be evaluated. [See U.S. Patent No. 10,106,607, the entirety of which is incorporated herein by reference.]

[0082] The present invention also provides a modified canine IgG-D comprising a hinge region from the following sources instead of its natural IgG-D hinge region: [ka]

[0083] Alternatively, the IgG-D hinge region can be modified by substituting a serine residue with a proline residue, i.e., [ka] It can be genetically modified by substituting naturally occurring serine residues with proline residues (P) that are underlined and in bold. Such modifications can result in 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 variants, the nucleic acid encoding the amino acid sequence of canine IgG-D can be modified to encode modified IgG-D. The modified nucleic acid sequence is then cloned into an expression plasmid for protein expression.

[0084] The six complementarity-determining regions (CDRs) of the canine rat anti-canine antibody described herein may include canine antibody kappa(k) light chains or canine antibody lambda(l) light chains containing rat light chains LCDR1, LCDR2, and LCDR3, and canine antibody heavy chain IgG containing rat heavy chains HCDR1, HCDR2, and HCDR3.

[0085] nucleic acid The present invention further comprises nucleic acids encoding the antibody of the present invention (see, for example, the following examples). The present invention also includes nucleic acids encoding immunoglobulin polypeptides that, when compared using the BLAST algorithm, contain amino acid sequences that are 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 sequences of canine antibodies, with the exception of the unchanging CDRs provided herein, wherein the parameters of the algorithm are selected to give the maximum match between each sequence over the full length of each reference sequence. The present invention further provides nucleic acids encoding immunoglobulin polypeptides that, when compared using the BLAST algorithm, contain amino acid sequences that are 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, wherein the parameters of the algorithm are selected to give the maximum match between each sequence over the full length of each reference sequence.

[0086] As used herein, the percentage identity of nucleotide and amino acid sequences can be determined using C, MacVector (MacVector, Inc. Cary, NC 27519), Vector NTI (Informax, Inc. MD), Oxford Molecular Group PLC (1996), and Clustal W algorithms, along with default parameters for alignment and identity. These commercially available programs can also be used to determine sequence similarity using the same or similar default parameters. Alternatively, for example, an 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) pile-up program, which uses default parameters.

[0087] The following references concern the BLAST algorithm, which is often used for sequence analysis: BLAST ALGORITHMS: Altschul, SF, et al., J.Mol.Biol.215:403-410(1990); Gish, W., et al., Nature Genet.3:266-272(1993); Madden, TL, et al., Meth.Enzymol.266:131-141(1996); Altschul, SF, et al., Nucleic Acids Res.25:3389-3402(1997); Zhang, J., et al., Genome Res.7:649-656(1997); Wootton, JC, et al., Comput.Chem.17:149-163(1993); Hancock, JMet 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, suppl. 3. MODayhoff (ed.), pp. 345-352, (1978); Natl. Structure, vol. 5, suppl. 3.” (1978), MODayhoff (ed.), pp. 353-358 (1978), Natl. Biomed. Res. Found., Washington, DC; Altschul, SF, J. Mol. 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);andAltschul,SF “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).

[0088] Antibody protein engineering For example, but not limited to, the canine heavy chain constant region may be derived from IgG-B or modified cFc, e.g., IgG-Bm as used herein [see U.S. Patent No. 10,106,607, which is incorporated herein in whole by reference], and the canine light chain constant region may be derived from kappa or lambda.

[0089] Antibodies may be manipulated, for example, to include modifications to the canine framework and / or canine frame residues within the variable domain of the parental (i.e., rat) monoclonal antibody in order to improve the properties of the antibody.

[0090] The construction of canine-modified anti-canine IL-31 receptor alpha monoclonal antibodies can be carried out by determining the DNA sequences encoding the heavy and light chains of canine IgG. The DNA and protein sequences of canine heavy and light chains are publicly known in the art and can be obtained by searching the NCBI gene and protein database. As described above, for canine antibodies, there are four known IgG subtypes, namely IgG-A, IgG-B, IgG-C, and IgG-D, as well as two types of light chains, namely kappa and lambda.

[0091] Canine rat anti-canine IL-31 antibodies can be recombinantly produced by methods known in the art. Mammalian cell lines available as hosts for the expression of the antibodies or fragments disclosed herein are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). 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., Hep G2), A549 cells, 3T3 cells, HEK-293 cells, and several other cell lines. Mammalian host cells include human cells, mouse cells, rat cells, canine cells, monkey cells, porcine cells, goat cells, bovine cells, horse cells, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels. Other cell lines that may be used include insect cell lines, e.g., Sf9 cells, amphibian cells, bacterial cells, plant cells, and fungal cells. When a recombinant expression vector encoding a heavy chain or its antigen-binding portion or fragment, a light chain and / or its antigen-binding fragment is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow for antibody expression within the host cell, or more preferably, the secretion of the antibody into the culture medium in which the host cell is growing.

[0092] Antibodies can be recovered from culture media using standard protein purification methods. Furthermore, the expression of the antibodies of the present invention (or other parts thereof) from producing cell lines can be enhanced using several known techniques. For example, the glutamine synthetase gene expression system (GS system) is a common method for enhancing expression under specific conditions. The GS system is described in whole or in part in relation to European Patent No. 0216846, European Patent No. 0256055, and European Patent No. 0323997, and European Patent Application No. 89303964.4.

[0093] In certain embodiments, the antibody or antigen-binding fragment includes a heavy chain constant region, e.g., a canine constant region, e.g., IgG-A, IgG-B, IgG-C, and IgG-D canine heavy chain constant regions or variants thereof. In certain embodiments, the antibody or antigen-binding fragment includes a light chain constant region, e.g., a canine light chain constant region, e.g., a lambda or kappa canine light chain region or variants thereof. For example, but not limited to, the canine heavy chain constant region may be derived from IgG-B, and the canine light chain constant region may be derived from kappa.

[0094] Epitope Mapping The interaction between an antibody and its homologous protein antigen is mediated through the binding of specific amino acids (paratopes) of the antibody to specific amino acids (epitopes) of the target antigen. Epitopes are antigenic determinants that trigger a specific response by immunoglobulins. Epitopes consist of a group of amino acids on the surface of an antigen. A target protein may contain several epitopes that are recognized by different antibodies. Epitopes recognized by antibodies are classified as linear epitopes or conformational epitopes. Linear epitopes are formed by stretching a continuous sequence of amino acids within a protein, while conformational epitopes consist of amino acids that are discontinuous in the primary amino acid sequence (e.g., far apart) but come together during three-dimensional protein folding.

[0095] Epitope mapping refers to the process of identifying amino acid sequences (i.e., epitopes) recognized by antibodies on target antigens. Identifying epitopes recognized by monoclonal antibodies (mAbs) on target antigens has important applications. For example, it can aid in the development of new therapeutic agents, diagnostics, and vaccines. Epitope mapping can also assist in the selection of optimized therapeutic mAbs and help elucidate their mechanisms of action. Epitope information on the IL-31 receptor alpha can also elucidate unique epitopes and define the protective or pathogenic effects of vaccines. Epitope identification can also lead to the development of subunit vaccines based on chemical or genetic coupling of identified peptide epitopes with carrier proteins or other immunostimulants.

[0096] Epitope mapping can be performed using polyclonal or monoclonal antibodies, and several methods are used for epitope identification depending on the suspected properties of the epitope (i.e., linear allele conformation). Mapping linear epitopes is simpler and relatively easier. For this purpose, commercial services for linear epitope mapping often use peptide scanning. In this case, a series of overlapping short peptide sequences of the target protein are chemically synthesized and tested for their ability to bind to the antibody of interest. This strategy is rapid, high-throughput, and relatively inexpensive to perform. On the other hand, mapping discontinuous epitopes is relatively technically difficult and requires more specialized techniques such as X-ray cocrystallization of monoclonal antibodies with their target proteins, hydrogen-deuterium (H / D) exchange, mass spectrometry combined with enzymatic digestion, and several other methods known to those skilled in the art.

[0097] Epitope binding and cross-blocking antibodies The anti-canine IL-31RA antibody or its antigen-binding fragment of the present invention includes, in the case of canine IL-31RA, one of the antibodies disclosed herein, for example, a canine antibody, a 28F12 antibody that binds to an epitope comprising the amino acid sequence of SEQ ID NO: 101, and any antibody or antigen-binding fragment that (partially or completely) cross-blocks the antibody or fragment described herein for canine IL-31RA binding, or is (partially or completely) cross-blocked by the antibody or fragment described herein for canine IL-31RA binding, as well as any variant thereof, an antibody or its antigen-binding fragment that binds to the same epitope as the antibody or fragment described herein for canine IL-31RA binding.

[0098] Cross-blocking antibodies and antigen-binding fragments can be identified in standard binding assays (e.g., BIAcore®, ELISA, or flow cytometry, as illustrated below) based on their ability to cross-compete with, for example, the 28F12 antibody. For example, a standard ELISA assay can be used in which recombinant canine IL-31RA protein is immobilized on a plate, one of the antibodies is fluorescently labeled, and the ability of the unlabeled antibody to compete for binding with the labeled antibody is assessed. Additionally or alternatively, BIAcore® analysis can be used to assess the ability of antibodies to cross-compete. The ability of a test antibody to inhibit the binding of the 28F12 antibody to canine IL-31RA demonstrates that the test antibody can compete with the 28F12 antibody for binding to canine IL-31RA and, therefore, in some cases, can bind to the same epitope on canine IL-31RA to which the 28F12 antibody binds. Antibodies and fragments thereof that bind to the same epitopes as any of the anti-canine IL-31RA antibodies or fragments of the present invention also form part of the present invention.

[0099] Pharmaceutical composition and administration To prepare a pharmaceutical composition or sterile composition containing the antibodies of the present invention, these antibodies can be mixed with a pharmaceutically acceptable carrier or excipient. [See, for example, Remington's Pharmaceutical Sciences and USPharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984)].

[0100] Formulations of therapeutic and diagnostic agents can be prepared by mixing them with acceptable carriers, excipients, or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions, or suspensions. [For example, 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] Forms: See Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY. In one embodiment, the antibody of the present invention is diluted to an appropriate concentration in a sodium acetate solution pH 5-6, and NaCl or sucrose is added for tonicity. Additional agents such as polysorbate 20 or polysorbate 80 may be added to enhance stability.

[0101] The toxicity and therapeutic efficacy of an antibody composition administered alone or in combination with another agent can be determined, for example, by standard pharmaceutical procedures for cell culture or experimental animals to determine LD 50 (the dose lethal to 50% of the population) and ED 50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic effect and the therapeutic effect is the therapeutic index (LD 50 / ED 50 ). In certain embodiments, antibodies with a high therapeutic index are desirable. The data obtained from these cell culture assays and animal tests can be used to devise a range of dosages for use in dogs. The dosage of such a compound preferably lies within a range of circulating concentrations that have little or no toxicity and include ED 50 . The dosage can vary within this range depending on the dosage form used and the route of administration.

[0102] The mode of administration can vary. Suitable routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal or intraarterial. In certain embodiments, the antibodies of the invention can be administered by invasive routes such as injection. In further embodiments of the invention, the antibodies or pharmaceutical compositions thereof of the invention are administered intravenously, subcutaneously, intramuscularly, intraarterially or by inhalation, aerosol delivery. Administration by non-invasive routes (e.g., oral; e.g., pills, capsules or tablets) is also within the scope of the invention.

[0103] The compositions may be administered using medical devices known in the art. For example, the pharmaceutical compositions of the present invention may be administered by injection using a subcutaneous needle, including a pre-filled syringe or an automated injection device. The pharmaceutical compositions disclosed herein may also be administered using needleless subcutaneous injection devices, such as those disclosed in U.S. Patent Nos. 6,620,135, 6,096,002, 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556.

[0104] The pharmaceutical compositions disclosed herein may also be administered by infusion. Examples of well-known implant and module configurations for administering pharmaceutical compositions include U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing a drug at a controlled rate; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering a drug at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; and U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multiple chamber compartments. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

[0105] Alternatively, the antibody of the present invention may be administered topically rather than systemically, often in the form of a depot formulation or a sustained-release formulation.

[0106] The administration regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic antibody, the level of symptoms, the immunogenicity of the therapeutic antibody, and the accessibility of target cells in the biological matrix. Preferably, the administration regimen delivers sufficient therapeutic antibody to bring about improvement in the target disease / condition while simultaneously minimizing undesirable side effects. Therefore, the amount of biologic delivered depends in part on the specific therapeutic antibody and the severity of the condition being treated. Guidelines are available for selecting the appropriate dose of therapeutic antibody [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 See 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)].

[0107] The determination of the appropriate dose is made by a veterinarian, for example, using parameters or factors known or suspected in the art to affect the treatment. Generally, the dose is started at a somewhat lower amount than the optimal dose and then gradually increased until the desired or optimal effect is achieved against any negative side effects. Important diagnostic measures include symptomatic diagnostic measures.

[0108] The antibodies provided herein may be provided by serial infusion or by doses administered, for example, daily, 1 to 7 times per week, weekly, bi-weekly, monthly, bi-monthly, quarterly, semi-annually, or annually. The doses may be provided, for example, intravenously, subcutaneously, topically, orally, intranasally, rectally, intramuscularly, intracerebrally, intraspinally, or by inhalation. The total weekly dose is generally at least 0.05 μg / kg body weight, and more generally 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 / ml, 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 See Immunol.Immunother.52:133-144(2003). Doses may also be provided to achieve predetermined target concentrations of the antibody of the present invention in canine serum, e.g., 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg / ml or higher. In other embodiments, the antibody of the present invention is administered subcutaneously or intravenously at doses of 10, 20, 50, 80, 100, 200, 500, 1000 or 2500 mg / subject, weekly, bi-weekly, "every four weeks", monthly, bi-monthly, or quarterly.

[0109] As used herein, “inhibit,” “treat,” or “treatment” includes delaying the onset of symptoms associated with a disorder and / or reducing the severity of symptoms of such disorder. The terms further include improving existing uncontrolled or undesirable symptoms, preventing additional symptoms, and improving or preventing the underlying cause of such symptoms. Accordingly, the terms mean that a beneficial outcome was given to a vertebrate subject (e.g., a dog) having a disorder, condition and / or symptoms, or being at risk of developing such disorder, disease or symptoms.

[0110] As used herein, the terms “therapeutic effective dose,” “therapeutic effective dosage,” and “effective dose” refer to the amount of the antibody of the present invention that, when administered alone or in combination with additional therapeutic agents to cells, tissues, or subjects, such as dogs, is effective in causing one or more symptoms of a disease or condition, or a measurable improvement in the progression of such a disease or condition. The therapeutic effective dose further refers to the amount of antibody sufficient to result in at least partial improvement of symptoms, e.g., treatment, cure, prevention, or improvement of the associated medical condition, or an increase in the rate of treatment, cure, prevention, or improvement of such a condition. When applied to combinations, the therapeutic effective dose refers to the combined amount of active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective dose of a therapeutic agent results in 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% of a diagnostic measure or parameter. An effective dose may also result in an improvement in a subjective measure when the subjective measure is used to assess the severity of the condition.

[0111] [Examples] [Example 1] IL-31 receptor alpha Nucleotide sequence The nucleotide sequence of Sequence ID No. 1 encodes the extracellular domain of canine IL-31 receptor alpha (cIL-31RA) fused to the HIS tag. The canine IL-31RA ECD HIS-tagged protein contains the amino acid sequence of Sequence ID No. 2. The nucleotide sequences were prepared by chemical synthesis and then cloned into expression plasmids suitable for the production of the corresponding proteins in eukaryotic cells, either HEK-293 or CHO cells. [ka]

[0112] [Example 2] Expression and purification of IL-31 receptor alpha-ECD Following the manufacturer's recommendations, a plasmid containing the nucleotide sequence of SEQ ID NO: 1 was transfected into HEK-293 or CHO cells using electroporation via a MaxCyte instrument. Several days after transfection, the supernatants of the transfected cells and untransfected controls were collected and centrifuged to remove cell debris. Following the manufacturer's recommendations, IL-31RA with the HIS tag was purified from the cell culture medium by passing the clarified recovered fluid from the transfected cells through a nickel column. The purified protein was quantified by measuring the absorbance of ultraviolet light at 280 nm. [ka]

[0113] [Example 3] Binding of canine IL-31RA to biotinylated canine IL-31 protocol 1. Coat one or more immunosuppression plates with IL-31RA protein by diluting it to 1 μg / mL in phosphate-buffered saline (PBS). Add 100 μL / well. Incubate one or more plates overnight at 2–7°C. Wash the plate three times with 2.275 μL / well of phosphate-buffered saline + TWEEN® 20 (PBST). 3. Block the plate with 200 μL / well blocking buffer [1% skim milk powder in PBST (NFDM)] for 30-45 minutes at 36 ± 2°C while gently shaking (120 ± 20 RPM). Wash the plate three times with 4.275 μL / well of PBST. 5. Dilute biotinylated IL-31 (10 μg / mL) 3-fold with 1% NFDM in PBST on a dilution plate, and transfer 100 μL / well to one or more immunosorbent plates. Incubate at 36 ± 2°C for 30–45 minutes with gentle shaking (120 ± 20 RPM). Wash the plate three times with 6.275 μL / well of PBST. 7. Dilute horseradish peroxidase-streptavidin (HRP-streptavidin) with 1% NFDM in PBST to a final dilution of 1:1000. Add 8,100 μL / well of HRP-streptavidin to one or more immunoassay plates and incubate at 36±2°C for 30–45 minutes with gentle shaking (120±20 RPM). Wash the plate three times with 9.275 μL / well of PBST. 10. Immediately before use, combine equal amounts of preheated TMP 2-component substrate. Add 11,100 μL / well of the prepared 3,3',5,5'-tetramethylbenzidine (TMP) substrate to one or more immunoassay plates and incubate in the dark at 36±2°C for 10–15 minutes with gentle shaking (120±20 RPM). The reaction is stopped by adding 12,100 μL / well of 1M H3PO4. 13. Use a microplate reader to read the plate at a wavelength of 450 nm and a reference wavelength of 540 nm.

[0114] [Example 4] Monoclonal antibody against canine IL-31 receptor alpha Monoclonal antibodies (mAbs) against canine IL-31RA were induced by immunizing two Lewis rats multiple times with canine IL-31RA ECD over a period of 3–4 weeks (using 10 μg or 25 μg of antigen / rat each time). After immunization, serum was collected from each rat and tested for canine IL-31RA by ELISA. Hybridomas were created by fusing rat lymph node cells with the highest IL-31RA ECD reactivity to myeloma SP2 / 0 cell lines. Approximately 10 days after fusion, the supernatant obtained from the growing hybridomas was screened by ELISA on plates coated with IL-31RA ECD protein using the protocol described below. As illustrated in Figures 2A–2E below, this ELISA selected approximately 260 clones showing potential binding to IL-31RA, with rat IgG2a / kappa used as a negative control. The majority of clones had an OD450 > 1.

[0115] ELISA procedure: 1. Coat a 96-well half-area plate with IL-31RA (1 μg / mL in PBS buffer), 25 μL / well. 2. Incubate the plate at 4°C overnight. 3. Wash the plate three times using PBST (PBS + 0.05% Tween® 20). 4. Block the plate at room temperature for 30 minutes using blocking buffer (PBS containing 5% fetal bovine serum (FBS)) at 25 ul / well. Transfer 5.25 ul / well of hybridoma supernatant to a 96-well plate and incubate at room temperature for 60 minutes. 6. Wash the plate three times using PBST. Add 7.25 ul / well of anti-rat HRP, diluted 1:4000 in blocking buffer, to the plate and incubate at room temperature for 60 minutes. 8. Wash the plate five times with PBST. 9. Add the TMB reagent to the plate for a colorimetric reaction lasting 9.2 to 3 minutes. The reaction is stopped using 10.0.16M sulfuric acid. 11. Read the plate using a plate reader. Fourteen rat antibodies were selected that were induced against canine IL-31RA, which binds to IL-31. The heavy chain and light chain variable regions of the rat antibodies are shown below. In Example 5 below, these antibodies were further tested for their ability to block the binding of canine IL-31RA to canine IL-31. [ka] TIFF0007905331000009.tif212168TIFF0007905331000010.tif214170TIFF0007905331000011.tif37167

[0116] [Example 5] Blocking activity of anti-IL-31 receptor alpha antibody The blocking ELISA described below evaluated the ability of anti-canine IL-31RA hybridoma supernatant to block the binding of IL-31 to IL-31RA.

[0117] protocol 1. Coat a 96-well half-area plate with IL-31RA (1 μg / mL in PBS buffer), 25 μL / well. 2. Incubate the plate at 4°C overnight. 3. Wash the plate three times with PBST (PBS + 0.05% Tween® 20). 4. Block the plate at room temperature for 30 minutes using blocking buffer (PBS containing 5% FBS), 25 µl / well. Transfer 5.25 ul / well of hybridoma supernatant to a 96-well plate and incubate at room temperature for 60 minutes. 6. Wash the plate three times using PBST. Transfer 7.25 μL / well of biotinylated IL31 (0.5 μg / mL in blocking buffer) and incubate at room temperature for 60 minutes. 8. Wash the plate three times using PBST. Add 9.25 μl / well of streptavidin-HRP, diluted 1:5000 in blocking buffer, to the plate and incubate at room temperature for 60 minutes. 10. Wash the plate five times using PBST. 11. Add the TMB reagent to the plate for a colorimetric reaction lasting 2-3 minutes. The reaction is stopped using 12.0.16M sulfuric acid. 13. Read the plate using a plate reader.

[0118] result: Of the approximately 260 clones that showed binding to IL-31RA, only 20 to 25 clones also showed potential blockade of canine IL-31 binding to canine IL-31RA. Among these, a specific group of three rat anti-canine IL-31RA antibodies (44E2, 4G7, and 28F12) were identified that both bound to IL-31RA and blocked IL-31 binding to IL-31RA. See Figure 3, which uses rat IgG2a and rat IgG2b as negative controls. These three antibodies appear to show a moderate degree of homology in the amino acid sequences of their respective CDRs. These amino acid sequences are also shown in Table 3 below. [ka] TIFF0007905331000013.tif7156

[0119] A second group of six rat anti-canine IL-31RA antibodies that bind to canine IL-31RA and block the binding of canine IL-31 to canine IL-31RA was identified as containing a set of CDRs with significant amino acid sequence similarity. These amino acid sequences are also shown in Table 3 below. [ka] TIFF0007905331000015.tif81156

[0120] [Example 6] Canine antibodies The overall process for producing canine heavy and light chains, which can be mixed in various combinations to produce canine anti-canine IL-31 receptor alpha mAb, involves the following scheme: i) Identify the DNA sequences of the VH and VL domains containing the CDR of the desired anti-IL-31 receptor alpha mAb. ii) Identify the H-chain and L-chain CDRs of the desired anti-IL-31RA mAb. iii) Identify sequences suitable for the H and L chains of canine IgG. iv) Identify the DNA sequences encoding the endogenous CDRs in the H and L strands of canine IgG with the above sequences. v) Replace the DNA sequences encoding the endogenous canine H chain CDR and endogenous canine L chain CDR with the DNA sequence encoding the desired anti-IL-31RA CDR. Furthermore, some canine framework residues may be replaced with residues selected from the desired anti-IL-31 receptor alpha mAb framework region. vi) Synthesize the DNA from step (v), clone it into a suitable expression plasmid, and transfect HEK 293 cells with the plasmid containing the desired canine H and canine L chains. vii) Purify the expressed canine antibody from the HEK 293 supernatant. viii) Test the binding of purified canine antibodies to the alpha chain of the canine IL-31 receptor.

[0121] The application of the process outlined above can yield the following sets of canine H chain and canine L chain sequences. The corresponding sequence numbers are listed in Table 5 below.

[0122] Figures 6A to 6C show the binding of canine anti-canine IL-31RA antibodies containing either a lambda (L) light chain or a kappa (K) light chain, as evaluated by ELISA. The results indicate that canine anti-canine IL-31RA antibodies bind to canine IL-31RA.

[0123] Figures 7A–7C are plots showing the inhibition of cIL-31-mediated STAT-3 phosphorylation by cIL-31RA antibodies using the assay described in Example 6 below. Three different canine monoclonal anti-canine IL-31RA antibodies, called c10A12, c28F12, and c44E2, were evaluated for their ability to inhibit STAT-3 phosphorylation. The data show that all three antibodies result in dose-dependent inhibition of STAT-3 phosphorylation in the presence of IL-31. [ka] TIFF0007905331000017.tif212168TIFF0007905331000018.tif214168TIFF0007905331000019.tif90166

[0124] [Example 7] STAT-3 assay Stat-3 is known to be activated by IL-31 in cells containing a heterodimer receptor for IL-31. To develop an assay to evaluate STAT-3 activation by canine IL-31, nucleotide sequences encoding IL-31RA and OSMR, respectively, were prepared by chemical synthesis and then cloned into the expression vector pcDNA3.1. Vectors containing the nucleotide sequences of IL-31RA and OSMR, respectively, were co-transfected into Ba / f3 cells, and the transfected cells, indicated as "Ba / f3-OI," were grown as a pool under antibiotic selectivity. The ability of canine IL-31 to induce STAT-3 activation was tested as follows.

[0125] material: Cell line: Ba / f3-OI stable pooled cells Growth medium containing mouse IL-3 or canine IL-31 (cIL-31) RPMI 1640 435ml (ThermoFisher, 12633-020) FBS 50mL (SAFC catalog number 12003c-500mL) 2-Mercaptoethanol (50mM) 0.5mL (Gibco 31350-010) 100X Pen Strep 5mL(Gibco 15140-122 Lot1734040) 200mM L-Glu 10ml(Gibco 25030-081 Lot1677185) 500 ng / mL Geneticin G418 (Gibco or Sigma), 5 ng / mL mIL-3, or 100 ng / mL cIL-31 Starvation medium: Growth medium that does not contain mRNA-3 and cIL-31. p-STAT3(Tyr705) Assay Kit: PerkinElmer, ALSU-PST3-A-HV

[0126] procedure cell culture 1. Thaw the vial of Ba / f3-OI cells and grow the cells in growth medium containing mRNA in a 37°C CO2 shaker at 125 rpm. 2. Before setting up the cell-based assay, passage the cells 2-3 times to obtain cells with a viability of ≥90%. 3. To set up the assay, harvest the cells and 1 × 10 7 Resuspend in starvation medium until the number of viable cells / mL reaches the desired level. 4. Plate the cells in a 96-well plate, 50 μL / well (approximately 5 × 10⁶). 5 Dispense into the cells / well. 5. Dilute cIL-31 three-fold with starvation medium in a dilution plate, then transfer 50 μL of each of the serially diluted cIL-31 aliquots to a cell plate. 6. Incubate the cell plate in a 37°C CO2 shaker for 15-30 minutes at 125 rpm for 1-2 hours. AlphaLISA assay as directed by the manufacturer: 7. Centrifuge the cells, aspirate the supernatant, and add 50-100 μL / well of 1× lysis buffer. Incubate at room temperature for 10 minutes while shaking at 1000 rpm. Transfer 8.30 μL of cell lysate to a half-area plate, or freeze and store at -80°C for subsequent testing. 9. SureFire Assay: Add 15 μL / well of acceptor mix to the cell lysate. Seal the plate, agitate at 1000 rpm for 2 minutes, then incubate at room temperature for 1-2 hours. Add 10.15 μL / well of donor mix to the cell lysate. Seal and mix at 1000 rpm for 2 minutes, then incubate at room temperature for 1-2 hours (plates can be stored overnight at 4°C; incubate at room temperature for 1 hour, then read the plate the next day). 11. Read the plate at 520-620 nm using the Alpha plate reader.

[0127] result: As shown in Figure 4, canine IL-31 stimulates STAT-3 activation in Ba / f3-OI cells in a dose-dependent manner.

[0128] [Example 8] Biological activity of anti-canine IL-31RA antibodies The ability of anti-canine IL-31RA mAbs to inhibit STAT-3 activation in Ba / f3-OI cells is evaluated as follows: 1. Thaw the vial of Ba / f3-OI cells and grow them in growth medium containing mRNA in a 37°C CO2 shaker at 125 rpm. 2. Before setting up the cell-based assay, passage the cells 2-3 times to obtain cells with a viability of ≥90%. 3. To set up the assay, harvest the cells and 1 × 10 7 Resuspend in starvation medium until the number of viable cells / mL reaches the desired level. 4. Plate the cells in a 96-well plate, 50 μL / well (approximately 5 × 10⁶). 5 Dispense into the cells / well. Dilute the antibody three-fold, starting at a concentration of 200 μg / mL, in a single row of starvation medium on a 5.96-well plate. Then, add 5–10 μL of cIL-31 to each well to a final concentration of 100 ng / mL. Transfer 6.50 μL of diluted antibody and cIL-31 mixture to each well of the cell plate and mix gently. 7. Incubate the cell plate in a 37°C CO2 shaker at 125 rpm for 1-2 hours. AlphaLISA assay as directed by the manufacturer: (See Example 7)

[0129] result: As illustrated in Figure 5, both antibodies 4G7 and 44E2 inhibit the ability of canine IL-31 to stimulate STAT-3 activation in Ba / f3-OI cells.

[0130] [Example 9] Mapping of canine IL-31 receptor alpha epitopes using mass spectrometry Using a method based on chemical crosslinking and mass spectrometry detection, epitopes recognized by anti-canine IL-31 receptor alpha mAbs were identified [CovalX Instrument Incorporated, 999 Broadway, Suite 305, Saugus, MA 01906-4510]. Application of this technique to epitope mapping of the canine IL-31 receptor alpha chain identified epitopes recognized by the mAbs listed in Table 6. Results obtained from epitope mapping of canine IL-31 receptor alpha with the three antibodies disclosed herein indicate that the mAbs recognize specific peptide epitopes present within the extracellular domain of canine IL-31 receptor alpha (see Table 6 below).

[0131] Notably, the epitopes identified for each of the three monoclonal antibodies (mAbs) tested were remarkably different. As shown in Table 6 below, the data indicate that the 28F12 antibody binds to a single epitope containing the amino acid sequence of SEQ ID NO: 101, and that the antibody binds to arginine (R) residues at positions 215 and 225, and lysine (K) residues at position 233, of amino acid sequence SEQ ID NO: 2. The data further demonstrate that the 44E2 antibody binds to two distinct epitopes: a first containing the amino acid sequence of SEQ ID NO: 102 (where the antibody binds to a threonine (T) residue at position 408 of SEQ ID NO: 2), and a second epitope containing the amino acid sequence of SEQ ID NO: 103, which has two distinct parts, in one of which the antibody binds to serine (S) residues at positions 464 and 472 and a tyrosine (Y) residue at position 471 of SEQ ID NO: 2, and in the other part the antibody binds to a second epitope where the antibody binds to a threonine (T) residue at position 487 of SEQ ID NO: 2. The data further demonstrate that the 10A12 antibody binds to two distinct epitopes: a first containing the amino acid sequence of SEQ ID NO: 104 (where the antibody binds to tyrosine (Y) residues at positions 31, 34, and 42 of amino acid sequence SEQ ID NO: 2, and to a threonine (T) residue at position 39), and a second epitope containing the amino acid sequence of SEQ ID NO: 105 (where the antibody binds to a lysine (K) residue at position 89, a serine (S) residue at position 90, a threonine (T) residue at position 93, and a tyrosine (Y) residue at position 94 of amino acid sequence SEQ ID NO: 2).

[0132] Sequence List

[0133] [Table 2]

[0134] [Table 3]

[0135] [Table 4]

[0136] Table 5

[0137] Table 6

[0138] Table 7

Claims

1. An isolated mammalian antibody or its antigen-binding fragment that binds to the canine interleukin-31 receptor alpha (canine IL-31RA), The antibody comprises a set of six complementarity-determining regions (CDRs), three of which are heavy chain CDRs: CDR heavy 1 (HCDR1), CDR heavy 2 (HCDR2), and CDR heavy 3 (HCDR3), and three of which are light chain CDRs: CDR light 1 (LCDR1), CDR light 2 (LCDR2), and CDR light 3 (LCDR3). The set of six CD-Rs is selected from the group of sets consisting of (i), (ii), and (iii), In set (i), HCDR1 contains the amino acid sequence of SEQ ID NO: 25, HCDR2 contains the amino acid sequence of SEQ ID NO: 26, HCDR3 contains the amino acid sequence of SEQ ID NO: 27, LCDR1 contains the amino acid sequence of SEQ ID NO: 28, LCDR2 contains the amino acid sequence of SEQ ID NO: 29, LCDR3 contains the amino acid sequence of SEQ ID NO: 30, In set (ii), HCDR1 contains the amino acid sequence of SEQ ID NO: 13, HCDR2 contains the amino acid sequence of SEQ ID NO: 14, HCDR3 contains the amino acid sequence of SEQ ID NO: 15, LCDR1 contains the amino acid sequence of SEQ ID NO: 16, LCDR2 contains the amino acid sequence of SEQ ID NO: 17, LCDR3 contains the amino acid sequence of SEQ ID NO: 18, In set (iii), HCDR1 contains the amino acid sequence of SEQ ID NO: 19, HCDR2 contains the amino acid sequence of SEQ ID NO: 20, HCDR3 contains the amino acid sequence of SEQ ID NO: 21, LCDR1 contains the amino acid sequence of SEQ ID NO: 22, LCDR2 contains the amino acid sequence of SEQ ID NO: 23, LCDR3 is an isolated mammalian antibody or its antigen-binding fragment containing the amino acid sequence of SEQ ID NO:

24.

2. HCDR1 contains the amino acid sequence of SEQ ID NO: 25, HCDR2 contains the amino acid sequence of SEQ ID NO: 26, HCDR3 contains the amino acid sequence of SEQ ID NO: 27, LCDR1 contains the amino acid sequence of SEQ ID NO: 28, LCDR2 contains the amino acid sequence of SEQ ID NO: 29, The isolated mammalian antibody or its antigen-binding fragment according to claim 1, wherein LCDR3 comprises the amino acid sequence of SEQ ID NO:

30.

3. The isolated mammalian antibody or its antigen-binding fragment according to claim 2, wherein when the antibody binds to canine IL-31RA, it binds to an epitope composed of the amino acid sequence of SEQ ID NO:

101.

4. HCDR1 contains the amino acid sequence of SEQ ID NO: 13, HCDR2 contains the amino acid sequence of SEQ ID NO: 14, HCDR3 contains the amino acid sequence of SEQ ID NO: 15, LCDR1 contains the amino acid sequence of SEQ ID NO: 16, LCDR2 contains the amino acid sequence of SEQ ID NO: 17, The isolated mammalian antibody or its antigen-binding fragment according to claim 1, wherein LCDR3 comprises the amino acid sequence of SEQ ID NO:

18.

5. The isolated mammalian antibody or its antigen-binding fragment according to claim 4, wherein when the antibody is bound to canine IL-31RA, it binds to an epitope composed of an amino acid sequence selected from the group consisting of SEQ ID NO: 102, SEQ ID NO: 103, and both SEQ ID NO: 102 and SEQ ID NO:

103.

6. HCDR1 contains the amino acid sequence of SEQ ID NO: 19, HCDR2 contains the amino acid sequence of SEQ ID NO: 20, HCDR3 contains the amino acid sequence of SEQ ID NO: 21, LCDR1 contains the amino acid sequence of SEQ ID NO: 22, LCDR2 contains the amino acid sequence of SEQ ID NO: 23, The isolated mammalian antibody or its antigen-binding fragment according to claim 1, wherein LCDR3 comprises the amino acid sequence of SEQ ID NO:

24.

7. The isolated mammalian antibody or antigen-binding fragment according to any one of claims 1 to 6, wherein the antibody and its antigen-binding fragment bind to canine IL-31RA and block the binding of canine IL-31RA to canine interleukin-31 (IL-31).

8. The isolated mammalian antibody or its antigen-binding fragment according to claim 7, which is a canine antibody or a canine antigen-binding fragment thereof.

9. An isolated mammalian antibody or its antigen-binding fragment according to claim 8, comprising a hinge region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO:

82.

10. An isolated mammalian antibody or its antigen-binding fragment according to claim 9, comprising a heavy chain containing a modified canine IgG-B (IgG-Bm) having the amino acid sequence of SEQ ID NO:

78.

11. The isolated mammalian antibody or its antigen-binding fragment according to claim 8, wherein the canine IL-31RA antibody comprises a light chain containing an amino acid sequence selected from the group consisting of SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, and SEQ ID NO: 93, and a heavy chain containing an amino acid sequence selected from the group consisting of SEQ ID NO: 88 and SEQ ID NO:

89.

12. The aforementioned canine IL-31RA antibody, A light chain containing the amino acid sequence of SEQ ID NO: 92, and a heavy chain containing the amino acid sequence of SEQ ID NO: 88, A light chain containing the amino acid sequence of SEQ ID NO: 93, and a heavy chain containing the amino acid sequence of SEQ ID NO: 88, A light chain containing the amino acid sequence of SEQ ID NO: 92, and a heavy chain containing the amino acid sequence of SEQ ID NO: 89, or An isolated mammalian antibody or its antigen-binding fragment according to claim 11, comprising a light chain containing the amino acid sequence of SEQ ID NO: 93 and a heavy chain containing the amino acid sequence of SEQ ID NO:

89.

13. The isolated mammalian antibody or its antigen-binding fragment according to claim 8, wherein the canine IL-31RA antibody comprises a light chain containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 97, SEQ ID NOs. 98, and SEQ ID NOs. 99, and a heavy chain containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 95, SEQ ID NOs. 96, and SEQ ID NOs.

100.

14. The aforementioned canine IL-31RA antibody, A light chain containing the amino acid sequence of SEQ ID NO: 97, and a heavy chain containing the amino acid sequence of SEQ ID NO: 95, A light chain containing the amino acid sequence of SEQ ID NO: 98, and a heavy chain containing the amino acid sequence of SEQ ID NO: 95, A light chain containing the amino acid sequence of SEQ ID NO: 99, and a heavy chain containing the amino acid sequence of SEQ ID NO: 95, A light chain containing the amino acid sequence of SEQ ID NO: 97, and a heavy chain containing the amino acid sequence of SEQ ID NO: 96, A light chain containing the amino acid sequence of SEQ ID NO: 98, and a heavy chain containing the amino acid sequence of SEQ ID NO: 96, A light chain containing the amino acid sequence of SEQ ID NO: 99, and a heavy chain containing the amino acid sequence of SEQ ID NO: 96, A light chain containing the amino acid sequence of SEQ ID NO: 97, and a heavy chain containing the amino acid sequence of SEQ ID NO: 100, A light chain containing the amino acid sequence of SEQ ID NO: 98, and a heavy chain containing the amino acid sequence of SEQ ID NO: 100, or A light chain containing the amino acid sequence of SEQ ID NO: 99, and a heavy chain containing the amino acid sequence of SEQ ID NO:

100. The isolated mammalian antibody or its antigen-binding fragment according to claim 13, comprising:

15. An isolated mammalian antibody or its antigen-binding fragment that binds to the canine interleukin-31 receptor alpha (canine IL-31RA) and, when bound to canine IL-31RA, binds to an epitope composed of an amino acid sequence selected from the group consisting of SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, or any combination thereof, The isolated mammalian antibody or its antigen-binding fragment according to any one of claims 8 to 14, wherein the isolated mammalian antibody or its antigen-binding fragment binds to canine IL-31RA and blocks the binding of canine IL-31RA to canine IL-31.

16. Isolated nucleic acids encoding polypeptides comprising three HCDRs: HCDR1, HCDR2, and HCDR3, and polypeptides comprising a set of three LCDRs: LCDR1, LCDR2, and LCDR3, (i) HCDR1 contains the amino acid sequence of SEQ ID NO: 13, HCDR2 contains the amino acid sequence of SEQ ID NO: 14, HCDR3 contains the amino acid sequence of SEQ ID NO:

15. (ii) HCDR1 contains the amino acid sequence of SEQ ID NO: 19, HCDR2 contains the amino acid sequence of SEQ ID NO: 20, HCDR3 contains the amino acid sequence of SEQ ID NO: 21, or (iii) HCDR1 contains the amino acid sequence of SEQ ID NO: 25, HCDR2 contains the amino acid sequence of SEQ ID NO: 26, HCDR3 contains the amino acid sequence of SEQ ID NO: 27, (iv) LCDR1 contains the amino acid sequence of SEQ ID NO: 16, LCDR2 contains the amino acid sequence of SEQ ID NO: 17, LCDR3 contains the amino acid sequence of SEQ ID NO:

18. (v) LCDR1 contains the amino acid sequence of SEQ ID NO: 22, LCDR2 contains the amino acid sequence of SEQ ID NO: 23, LCDR3 contains the amino acid sequence of SEQ ID NO: 24, or (vi) LCDR1 contains the amino acid sequence of SEQ ID NO: 28, LCDR2 contains the amino acid sequence of SEQ ID NO: 29, LCDR3 is an isolated nucleic acid containing the amino acid sequence of SEQ ID NO:

30.

17. An isolated nucleic acid encoding both the heavy chain and the light chain of an isolated mammalian antibody or its antigen-binding fragment according to claim 8.

18. An expression vector comprising the isolated nucleic acid according to claim 16.

19. An expression vector comprising the isolated nucleic acid described in claim 17.

20. A host cell comprising the expression vector according to claim 18 or claim 19.

21. An isolated nucleic acid encoding a polypeptide comprising three HCDRs: HCDR1, HCDR2, and HCDR3, (i) HCDR1 contains the amino acid sequence of SEQ ID NO: 13, HCDR2 contains the amino acid sequence of SEQ ID NO: 14, HCDR3 contains the amino acid sequence of SEQ ID NO:

15. (ii) HCDR1 contains the amino acid sequence of SEQ ID NO: 19, HCDR2 contains the amino acid sequence of SEQ ID NO: 20, HCDR3 contains the amino acid sequence of SEQ ID NO: 21, or (iii) HCDR1 contains the amino acid sequence of SEQ ID NO: 25, HCDR2 contains the amino acid sequence of SEQ ID NO: 26, HCDR3 is an expression vector containing an isolated nucleic acid that includes the amino acid sequence of SEQ ID NO: 27, and A set of three LCDRs: isolated nucleic acids encoding polypeptides comprising LCDR1, LCDR2, and LCDR3, (iv) LCDR1 contains the amino acid sequence of SEQ ID NO: 16, LCDR2 contains the amino acid sequence of SEQ ID NO: 17, LCDR3 contains the amino acid sequence of SEQ ID NO:

18. (v) LCDR1 contains the amino acid sequence of SEQ ID NO: 22, LCDR2 contains the amino acid sequence of SEQ ID NO: 23, LCDR3 contains the amino acid sequence of SEQ ID NO: 24, or (vi) LCDR1 contains the amino acid sequence of SEQ ID NO: 28, LCDR2 contains the amino acid sequence of SEQ ID NO: 29, LCDR3 contains an expression vector containing an isolated nucleic acid that includes the amino acid sequence of SEQ ID NO:

30. host cell.

22. A pharmaceutical composition comprising a canine antibody or its antigen-binding fragment according to claim 8, and a pharmaceutically acceptable carrier or diluent.

23. A method for assisting in blocking pruritus associated with atopic dermatitis, comprising administering a therapeutically effective amount of the pharmaceutical composition described in claim 22 to a subject in need thereof, wherein the subject is a non-human animal.

24. A method for producing an antibody or antigen-binding fragment that binds to canine IL-31RA, Culture the host cells described in claim 20 under conditions suitable for protein expression, and A method comprising purifying the antibody or its antigen-binding fragment from the host cells and culture medium.

25. A method for producing an antibody or antigen-binding fragment that binds to canine IL-31RA, Culture the host cells described in claim 21 under conditions suitable for protein expression, and A method comprising purifying the antibody or its antigen-binding fragment from the host cells and culture medium.

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