Caninized antibody against canine interleukin-31 receptor alpha II

Abstract

The present invention provides a caninized mouse antibody against canine IL-31 receptor alpha, which has high binding affinity to canine IL-31 receptor alpha and can block 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] Reference to Electronically Submitted Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML file created on December 9, 2022 is named 25365.xml. This Sequence Listing, submitted via EFS-Web, is a part of the present specification and is incorporated herein by reference in its entirety.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 341,443, filed May 13, 2022, U.S. Patent Application No. 63 / 290,259, filed December 16, 2021, and U.S. Patent Application No. 63 / 290,256, filed December 16, 2021, the subject matter of which is incorporated herein by reference in its entirety.

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

[0004] The immune system comprises a network of resident and recirculating specialized cells that work in concert to protect the host against infection and cancer. The ability of the immune system to perform this function depends largely on the biological activity of a group of proteins secreted by white blood cells 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). IL-4, IL-13, IL-22, and IL-31 are key cytokines for the generation of immune responses necessary for protection against extracellular pathogens (e.g., parasites present in tissues or lumina), but 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 by immune system dysregulation and epidermal barrier abnormalities in humans. The pathological and immunological attributes of atopic dermatitis have been the subject of extensive investigation [reviewed in Rahman et al. Inflammation & Allergy-drug target 10:486-496 (2011) and Harskamp et al., Seminar in Cutaneous Medicine and Surgery 32:132-139 (2013)]. Atopic dermatitis is also a common condition in companion animals, especially dogs, with its prevalence estimated to be approximately 10-15% of the canine population. The pathogenesis of atopic dermatitis in dogs and cats [reviewed in Nuttall et al., Veterinary Records 172(8):201-207 (2013)] shows significant similarities to the pathogenesis of atopic dermatitis in humans, including skin infiltration by a variety of immune cells and CD4+ receptors, including a predominance of IL-31, IL-4, and IL-13. + This includes a Th2-polarizing cytokine environment. Furthermore, IL-22 is involved in the excessive epidermal 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] Pharmaceutical agents that have either been proven and / or shown to have promise in aiding in the treatment of atopic dermatitis include Janus kinase (JAK) inhibitors (see, e.g., U.S. Pat. Nos. 8,133,899; 8,987,283; WO 2018 / 108969), spleen tyrosine kinase (SYK) inhibitors (see, e.g., U.S. Pat. No. 8,759,366), and antagonists to chemoattractant receptor homologous molecules expressed on TH2 cells (see, e.g., U.S. Pat. Nos. 7,696,222, 8,546,422, 8,637,541, and 8,546,422).

[0008] However, despite some recent successes in treating atopic dermatitis, there remains a need to design alternative and / or better therapies that can address one or more of the symptoms of canine atopic dermatitis.

[0009] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] U.S. Pat. No. 8,790,651 [Patent Document 2] U.S. Patent No. 10,093,731 [Patent Document 3] U.S. Pat. No. 8,133,899 [Patent Document 4] U.S. Pat. No. 8,987,283 [Patent Document 5] International Publication No. 2018 / 108969 [Patent Document 6] U.S. Pat. No. 8,759,366 [Patent Document 7] U.S. Patent No. 7,696,222 [Patent Document 8] U.S. Pat. No. 8,546,422 [Patent Document 9] U.S. Pat. No. 8,637,541 [Non-patent literature]

[0011] [Non-Patent Document 1] Rahman et al.Inflammation&Allergy-drug target 10:486-496(2011) [Non-Patent Document 2] Harskamp et al.,Seminar in Cutaneous Medicine and Surgery 32:132-139(2013) [Non-Patent Document 3] Nuttall et al., Veterinary Records 172(8):201-207(2013) [Non-Patent Document 4] Ruzicka,et al.,New England Journal of Medicine,376(9),826-835(2017) Summary of the Invention

[0012] The present invention provides new mammalian antibodies, including caninized mouse antibodies, against canine-derived 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 antigen-binding fragment thereof binds to canine IL-31RA. In more specific embodiments, the mammalian antibody or antigen-binding fragment also blocks binding of canine IL-31RA to canine interleukin-31. In certain embodiments, the mammalian antibody is an antibody against canine IL-31RA. In more specific embodiments, the mammalian antibody is a caninized antibody. In even more specific embodiments, the caninized antibody is a caninized mouse antibody against canine IL-31RA.

[0013] Thus, the present invention provides a mammalian antibody, or antigen-binding fragment thereof, that binds to canine interleukin-31 receptor alpha and that comprises a heavy chain comprising a set of three heavy chain complementarity determining regions (HCDRs), CDR heavy 1 (HCDR1), CDR heavy 2 (HCDR2) and CDR heavy 3 (HCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:3, HCDR2 comprises the amino acid sequence of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13, and HCDR3 comprises the amino acid sequence of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:23.

[0014] In certain embodiments, the mammalian antibody or antigen-binding fragment further comprises a light chain comprising a set of three light chain complementarity determining regions (LCDRs), CDR light 1 (LCDR1), CDR light 2 (LCDR2), and CDR light 3 (LCDR3), wherein LCDR1 comprises the amino acid sequence of SEQ ID NO:30 or SEQ ID NO:31, LCDR2 comprises the amino acid sequence of SEQ ID NO:38, SEQ ID NO:39, or SEQ ID NO:40, and LCDR3 comprises the amino acid sequence of SEQ ID NO:45.

[0015] In a particular embodiment, upon binding to canine IL-31RA, the mammalian antibody or antigen-binding fragment thereof binds to one or more epitopes comprised by the amino acid sequence of SEQ ID NO: 97, SEQ ID NO: 103, SEQ ID NO: 99, or SEQ ID NO: 102. In a more particular embodiment, upon binding to canine IL-31RA, the mammalian antibody or antigen-binding fragment thereof binds to two epitopes, one comprised by the amino acid sequence of SEQ ID NO: 97 and the other comprised by the amino acid sequence of SEQ ID NO: 103. In another embodiment, upon binding to canine IL-31RA, the mammalian antibody or antigen-binding fragment thereof binds to two epitopes, one comprised by the amino acid sequence of SEQ ID NO: 99 and the other comprised by the amino acid sequence of SEQ ID NO: 102.

[0016] In a particular embodiment, upon binding to canine IL-31RA, the mammalian antibody or antigen-binding fragment thereof binds to an epitope comprised by the amino acid sequence of SEQ ID NO: 97. In another embodiment, upon binding to canine IL-31RA, the mammalian antibody or antigen-binding fragment thereof binds to an epitope comprised by the amino acid sequence of SEQ ID NO: 103. In yet another embodiment, upon binding to canine IL-31RA, the mammalian antibody or antigen-binding fragment thereof binds to an epitope comprised by the amino acid sequence of SEQ ID NO: 99. In yet another embodiment, upon binding to canine IL-31RA, the mammalian antibody or antigen-binding fragment thereof binds to an epitope comprised by the amino acid sequence of SEQ ID NO: 102.

[0017] In a related embodiment, when bound to canine IL-31RA, the antibody 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 more preferably 3 to 8 or more amino acid residues within the amino acid sequence of SEQ ID NO: 97 or SEQ ID NO: 103, or both SEQ ID NO: 97 and SEQ ID NO: 103, and / or SEQ ID NO: 99 or SEQ ID NO: 102, or both SEQ ID NO: 99 and SEQ ID NO: 102.

[0018] In certain embodiments, the mammalian antibody or antigen-binding fragment thereof comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 3, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 20. In more particular embodiments of this kind, the mammalian antibody or antigen-binding fragment thereof further comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 30, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 40, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 45.

[0019] In other embodiments, the mammalian antibody or antigen-binding fragment thereof comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 3, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 21. In more particular embodiments of this type, the mammalian antibody or antigen-binding fragment thereof further comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 30, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 40, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 45.

[0020] In yet other embodiments, the mammalian antibody or antigen-binding fragment thereof comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 3, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 22. In more particular embodiments of this type, the mammalian antibody or antigen-binding fragment thereof further comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 30, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 38, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 45.

[0021] In yet other embodiments, the mammalian antibody or antigen-binding fragment thereof comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 3, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 23. In more particular embodiments of this type, the mammalian antibody or antigen-binding fragment thereof further comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 31, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 39, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 45.

[0022] In preferred embodiments, the antibodies and antigen-binding fragments thereof bind to canine IL-31RA and block binding of canine IL-31RA to canine interleukin-31. In certain embodiments, the mammalian antibody to canine IL-31RA is a murine antibody. In certain embodiments, the mammalian antibody to canine IL-31RA is a caninized antibody. In more certain embodiments, the caninized antibody to canine IL-31RA is a caninized murine antibody.

[0023] The caninized antibody of the present invention comprises a canine fragment crystallizable region (cFc region). The caninized antibody of the present invention also comprises a canine light chain constant region. In a specific embodiment, the canine light chain constant region is a Kappa canine light chain constant region. In a more specific embodiment, the Kappa canine light chain constant region comprises the amino acid sequence of SEQ ID NO: 127.

[0024] Furthermore, the caninized antibody or antigen-binding fragment thereof can comprise a heavy chain comprising a cFc region and a hinge region. The hinge region is preferably a canine hinge region. The canine hinge region can comprise a naturally occurring IgG-A hinge region, IgG-B hinge region, IgG-C hinge region, or IgG-D hinge region. Alternatively, the hinge region is a corresponding modified canine hinge region. In a particular embodiment, the hinge region is an IgG-A hinge region comprising an amino acid sequence comprising at least 90%, 95%, or 100% identity with the amino acid sequence of SEQ ID NO: 112. In another embodiment, the hinge region is an IgG-B hinge region comprising an amino acid sequence comprising at least 90%, 95%, or 100% identity with the amino acid sequence of SEQ ID NO: 113. In yet other embodiments, the hinge region is an IgG-C hinge region comprising an amino acid sequence that comprises at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 114. In yet other embodiments, the hinge region is a modified IgG-D hinge region comprising the amino acid sequence of SEQ ID NO: 115.

[0025] Similarly, the canine Fc region can be IgG-A, IgG-B, IgG-C, IgG-D, or modifications thereof. In certain embodiments, the caninized antibody or antigen-binding fragment thereof comprises IgG-Bm. In certain embodiments, the caninized antibody or antigen-binding fragment thereof comprises IgG-A comprising an amino acid sequence having at least 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 116. In other embodiments, the caninized antibody or antigen-binding fragment thereof comprises IgG-B comprising an amino acid sequence having at least 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 110. In yet other embodiments, the caninized antibody or antigen-binding fragment thereof comprises IgG-C comprising an amino acid sequence having at least 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 117. In yet other embodiments, the caninized antibody or antigen-binding fragment thereof comprises an IgG-D comprising an amino acid sequence having at least 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 118. In yet other embodiments, the caninized antibody or antigen-binding fragment thereof comprises an IgG-Bm comprising an amino acid sequence having at least 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 111, in which both the aspartic acid residue (D) at position 31 of SEQ ID NO: 110 and the asparagine residue (N) at position 63 of SEQ ID NO: 110 remain substituted with an alanine residue (A) in the sequence of IgG-Bm.

[0026] In certain embodiments, the caninized antibody, or antigen-binding fragment thereof, comprises a canine IgG-D, but the naturally occurring IgG-D hinge region is replaced by a hinge region comprising an amino acid sequence comprising at least 90%, 95% or 100% identity to the amino acid sequence of SEQ ID NO: 112. In other embodiments, the caninized antibody comprises a heavy chain comprising IgG-D, but the naturally occurring IgG-D hinge region is replaced by a hinge region comprising an amino acid sequence having at least 90%, 95% or 100% identity to the amino acid sequence of SEQ ID NO: 113. In yet other embodiments, the caninized antibody comprises a heavy chain comprising IgG-D, but the naturally occurring IgG-D hinge region is replaced by a hinge region comprising an amino acid sequence having at least 90%, 95% or 100% identity to the amino acid sequence of SEQ ID NO: 114. In yet other embodiments, the caninized antibody comprises a heavy chain comprising an IgG-D, but the naturally occurring IgG-D hinge region has been replaced by a hinge region comprising the amino acid sequence of SEQ ID NO:115.

[0027] In certain embodiments of the composition, the caninized antibody against canine IL-31RA comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 79, SEQ ID NO: 80, or SEQ ID NO: 81, and a light chain comprising the amino acid sequence of SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, or SEQ ID NO: 85. The invention further provides antigen-binding fragments of these caninized antibodies.

[0028] In certain embodiments, the caninized antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 84 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 80. In other embodiments, the caninized antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 84 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 81. In yet other embodiments, the caninized antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 84 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 79.

[0029] In yet other embodiments, the caninized antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 85 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 80. In yet other embodiments, the caninized antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 85 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 81. In yet other embodiments, the caninized antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 85 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 79.

[0030] In yet other embodiments, the caninized antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 83 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 80. In yet other embodiments, the caninized antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 83 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 81. In yet other embodiments, the caninized antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 83 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 79.

[0031] In yet other embodiments, the caninized antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 82 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 80. In yet other embodiments, the caninized antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 82 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 81. In yet other embodiments, the caninized antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 82 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 79.

[0032] In certain embodiments, the mammalian antibody (e.g., caninized antibody), when bound to canine IL-31RA, binds to an epitope comprised by amino acids of SEQ ID NO:97 or SEQ ID NO:103, or both SEQ ID NO:97 and SEQ ID NO:103. In certain embodiments, identification of the epitope is based on chemical cross-linking and mass spectrometry detection. In related embodiments, the mammalian antibody, when bound to canine IL-31RA, binds to at least one amino acid residue, preferably 1-3 amino acid residues, more preferably 2-5 amino acid residues, and / or more preferably 3-8 or more amino acid residues within the amino acid sequence of SEQ ID NO:97 or SEQ ID NO:103, or both SEQ ID NO:97 and SEQ ID NO:103.

[0033] In certain embodiments, a mammalian antibody that binds to SEQ ID NO:97 is selected from the group consisting of a tyrosine residue at position 94 of SEQ ID NO:2, i.e., Y 94 In another embodiment, the mammalian antibody binds to the lysine residue at position 102 of SEQ ID NO:2, i.e., K 102 In yet another embodiment, the mammalian antibody binds to the lysine residue at position 112 of SEQ ID NO:2, i.e., K 112 In yet another embodiment, the mammalian antibody binds to a tyrosine residue at position 94 of SEQ ID NO:2 and a lysine residue at position 102 of SEQ ID NO:2. In yet another embodiment, the mammalian antibody binds to a lysine residue at position 102 of SEQ ID NO:2 and a lysine residue at position 112 of SEQ ID NO:2. In yet another embodiment, the mammalian antibody binds to a tyrosine residue at position 94 of SEQ ID NO:2 and a lysine residue at position 112 of SEQ ID NO:2. In yet another embodiment, the mammalian antibody binds to a tyrosine residue at position 94, a lysine residue at position 102 ...

[0034] In a related embodiment, the mammalian antibody that binds to SEQ ID NO: 103 is selected from the group consisting of the arginine residue at position 183 of SEQ ID NO: 2, i.e., R 183 In another embodiment, the mammalian antibody binds to the serine residue at position 193 of SEQ ID NO:2, i.e., S 193 In yet another embodiment, the mammalian antibody binds to the threonine residue at position 202 of SEQ ID NO:2, i.e., T 202 In yet another embodiment, the mammalian antibody binds to an arginine residue at position 183 of SEQ ID NO:2 and a serine residue at position 193 of SEQ ID NO:2. In yet another embodiment, the mammalian antibody binds to a serine residue at position 193 of SEQ ID NO:2 and a threonine residue at position 202 of SEQ ID NO:2. In yet another embodiment, the mammalian antibody binds to an arginine residue at position 183 of SEQ ID NO:2 and a threonine residue at position 202 of SEQ ID NO:2. In yet another embodiment, the mammalian antibody binds to an arginine residue at position 183 of SEQ ID NO:2 and a serine residue at position 193 of SEQ ID NO:2 and a threonine residue at position 202 of SEQ ID NO:2. The invention further provides antigen-binding fragments of these mammalian antibodies.

[0035] The present invention also provides nucleic acids, including isolated nucleic acids, encoding either a set of three HCDRs or three LCDRs; a heavy chain variable region of a caninized antibody or antigen-binding fragment thereof; a heavy chain of a caninized antibody or antigen-binding fragment thereof, a light chain variable region of a caninized antibody or antigen-binding fragment thereof, and / or a light chain of a caninized antibody or antigen-binding fragment thereof. The present invention further provides pairs of nucleic acids, one of which comprises a nucleotide sequence encoding a light chain of a specific caninized antibody of any one of the antibodies of the present invention, and the other of which comprises a nucleotide sequence encoding a heavy chain of that (said) specific caninized antibody. The present invention further provides expression vectors comprising such pairs of nucleic acids, or individual nucleic acids of the present invention. The present invention further provides pairs of expression vectors, one of which comprises a nucleic acid comprising a nucleotide sequence encoding a light chain of a specific caninized antibody of any one of the antibodies of the present invention, and the other of which comprises a nucleic acid comprising a nucleotide sequence encoding a heavy chain of that (said) specific caninized antibody.

[0036] Thus, the present invention further provides nucleic acids encoding the 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 (including caninized antibody) of the present invention. In more particular embodiments, the nucleic acid encodes HCDR1 comprising the amino acid sequence of SEQ ID NO:3; HCDR2 comprising the amino acid sequence of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13; HCDR3 comprising the amino acid sequence of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:23; or any combination thereof.

[0037] The present invention further provides nucleic acids encoding the set of three light chain complementarity determining regions (CDRs), CDR light 1 (LCDR1), CDR light 2 (LCDR2) and CDR light 3 (LCDR3) of a mammalian antibody (including caninized antibody) or antigen-binding fragment thereof of the present invention. In more particular embodiments of such, the nucleic acid encodes LCDR1 comprising the amino acid sequence of SEQ ID NO:30 or SEQ ID NO:31; LCDR2 comprising the amino acid sequence of SEQ ID NO:38, SEQ ID NO:39, or SEQ ID NO:40; and LCDR3 comprising the amino acid sequence of SEQ ID NO:45; or any combination thereof.

[0038] The present invention further provides nucleic acids encoding any of the heavy chains of the mammalian antibodies (including caninized antibodies) or antigen-binding fragments thereof of the present invention. The present invention also provides nucleic acids encoding any of the light chains of the mammalian antibodies (including caninized antibodies) or antigen-binding fragments thereof of the present invention. Furthermore, the present invention provides expression vectors comprising and capable of expressing one or more of the nucleic acids of the present invention, and host cells comprising one or more of such expression vectors.

[0039] The invention further provides pharmaceutical compositions comprising the caninized antibodies and antigen-binding fragments thereof of the invention together with a pharma- ceutically acceptable carrier and / or diluent.The invention further provides pharmaceutical compositions comprising the nucleic acids of the invention together with a pharma- ceutically acceptable carrier and / or diluent, and / or expression vectors comprising one or more of the nucleic acids of the invention together with a pharma- ceutically acceptable carrier and / or diluent.

[0040] The present invention also provides a method for treating atopic dermatitis, comprising administering one of the above-mentioned pharmaceutical compositions to an animal subject having atopic dermatitis.In certain embodiments, the animal subject is a dog.The present invention also provides a method for helping block the pruritus associated with atopic dermatitis in an animal subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention to an animal subject in need thereof.In certain embodiments, the animal subject is a dog.

[0041] In addition, the invention provides methods for producing a caninized antibody or antigen-binding fragment thereof that binds canine IL-31RA. In certain embodiments, the methods comprise culturing a host cell comprising one or more expression vectors encoding and expressing a caninized antibody light chain of the invention and a caninized antibody heavy chain in a culture medium under conditions in which the nucleic acids are expressed, thereby producing a polypeptide comprising a caninized antibody light chain of the invention and / or a caninized antibody heavy chain. The polypeptide is then recovered from the host cell or medium. In certain embodiments, a polypeptide comprising a caninized antibody light chain of the invention and a polypeptide comprising a caninized antibody heavy chain are combined with each other under conditions favoring the formation of a caninized antibody.

[0042] These and other aspects of the present invention will be better understood by reference to the following brief description and detailed description of the drawings. [Brief description of the drawings]

[0043] [Figure 1] Binding of IL-31 to IL-31RA. The extracellular domain (ECD) of canine IL-31RA was tested for its ability to bind to canine IL-31. The results show that IL-31RA ECD binds to biotinylated canine IL-31 in a dose-dependent manner, with an EC50 of 0.55 ng / ml. [Figure 2A] FIG. 1 shows binding of xIL-31RA monoclonal antibody (mABS) to IL-31RA. [Figure 2B]Figure 2 shows the binding of xIL-31RA monoclonal antibodies (mABS) to IL-31RA. Selected mouse mAbs were tested for their reactivity to canine IL-31RA. The results show that the selected mouse mAbs bind to canine IL-31RA in a dose-dependent manner. All ten mouse monoclonal antibodies have strong binding reactivity to canine IL-31RA. Figure 2A shows mouse mAbs: 51F8 (●), 74H10 (■), 100H8 (▲), 209G5 (▼), 224G3 (◆), and isotype control (o). Figure 2B shows mouse mAbs: 55B3 (●), 65G9 (■), 85C10 (▲), 218D9 (▼), 227E7 (◆), and isotype control (o). [Diagram 3] 1 shows blocking of IL-31 binding to IL-31RA by monoclonal antibodies (mABS) against IL-31RA. Selected mouse mAbs (anti-canine IL-31RA) were tested for their ability to block binding of IL-31 to IL-31RA / OSMR by flow cytometry. FACS results show that 10 mouse mAbs can block IL-31 binding to IL-31RA / OSMR complexes displayed on CHO-IL-31RA / OSMR cells. Antibodies 51F8, 74H10, 100H8, 209G5, and 218D9 showed excellent blocking activity. [Figure 4] FIG. 1 shows the induction of STAT-3 phosphorylation by IL-31. Ba / f3-OI cells expressing the IL-31 receptor complex were tested for IL-31-induced STAT-3 phosphorylation. The results show that STAT-3 phosphorylation was induced by IL-31 in a dose-dependent manner in Baf3-OI cells (■), implying that (i) the canine IL-31 receptor complex is successfully expressed on the cell surface, (ii) the binding of canine IL-31 to the IL-31 receptor can stimulate endogenous STAT3 phosphorylation, and (iii) subsequently initiate its downstream signaling pathway. Ba / f3 cells (o) were used as a control. [Figure 5A]FIG. 1 shows inhibition of IL-31-mediated STAT-3 phosphorylation in Ba / f3-OI cells by selected xIL-31RA antibodies. [Figure 5B] Figure 5 shows inhibition of IL-31-mediated STAT-3 phosphorylation in Ba / f3-OI cells by selected xIL-31RA antibodies. The results show that selected mAbs inhibit IL-31-mediated STAT-3 phosphorylation in Ba / f3-OI cells in a dose-dependent manner. Figure 5A shows mouse mAbs 209G5 (■), 218D9 (▲), 85C10 (▼), and IL-31 protein (◆). Figure 5B shows mouse mAbs 100H8 (hexagons), 74H10 (■), 85C10 (▲), 51F8 (▼), and cIL-31 protein (◆). [Figure 6A] FIG. 1 provides the epitope on canine IL-31RA for antibody 100H8. [Figure 6B] FIG. 1 provides the epitope on canine IL-31RA for antibody 51F8. [Figure 6C] FIG. 1 provides the epitope on canine IL-31RA for antibody 218D9. [Figure 6D] FIG. 1 provides the epitope on canine IL-31RA for antibody 85C10. [Figure 6E]

[0023] Figure 6 provides the epitope on canine IL-31RA for antibody 224G3. Figure 6A shows the epitope of 100H8, which comprises the amino acid sequence of SEQ ID NO:97 (within SEQ ID NO:119) and SEQ ID NO:103 (within SEQ ID NO:120), respectively. Figure 6B shows the epitope of 51F8, which comprises the amino acid sequence of SEQ ID NO:98 (within SEQ ID NO:121) and SEQ ID NO:100 (within SEQ ID NO:122), respectively. Figure 6C shows the epitope of 218D9, which comprises the amino acid sequence of SEQ ID NO:104 (within SEQ ID NO:123) and SEQ ID NO:105 (within SEQ ID NO:124), respectively. Figure 6D shows the epitope of 85C10, which comprises the amino acid sequence of SEQ ID NO:106 (within SEQ ID NO:125) and SEQ ID NO:108 (within SEQ ID NO:126), respectively. Figure 6E shows the epitope of 224G3, which comprises the amino acid sequence of SEQ ID NO: 109 (also in SEQ ID NO: 126). The positions of the binding residues of the amino acid sequence of SEQ ID NO: 2 for each epitope on the cIL-31R ECD antigen are also shown. [Figure 7A] 1 provides plots of the binding activity of identified mouse-canine chimeric or caninized antibodies to canine IL-31RA. [Figure 7B] 1 provides plots of the binding activity of identified mouse-canine chimeric or caninized antibodies to canine IL-31RA. [Figure 7C] 1 provides plots of the binding activity of identified mouse-canine chimeric or caninized antibodies to canine IL-31RA. [Figure 7D] 1 provides plots of the binding activity of identified mouse-canine chimeric or caninized antibodies to canine IL-31RA. [Figure 7E]7A provides plots of the binding activity of identified mouse-canine chimeric or caninized antibodies to canine IL-31RA. The results show that the caninized antibodies have similar binding affinity to their corresponding parental antibodies (represented by mouse-canine chimeric antibodies). Figure 7A shows binding plots of monoclonal 51F8 antibodies: chimeric 51F8 (●), c51F8VH3VL6 (■), c51F8VH3VL7 (▲), c51F8VH4VL6 (▼), and c51F8VH4VL7 (◆), as well as an isotype control (o). Figure 7B shows binding plots of monoclonal 100H8 antibodies: chimeric 100H8 (●), c100H8VH5VL4 (■), and c100H8VH7VL4 (▲). Figure 7C shows binding plots for monoclonal 85C10 antibodies: chimeric 85C10 (●), c85C10VH3VL2 (■), and c85C10VH1VL2 (▲). Figure 7D shows binding plots for monoclonal 218D9 antibodies: chimeric 218D9 (●), c218D9VH3VL2 (■), c218D9VH3VL3 (▲), c218D9VH4VL2 (▼), and c218D9VH4VL3 (◆), as well as an isotype control (o). Figure 7E shows binding plots for monoclonal 224G3 antibodies: m224G3 Chim (●), c224G3VH2VL2 (■), and c224G3VH2VL3 (▲). EC50s are provided in the table.

[0044] The term "chimeric" before an antibody number means that the antibody is a mouse-dog chimeric antibody, such as chimeric 218D9 or chimeric 51F8. Furthermore, an "m" followed by "Chim" before an antibody number means that the antibody is a mouse-dog chimeric antibody, such as m224G3 Chim. A lowercase "c" before an antibody number means that it is a caninized antibody, such as c218D9VH4VL2. [Figure 8]Figure 1 shows blocking of IL-31 binding to IL-31RA by inhibition of IL-31-mediated STAT-3 phosphorylation in Ba / f3-OI cells. The results show that caninized 218D9 antibodies can inhibit IL-31-mediated STAT-3 phosphorylation in Ba / f3-OI cells in a dose-dependent manner, and constructs c218D9VH3VL3 and c218D9VH4VL3 have the same inhibitory activity as the parental mouse-canine chimeric 218D9 antibody: chimeric 218D9 (●), c218D9VH3VL2 (■), c218D9VH3VL3 (▲), c218D9VH4VL2 (▼), and c218D9VH4VL3 (◆); and IL-31 only control (o). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] In response to the need for better treatments for atopic dermatitis, the present invention provides formulations and methodologies that can achieve significant benefits against the skin inflammation associated with atopic dermatitis.

[0046] Abbreviation The following abbreviations are used throughout the detailed description and examples of the present invention:

[0047] ADCC antibody-dependent cytotoxicity CDC Complement-dependent cytotoxicity CDR Complementarity determining region in an immunoglobulin variable region, as defined using the Kabat numbering system EC50 Concentration that results in 50% efficacy or binding ELISA Enzyme-Linked Immunosorbent Assay FR Antibody framework region: immunoglobulin variable region excluding the CDR regions IC50 Concentration that produces 50% inhibition IgG Immunoglobulin G The immunoglobulin alignment and numbering system pioneered by Elvin A. Kabat [Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)] mAb Monoclonal antibody (also Mab or MAb) V region The segment of an IgG chain that is variable in sequence among different antibodies. VH immunoglobulin heavy chain variable region VL immunoglobulin light chain variable region

[0048] definition So that the present invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere herein, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0049] As used in this specification, including the appended claims, the singular forms of words such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0050] "Administration" and "treatment," as applied to an animal, e.g., a canine subject, a cell, a tissue, an organ, or a biological fluid, refer to contact of an exogenous medicinal, therapeutic, diagnostic agent, or composition with an animal, e.g., a canine subject, a cell, a tissue, an organ, or a biological fluid. Treatment of a cell encompasses contact of a reagent to a cell, as well as contact of a reagent to a fluid where the fluid is in contact with a cell.

[0051] "Administration" and "treatment" also refer to in vitro and ex vivo treatments, for example, of a cell, with a reagent, diagnostic, binding compound, or another cell. The term "subject" includes any organism, preferably an animal, more preferably a mammal (e.g., a dog or cat), most preferably a dog.

[0052] "Treat" or "treatment" refers to administering, internally or externally, a therapeutic agent, such as a composition containing any of the antibodies of the present invention, to a canine subject or patient having one or more symptoms or suspected of having a condition for which the agent has therapeutic activity. Typically, the agent is administered in an amount effective to alleviate and / or ameliorate one or more disease / condition symptoms in the treated subject or population, whether by inducing regression of such symptom(s) by any clinically measurable extent or by inhibiting the progression of such symptoms. The amount of therapeutic agent effective to alleviate any particular disease / condition symptom (also referred to as a "therapeutically effective amount") may vary depending on factors such as the disease / condition, the age and weight of the patient (e.g., dog), and the ability of the pharmaceutical composition to elicit a desired response in the subject. Whether a disease / condition symptom has been alleviated or improved can be assessed by any clinical measurement routinely used by a veterinarian or other skilled health care provider to assess the severity or progression of the condition. Although an embodiment of the invention (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the symptom(s) of the target disease / condition in every subject, it should alleviate the symptom(s) of the target disease / condition in a statistically significant number of subjects as determined by any statistical test known in the art, e.g., Student's t-test, chi-squared test, Mann-Whitney U test, Kruskal-Wallis test (H test), Jonkheel-Terpstra test, and Wilcoxon test.

[0053] "Treatment" as applied to a human, veterinary (e.g., dog), or research subject refers to therapeutic treatment as well as research and diagnostic uses. "Treatment" as applied to a human, veterinary (e.g., dog), or research subject, or cell, tissue, or organ, encompasses contacting an antibody of the invention with, for example, a dog or other animal subject, cell, tissue, physiological compartment, or physiological fluid.

[0054] As used herein, the term "dog" includes all domesticated dogs, Canis lupus familiaris or Canis familiaris, unless otherwise specified.

[0055] As used herein, the term "cat" refers to any member of the Felidae family. Members of this family include wild, zoo, and domestic members, including domestic cats, purebred and / or mixed breed companion cats, show cats, laboratory cats, cloned cats, and wild or feral cats.

[0056] As used herein, the term "canine frame" refers to the amino acid sequences of the heavy and light chains of a canine antibody other than the hypervariable region residues defined herein as CDR residues. For caninized antibodies, in most embodiments, the amino acid sequences of the native canine CDRs are replaced in both chains with the corresponding foreign CDRs (e.g., from a mouse or rat antibody). The heavy and / or light chains of the canine antibody may contain some foreign non-CDR residues to preserve the configuration of the foreign CDRs in the caninized antibody and / or to modify Fc function, e.g., as exemplified below and / or as disclosed in U.S. Pat. No. 10,106,607, which is incorporated herein by reference in its entirety.

[0057] The "fragment crystallizable region", abbreviated as "Fc" or interchangeably used "Fc region", corresponds to the CH3-CH2 portion of an antibody that interacts with cell surface receptors called Fc receptors. The canine fragment crystallizable region (cFc region) of each of the four canine IgGs was first described by Tang et al. [Vet. Immunol. Immunopathol. 80:259-270 (2001); Bergeron et al., Vet. Immunol. Immunopathol. 157:31-41 (2014) and see also U.S. Pat. No. 10,106,607].

[0058] As used herein, canine Fc (cFc) "IgG-Bm" is a canine IgG-B Fc that contains two amino acid residue substitutions, D31A and N63A, as in the amino acid sequence of SEQ ID NO: 111 of IgG-B (see below) and does not contain the c-terminal lysine ("K"). The aspartic acid residue (D) at position 31 of SEQ ID NO: 110 and the asparagine residue (N) at position 63 of SEQ ID NO: 110 are both replaced with an alanine residue (A) in IgG-Bm. These two amino acid residue substitutions serve to significantly reduce the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of naturally occurring canine IgG-B [see U.S. Pat. No. 10,106,607, the entire contents of which are incorporated herein by reference]. Further amino acid substitutions for IgG-Bm are also envisioned, which may include amino acid substitutions in parallel with those that can be made in IgG-B to promote heterodimer formation in bispecific antibodies. The amino acid sequence of IgG-B, SEQ ID NO:110, is below.

[0059] TIFF2024546759000002.tif83163

[0060] The amino acid sequence of IgG-Bm, SEQ ID NO:111, is provided below. TIFF2024546759000003.tif28162

[0061] As used herein, a "substitution of an amino acid residue" in the amino acid sequence of an antibody by another amino acid residue is equivalent to, for example, a "substitution of 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., an alanine can be deliberately replaced with a serine at a particular position in the amino acid sequence by, for example, recombinant DNA techniques. Alternatively, a particular amino acid residue or set of amino acid residues of an antibody can be replaced by one or more amino acid residues, for example, based on the ability of an antibody produced by a cell to bind to a given region on its antigen, e.g., one that contains an epitope or part thereof, and / or through a more natural selection process in order to include a particular CDR that retains the same canonical structure as the CDR being replaced. Such substitutions / replacements can result in "variant" CDRs and / or variant antibodies.

[0062] As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological activity. Antibodies can be monomeric, dimeric, or larger multimeric. It is therefore used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), caninized antibodies, full canine antibodies, chimeric antibodies, and camelized single domain antibodies. A "parent antibody" is an antibody obtained by exposing the immune system to an antigen prior to modification of the antibody for its intended use, such as caninization of the antibody for use as a canine therapeutic antibody.

[0063] As used herein, an antibody of the invention that "blocks" or "blocking" or "blocks the binding of" e.g. a canine receptor to its binding partner (ligand), is an antibody that blocks (partially or completely) the binding of a canine receptor to its canine ligand, or vice versa, as determined by standard binding assays (e.g., BIACore®, ELISA or flow cytometry).

[0064] Typically, an antibody or antigen-binding fragment of the invention retains at least 10% of its canine antigen-binding activity (when compared to the parent antibody) when that activity is expressed on a molar basis. Preferably, an antibody or antigen-binding fragment of the 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 contemplated that the antibody or antigen-binding fragment of the invention may include conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "function-conservative variants" of an antibody) that do not substantially alter its biological activity.

[0065] "Isolated antibody" refers to a purified state, and in that context means that the molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cellular debris and growth medium. In general, the term "isolated" is not intended to refer to the complete absence of such materials, or the absence of water, buffers, or salts, unless present in amounts that would substantially interfere with experimental or therapeutic uses of the binding compounds described herein.

[0066] As used herein, an antibody is said to specifically bind to a polypeptide containing a given antigen sequence (in this case, 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 canine IL-31RA sequence. For example, an antibody that specifically binds to a polypeptide containing canine IL-31RA may bind to a FLAG®-tagged form of canine IL-31RA, but does not bind to other FLAG®-tagged canine proteins.

[0067] As used herein, unless otherwise indicated, "antibody fragment" or "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to an antigen bound by the full-length antibody (e.g., canine IL-31RA), e.g., a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments.

[0068] An antibody, or a binding compound derived from the antigen-binding site of an antibody, binds to that canine antigen, or a variant or mutein thereof, with "specificity" if it has an affinity for that canine antigen, or a variant or mutein thereof, that is at least 10 times greater, more preferably at least 20 times greater, and even more preferably at least 100 times greater, than it has affinity for any other canine antigen tested.

[0069] As used herein, a "chimeric antibody" is an antibody having variable domains derived from a first antibody and constant domains derived from a second antibody, the first and second antibodies being from different species (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Typically, the variable domains are obtained from an antibody from an experimental animal such as a rodent (the "parent antibody"), and the constant domain sequences are obtained from an animal subject antibody, e.g., human or canine, such that the resulting chimeric antibody is less likely to provoke an adverse immune response in a human or canine subject, respectively, than the parent (e.g., rodent) antibody.

[0070] As used herein, the term "caninized antibody" refers to a form of an antibody that contains sequences from both canine and non-canine (e.g., murine) antibodies. In general, a caninized antibody contains substantially all of at least one or more, typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-canine immunoglobulin (e.g., including six CDRs as exemplified below), and all or substantially all of the framework (FR) regions (and typically all or substantially all of the remaining frame) of a canine immunoglobulin sequence. As exemplified herein, a caninized antibody contains both three heavy chain CDRs and three light chain CDRs from a murine (mouse) anti-canine antigen antibody, along with a canine frame or modified canine frame. The modified canine frame contains one or more amino acid changes exemplified herein that further optimize the effectiveness of the caninized antibody, for example, to increase its binding to the canine antigen and / or its ability to block binding of the canine antigen to its natural binding partner.

[0071] The variable regions of each light / heavy chain pair form the antibody binding site. Thus, an intact antibody generally has two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites are generally the same. Typically, both the heavy and light chain variable domains contain three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FRs). The CDRs are usually aligned by the framework regions, allowing binding to a specific epitope. 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 assignment of amino acids to each domain is generally described in Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5 th ed.;NIH Publ.No.91-3242(1991);Kabat,Adv.Prot.Chem.32:1-75(1978);Kabat,et al.,J.Biol.Chem.252:6609-6616(1977);Chothia,et al., J. Mol. Biol. 196:901-917 (1987) or Chothia, et al., Nature 342:878-883 (1989)].

[0072] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody which are involved in antigen binding. The hypervariable region comprises amino acid residues from the "complementarity determining regions" or "CDRs" (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), which defines antibody CDR regions by sequence, and Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987), which defines antibody CDR regions by structure]. As used herein, the term "framework" or "FR" residues refers to those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0073] There are four known IgG heavy chain subtypes of canine IgG, which are called IgG-A, IgG-B, IgG-C, and IgG-D. The two known light chain subtypes are called lambda and kappa. In certain embodiments of the invention, in addition to binding to canine IL-31RA, a canine or caninized antibody to that antigen of the invention optimally has the following two attributes:

[0074] 1) lack of effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC); and 2) It is easily purified on a large scale using industry standard techniques such as those based on Protein A chromatography.

[0075] None of the naturally occurring canine IgG isotypes meets both criteria. For example, IgG-B can be purified using Protein A, but has high levels of ADCC activity. On the other hand, IgG-A binds weakly to Protein A, but also exhibits ADCC activity. Furthermore, IgG-D does not exhibit ADCC activity, but neither IgG-C nor IgG-D can be purified on a Protein A column. (IgG-C has significant ADCC activity). One way in which the present invention addresses these issues in certain embodiments is by providing modified canine IgG-B antibodies of the invention specific for the antigens of the invention that lack effector functions such as ADCC and can be easily purified using industry standard Protein A chromatography.

[0076] As used herein, an "antipruritic agent" is a compound, polymer, and / or formulation that tends to inhibit, reduce, and / or prevent itch. Antipruritic agents are colloquially referred to as anti-itch agents.

[0077] As used herein, an "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 a specific protein in the IL-31 signaling pathway, such as IL-31 or its receptor IL-31RA. 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, disrupting and / or preventing successful signaling of this pathway, thereby inhibiting, reducing and / or preventing pruritus caused by the IL-31 signaling pathway.

[0078] "Homology" as used herein refers to the sequence similarity between two polynucleotide sequences or two polypeptide sequences when they are optimally aligned. If both positions of the two compared sequences are occupied by the same base or amino acid residue, for example, if each position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology is the number of homologous positions shared by the two sequences divided by the total number of positions compared times 100. For example, if 6 out of 10 positions in the two sequences are identical or homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, the comparison is performed when the two sequences are aligned to give the maximum percentage of homology.

[0079] 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, an amino acid sequence is 100% "identical" to a second amino acid sequence if the amino acid residues of both sequences are identical. Thus, an amino acid sequence is 50% "identical" to a second amino acid sequence if 50% of the amino acid residues of the two amino acid sequences are identical. Sequence comparison is performed over contiguous blocks of amino acid residues contained in a given protein, e.g., a protein or portion of a polypeptide being compared. In certain embodiments, selected deletions or insertions that would otherwise change the correspondence between the two amino acid sequences are taken into account.

[0080] Sequence similarity includes identical residues and non-identical biochemically related amino acids that share similar properties and may be interchangeable.

[0081] "Conservatively modified variants" or "conservative substitutions" refer to the substitution of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), such that changes can be made frequently without altering the biological activity of the protein. Those skilled in the art will generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity [see, e.g., Watson et al., Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.; 1987)]. Moreover, substitution of structurally or functionally similar amino acids is unlikely to destroy biological activity. Exemplary conservative substitutions are shown directly in Table A below.

[0082] [Table 1]

[0083] Function-conservative variants of the antibodies of the invention are also contemplated by the present invention. As used herein, "function-conservative variant" refers to an antibody or fragment in which one or more amino acid residues have been altered without altering the desired properties, such as antigen affinity and / or specificity. Such variants include, but are not limited to, replacing amino acids with those having similar properties, such as the conservative amino acid substitutions in Table A above.

[0084] An "isolated nucleic acid molecule" means DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or any combination thereof, that is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature or linked to a polynucleotide with which it is not linked in nature. For purposes of this disclosure, it should be understood that a "nucleic acid molecule comprising" a particular nucleotide sequence does not encompass an intact chromosome. An isolated nucleic acid molecule "comprising" a specified nucleic acid sequence may, in addition to the specified sequence, include coding sequences for up to 10 or even up to 20 or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequence, and / or may include vector sequences.

[0085] The invention provides isolated caninized antibodies of the invention, methods of using the antibodies in treating conditions, e.g., treating atopic dermatitis in dogs. In dogs, there are four IgG heavy chains, designated A, B, C, and D. These heavy chains represent four different subclasses of canine IgG, designated IgG-A (or IgGA), IgG-B (or IgGB), IgG-C (or IgGC), and IgG-D (or IgGD). Each of the two heavy chains consists of one variable domain (VH) and three constant domains, designated CH-1, CH-2, and CH-3. The CH-1 domain is connected to the CH-2 domain via an amino acid sequence designated the "hinge" or alternatively the "hinge region".

[0086] 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 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). Canine antibodies also contain two types of light chains, kappa and lambda. The DNA and amino acid sequences of these light chains are available from the GenBank Database. For example, the kappa light chain amino acid sequence has accession number ABY57289.1, and the lambda light chain has accession number ABY55569.1.

[0087] The known amino acid sequences of the four unmodified canine Fc's are as follows:

[0088] cIgG-A [SEQ ID NO: 116] TIFF2024546759000005.tif28162

[0089] cIgG-B [SEQ ID NO: 110] TIFF2024546759000006.tif28164

[0090] cIgG-C [SEQ ID NO: 117] TIFF2024546759000007.tif30162

[0091] cIgG-D [SEQ ID NO: 118] TIFF2024546759000008.tif28163

[0092] The amino acid sequence of the Kappa Inguinea light chain constant region is [SEQ ID NO: 127].

[0093] TIFF2024546759000009.tif15163

[0094] In the present invention, the amino acid sequence of each of the four canine IgG Fc fragments is based on the identified boundaries of the CH1 and CH2 domains as determined by Tang et al., supra. Caninized mouse anti-canine antibodies that bind to canine IL-31RA include, but are not limited to, antibodies of the invention that comprise a canine IgG-A, IgG-B, IgG-C, and IgG-D heavy chain and / or a canine kappa or lambda light chain with mouse anti-canine IL-31RA CDRs. Thus, the present invention provides caninized mouse anti-canine antibodies of the invention, including isolated caninized mouse anti-canine antibodies that bind to canine IL-31RA and preferably also block binding of that canine IL-31RA to canine IL-31.

[0095] Accordingly, the invention further provides caninized murine antibodies and methods of using the antibodies of the invention in treating conditions, such as, for example, atopic dermatitis in dogs.

[0096] The invention further provides full-length caninized heavy chains that can be matched with corresponding light chains to make caninized antibodies. Accordingly, the invention further provides caninized mouse anti-canine antigen antibodies of the invention (including isolated caninized mouse anti-canine antibodies) and methods of using the antibodies of the invention in treating conditions, such as treating atopic dermatitis in dogs.

[0097] The invention also provides antibodies of the invention comprising a canine fragment crystallizable region (cFc region), wherein the cFc region has been genetically modified to enhance, reduce, or eliminate one or more effector functions. In one aspect of the invention, the genetically modified cFc region reduces or eliminates one or more effector functions. In another aspect of the invention, the genetically modified cFc region enhances one or more effector functions. In certain embodiments, the genetically modified cFc region is a genetically modified canine IgGB Fc region. In another such embodiment, the genetically modified cFc region is a genetically modified canine IgGC Fc region. In a particular embodiment, the effector function is antibody-dependent cellular cytotoxicity (ADCC), which is enhanced, reduced, or eliminated. In another embodiment, the effector function is complement-dependent cytotoxicity (CDC), which is enhanced, reduced, or eliminated. In yet another embodiment, the cFc region has been genetically modified to enhance, reduce, or eliminate both ADCC and CDC.

[0098] In order to generate variants of canine IgG lacking effector function, a number of mutant canine IgGB heavy chains were generated. These variants may contain one or more of the following single or combination substitutions in the Fc portion of the heavy chain amino acid sequence: P4A, D31A, N63A, G64P, T65A, A93G, and P95A. The variant heavy chains (i.e., containing such amino acid substitutions) are cloned into an expression plasmid and transfected into HEK293 cells along with a plasmid containing a gene encoding the light chain. Intact antibodies are expressed and purified from HEK293 cells, and then expressed as Fc B heavy chains. γ Binding to RI and C1q can be assessed to assess their potential for mediating immune effector functions. [See U.S. Pat. No. 10,106,607, the entire contents of which are incorporated herein by reference.] The present invention also provides a modified canine IgG-D which comprises, in place of its native IgG-D hinge region, a hinge region from:

[0099] IgG-A: FNECRCTDTPPCPVPEP SEQ ID NO: 112 IgG-B: PKRENGRVPRPPDCPKCPAPEM SEQ ID NO: 113; or IgG-C:AKECECKCNCNNCPCPGCGL SEQ ID NO: 114

[0100] Alternatively, the IgG-D hinge region may be modified by replacing the serine residue with a proline residue, i.e., PKESTCKCI of SEQ ID NO: 115. P Canine IgG-D can be genetically modified by substitution with PCPVPES (substituting the underlined and bolded proline residue (P) with a naturally occurring serine residue). Such modifications can result in a canine IgG-D lacking fab arm exchange. Modified canine IgG-D can be constructed using standard methods of recombinant DNA technology [e.g., Maniatis et al., Molecular Cloning, A Laboratory Manual (1982)]. To construct these variants, a nucleic acid encoding the amino acid sequence of canine IgG-D can be modified to encode the modified IgG-D. The modified nucleic acid sequence is then cloned into an expression plasmid for protein expression.

[0101] The six complementarity determining regions (CDRs) of the caninized mouse anti-canine antibodies described herein can include a canine antibody kappa (k) or lambda (l) light chain comprising mouse light chain LCDR1, LCDR2 and LCDR3, and a canine antibody heavy chain IgG comprising mouse heavy chain HCDR1, HCDR2 and HCDR3.

[0102] nucleic acid The invention further includes nucleic acids encoding the antibodies of the invention (see, eg, the Examples below).

[0103] Also included in the present invention are nucleic acids encoding immunoglobulin polypeptides comprising 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 the caninized antibodies provided herein, excluding the unchanged CDRs, when the comparison is performed by the BLAST algorithm, and the parameters of the algorithm are selected to give the maximum match between the respective sequences over the entire length of the respective reference sequences. The present invention further provides nucleic acids encoding immunoglobulin polypeptides comprising 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, when the comparison is performed using the BLAST algorithm, and the parameters of the algorithm are selected to give the maximum match between the respective sequences over the entire length of the respective reference sequences, also included in the present invention.

[0104] As used herein, percent nucleotide and amino acid sequence identity can be determined using the C, MacVector (MacVector, Inc. Cary, NC 27519), Vector NTI (Informax, Inc. MD), Oxford Molecular Group PLC (1996) and Clustal W algorithms with default alignment and identity parameters. These commercially available programs can also be used to determine sequence similarity using the same or similar default parameters. Alternatively, Advanced Blast searches can be used under default filter conditions, for example, using the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program with default parameters.

[0105] The following references relate to the BLAST algorithm, which is often used for sequence analysis: BLAST ALGORITHMS: Altschul, S. F., et al., J. Mol. Biol. 215:403-410 (1990); Gish, W., et al., Nature Genet. 3:266-272 (1993); Madden, T. L., et al., Meth. Enzymol. 266:131-141 (1996); Altschul, S. F., et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang, J., et al., Genome Res. 7:649-656 (1997); Wootton, J. C., et al., Comput. Chem. 17:149-163 (1993); Hancock, J. Met al.,Comput.Appl.Biosci.10:67-70(1994);ALIGNMENT SCORING SYSTEMS:Dayhoff,MO,et al.,“A model of evolutionary change in proteins.”in Atlas of Protein Sequence and Structure, vol. 5, 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); and Altschul, 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). .

[0106] Antibody Protein Engineering By way of example and not limitation, the dog heavy chain constant region can be derived from IgGA, IgG-B, IgGC, IgGD, or modified cFc, such as IgG-Bm, as used herein [see U.S. Pat. No. 10,106,607, which is incorporated by reference in its entirety], and the dog light chain constant region can be derived from kappa or lambda.

[0107] Antibodies can be engineered to contain modifications to the canine framework and / or canine frame residues within the variable domains of a parent (ie, murine) monoclonal antibody, e.g., to improve the properties of the antibody.

[0108] The construction of caninized anti-canine IL-31 receptor alpha monoclonal antibodies can be carried out by determining the DNA sequence encoding the heavy and light chains of canine IgG. The DNA and protein sequences of canine heavy and light chains are known in the art and can be obtained by searching the NCBI gene and protein database. As mentioned above, for canine antibodies, there are four known IgG subtypes: IgG-A, IgG-B, IgG-C and IgG-D, and two types of light chains: kappa and lambda.

[0109] Caninized mouse anti-canine IL-31RA 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 many other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, 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 are insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells, and fungal cells. When recombinant expression vectors encoding the heavy chain or antigen-binding portion or fragment thereof, and the light chain and / or antigen-binding fragment thereof are introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow for expression of the antibody in the host cell, or, more preferably, secretion of the antibody into the culture medium in which the host cell is growing.

[0110] The antibody can be recovered from the culture medium using standard protein purification methods. Furthermore, expression of the antibody of the present invention (or other moieties therefrom) from the production cell line can be enhanced using several known techniques. For example, the glutamine synthetase gene expression system (GS system) is a common approach to enhance expression under certain conditions. The GS system is discussed in whole or in part in connection with European Patent Nos. 0216846, 0256055, and 0323997, as well as European Patent Application No. 89303964.4.

[0111] In certain embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region, e.g., a canine constant region, e.g., an IgG-A, IgG-B, IgG-C, and IgG-D canine heavy chain constant region or a variant thereof. In certain embodiments, the antibody or antigen-binding fragment comprises a light chain constant region, e.g., a canine light chain constant region, e.g., a lambda or kappa canine light chain region or a variant thereof. By way of example and not limitation, the canine heavy chain constant region can be from IgG-B and the canine light chain constant region can be from kappa.

[0112] Epitope mapping The interaction of an antibody with its cognate protein antigen is mediated through the binding of a specific amino acid (paratope) of the antibody with a specific amino acid (epitope) of the target antigen. An epitope is an antigenic determinant that triggers a specific reaction by an immunoglobulin. An epitope consists of a group of amino acids on the surface of an antigen. A protein of interest may contain several epitopes recognized by different antibodies. Epitopes recognized by antibodies are classified as linear epitopes or conformational epitopes. Linear epitopes are formed by stretches of contiguous sequences of amino acids in a protein, whereas conformational epitopes consist of amino acids that are discontinuous (e.g., far apart) in the primary amino acid sequence but are brought together upon three-dimensional protein folding.

[0113] Epitope mapping refers to the process of identifying the amino acid sequences (i.e., epitopes) recognized by antibodies on their target antigens. Identification of epitopes recognized by monoclonal antibodies (mAbs) on target antigens has important applications. For example, it can aid in the development of new therapeutics, diagnostics, and vaccines. Epitope mapping can also aid in the selection of optimized therapeutic mAbs and aid in elucidating their mechanism 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 to carrier proteins or other immunostimulants.

[0114] Epitope mapping can be performed using polyclonal or monoclonal antibodies, and several methods are used for epitope identification depending on the suspected nature of the epitope (i.e., linear allelic conformation). Mapping of linear epitopes is more direct and relatively easy to perform. For this purpose, commercial services for linear epitope mapping often use peptide scanning. In this case, an overlapping set of 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 of discontinuous epitopes is more technically challenging and requires more specialized techniques such as X-ray co-crystallography of a monoclonal antibody with its target protein, hydrogen-deuterium (H / D) exchange, mass spectrometry combined with enzymatic digestion, as well as several other methods known to those skilled in the art.

[0115] Epitope-binding and cross-blocking antibodies Anti-canine IL-31RA antibodies or antigen-binding fragments thereof of the invention include any antibody or antigen-binding fragment thereof that binds to the same epitope in canine IL-31RA as one of the antibodies disclosed herein, e.g., the 218D9 antibody that binds to an epitope comprising the amino acid sequence of either SEQ ID NO: 104, SEQ ID NO: 105, or both SEQ ID NO: 104 and SEQ ID NO: 105, or the 51F8 antibody that binds to an epitope comprising the amino acid sequence of either SEQ ID NO: 98, SEQ ID NO: 100, or both SEQ ID NO: 98 and SEQ ID NO: 100, etc. (including caninized antibodies and any antibodies or antigen-binding fragments thereof that are cross-blocked (partially or fully) or cross-blocked (partially or fully) by the antibodies or fragments described herein for canine IL-31RA binding) and any variants thereof.

[0116] Cross-blocking antibodies and antigen-binding fragments can be identified based on their ability to cross-compete with, for example, the 100H8 or 74H10 antibodies in standard binding assays (e.g., BIACore®, ELISA, or flow cytometry as exemplified below). 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 with the binding of the labeled antibody is evaluated. Additionally or alternatively, BIAcore® analysis can be used to evaluate the ability of the antibodies to cross-compete. For example, the ability of a test antibody to inhibit the binding of 100H8 or 74H10 antibodies to canine IL-31RA demonstrates that the test antibody can compete with 100H8 or 74H10 antibodies for binding to canine IL-31RA, and thus, in some cases, bind to the same epitope on canine IL-31RA as the 100H8 and / or 74H10 antibodies bind.

[0117] Antibodies and fragments thereof that bind to the same epitope as any of the anti-canine IL-31RA antibodies or fragments of the invention also form part of the invention.

[0118] Pharmaceutical Compositions and Administration To prepare pharmaceutical or sterile compositions containing the antibodies of the invention, the antibodies can be mixed with a pharma- ceutically acceptable carrier or excipient. [See, e.g., Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984)].

[0119] Formulations of therapeutic and diagnostic agents can be prepared, for example, by mixing with acceptable carriers, excipients, or stabilizers in the form of lyophilized powders, slurries, aqueous solutions, or suspensions [see, 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: See, for example, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY; Weiner and Kotkoskie (2000) Excipient Forms: Disperse Systems, Marcel Dekker, NY. In one embodiment, the antibody of the present invention is diluted to an appropriate concentration in 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.

[0120] The toxicity and therapeutic efficacy of an antibody composition administered alone or in combination with another agent can be determined, for example, by the LD 50 (the dose lethal to 50% of the population) and ED 50 The dose that is therapeutically effective in 50% of the population can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects is called the therapeutic index (LD 50 / ED 50 In certain embodiments, antibodies that exhibit a high therapeutic index are desirable. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosages for use in dogs. The dosage of such compounds is preferably within the ED range with little or no toxicity. 50 The dosage may vary within this range depending upon the dosage form and route of administration used.

[0121] The mode of administration may vary. Suitable routes of administration include oral, rectal, mucosal, intestinal, parenteral, intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intracerebroventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, dermal, transdermal, or intraarterial. In certain embodiments, the antibody of the present invention may be administered by an invasive route, such as injection. In further embodiments of the present invention, the antibody of the present invention or a pharmaceutical composition thereof is administered intravenously, subcutaneously, intramuscularly, intraarterially, or by inhalation, aerosol delivery. Administration by non-invasive routes (e.g., oral; e.g., pill, capsule, or tablet) is also within the scope of the present invention.

[0122] The composition can be administered using medical devices known in the art.For example, the pharmaceutical composition of the present invention can be administered by injection with a hypodermic needle, including, for example, a prefilled syringe or an autoinjector.The pharmaceutical composition disclosed herein can also be administered using a needleless hypodermic injection device, such as the device disclosed in U.S. Patent No. 6,620,135; U.S. Patent No. 6,096,002; U.S. Patent No. 5,399,163; U.S. Patent No. 5,383,851; U.S. Patent No. 5,312,335; U.S. Patent No. 5,064,413; U.S. Patent No. 4,941,880; U.S. Patent No. 4,790,824 or U.S. Patent No. 4,596,556.

[0123] The pharmaceutical composition disclosed herein can also be administered by injection. Examples of well-known implants and modules for administering pharmaceutical compositions include U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing pharmaceuticals at a controlled rate, U.S. Patent No. 4,447,233, which discloses a pharmaceutical infusion pump for delivering pharmaceuticals at a precise infusion rate, U.S. Patent No. 4,447,224, which discloses a variable flow rate implantable infusion device for continuous drug delivery, and U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system with multi-chamber compartments. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0124] Alternatively, the antibodies of the invention can be administered in a local rather than systemic manner, often in a depot or sustained release formulation.

[0125] The dosing 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 the target cells in the biological matrix. Preferably, the dosing regimen delivers enough therapeutic antibody to bring about improvement of the target disease / symptom, while minimizing undesirable side effects. Thus, the amount of biologic delivered depends, in part, on the particular therapeutic antibody and the severity of the condition being treated. Guidance on selecting appropriate doses of therapeutic antibodies is available [e.g., Wawrzynczak Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK (1996); Kresina (ed.) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY (1991); Bach (ed.) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY (1993); Baert, et al. New Engl. J. Med. 348:601-608 (2003); Milgrom et al. New Engl. J. Med. 341:1966-1973 (1999); Slamon et al. New Engl. J. Med. 344:783-792 (2001); Beniaminovitz et al. New Engl. J. Med. 344:783-792 (2001); 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)].

[0126] Determination of appropriate dosages is performed by a veterinarian, for example, using parameters or factors known or suspected in the art to affect treatment. Generally, dosages are started at amounts somewhat less than the optimum dose and then increased in small increments until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms.

[0127] Antibodies provided herein can be provided by continuous infusion or by doses administered, for example, daily, 1 to 7 times per week, weekly, biweekly, monthly, bimonthly, quarterly, semi-annually, yearly, etc. Doses can be provided, for example, intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, intraspinally, or by inhalation. The total weekly dose is generally at least 0.05 μg / kg body weight, more 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 Immunol. Immunother. 52:133-144 (2003)]. Dosages may also be provided to achieve a predetermined target concentration of the antibody of the invention in the serum of the dog, e.g., 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg / ml or more. In other embodiments, the antibody of the invention is administered subcutaneously or intravenously at 10, 20, 50, 80, 100, 200, 500, 1000 or 2500 mg / subject weekly, biweekly, "every 4 weeks", monthly, bimonthly or quarterly.

[0128] As used herein, "inhibit" or "treat" or "treatment" includes postponing the onset of symptoms associated with a disorder and / or reducing the severity of symptoms of such a disorder. The term further includes ameliorating existing uncontrolled or undesirable symptoms, preventing further symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the term refers to the conferring of a beneficial result to a vertebrate subject (e.g., a dog) having a disorder, condition and / or symptom or having a potential for developing such a disorder, disease or condition.

[0129] As used herein, the terms "therapeutically effective amount", "therapeutically effective dose" and "effective amount" refer to an amount of an antibody of the invention that is effective when administered alone or in combination with an additional therapeutic agent to a cell, tissue or subject, e.g., a dog, to cause a measurable improvement in one or more symptoms of a disease or condition or the progression of such a disease or condition. A therapeutically effective dose further refers to an amount of an antibody sufficient to cause at least a partial improvement of a symptom, e.g., treatment, cure, prevention or amelioration of an associated medical condition, or an increase in the rate of treatment, cure, prevention or amelioration of such a condition. When applied to a combination, a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent will usually result in at least a 10% improvement in a diagnostic measure or parameter, which is at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably at least 50%. An effective amount can also result in an improvement in a subjective measure when a subjective measure is used to assess the severity of the condition.

[0130] [Example] [Example 1] IL-31 receptor alpha Nucleotide sequence The nucleotide sequence of SEQ ID NO: 1 encodes the extracellular domain of the canine IL-31 receptor alpha (cIL-31RA) fused to a HIS tag. The canine IL-31RA ECD HIS-tagged protein comprises the amino acid sequence of SEQ ID NO: 2. The nucleotide sequence was prepared by chemical synthesis and then cloned into an expression plasmid suitable for production of the corresponding protein in eukaryotic cells, either HEK-293 cells or CHO cells.

[0131] Canine IL-31RA ECD-10His: [SEQ ID NO: 1] TIFF2024546759000010.tif140164

[0132] [Example 2] Expression and purification of IL-31 receptor alpha ECD 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 according to the manufacturer's recommendations. A few days after transfection, the supernatants of the transfected cells and non-transfected controls were harvested and spun down to remove cell debris. IL-31RA with a HIS tag was purified from the cell culture fluid by passing the clarified and collected fluid from the transfected cells through a nickel column according to the manufacturer's recommendations. The purified protein was quantified by measuring the absorbance of ultraviolet light at 280 nm.

[0133] Canine IL-31RA ECD-10His: [SEQ ID NO: 2] TIFF2024546759000011.tif56163

[0134] [Table 2]

[0135] [Example 3] Binding of canine IL-31RA to biotinylated canine IL-31: protocol 1) Coat immunoplate(s) with IL-31RA protein by diluting to 1 μg / mL in phosphate buffered saline (PBS). Add 100 μL / well. Incubate plate(s) overnight at 2-7°. 2) Wash the plate three times with 275 μL / well of Phosphate Buffered Saline + TWEEN® 20 (PBST). 3) Block the plate with 200 µL / well of blocking buffer [1% nonfat dry milk (NFDM)] in PBST for 30-45 min at 36 ± 2 °C with gentle shaking (120 ± 20 RPM). 4) Wash the plate 3 times with 275 μL / well of PBST. 5) Dilute biotinylated IL-31 (10 μg / mL) 3-fold in 1% NFDM in PBST onto the dilution plate and transfer 100 μL / well to the immunoplate(s). Incubate with gentle shaking (120 ± 20 RPM) at 36 ± 2°C for 30-45 minutes. 6) Wash plate 3 times with 275 μL / well PBST. 7) Dilute horseradish peroxidase-streptavidin (HRP-streptavidin) in 1% NFDM in PBST to a final dilution of 1:1000. 8) Add 100 μL / well of HRP-streptavidin to the immunoplate(s) and incubate with gentle shaking (120±20 RPM) at 36±2°C for 30-45 minutes. 9) Wash plate 3 times with 275 μL / well PBST. 10) Just prior to use, combine equal amounts of pre-heated TMP 2-component substrate. 11) Add 100 μL / well of prepared 3,3',5,5'-tetramethylbenzidine (TMP) substrate to the immunoplate(s) and incubate in the dark with gentle shaking (120±20 RPM) at 36±2°C for 10-15 minutes. 12) Stop the reaction by adding 100 μL / well of 1M H3PO4. 13) Read the plate using a microplate reader at a wavelength of 450 nm with a reference wavelength of 540 nm.

[0136] The extracellular domain (ECD) of canine IL-31RA was tested for its ability to bind to canine IL-31 (see FIG. 1). The results show that IL-31RA ECD binds to biotinylated canine IL-31 in a dose-dependent manner, with an EC50 of 0.55 ng / ml.

[0137] [Example 4] Monoclonal antibody against canine IL-31 receptor alpha Monoclonal antibodies (mAbs) against canine IL-31RA were generated by immunizing mice multiple times with canine IL-31RA ECD. Mice were immunized with IL-31RA ECD in GS's proprietary adjuvant via intraperitoneal route three times on days 0, 14, and 28, using 50 μg per mouse for the first immunization and 25 μg per mouse for subsequent boosts. On day 48, mice were immunized once more with 25 μg of antigen, and 4 days later their spleen cells were fused with the myeloma SP2 / 0 cell line to produce hybridomas secreting antibodies. At various times after immunization, serum was collected from the mice and tested against canine IL-31RA by ELISA. Spleen cells from mice with the highest IL-31RA ECD reactivity were fused with the myeloma SP2 / 0 cell line to generate hybridomas. Approximately 14 days after fusion, supernatants from growing hybridomas were screened by flow cytometry using cells expressing IL-31RA protein. Hybridoma reactivity was confirmed by ELISA as follows:

[0138] ELISA procedure: 1) Coat 96-well plates 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 with blocking buffer [PBS containing 5% fetal bovine serum (FBS)], 25 μl / well, for 30 minutes at room temperature. 5) Transfer 25 μl / well of hybridoma supernatant to a 96-well plate and incubate at room temperature for 60 minutes. 6) Wash the plate 3 times with PBST. 7) Add 25 μl / well of anti-mouse HRP, 1:4000 dilution in blocking buffer to the plate and incubate at room temperature for 60 minutes. 8) Wash the plate 5 times with PBST. 9) Add TMB-based reagent to the plate for a colorimetric reaction for 2-3 minutes. 10) Stop the reaction with 0.16M sulfuric acid. 11) Read the plate in a plate reader.

[0139] The selected mouse mAbs were tested for their reactivity to canine IL-31RA, as shown in Figures 2A-2B. The results show that the selected mouse mAbs bind to canine IL-31RA in a dose-dependent manner. As shown in Figures 2A-2B, ten (10) mouse monoclonal antibodies with strong binding reactivity to canine IL-31RA were obtained.

[0140] The amino acid sequences of the heavy and light chain variable regions of these 10 mouse monoclonal antibodies are shown below.

[0141] 100H8VH [SEQ ID NO: 52] TIFF2024546759000013.tif15163100H8VL[SEQ ID NO:53] TIFF2024546759000014.tif15162222E7VH [SEQ ID NO: 54] TIFF2024546759000015.tif15163222E7VL[SEQ ID NO:55] TIFF2024546759000016.tif1416355B3VH [SEQ ID NO: 56] TIFF2024546759000017.tif1516355B3VL[SEQ ID NO:57] TIFF2024546759000018.tif1616174H10VH[SEQ ID NO:58] TIFF2024546759000019.tif1416274H10VL[SEQ ID NO:59] TIFF2024546759000020.tif15164209G5VH[SEQ ID NO:60] TIFF2024546759000021.tif14162209G5VL[SEQ ID NO:61] TIFF2024546759000022.tif1516351F8VH [SEQ ID NO: 62] TIFF2024546759000023.tif1516351F8VL[SEQ ID NO:63] TIFF2024546759000024.tif1516265G9VH [SEQ ID NO: 64] TIFF2024546759000025.tif1516265G9VL[SEQ ID NO:65] TIFF2024546759000026.tif1516285C10VH[SEQ ID NO:66] TIFF2024546759000027.tif1516285C10VL[SEQ ID NO:67] TIFF2024546759000028.tif15163218D9VH [SEQ ID NO: 68] TIFF2024546759000029.tif15162218D9VL[SEQ ID NO:69] TIFF2024546759000030.tif13162224G3VH[SEQ ID NO:70] TIFF2024546759000031.tif16163224G3VL[SEQ ID NO:71] TIFF2024546759000032.tif14162

[0142] Table 2 below provides the association rate constant (ka), dissociation rate constant (kd) and dissociation constant (KD) analyzed by Octet Kinetics (see also Example 9 below). These constants reflect the binding affinity of individual monoclonal antibodies to canine IL-31RA. [Table 3]

[0143] The results show that the selected mAbs have low nanomolar to sub-picomolar binding affinities ranging from about 0.85 nM to about 1 pM.

[0144] The set of six CDRs for each of the ten monoclonal antibodies listed above is provided below in Table 3. Additionally, the canonical structures for each of these CDRs are provided below in Table 4. [Table 4]

[0145] [Table 5]

[0146] Of these 10 sets of CDRs in Table 3 above, a particular group of four anti-canine IL-31RA antibodies that bind IL-31RA were identified as containing significant amino acid sequence similarity.

[0147] TIFF2024546759000036.tif86141TIFF2024546759000037.tif89141

[0148] In fact, all HCDR1s of this group of four antibodies have the same amino acid sequence (SEQ ID NO:3). The four HCDR2s are different, but they are not very different. In particular, the HCDR2 of 100H8 differs from the other three HCDR2s by having an isoleucine residue at position 9 instead of a threonine residue. 74H10 further differs from 100H8 by having an aspartic acid residue at its C-terminus instead of a glycine residue. Like 100H8, the other three HCDR2s have a glycine residue at their C-terminus. Unlike 100H8, the other three HCDRs have three additional amino acid residues (FDV) at their C-terminus. Both 74H10 and 55B3 have a leucine at their position 4 instead of a valine residue like 100H8, while 222E7 has a glutamine residue. Finally, both 100H8 and 74H10 have a proline residue at position 3, whereas both 222E7 and 55B3 have a glutamine residue at position 3.

[0149] Three of the four LCDR1s in this group of four antibodies have the same amino acid sequence (SEQ ID NO: 30), but the LCDR1 of 55B3 shares their first four amino acid residues, whereas its LCDR1 differs from the other three LCDR1s in its remaining seven amino acid residues. The LCDR2s of 100H8 and 74H10 have the same amino acid sequence (SEQ ID NO: 40), but the LCDR2s of 55B3 and 222E7 both differ from the other two LCDR2s by having an asparagine residue at position 3 instead of the aspartic acid residues of 100H8 and 74H10. Furthermore, the LCDR2 of 222E7 has a valine residue at position 2, rather than the alanine residue found in the LCDR2s of the three other antibodies. Notably, all four LCDR3s in this group of four antibodies have the same amino acid sequence (SEQ ID NO: 45).

[0150] The remaining six antibodies against canine IL-31RA detailed above, namely 65G9, 85C10, 224G3, 51F8, 209G5 and 218D9, can be further decomposed into two pairs of antibodies with significant identity in their respective CDR amino acid sequences and two antibodies that are relative outliers. Thus, there is considerable amino acid sequence identity between the set of six CDRs of antibody 65G9 and the set of six CDRs of antibody 85C10 (see Table 3). Consistently, both antibodies 65G9 and 85C10 bind to two linear sequences within the C-terminal region of IL-31RA-ECD, one of which is the same (SEQ ID NO: 106; see Table 6 and Figure 6D) and the other of which has substantial overlap (compare SEQ ID NO: 107 with SEQ ID NO: 108; see Table 6). In particular, one of the outliers, antibody 244G3, binds to an epitope comprising a single linear sequence in the C-terminal region of IL-31RA-ECD (see Figure 6E) contained in the second linear sequence of antibody 85C10 (compare SEQ ID NO:109 with SEQ ID NO:108; see Table 6).

[0151] The second group contains antibodies 51F8 and 209G5, which also have significant amino acid sequence identity between their respective six CDR sets (see Table 3). Consistently, antibodies 51F8 and 209G5 both bind to two linear sequences within the N-terminal region of IL-31RA-ECD, one of which is identical (SEQ ID NO:98; see Table 6 and FIG. 6B), while the second linear sequence of IL-31RA-ECD to which antibody 209G5 binds (SEQ ID NO:101) is within the amino acid sequence of the second linear sequence of IL-31RA-ECD to which antibody 51F8 binds (SEQ ID NO:100; see Table 6).

[0152] Another outlier, antibody 218D9, binds to two linear amino acid sequences located in the central portion of the amino acid sequence of IL-31RA-ECD (SEQ ID NOs: 104 and 105; see FIG. 6C and Table 6). This antibody proved to be both a strong binder of IL-31RA and a good blocker of the binding of IL-31RA to IL-31.

[0153] [Example 5] Blocking activity of anti-IL-31 receptor alpha antibodies The ability of anti-canine IL-31RA hybridoma supernatants to block the binding of IL-31 to IL-31RA was assessed in a blocking ELISA as described below.

[0154] protocol 1) Coat a 96-well half-area plate with 25 μL / well of IL-31RA (1 μg / mL in PBS buffer). 2) Incubate the plate at 4°C overnight. 3) Wash the plate three times with PBST (PBS + 0.05% Tween 20). 4) Block plate with blocking buffer (PBS with 5% FBS), 25 ul / well, for 30 minutes at room temperature. 5) Transfer 25 μl / well of hybridoma supernatant to a 96-well plate and incubate at room temperature for 60 minutes. 6) Wash the plate 3 times with PBST. 7) Add 25 μL / well of biotinylated IL31 (0.5 μg / mL in blocking buffer) and incubate for 60 min at room temperature. 8) Wash the plate 3 times with PBST. 9) Add 25 μl / well of Streptavidin-HRP, 1:5000 dilution in Blocking Buffer to the plate and incubate at room temperature for 60 minutes. 10) Wash the plate 5 times with PBST. 11) Add TMB-based reagent to the plate for a colorimetric reaction for 2-3 minutes. 12) Stop the reaction with 0.16M sulfuric acid. 13) Read the plate in a plate reader.

[0155] result: Of the approximately 2,000 hybridoma clones initially identified, approximately 300 were found to have binding affinity for IL-31RA, and about 10 of such clones also showed significant blocking of the binding of canine IL-31 to canine IL-31RA (see Examples below).

[0156] [Example 6] FACS assay to test the blocking activity of monoclonal antibodies against canine IL31-RA To develop a cell-based assay to evaluate the binding and blocking of canine IL-31 by anti-canine IL-31RA antibodies, the nucleotide sequences of canine IL-31RA with a c-terminal Flag tag and OSMR with a c-terminal HA tag were prepared by chemical synthesis and then cloned into lentiviral vectors Lenti-puro and Lenti-Hygro, respectively. The lentiviruses Lenti-puro-IL31RA-Flag and Lenti-Hygro-OSMR-HA prepared from Lenti-X293T cells were co-transfected into CHO-k1 cells. CHO stable cell pools co-expressing canine IL-31RA and OSMR were selected by FACS with anti-flag and anti-HA antibodies. Single cell clones were isolated from the stable pools. The developed CHO-IL-31RA / OSMR expressing cell lines are applied to screen anti-canine IL-31RA monoclonal antibodies for blocking IL-31 at its receptor complex IL-31RA / OSMR.

[0157] material Cell line: CHO-IL-31RA / OSMR stable cell line Cell growth medium: F-12K medium containing 10% FBS, 8 μg / ml puromycin and 200 μg / ml hygromycin Recombinant canine IL-31-His protein (0.5 μg / ml) FACS buffer: PBS Isotype control: Mouse IgG (Genscript, A01007) (3 μg / ml) Secondary antibody: Mouse anti-His tag antibody, 1 μg / ml (Genscript, A01802) Flow cytometer: BD FACSCanto

[0158] Flow cytometry procedure 1) CHO-IL-31RA / OSMR cells were grown in Growth Medium in T75 flasks. 2) Trypsinize to detach the cells, then resuspend the cells in 5 mL of fresh growth medium. 3) Spin down the cells at 300g for 3 minutes, discard the supernatant, and wash the cells twice with PBS. 4) Resuspend cells in PBS at 2 x 106 cells / mL. 5) Cells were plated into 96-well assay plates at 50 μl / well. 6) Anti-IL-31RA antibody (20 μg / mL) or isotype control is mixed with IL-31 (1 μg / ml), and then 50 μl of the mixture is transferred to each well of the assay plate. 7) After incubation at 4°C for 40 minutes, the cells were washed twice with 150 μl of cold PBS. 8) Add 100 μl of secondary antibody (1 μg / mL) to each well of the assay plate. 9) After incubation at 4°C for 40 minutes, the cells were washed twice with 150 μl of cold PBS. 10) Cells are resuspended in 100 μl / well of cold PBS and read by flow cytometry.

[0159] As shown in Figure 3, FACS results indicate that 9 of 10 mouse anti-canine IL-31RA mAbs can significantly block IL-31 binding to the IL-31RA / OSMR complex displayed on CHO-IL-31RA / OSMR cells. The lead antibodies are 51F8, 74H10, 100H8, 209G5 and 218D9.

[0160] [Example 7] STAT-3 assay Stat-3 is known to be activated by IL-31 in cells containing a heterodimeric receptor for IL-31. To develop an assay to evaluate the activation of STAT-3 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 IL-31RA and OSMR nucleotide sequences, respectively, were co-transfected into Ba / f3 cells, and the transfected cells, designated as "Ba / f3-OI", were grown as a pool under antibiotic selection. The ability of canine IL-31 to induce STAT-3 activation was tested as follows.

[0161] material: Cell line: Ba / f3-OI stable pool cells Growth medium containing mouse IL-3 or canine IL-31 (cIL-31): RPMI 1640 435ml(ThermoFisher,12633-020) FBS 50mL (SAFC Catalog No. 12003c-500mL) 2-Mercaptoethanol (50 mM) 0.5 mL (Gibco 31350-010) 100X Pen Strep 5mL(Gibco15140-122 Lot1734040) 200mM L-Glu 10ml(Gibco 25030-081 Lot1677185) 500ng / mL Geneticin G418 (Gibco or Sigma) 5ng / mL mIL-3 or 100ng / mL cIL-31 Starvation medium: growth medium without mIL-3 and cIL-31 p-STAT3 (Tyr705) assay kit: PerkinElmer, ALSU-PST3-A-HV

[0162] procedure: cell culture 1) Thaw a vial of Ba / f3-OI cells and grow the cells in growth medium containing mIL-3 at 37° C. in a CO 2 shaker at 125 rpm. 2) Passage the cells for 2-3 passages to obtain cells with a viability of 90% or higher before setting up the cell-based assay. 3) To set up the assay, harvest cells at 1 x 10 7 Resuspend in starvation medium to viable cells / mL. 4) Plate the cells in a 96-well plate, 50 μL / well (approximately 5 × 10 5 Dispense into 100 µL of cultured cells (cells / well). 5) Dilute cIL-31 3-fold with starvation medium in the dilution plate and then transfer 50 μL of each serially diluted cIL-31 aliquot to the cell plate. 6) Incubate the cell plate at 37°C in a CO2 shaker at 125 rpm for 1-2 hours, 15-30 minutes.

[0163] AlphaLISA assay according to manufacturer's instructions: 7) Spin down the cells, aspirate the supernatant, and add 50-100 μL / well of 1x lysis buffer. Incubate at room temperature for 10 minutes with shaking at 1000 rpm.

[0164] 8) Remove 30 μL of cell lysate to a ½ area plate or store frozen at −80° C. for future testing.

[0165] SUREFIRE Assay: 8) Add 15 μL / well of acceptor mix to the cell lysate. Seal the plate, agitate at 1000 rpm for 2 min, then incubate at room temperature for 1-2 h. 9) Add 15 μL / well of donor mix to the cell lysate. Seal and agitate at 1000 rpm for 2 min, then incubate at room temperature for 1-2 h (plates can be stored at 4°C overnight. After 1 h incubation at room temperature, read plates the next day) 10) Read the plate on an alpha plate reader at 520-620 nm.

[0166] result: FIG. 4 shows the induction of STAT-3 phosphorylation by canine IL-31, which stimulates the activation of STAT-3 in Ba / f3-OI cells in a dose-dependent manner. Ba / f3 cells were used as a control. Ba / f3-OI cells expressing IL-31 receptor complex were tested for IL-31-induced STAT-3 phosphorylation. The results show that STAT-3 phosphorylation was induced by IL-31 in Baf3-OI cells in a dose-dependent manner, implying that (i) canine IL-31 receptor complex is successfully expressed on the cell surface, (ii) canine IL-31 binding to IL-31 receptor can stimulate endogenous STAT3 phosphorylation, and (iii) subsequently initiate its downstream signaling pathway.

[0167] [Example 8] Biological activity of anti-canine IL-31RA antibodies The ability of anti-canine IL-31RA mAbs to inhibit activation of STAT-3 in Ba / f3-OI cells is assessed as follows.

[0168] 1) Thaw a vial of Ba / f3-OI cells and grow the Ba / f3-OI cells in growth medium containing mIL-3 in a 37° C. CO 2 shaker at 125 rpm. 2) Passage the cells for 2-3 passages to obtain cells with a viability of 90% or higher before setting up the cell-based assay. 3) To set up the assay, harvest cells at 1 x 10 7 Resuspend in starvation medium to viable cells / mL. 4) Plate the cells in a 96-well plate, 50 μL / well (approximately 5 × 10 5 Dispense into 100 µL of cultured cells (cells / well). 5) Dilute the antibodies 3-fold starting at a concentration of 200 μg / mL in starvation medium in a row on a 96-well plate. Then add 5 μL of cIL-31 to each well for a final concentration of 100 ng / mL. 6) Transfer 50 μL of the diluted antibody and cIL-31 mixture to each well of the cell plate and mix gently. 7) Incubate the cell plate for 15-30 minutes in a CO2 shaker at 37 °C at 125 rpm. AlphaLISA Assay according to manufacturer's instructions: (See Example 7)

[0169] As illustrated in Figure 5A for monoclonal antibodies 209G5, 218D9 and 85C10, in Figure 5B for monoclonal antibodies 100H8, 74H10, 51F8 and 85C10, and again in Figure 8 for various constructs of 218D9, all of the IL-31RA mAbs tested inhibit canine IL-31-mediated STAT-3 phosphorylation in Ba / f3-OI cells, but there is no inhibition in the absence of these antibodies (labeled IL-31). From Figure 8, the IC50 of the various 218D9 antibody constructs was calculated to be approximately 2.2nM for the chimeric antibody; 230nM for c218D9VH3VL2; 8.3nM for c218D9VH4VL2; 2.9nM for c218D9VH4VL3; and 2.5nM for c218D9VH3VL3.

[0170] [Example 9] In vitro binding of anti-canine IL-31RA monoclonal antibodies to the canine IL-31RA receptor All kinetic measurements were performed with an Octet HTX using SA biosensors and Data Acquisition 12.0 software. Biotin-labeled antigen (canine IL-31RA) was loaded onto pre-rehydrated SA biosensors at a concentration of 1 μg / mL for 120 seconds. The biosensors were then placed into Octet Kinetics Buffer (PBS + 0.02% Tween 20, 0.1% BSA) for the blocking phase for 120 seconds. The antigen-loaded biosensors were then placed into 2-fold serial dilutions of anti-IL-31RA monoclonal antibodies from 100 nM to 3.13 nM in Octet Kinetics Buffer for 300 seconds for the association phase. The last well was buffer only, and that sensor was used for subtraction of the reference sensor. Finally, the biosensors were placed into Octet Kinetics Buffer for the dissociation phase for 300 seconds. Analysis was performed using Data Analysis 12.0 software, and curves were fitted using a 1:1 binding model.

[0171] Binding affinity measurements show that all monoclonal antibodies tested have low nanomolar to sub-picomolar binding affinities ranging from about 1.5 nM to about 1 pM (see Table 2 above). Two of the top blocking antibodies: 51F8 and 218D9, had KDs of about 0.2 nM and about 0.07 nM, respectively (see Table 5B below).

[0172] [Example 10] Caninized Antibody The DNA and protein sequences of canine heavy and light chains are known and can be obtained by searching the NCBI gene and protein database. As mentioned above, for canine antibodies, there are four known IgG subtypes: IgG-A, IgG-B, IgG-C and IgG-D, and two types of light chains: kappa and lambda. Without being bound to any particular approach, the process of producing caninized heavy and light chains, which can be mixed in different combinations to produce caninized anti-canine IL-31 receptor alpha mAbs, includes the following scheme: The DNA sequences of the VH and VL domains containing the CDRs of the desired anti-IL-31 receptor alpha mAb are identified. i) Identifying the heavy and light chain CDRs of the desired anti-IL-31RA mAb. ii) Identifying suitable sequences for the heavy and light chains of canine IgG. iii) Identifying the DNA sequences encoding the endogenous CDRs of the heavy and light chains of the canine IgG of the above sequences. iv) The DNA sequences encoding the endogenous canine heavy and light chain CDRs are replaced with DNA sequences encoding the desired anti-IL-31RA CDRs. Additionally, some canine framework residues may be replaced with residues selected from the desired anti-IL-31 receptor alpha mAb framework regions. v) synthesizing the DNA from step (v), cloning it into an appropriate expression plasmid, and transfecting the plasmid containing the desired caninized heavy and light chains into HEK293 cells. vi) Purify expressed caninized antibodies from HEK293 supernatants. vii) Testing the purified caninized antibodies for binding to the canine IL-31 receptor alpha chain.

[0173] Application of the steps outlined above can result in the set of caninized heavy and light chain amino acid sequences provided below.

[0174] c51F8VH1-cIgGBm [SEQ ID NO: 72] TIFF2024546759000038.tif49162

[0175] c51F8VH2-cIgGBm [SEQ ID NO: 73] TIFF2024546759000039.tif48162

[0176] c51F8VH3-cIgGBm [SEQ ID NO: 74] TIFF2024546759000040.tif50162

[0177] c51F8VH4-cIgGBm [SEQ ID NO: 75] TIFF2024546759000041.tif62162

[0178] c51F8VL1-cCK [SEQ ID NO: 76] TIFF2024546759000042.tif30152

[0179] c51F8VL6-cCK [SEQ ID NO: 77] TIFF2024546759000043.tif28161

[0180] c51F8VL7-cCK [SEQ ID NO: 78] TIFF2024546759000044.tif29161

[0181] c100H8VH4-cIgGBm [SEQ ID NO: 79] TIFF2024546759000045.tif56161

[0182] c100H8VH7-cIgGBm [SEQ ID NO: 80] TIFF2024546759000046.tif40163TIFF2024546759000047.tif13161

[0183] c100H8VH8-cIgGBm [SEQ ID NO: 81] TIFF2024546759000048.tif55161

[0184] c100H8VL1-cCK [SEQ ID NO: 82] TIFF2024546759000049.tif27161

[0185] c100H8VL2-cCK [SEQ ID NO: 83] TIFF2024546759000050.tif28160

[0186] c100H8VL3-cCK [SEQ ID NO: 84] TIFF2024546759000051.tif26162

[0187] c100H8VL4-cCK [SEQ ID NO: 85] TIFF2024546759000052.tif28160

[0188] c218D9VH1-cIgGBm [SEQ ID NO: 86] TIFF2024546759000053.tif56161

[0189] c218D9VH2-cIgGBm [SEQ ID NO: 87] TIFF2024546759000054.tif55161

[0190] c218D9VH3-cIgGBm [SEQ ID NO: 88] TIFF2024546759000055.tif55151

[0191] c218D9VH4-cIgGBm [SEQ ID NO: 89] TIFF2024546759000056.tif54163

[0192] c218D9VL1-cCK [SEQ ID NO: 90] TIFF2024546759000057.tif28160

[0193] c218D9VL2-cCK [SEQ ID NO: 91] TIFF2024546759000058.tif28161

[0194] c218D9VL3-cCK [SEQ ID NO: 92] TIFF2024546759000059.tif28161

[0195] c218D9VH1 [SEQ ID NO: 93] TIFF2024546759000060.tif14161

[0196] c218D9VH2 [SEQ ID NO: 94] TIFF2024546759000061.tif13161

[0197] c218D9VH3 [SEQ ID NO: 95] TIFF2024546759000062.tif14161

[0198] c218D9VH4 [SEQ ID NO: 96] TIFF2024546759000063.tif13161

[0199] c218D9VL1 [SEQ ID NO: 128] TIFF2024546759000064.tif13160

[0200] c218D9VL2 [SEQ ID NO: 129] TIFF2024546759000065.tif14161

[0201] c218D9VL3 [SEQ ID NO: 130] TIFF2024546759000066.tif14161

[0202] [Example 11] Reactivity of caninized antibodies to canine IL-31RA The caninized antibodies were tested for reactivity with canine IL-31RA as follows: 1. Coat 200 ng / well of IL-31RA onto immune plates and incubate plates at 4° C. overnight. 2. Wash the plate three times with phosphate buffered saline (PBS) containing 0.05% Tween 20 (PBST). 3. Block the plate with 0.5% bovine serum albumin (BSA) in PBS for 45-60 minutes at room temperature. 4. Wash the plate 3 times with PBST. 5. In each row or column of the dilution plate, dilute the caninized antibody 3-fold starting at 0.3 μg / mL. 6. Transfer the diluted caninized antibodies to each column or row of the immunoplate and incubate the plate at room temperature for 45-60 minutes. 7. Wash the plate 3 times with PBST. 8. Add 1:4000 diluted horseradish peroxidase-labeled anti-dog IgG Fc to each well of the plate, then incubate the plate at room temperature for 45-60 minutes. 9. Wash the plate 3 times with PBST. 10. Add 3,3',5,5'-tetramethylbenzidine (TMB) substrate to each well of the plate and incubate the plate at room temperature for 10-15 minutes to allow color development. 11. Stop the reaction by adding 100 μL of 1.5 M phosphoric acid to each well. Read the plate at 450 nm with a reference wavelength of 540 nm.

[0203] The caninized antibodies were tested for their reactivity against canine IL-31RA, as shown in Figures 7A-7E. The results show that the caninized antibodies have similar binding affinities to their corresponding parent antibodies (represented by the chimeric antibodies).

[0204] Table 5A below provides the relative binding affinities of different caninized antibodies (EC50) to their corresponding mouse-canine chimeric antibodies (see Figures 7A-7E). While these are just relative numbers, caninized antibody 218D9 stands out as an antibody that has essentially the same binding affinity as its parent chimeric mouse antibody.

[0205] The term "chimeric" before an antibody number means that the antibody is a mouse-dog chimeric antibody, such as chimeric 218D9 or chimeric 51F8. Furthermore, an "m" followed by "Chim" before an antibody number means that the antibody is a mouse-dog chimeric antibody, such as m224G3 Chim. A lowercase "c" before an antibody number means that it is a caninized antibody, such as c218D9VH4VL2.

[0206] [Table 6]

[0207] Table 5B below shows the binding constants of the 51F8 and 218D9 caninized antibodies. The results again show that the caninized 218D9 antibodies have essentially the same binding affinity as their parent chimeric mouse antibodies, while the caninized 51F8 antibodies have a slightly weaker binding affinity than their parent chimeric mouse antibodies.

[0208] [Table 7]

[0209] [Example 12] Mapping of canine IL-31 receptor alpha epitopes using mass spectrometry A method based on chemical cross-linking and mass spectrometry detection was used to identify the epitopes recognized by anti-canine IL-31 receptor alpha mAb (located at CovalX Instrument Incorporated, 999 Broadway, Suite 305, Saugus, MA 01906-4510). Application of this technology to epitope mapping of the canine IL-31 receptor alpha chain led to the identification of epitopes recognized by the mAbs listed in Table 6 below. Results from epitope mapping of canine IL-31 receptor alpha with the 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). Results from epitope mapping of canine IL-31RA with the eight antibodies included in Table 6 indicate that the mAbs recognize specific peptide epitopes present within the extracellular domain of canine IL-31RA. Notably, seven epitopes distributed from the N-terminus to the C-terminus of IL-31RA-ECD were identified as bound by the eight antibodies. Antibodies 100H8, 51F8, 209G5 and 55B3 share an epitope toward the N-terminus, and antibodies 65G9, 85C10 and 224G3 share an epitope toward the C-terminus of IL-31RA-ECD. Antibody 218D9 has two unique epitopes located in the central part of IL-31RA-ECD. As shown by the functional results, all eight monoclonal antibodies blocked STAT-3 phosphorylation mediated by IL-31, implying that the seven epitopes are important in the interaction of canine IL-31RA with its ligand. This indicates the complexity of the interaction between canine IL-31 and the canine IL-31 receptor complex.Five of the eight monoclonal antibodies tested were caninized and both the location of their respective epitopes on the IL-31RA ECD and the identified binding amino acid residues are shown in Figures 6A-6E and included in Table 6 below [see also Figures 6A-6E, which provide the epitopes on canine IL-31RA for antibodies 100H8, 51F8, 218D9, 85C10 and 224G3, respectively, and the binding residue locations of the amino acid sequence of SEQ ID NO:2 for each epitope on the cIL-31R ECD antigen].

[0210] [Table 8] [Table 9]

[0211] TIFF2024546759000071.tif235145TIFF2024546759000072.tif230152TIFF2024546759000073.tif234150TIFF2024546759000074.tif219151TIFF2024546759000075.tif233151TIFF2024546759000076.tif217151TIFF2024546759000077.tif236151TIFF2024546759000078.tif225150TIFF2024546759000079.tif236150TIFF2024546759000080.tif237151TIFF2024546759000081.tif227149TIFF2024546759000082.tif231149TIFF2024546759000083.tif232149TIFF2024546759000084.tif226149TIFF2024546759000085.tif230150TIFF2024546759000086.tif232151TIFF2024546759000087.tif224149TIFF2024546759000088.tif228149TIFF2024546759000089.tif234150TIFF2024546759000090.tif227151TIFF2024546759000091.tif229150TIFF2024546759000092.tif230147TIFF2024546759000093.tif231150TIFF2024546759000094.tif234151TIFF2024546759000095.tif227150TIFF2024546759000096.tif237147TIFF2024546759000097.tif227149TIFF2024546759000098.tif225149TIFF2024546759000099.tif226156TIFF2024546759000100.tif236150TIFF2024546759000101.tif225152TIFF2024546759000102.tif219150TIFF2024546759000103.tif235150TIFF2024546759000104.tif226149TIFF2024546759000105.tif219151TIFF2024546759000106.tif226150TIFF2024546759000107.tif234151TIFF2024546759000108.tif217148TIFF2024546759000109.tif227148TIFF2024546759000110.tif226152TIFF2024546759000111.tif226141TIFF2024546759000112.tif234143TIFF2024546759000113.tif233142TIFF2024546759000114.tif219143TIFF2024546759000115.tif227143TIFF2024546759000116.tif232146TIFF2024546759000117.tif225141TIFF2024546759000118.tif218140TIFF2024546759000119.tif233144TIFF2024546759000120.tif234141TIFF2024546759000121.tif226141TIFF2024546759000122.tif225142TIFF2024546759000123.tif226142TIFF2024546759000124.tif226143TIFF2024546759000125.tif218141TIFF2024546759000126.tif233141TIFF2024546759000127.tif225142TIFF2024546759000128.tif219141TIFF2024546759000129.tif226139TIFF2024546759000130.tif234141TIFF2024546759000131.tif226141TIFF2024546759000132.tif219141TIFF2024546759000133.tif234141TIFF2024546759000134.tif226142TIFF2024546759000135.tif225141TIFF2024546759000136.tif226141TIFF2024546759000137.tif225143TIFF2024546759000138.tif226142TIFF2024546759000139.tif226143TIFF2024546759000140.tif224142TIFF2024546759000141.tif229145TIFF2024546759000142.tif234142TIFF2024546759000143.tif235142TIFF2024546759000144.tif217141TIFF2024546759000145.tif226141TIFF2024546759000146.tif233142TIFF2024546759000147.tif233141TIFF2024546759000148.tif225140TIFF2024546759000149.tif223141TIFF2024546759000150.tif232143TIFF2024546759000151.tif220141TIFF2024546759000152.tif234142TIFF2024546759000153.tif225143.

Claims

1. 1. An isolated mammalian antibody or antigen-binding fragment thereof that binds to canine interleukin-31 receptor alpha (canine IL-31RA), comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequences: CDR heavy 1 (HCDR1), CDR heavy 2 (HCDR2), and CDR heavy 3 (HCDR3), and the light chain comprises the amino acid sequences: CDR light 1 (LCDR1), CDR light 2 (LCDR2), and CDR light 3 (LCDR3); where: (i) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 3; (ii) the HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13; (iii) the HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23; (iv) the LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 30 and SEQ ID NO: 31; (v) the LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40; and (vi) the LCDR3 comprises the amino acid sequence of SEQ ID NO: 45; An isolated mammalian antibody or antigen-binding fragment thereof.

2. when bound to canine IL-31RA, the antibody binds to an epitope contained in an amino acid sequence selected from the group consisting of SEQ ID NO:97, SEQ ID NO:103, SEQ ID NO:99, SEQ ID NO:102, or any combination thereof.

2. The isolated mammalian antibody or antigen-binding fragment thereof of claim 1.

3. 3. The isolated mammalian antibody or antigen-binding fragment thereof of claim 2, wherein, upon binding to canine IL-31RA, the antibody binds to an epitope encompassed by the amino acid sequences of SEQ ID NO:97 and SEQ ID NO:

103.

4. 3. The isolated mammalian antibody or antigen-binding fragment thereof of claim 2, wherein, upon binding to canine IL-31RA, the antibody binds to an epitope encompassed by the amino acid sequences of SEQ ID NO:99 and SEQ ID NO:

102.

5. (i) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 3; (ii) the HCDR2 comprises the amino acid sequence of SEQ ID NO: 10; (iii) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 20; (iv) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 30; (v) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 40, and (vi) the LCDR3 comprises the amino acid sequence of SEQ ID NO: 45; 4. The isolated mammalian antibody or antigen-binding fragment thereof of claim 1 or 3.

6. (i) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 3; (ii) the HCDR2 comprises the amino acid sequence of SEQ ID NO: 11; (iii) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 21; (iv) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 30; (ii) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 40; and (iii) the LCDR3 comprises the amino acid sequence of SEQ ID NO: 45; An isolated mammalian antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

7. (i) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 3; (ii) the HCDR2 comprises the amino acid sequence of SEQ ID NO: 12; (iii) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 22; (iv) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 30; (v) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 38, and (vi) the LCDR3 comprises the amino acid sequence of SEQ ID NO: 45; An isolated mammalian antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

8. (i) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 3; (ii) the HCDR2 comprises the amino acid sequence of SEQ ID NO: 13; (iii) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 23; (iv) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 31; (v) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 39, and (vi) the LCDR3 comprises the amino acid sequence of SEQ ID NO: 45; 10. The isolated mammalian antibody or antigen-binding fragment thereof of claim 1 or 4.

9. The isolated mammalian antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the isolated mammalian antibody or antigen-binding fragment thereof binds to canine IL-31RA and blocks binding of canine IL-31RA to canine interleukin-31 (IL-31).

10. The isolated mammalian antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the mammalian antibody or antigen-binding fragment thereof is a caninized antibody or caninized antigen-binding fragment thereof.

11. 11. The isolated mammalian antibody or antigen-binding fragment thereof of claim 10, wherein the heavy chain further comprises a hinge region comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, and SEQ ID NO:

115.

12. 12. The isolated mammalian antibody or antigen-binding fragment thereof of claim 10 or 11, wherein the heavy chain further comprises a canine fragment crystallizable region (cFc region), and the cFc region comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:110, SEQ ID NO:116, SEQ ID NO:117, and SEQ ID NO:

118.

13. 12. The isolated mammalian antibody or antigen-binding fragment thereof of claim 10 or 11, wherein the heavy chain further comprises a canine fragment crystallizable region (cFc region), wherein the cFc region comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 111, wherein the cFc region comprises an alanine residue (A) at both positions 31 and 63 of SEQ ID NO:

111.

14. An isolated mammalian antibody or antigen-binding fragment thereof that binds to canine IL31RA, comprising a heavy chain and a light chain; wherein the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, and SEQ ID NO: 85, and the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO:

81.

15. 15. The isolated mammalian antibody or antigen-binding fragment thereof of claim 14, wherein, upon binding to canine IL-31RA, the antibody binds to an epitope encompassed by the amino acid sequences of SEQ ID NO:97 and SEQ ID NO:

103.

16. The IL-31RA antibody is a light chain comprising the amino acid sequence of SEQ ID NO: 84 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 80; a light chain comprising the amino acid sequence of SEQ ID NO: 84 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 81; a light chain comprising the amino acid sequence of SEQ ID NO: 84 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 79; a light chain comprising the amino acid sequence of SEQ ID NO: 85 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 80; a light chain comprising the amino acid sequence of SEQ ID NO: 85 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 81; a light chain comprising the amino acid sequence of SEQ ID NO: 85 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 79; a light chain comprising the amino acid sequence of SEQ ID NO: 83 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 80; a light chain comprising the amino acid sequence of SEQ ID NO: 83 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 81; a light chain comprising the amino acid sequence of SEQ ID NO: 83 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 79; a light chain comprising the amino acid sequence of SEQ ID NO: 82 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 80; a light chain comprising the amino acid sequence of SEQ ID NO: 82 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 81; a light chain comprising the amino acid sequence of SEQ ID NO: 82 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 79; 16. The isolated mammalian antibody or antigen-binding fragment thereof of claim 14 or 15, comprising:

17. An isolated nucleic acid encoding: (a) the heavy chain of an isolated mammalian antibody or antigen-binding fragment thereof according to any one of claims 10 to 16; (b) the light chain of the isolated mammalian antibody or antigen-binding fragment thereof of any one of claims 10 to 16; (c) both the heavy and light chains of the isolated mammalian antibody or antigen-binding fragment thereof of any of claims 10-16.

18. 18. An expression vector comprising the isolated nucleic acid of claim 17.

19. A host cell comprising the expression vector of claim 18.

20. A pharmaceutical composition comprising the isolated mammalian antibody or antigen-binding fragment thereof of any of claims 10 to 16 and a pharmaceutically acceptable carrier or diluent.

21. A method for helping to block pruritus associated with atopic dermatitis in a non-human animal subject, comprising administering to the animal subject a therapeutically effective amount of an isolated mammalian antibody or antigen-binding fragment thereof described in any of claims 10 to 16, or a pharmaceutical composition described in claim 20.

22. 1. A method for producing an isolated mammalian antibody or antigen-binding fragment thereof that binds canine IL-31RA, the method comprising: Culturing the host cell of claim 19 under conditions suitable for protein expression, and purifying the antibody or antigen-binding fragment thereof from the host cell and culture medium. A method comprising:

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