Anti-IL31 antibodies for animals

Abstract

Various embodiments are provided relating to anti-IL31 antibodies with enhanced binding to canine IL31 and feline IL31. Such antibodies can be used in methods of treating IL31-induced conditions in companion animals such as dogs and cats. Antibodies with enhanced binding to canine IL31 and feline IL31 are provided. Antibody heavy and light chains capable of forming antibodies that bind to canine and feline IL31 are also provided.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of U.S. Provisional Application No. 62 / 893,799, filed August 29, 2019, and U.S. Provisional Application No. 62 / 894,526, filed August 30, 2019, each of which is incorporated herein by reference in its entirety for all purposes.

[0002] [Field] The present invention relates to isolated anti-IL31 antibodies, e.g., with enhanced binding to canine IL31 and feline IL31, and methods of using the antibodies to, e.g., treat IL31-induced conditions or reduce IL31 signaling function in cells in companion animals, e.g., dogs and cats. [Background technology]

[0003] Interleukin-31 (IL-31) is a cytokine produced predominantly by Th2 cells and is understood to be involved in promoting skin diseases such as pruritus and other forms of allergic disorders (e.g., atopic dermatitis). IL-31 functions by binding to its receptor complex (IL-31 receptor A (IL-31Ra) and oncostatin M receptor (OSMR) subunit complex) and activating downstream activities, such as activation of JAK kinases and subsequent phosphorylation and activation of STAT1, STAT3, and STAT5. Activation of this pathway is thought to cause many of the clinical problems associated with dermatitis and other disorders. Companion animals, such as cats, dogs, and horses, suffer from many skin diseases similar to those in humans, including atopic dermatitis. However, IL31 sequences vary widely among humans, cats, dogs, and horses. Thus, there remains a need for methods and compounds that can be used to specifically bind companion animal IL31 to treat IL31-induced conditions and reduce IL31 signaling. Summary of the Invention

[0004] Embodiment 1. An isolated antibody that binds to canine IL31 or feline IL31, comprising: a) a heavy chain comprising a CDR-H3 sequence having the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15; and / or b) a light chain comprising a CDR-L1 sequence having the amino acid sequence of SEQ ID NO: 20; and / or c) a light chain comprising a CDR-L3 sequence having the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 24 an antibody, Embodiment 2. An isolated antibody that binds to canine IL31 or feline IL31, a) a heavy chain comprising a CDR-H1 sequence having the amino acid sequence of SEQ ID NO: 11, a CDR-H2 sequence having the amino acid sequence of SEQ ID NO: 12, and a CDR-H3 sequence having the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15; and / or b) a light chain comprising a CDR-L1 sequence having the amino acid sequence of SEQ ID NO: 20, a CDR-L2 sequence having the amino acid sequence of SEQ ID NO: 21, and a CDR-L3 sequence having the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. an antibody, Embodiment 3. The antibody has a 5×10 -8 Less than M, 1 x 10 -8 Less than M, 5 x 10 -9 Less than M, 1 x 10 -9 Less than M, 5 x 10 -10 Less than M, 1 x 10 -10 Less than M, 5 x 10 -11 Less than M, 1 x 10 -11 Less than M, 5 x 10 -12 Less than M or 1 x 10 -12 10. The antibody of any one of the preceding embodiments, which binds to canine IL31 or feline IL31 with a dissociation constant (Kd) of less than M. Embodiment 4. The antibody of any one of the preceding embodiments, wherein the antibody reduces IL31 signaling function in a companion animal species as measured by a reduction in STAT-3 phosphorylation. Embodiment 5. The antibody of embodiment 4, wherein the companion animal species is dog or cat. Embodiment 6. The antibody of any one of the preceding embodiments, wherein the antibody binds to canine IL31 or feline IL31 as determined by immunoblot analysis and / or biolayer interferometry. Embodiment 7. The antibody of any one of the preceding embodiments, wherein the antibody competes with monoclonal M14 antibody for binding to canine IL31. Embodiment 8. The antibody of any one of the preceding embodiments, wherein the antibody competes with the monoclonal M14 antibody for binding to feline IL31. Embodiment 9. The antibody of any one of the preceding embodiments, wherein the antibody does not bind to human IL31 as determined by immunoblot analysis and / or biolayer interferometry. Embodiment 10. The antibody of any one of the preceding embodiments, wherein the antibody is a monoclonal antibody. Embodiment 11. The antibody of any one of the preceding embodiments, wherein the antibody is a caninized, feline, or chimeric antibody. Embodiment 12. An antibody of any one of the preceding embodiments, further comprising one or more of: (a) the variable region heavy chain framework 1 (HC-FR1) sequence of SEQ ID NO: 16; (b) the HC-FR2 sequence of SEQ ID NO: 17; (c) the HC-FR3 sequence of SEQ ID NO: 18; (d) the HC-FR4 sequence of SEQ ID NO: 19; (e) the variable region light chain framework 1 (LC-FR1) sequence of SEQ ID NO: 25; (f) the LC-FR2 sequence of SEQ ID NO: 26; (g) the LC-FR3 sequence of SEQ ID NO: 27; or (h) the LC-FR4 sequence of SEQ ID NO: 28. Embodiment 13. An antibody of any one of embodiments 1 to 11, further comprising one or more of: (a) the variable region heavy chain framework 1 (HC-FR1) sequence of SEQ ID NO: 55 or SEQ ID NO: 56; (b) the HC-FR2 sequence of SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59; (c) the HC-FR3 sequence of SEQ ID NO: 60 or SEQ ID NO: 61; (d) the HC-FR4 sequence of SEQ ID NO: 62 or SEQ ID NO: 63; (e) the variable region light chain framework 1 (LC-FR1) sequence of SEQ ID NO: 64 or SEQ ID NO: 65; (f) the LC-FR2 sequence of SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68; (g) the LC-FR3 sequence of SEQ ID NO: 69 or SEQ ID NO: 70; or (h) the LC-FR4 sequence of SEQ ID NO: 71 or SEQ ID NO: 72. Embodiment 14. The antibody comprises: a) (i) a variable heavy chain sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; (ii) a variable light chain sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; or (iii) a variable heavy chain sequence such as (i) and a variable light chain sequence such as (ii); or b) (i) a variable heavy chain sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:90; (ii) a variable light chain sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54; or (iii) a variable heavy chain sequence such as (i) and a variable light chain sequence such as (ii). 10. The antibody of any one of the preceding embodiments, comprising: Embodiment 15. The antibody of any one of the preceding embodiments, wherein the antibody comprises a variable heavy chain sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:90. Embodiment 16. The antibody of any one of the preceding embodiments, wherein the antibody comprises the variable light chain sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54. Embodiment 17. The antibody comprises: a) the variable heavy chain sequence of SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8; and the variable light chain sequence of SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11; or b) the variable heavy chain sequence of SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:90; and the variable light chain sequence of SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54. 10. The antibody of any one of the preceding embodiments, comprising: Embodiment 18. The antibody of any one of the preceding embodiments, wherein the antibody comprises a canine or feline constant heavy chain region and / or a canine or feline constant light chain region. Embodiment 19. The antibody of any one of the preceding embodiments, wherein the antibody comprises (a) a dog heavy chain constant region selected from an IgG-A, IgG-B, IgG-C, and IgG-D constant region; or (b) a feline heavy chain constant region selected from an IgG1, IgG2a, and IgG2b constant region. Embodiment 20. The antibody a) (i) a heavy chain amino acid sequence of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, or SEQ ID NO:45; (ii) a light chain amino acid sequence of SEQ ID NO:46, SEQ ID NO:47, or SEQ ID NO:48; or (iii) a heavy chain amino acid sequence as in (i) and a light chain amino acid sequence as in (ii); or b) (i) a heavy chain amino acid sequence of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ ID NO:91; (ii) a light chain amino acid sequence of SEQ ID NO:76, SEQ ID NO:77, or SEQ ID NO:78; or (iii) a heavy chain amino acid sequence such as (i) and a light chain amino acid sequence such as (ii). 10. The antibody of any one of the preceding embodiments, comprising: Embodiment 21. An isolated antibody comprising the variable heavy chain amino acid sequence of SEQ ID NO:3 and / or the variable light chain amino acid sequence of SEQ ID NO:4. Embodiment 22. An isolated antibody comprising the variable heavy chain amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. Embodiment 23. An isolated antibody comprising the variable light chain amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. Embodiment 24. The isolated antibody of embodiment 21, wherein the antibody comprises the variable light chain amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. Embodiment 25. An isolated antibody comprising the variable heavy chain amino acid sequence of SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:90. Embodiment 26. An isolated antibody comprising the variable light chain amino acid sequence of SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54. Embodiment 27. The isolated antibody of any one of embodiments 1 to 25, wherein the antibody comprises the variable light chain amino acid sequence of SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54. Embodiment 28. The antibody of any one of the preceding embodiments, wherein the antibody is an antibody fragment selected from Fv, scFv, Fab, Fab', F(ab')2, and Fab'-SH. Embodiment 29. The antibody of any one of the preceding embodiments, wherein the antibody is bispecific, and the antibody binds to IL31 and one or more antigens selected from IL17, TNFα, CD20, CD19, CD25, IL4, IL13, IL23, IgE, CD11α, IL6R, α4-Intergrin, IL12, IL1β, or BlyS. Embodiment 30. An isolated nucleic acid encoding the antibody of any one of embodiments 1 to 29. Embodiment 31. A host cell comprising the nucleic acid of embodiment 30. Embodiment 32. A method for producing an antibody, comprising culturing the host cell of embodiment 31 and isolating the antibody. Embodiment 33. A pharmaceutical composition comprising the antibody of any one of embodiments 1 to 29 and a pharmaceutically acceptable carrier. Embodiment 34. A method for treating a companion animal species having an IL31-induced condition, comprising administering to the companion animal species a therapeutically effective amount of the antibody of any one of embodiments 1 to 29 or the pharmaceutical composition of embodiment 33. Embodiment 35. The method of embodiment 33, wherein the companion animal species is a dog or a cat. Embodiment 36. The method of embodiment 34 or embodiment 35, wherein the IL31-induced condition is a pruritic or allergic condition. Embodiment 37. The method of any one of embodiments 34 to 36, wherein the IL31-induced condition is selected from atopic dermatitis, allergic dermatitis, pruritus, asthma, psoriasis, scleroderma, and eczema. Embodiment 38. The method of any one of embodiments 34 to 37, wherein the antibody or pharmaceutical composition is administered parenterally. Embodiment 39. The method of any one of embodiments 34 to 38, wherein the antibody or pharmaceutical composition is administered by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarterial, intrasynovial, intrathecal, or inhalation route. Embodiment 40. The method of any one of embodiments 34 to 39, wherein the method comprises administering a Jak inhibitor, a PI3K inhibitor, an AKT inhibitor, or a MAPK inhibitor in combination with the antibody or pharmaceutical composition. Embodiment 41. The method of any one of embodiments 34 to 40, wherein the method comprises administering one or more antibodies selected from an anti-IL17 antibody, an anti-TNFα antibody, an anti-CD20 antibody, an anti-CD19 antibody, an anti-CD25 antibody, an anti-IL4 antibody, an anti-IL13 antibody, an anti-IL23 antibody, an anti-IgE antibody, an anti-CD11α antibody, an anti-IL6R antibody, an anti-α4-integrin antibody, an anti-IL12 antibody, an anti-IL1β antibody, and an anti-BlyS antibody in combination with the antibody or pharmaceutical composition. Embodiment 42. A method for reducing IL31 signaling function in a cell, comprising exposing the cell to an antibody of any one of embodiments 1 to 29 or the pharmaceutical composition of embodiment 33 under conditions that allow binding of the antibody to extracellular IL31, thereby reducing binding to the IL31 receptor and / or reducing IL31 signaling function by the cell. Embodiment 43. The method of embodiment 42, wherein the cells are exposed to the antibody or pharmaceutical composition ex vivo. Embodiment 44. The method of embodiment 42, wherein the cells are exposed to the antibody or pharmaceutical composition in vivo. Embodiment 45. The method of any one of embodiments 42 to 44, wherein the cell is a canine or feline cell. Embodiment 46. A method for detecting IL31 in a sample from a companion animal species, comprising contacting the sample with an antibody of any one of embodiments 1 to 29 or the pharmaceutical composition of embodiment 33 under conditions that allow binding of the antibody to IL31, and detecting whether a complex is formed between the antibody and IL31 in the sample. Embodiment 47. The method of embodiment 46, wherein the sample is a biological sample obtained from a dog or cat. Embodiment 48. A method for identifying an IL31 antagonist, comprising contacting a modified cell line with a candidate IL31 antagonist, wherein the modified cell line is a mammalian cell line not derived from a dog or a cat, and the modified cell line expresses canine IL31Ra and / or feline IL31Ra. Embodiment 49. The method of embodiment 48, wherein the modified cell line is a HeLa cell line. Embodiment 50. The method of embodiment 48 or 49, wherein the modified cell line expresses canine IL31Ra or feline IL31Ra. Embodiment 51. The method of any one of embodiments 48 to 50, wherein the modified cell line expresses a polypeptide having the amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 93. Embodiment 52. The method of any one of embodiments 48 to 51, wherein the modified cell line does not express the canine or feline oncostatin M receptor (OSMR). Embodiment 53. The method of any one of embodiments 48 to 52, wherein the candidate IL31 antagonist is an IL31 antibody, a soluble IL31 receptor, an IL31Ra antibody, or a small molecule, an aptamer, or a peptide. Embodiment 54. The method of any one of embodiments 48 to 53, wherein the method comprises measuring IL31 signaling function. Embodiment 55. The method of embodiment 54, wherein the IL31 signaling function is measured by the level of STAT-1, STAT-3, and / or STAT-5 phosphorylation. Embodiment 56. The method of embodiments 48 to 55, wherein the candidate IL31 antagonist is identified by detecting a decrease in STAT-1, STAT-3, and / or STAT-5 phosphorylation. [Brief explanation of the drawings]

[0005] [Figure 1] FIG. 1 shows the binding analysis of six chimeric antibodies to feline IL-31 according to Example 2. Description of a given sequence

[0006] Table 1 provides a list of the sequences referenced herein. JPEG0007671275000001.jpg254170JPEG0007671275000002.jpg255170JPEG00076712750 00003.jpg255166JPEG0007671275000004.jpg255167JPEG0007671275000005.jpg255167 JPEG0007671275000006.jpg255169JPEG0007671275000007.jpg255168JPEG00076712750 00008.jpg255169JPEG0007671275000009.jpg255169JPEG0007671275000010.jpg196170 DETAILED DESCRIPTION OF THE INVENTION

[0007] Antibodies with enhanced binding to canine IL31 and feline IL31 are provided. Antibody heavy and light chains capable of forming antibodies that bind to canine and feline IL31 are also provided. In addition, antibodies, heavy and light chains comprising one or more specific complementarity determining regions (CDRs) are provided. Polynucleotides encoding antibodies to canine and feline IL31 are provided. Methods for producing or purifying antibodies to canine and feline IL31 are also provided. Therapeutic methods using antibodies to canine and feline IL31 are provided. Such methods include, but are not limited to, methods for treating IL31-induced conditions in companion animal species. Methods for detecting IL31 in samples from companion animal species are provided.

[0008] For the convenience of the reader, the following definitions are provided for terms used herein.

[0009] As used herein, numerical terms such as Kd are calculated based on scientific measurements and, therefore, are subject to appropriate measurement errors. In some instances, the numerical terms may include values ​​that are rounded to the nearest significant figure.

[0010] As used herein, "a" or "an" means "at least one" or "one or more" unless otherwise specified. As used herein, the term "or" means "and / or" unless expressly stated otherwise. In the context of a claim that is multiple dependent, the use of "or" when referring to another claim refers only to that alternative claim.

[0011] Exemplary Anti-IL31 Antibodies Antibodies with enhanced affinity to canine IL31 and feline IL31 are provided. The anti-IL31 antibodies provided herein include, but are not limited to, monoclonal antibodies, chimeric antibodies, caninized antibodies, and felineized antibodies.

[0012] Also provided herein are amino acid sequences of affinity-matured antibodies with enhanced binding to canine and feline IL-31. For example, the variable heavy chain CDRs (SEQ ID NOs: 11-15), variable light chain CDRs (SEQ ID NOs: 20-24), variable region heavy chain framework sequences (SEQ ID NOs: 16-19, 25-28, 55-72), and variable region light chain framework sequences (SEQ ID NOs: 11-14) for exemplary affinity-matured antibodies (caninized and feline) are provided. Exemplary amino acid sequences for the variable light chain, light chain, variable heavy chain, and heavy chain of exemplary affinity-matured antibodies (caninized and feline) are provided (e.g., SEQ ID NOs: 5-10, 29-54, 73-78).

[0013] The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific (e.g., bispecific T cell engagers) and trispecific antibodies), and antibody fragments (such as Fab, F(ab'), ScFv, minibodies, diabodies, triabodies, and tetrabodies), so long as they exhibit the desired antigen-binding activity. Canine, feline, and equine species have different types (classes) of antibodies shared by many mammals.

[0014] The term antibody includes, but is not limited to, fragments capable of binding antigen, such as Fv, single-chain Fv (scFv), Fab, Fab', dis-scFv, sdAb (single-domain antibodies), and (Fab')2 (including chemically conjugated F(ab')2). Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, the name of which reflects its ability to readily crystallize. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies from various species, such as mouse, human, cynomolgus monkey, dog, feline, and horse. Furthermore, for all antibody constructs provided herein, variants with sequences derived from other organisms are also contemplated. Thus, where a murine form of an antibody is disclosed, one of skill in the art would know how to convert an antibody based on murine sequences to feline, dog, horse, etc. Antibody fragments also include single-chain scFvs in any orientation, tandem di-scFvs, diabodies, tandem tri-sdcFvs, minibodies, etc. Antibody fragments also include nanobodies (sdAbs, antibodies with a single monomer domain, such as a heavy chain variable domain pair without a light chain). Antibody fragments can in some embodiments be said to be species specific (e.g., murine scFv or canine scFv). This indicates the sequence of at least a portion of the non-CDR regions, rather than the source of the construct. In some embodiments, the antibody comprises a label or is conjugated to a second moiety.

[0015] The terms "label" and "detectable label" refer to a moiety attached to an antibody or its analyte to render the reaction (e.g., binding) between members of a specific binding pair detectable. A labeled member of a specific binding pair is said to be "detectably labeled." Thus, the term "labeled binding protein" refers to a protein into which a label has been introduced that provides for the identification of the binding protein. In some embodiments, the label is a detectable marker that can produce a signal that is detectable by visual or instrumental means, e.g., by incorporation of a radiolabeled amino acid or attachment of a biotinyl moiety to the polypeptide that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected optically or colorimetrically). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C. 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153 Sm); chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic agents such as gadolinium chelates. Representative examples of labels commonly used in immunoassays include light-producing moieties, e.g., acridinium compounds, and fluorescence-producing moieties, e.g., fluorescein. In this regard, a moiety itself may not be detectably labeled but may become detectable upon reaction with yet another moiety.

[0016] The term "monoclonal antibody" refers to an antibody from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being raised against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is raised against a single determinant on the antigen. Thus, a sample of monoclonal antibody is capable of binding to the same epitope on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be made by recombinant DNA methods such as those described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554, for example.

[0017] An "amino acid sequence" refers to the sequence of amino acid residues in a peptide or protein. The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues can contain natural or unnatural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of this disclosure, "polypeptide" refers to a protein containing modifications such as deletions, additions, and substitutions (generally conservative in nature) relative to the native sequence, so long as the protein maintains a desired activity. These modifications may be deliberate, such as through site-directed mutagenesis, or may be accidental, such as through mutations in hosts producing the protein or errors during PCR amplification.

[0018] As used herein, "IL31" refers to any native IL31 resulting from expression and processing of IL31 in cells. Unless otherwise specified, the term includes IL31 from any vertebrate, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), and companion animals (e.g., dogs, cats, and horses). The term also includes naturally occurring variants of IL31, such as splice variants or allelic variants.

[0019] In some embodiments, canine IL31 comprises the amino acid sequence of SEQ ID NO: 79 or SEQ ID NO: 80. In some embodiments, feline IL31 comprises the amino acid sequence of SEQ ID NO: 81. In some embodiments, murine IL31 comprises the amino acid sequence of SEQ ID NO: 82.

[0020] The term "IL31-binding domain" of an antibody means the binding domain formed by the light and heavy chains of an anti-IL31 antibody that binds to IL31.

[0021] In some embodiments, the IL31-binding domain binds to canine IL31 or feline IL31 with higher affinity than it binds to human IL31. In some embodiments, the IL31-binding domain binds to IL31 of other companion animals, such as equine IL31. In some embodiments, the IL31-binding domain does not bind to human IL31.

[0022] As used herein, the term "epitope" refers to a site on a target molecule (e.g., an antigen, e.g., a protein, nucleic acid, sugar, or lipid) to which an antigen-binding molecule (e.g., an antibody, antibody fragment, or scaffold protein comprising an antibody binding region) binds. Epitopes often comprise chemically active surface groupings of molecules such as amino acids, polypeptides, or sugar side chains, and possess specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be formed from both contiguous or juxtaposed noncontiguous residues (e.g., amino acids, nucleotides, sugars, or lipid moieties) of a target molecule. Epitopes formed from contiguous residues (e.g., amino acids, nucleotides, sugars, or lipid moieties) are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes can include, but are not limited to, at least 3 residues, at least 5 residues, or 8-10 residues (e.g., amino acids or nucleotides). In some examples, the epitope length is less than 20 residues (e.g., amino acids or nucleotides), less than 15 residues, or less than 12 residues. Two antibodies may bind to the same epitope within an antigen if they exhibit competitive binding to the antigen. In some embodiments, an epitope may be identified by a predetermined minimum distance to CDR residues on an antigen-binding molecule. In some embodiments, an epitope may be identified by the above distance and further limited to those residues involved in binding (e.g., hydrogen bonding) between antibody and antigen residues. Epitopes can also be identified by a variety of scans; for example, an alanine or arginine scan may reveal one or more residues with which an antigen-binding molecule may interact. Unless explicitly indicated, identifying a set of residues as an epitope does not exclude other residues from being part of the epitope for a particular antibody. Rather, the existence of such a set represents a minimal series (or species set) of epitopes. Thus, in some embodiments, a set of residues identified as an epitope represents a minimal epitope associated with an antigen, rather than an exclusive list of epitope residues on the antigen.

[0023] The term "CDR" refers to a complementarity determining region as defined by at least one method of identification by one of skill in the art. In some embodiments, CDRs can be defined according to any of the Chothia numbering scheme, the Kabat numbering scheme, a combination of Kabat and Chothia, the AbM definition, the contact definition, or a combination of the Kabat, Chothia, AbM, or contact definitions. The various CDRs within an antibody can be represented by their appropriate number and chain type, including, but not limited to, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. The term "CDR" is used herein to also encompass "hypervariable regions" or HVRs, which include the hypervariable loops.

[0024] In some embodiments, the anti-IL31 antibody is a) a heavy chain comprising a CDR-H3 sequence having the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15; and / or b) a light chain comprising a CDR-L1 sequence having the amino acid sequence of SEQ ID NO: 20; and / or c) a light chain comprising a CDR-L3 sequence having the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 24 Includes.

[0025] In some embodiments, the anti-IL31 antibody is a) a heavy chain comprising a CDR-H1 sequence having the amino acid sequence of SEQ ID NO: 11, a CDR-H2 sequence having the amino acid sequence of SEQ ID NO: 12, and a CDR-H3 sequence having the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15; and / or b) a light chain comprising a CDR-L1 sequence having the amino acid sequence of SEQ ID NO: 20, a CDR-L2 sequence having the amino acid sequence of SEQ ID NO: 21, and a CDR-L3 sequence having the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. Includes.

[0026] As used herein, the term "variable region" refers to a region comprising at least three CDRs. In some embodiments, a variable region comprises three CDRs and at least one framework region ("FR"). The terms "heavy chain variable region" or "variable heavy chain" are used interchangeably and refer to a region comprising at least three heavy chain CDRs. The terms "light chain variable region" or "variable light chain" are used interchangeably and refer to a region comprising at least three light chain CDRs. In some embodiments, a variable heavy chain or variable light chain comprises at least one framework region. In some embodiments, an antibody comprises at least one heavy chain framework region selected from HC-FR1, HC-FR2, HC-FR3, and HC-FR4. In some embodiments, an antibody comprises at least one light chain framework region selected from LC-FR1, LC-FR2, LC-FR3, and LC-FR4. The framework region may be juxtaposed between the light chain CDRs or between the heavy chain CDRs. For example, the antibody may comprise a variable heavy chain having the following structure: (HC-FR1)-(CDR-H1)-(HC-FR2)-(CDR-H2)-(HC-FR3)-(CDR-H3)-(HC-FR4). The antibody may comprise a variable heavy chain having the following structure: (CDR-H1)-(HC-FR2)-(CDR-H2)-(HC-FR3)-(CDR-H3). The antibody may also comprise a variable light chain having the following structure: (LC-FR1)-(CDR-L1)-(LC-FR2)-(CDR-L2)-(LC-FR3)-(CDR-L3)-(LC-FR4). The antibody may also comprise a variable light chain having the following structure: (CDR-L1)-(LC-FR2)-(CDR-L2)-(LC-FR3)-(CDR-L3).

[0027] In some embodiments, the anti-IL31 antibody comprises one or more of: (a) the variable region heavy chain framework 1 (HC-FR1) sequence of SEQ ID NO: 16; (b) the HC-FR2 sequence of SEQ ID NO: 17; (c) the HC-FR3 sequence of SEQ ID NO: 18; (d) the HC-FR4 sequence of SEQ ID NO: 19; (e) the variable region light chain framework 1 (LC-FR1) sequence of SEQ ID NO: 25; (f) the LC-FR2 sequence of SEQ ID NO: 26; (g) the LC-FR3 sequence of SEQ ID NO: 27; or (h) the LC-FR4 sequence of SEQ ID NO: 28.

[0028] In some embodiments, the anti-IL31 antibody comprises one or more of: (a) the variable region heavy chain framework 1 (HC-FR1) sequence of SEQ ID NO:55 or SEQ ID NO:56; (b) the HC-FR2 sequence of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59; (c) the HC-FR3 sequence of SEQ ID NO:60 or SEQ ID NO:61; (d) the HC-FR4 sequence of SEQ ID NO:62 or SEQ ID NO:63; (e) the variable region light chain framework 1 (LC-FR1) sequence of SEQ ID NO:64 or SEQ ID NO:65; (f) the LC-FR2 sequence of SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68; (g) the LC-FR3 sequence of SEQ ID NO:69 or SEQ ID NO:70; or (h) the LC-FR4 sequence of SEQ ID NO:71 or SEQ ID NO:72.

[0029] As used herein, the term "constant region" refers to a region comprising at least three constant domains. The terms "heavy chain constant region" or "constant heavy chain" are used interchangeably and refer to a region comprising at least three heavy chain constant domains, CH1, CH2, and CH3. Non-limiting exemplary heavy chain constant regions include gamma, delta, alpha, epsilon, and mu. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody comprising a gamma constant region is an IgG antibody, an antibody comprising a delta constant region is an IgD antibody, an antibody comprising an alpha constant region is an IgA antibody, an antibody comprising a mu constant region is an IgM antibody, and an antibody comprising an epsilon constant region is an IgE antibody. A given isotype can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (containing a γ1 constant region), IgG2 (containing a γ2 constant region), IgG3 (containing a γ3 constant region), and IgG4 (containing a γ4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (containing an α1 constant region) and IgA2 (containing an α2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 and IgM2. The terms "light chain constant region" or "constant light chain" are used interchangeably and refer to the region containing the light chain constant domain CL. Non-limiting exemplary light chain constant regions include λ and κ. Deletions and changes within a domain that do not alter function are encompassed within the term "constant region" unless otherwise specified. Dogs, cats, and horses have antibody classes such as IgG, IgA, IgD, IgE, and IgM. Canine IgG antibody classes include IgG-A, IgG-B, IgG-C, and IgG-D. Within the feline IgG antibody class are IgG1a, IgG1b, and IgG2.

[0030] The term "chimeric antibody" or "chimera" refers to an antibody in which a portion of the heavy or light chain is derived from a particular source or species, while at least a portion of the remainder of the heavy or light chain is derived from a different source or species. In some embodiments, a chimeric antibody refers to an antibody that contains at least one variable region from a first species (e.g., mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (e.g., human, dog, cat, horse, etc.).

[0031] In some embodiments, the anti-IL31 antibody comprises a canine heavy chain constant region selected from an IgG-A, IgG-B, IgG-C, and IgG-D constant region. In some embodiments, the anti-IL31 antibody is (a) a canine IgG-A antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 83; (b) a canine IgG-B antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 84; (c) a canine IgG-C antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 85; or (d) a canine IgG-D antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 86.

[0032] In some embodiments, the anti-IL31 antibody comprises a feline heavy chain constant region selected from an IgG1, IgG2a, and IgG2b constant region.

[0033] A "caninized antibody" refers to an antibody in which at least one amino acid in a portion of a non-canine variable region has been replaced with the corresponding amino acid from a canine variable region. In some embodiments, a caninized antibody comprises at least one canine constant region (e.g., a gamma constant region, an alpha constant region, a delta constant region, an epsilon constant region, a mu constant region, etc.) or a fragment thereof. In some embodiments, a caninized antibody is an antibody fragment, such as an Fab, scFv, (Fab')2, etc. The term "caninized" also refers to forms of non-canine (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of antibodies) that comprise minimal non-canine immunoglobulin sequence. A caninized antibody can comprise a canine immunoglobulin (recipient antibody) in which residues from the recipient's CDRs have been replaced by residues from the CDRs of a non-canine species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the canine immunoglobulin are replaced by corresponding non-canine residues. Furthermore, caninized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance.

[0034] In some embodiments, the caninized variable chain is fused to a canine constant heavy chain or a canine constant light chain.

[0035] A "felineized antibody" refers to an antibody in which at least one amino acid in a portion of a non-feline variable region has been replaced with the corresponding amino acid from a feline variable region. In some embodiments, a felineized antibody comprises at least one feline constant region (e.g., a gamma constant region, an alpha constant region, a delta constant region, an epsilon constant region, a mu constant region, etc.) or a fragment thereof. In some embodiments, a felineized antibody is an antibody fragment, such as a Fab, scFv, or (Fab')2. The term "felineized" also refers to forms of non-feline (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of antibodies) that comprise minimal non-feline immunoglobulin sequence. A felineized antibody can comprise a feline immunoglobulin (recipient antibody) in which residues from the recipient's CDRs have been replaced by residues from the CDRs of a non-feline species (donor antibody), such as mouse, rat, or rabbit, that has the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the feline immunoglobulin are replaced by corresponding non-feline residues. Furthermore, felineized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance.

[0036] In some embodiments, the felinized variable chain is fused to a feline constant heavy chain or a feline constant light chain. The term "IgX Fc" means that the Fc region is derived from a particular antibody isotype (e.g., IgG, IgA, IgD, IgE, IgM, etc.), where "X" indicates the antibody isotype. Thus, "IgG Fc" indicates the Fc region of the gamma chain, "IgA Fc" indicates the Fc region of the alpha chain, "IgD Fc" indicates the Fc region of the delta chain, "IgE Fc" indicates the Fc region of the epsilon chain, "IgM Fc" indicates the Fc region of the mu chain, etc. In some embodiments, the IgG Fc region comprises a CH1, hinge, CH2, CH3, and CL1. "IgX-N-Fc" indicates that the Fc region is derived from a particular subclass of antibody isotype (such as canine IgG subclass A, B, C, or D; or feline IgG subclass 1, 2a, or 2b), where "N" indicates the subclass. In some embodiments, the IgX Fc or IgX-N-Fc region is derived from a companion animal, such as a dog or cat. In some embodiments, the IgG Fc region is isolated from a canine gamma heavy chain, such as IgG-A, IgG-B, IgG-C, or IgG-D. In some cases, the IgG Fc region is isolated from a feline gamma heavy chain, such as IgG1, IgG2a, or IgG2b. Antibodies comprising an IgG-A, IgG-B, IgG-C, or IgG-D Fc region may result in higher expression levels in recombinant production systems.

[0037] The term "affinity" refers to the strength of the total non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of a molecule X for its partner Y is generally measured by the dissociation constant (K D The affinity can be measured by common methods known in the art, such as immunoblotting, ELISA KD, KinEx A, biolayer interferometry (BLI), or surface plasmon resonance.

[0038] "K D "," "K dThe terms "," "Kd" or "Kd value" are used interchangeably to refer to the equilibrium dissociation constant of an antibody-antigen interaction. In some embodiments, the K D is measured by using a biolayer interference assay using a biosensor such as the Octet® system (Pall ForteBio LLC, Fremont, CA) according to the supplier's instructions. Briefly, biotinylated antigen is attached to the sensor tip, antibody binding is monitored for 90 seconds, and dissociation is monitored for 600 seconds. The buffer for the dilution and binding steps is 20 mM phosphate, 150 mM NaCl, pH 7.2. A buffer-only blank curve is subtracted to correct for drift. Data are fit to a 2:1 binding model using ForteBio data analysis software to determine the binding rate constant (k on ), dissociation rate constant (k off ), and K d Determine the equilibrium dissociation constant (K d ) is k off / k on The term "kon" refers to the rate constant for antibody binding to an antigen, and the term "koff" refers to the rate constant for antibody dissociation from the antibody / antigen complex.

[0039] The term "binding" to an antigen or epitope is a term well understood in the art, and methods for determining such binding are also well known in the art. A molecule is said to exhibit "binding" if it reacts with, associates with, or has affinity for a particular cell or substance, and the reaction, association, or affinity is detectable, for example, by one or more methods known in the art, such as immunoblot, ELISA KD, KinEx A, biolayer interferometry (BLI), surface plasmon resonance, etc.

[0040] "Surface plasmon resonance" can be used, for example, in BIAcore TMThe system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, NJ) demonstrates an optical phenomenon that allows for the analysis of real-time biospecific interactions by detecting changes in protein concentration within a biosensor matrix. For further description, see Jonsson et al. (1993) Ann. Biol. Clin. 51:19-26.

[0041] "Biolayer interferometry" refers to an optical analysis technique that analyzes the interference pattern of light reflected from a layer of immobilized proteins on a biosensor tip and an internal reference layer. Changes in the number of molecules bound to the biosensor tip cause shifts in the interference pattern that can be measured in real time. A non-limiting exemplary device for biolayer interferometry is the Octet® system (Pall ForteBio LLC). See, e.g., Abdiche et al., 2008, Anal. Biochem. 377:209-277.

[0042] In some embodiments, the anti-IL31 antibody has a titer of 5×10 as measured by biolayer interferometry. -6 Less than M, 1 x 10 -6 Less than M, 5 x 10 -7 Less than M, 1 x 10 -7 Less than M, 5 x 10 -8 Less than M, 1 x 10 -8 Less than M, 5 x 10 -9 Less than M, 1 x 10 -9 Less than M, 5 x 10 -10 Less than M, 1 x 10 -10 Less than M, 5 x 10 -11 Less than M, 1 x 10 -11 Less than M, 5 x 10 -12 Less than M or 1 x 10 -12 In some embodiments, the anti-IL31 antibody binds to canine IL31 or feline IL31 with a dissociation constant (Kd) of less than 5×10 M as measured by biolayer interferometry. -6 M and 1×10 -6 Between M, 5 x 10 -6 M and 5x10-7 Between M, 5 x 10 -6 M and 1×10 -7 Between M, 5 x 10 -6 M and 5x10 -8 Between M, 5 x 10 -6 M and 1×10 -8 Between M, 5 x 10 -6 M and 5x10 -9 Between M, 5 x 10 -6 M and 1×10 -9 Between M, 5 x 10 -6 M and 5x10 -10 Between M, 5 x 10 -6 M and 1×10 -10 Between M, 5 x 10 -6 M and 5x10 -11 Between M, 5 x 10 -6 M and 1×10 -11 Between M, 5 x 10 -6 M and 5x10 -12 Between M, 5 x 10 -6 M and 1×10 -12 Between M, 1 x 10 -6 M and 5x10 -7 Between M, 1 x 10 -6 M and 1×10 -7 Between M, 1 x 10 -6 M and 5x10 -8 Between M, 1 x 10 -6 M and 1×10 -8 Between M, 1 x 10 -6 M and 5x10 -9 Between M, 1 x 10 -6 M and 1×10 -9 Between M, 1 x 10 -6 M and 5x10 -10 Between M, 1 x 10 -6 M and 1×10 -10 Between M, 1 x 10 -6 M and 5x10 -11 Between M, 1 x 10 -6 M and 1×10 -11 Between M, 1 x 10 -6 M and 5x10 -12 Between M, 1 x 10 -6 M and 1×10 -12 Between M, 5 x 10 -7 M and 1×10 -7 Between M, 5 x 10 -7 M and 5x10-8 Between M, 5 x 10 -7 M and 1×10 -8 Between M, 5 x 10 -7 M and 5x10 -9 Between M, 5 x 10 -7 M and 1×10 -9 Between M, 5 x 10 -7 M and 5x10 -10 Between M, 5 x 10 -7 M and 1×10 -10 Between M, 5 x 10 -7 M and 5x10 -11 Between M, 5 x 10 -7 M and 1×10 -11 Between M, 5 x 10 -7 M and 5x10 -12 Between M, 5 x 10 -7 M and 1×10 -12 Between M, 1 x 10 -7 M and 5x10 -8 Between M, 1 x 10 -7 M and 1×10 -8 Between M, 1 x 10 -7 M and 5x10 -9 Between M, 1 x 10 -7 M and 1×10 -9 Between M, 1 x 10 -7 M and 5x10 -10 Between M, 1 x 10 -7 M and 1×10 -10 Between M, 1 x 10 -7 M and 5x10 -11 Between M, 1 x 10 -7 M and 1×10 -11 Between M, 1 x 10 -7 M and 5x10 -12 Between M, 1 x 10 -7 M and 1×10 -12 Between M, 5 x 10 -8 M and 1×10 -8 Between M, 5 x 10 -8 M and 5x10 -9 Between M, 5 x 10 -8 M and 1×10 -9 Between M, 5 x 10 -8 M and 5x10 -10 Between M, 5 x 10 -8 M and 1×10 -10 Between M, 5 x 10 -8 M and 5x10-11 Between M, 5 x 10 -8 M and 1×10 -11 Between M, 5 x 10 -8 M and 5x10 -12 Between M, 5 x 10 -8 M and 1×10 -12 Between M, 1 x 10 -8 M and 5x10 -9 Between M, 1 x 10 -8 M and 1×10 -9 Between M, 1 x 10 -8 M and 5x10 -10 Between M, 1 x 10 -8 M and 1×10 -10 Between M, 1 x 10 -8 M and 5x10 -11 Between M, 1 x 10 -8 M and 1×10 -11 Between M, 1 x 10 -8 M and 5x10 -12 Between M, 1 x 10 -8 M and 1×10 -12 Between M, 5 x 10 -9 M and 1×10 -9 Between M, 5 x 10 -9 M and 5x10 -10 Between M, 5 x 10 -9 M and 1×10 -10 Between M, 5 x 10 -9 M and 5x10 -11 Between M, 5 x 10 -9 M and 1×10 -11 Between M, 5 x 10 -9 M and 5x10 -12 Between M, 5 x 10 -9 M and 1×10 -12 Between M, 1 x 10 -9 M and 5x10 -10 Between M, 1 x 10 -9 M and 1×10 -10 Between M, 1 x 10 -9 M and 5x10 -11 Between M, 1 x 10 -9 M and 1×10 -11 Between M, 1 x 10 -9 M and 5x10 -12 Between M, 1 x 10 -9 M and 1×10 -12 Between M, 5 x 10 -10 M and 1×10-10 Between M, 5 x 10 -10 M and 5x10 -11 Between M, 1 x 10 -10 M and 5x10 -11 Between M, 1 x 10 -10 M and 1×10 -11 Between M, 1 x 10 -10 M and 5x10 -12 Between M, 1 x 10 -10 M and 1×10 -12 Between M, 5 x 10 -11 M and 1×10 -12 Between M, 5 x 10 -11 M and 5x10 -12 Between M, 5 x 10 -11 M and 1×10 -12 Between M, 1 x 10 -11 M and 5x10 -12 Between M or 1 x 10 -11 M and 1×10 -12 M. In some embodiments, the anti-IL31 antibody binds to canine IL31 or feline IL31 as determined by immunoblot analysis.

[0043] In some embodiments, the anti-IL31 antibody does not bind to human IL31 as determined by immunoblot analysis and / or biolayer interferometry.

[0044] In some embodiments, anti-IL31 antibodies are provided that compete with M14 for binding to IL31.

[0045] By "variant" is meant a biologically active polypeptide having at least about 50% amino acid sequence identity with a native sequence polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues have been added or deleted at the N- or C-terminus of the polypeptide.

[0046] In some embodiments, variants have at least about 50% amino acid sequence identity, at least about 60% amino acid sequence identity, at least about 65% amino acid sequence identity, at least about 70% amino acid sequence identity, at least about 75% amino acid sequence identity, at least about 80% amino acid sequence identity, at least about 85% amino acid sequence identity, at least about 90% amino acid sequence identity, at least about 95% amino acid sequence identity, at least about 97% amino acid sequence identity, at least about 98% amino acid sequence identity, or at least about 99% amino acid sequence identity to a native sequence polypeptide.

[0047] In some embodiments, the anti-IL31 antibody is a) (i) a variable heavy chain sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; (ii) a variable light chain sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; or (iii) a variable heavy chain sequence such as (i) and a variable light chain sequence such as (ii); or b) (i) a variable heavy chain sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:90; (ii) a variable light chain sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54; or (iii) a variable heavy chain sequence such as (i) and a variable light chain sequence such as (ii). Includes.

[0048] As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide, or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be performed using, for example, BLAST, BLAST-2, ALIGN, or MEGALINE. TM This can be achieved in a variety of ways within the skill of the art using publicly available computer software, such as (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0049] Amino acid substitutions can include, but are not limited to, replacing one amino acid in a polypeptide with another. Exemplary substitutions are shown in Table 2. Amino acid substitutions can be introduced into an antibody of interest and the products screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0050] JPEG0007671275000011.jpg174152

[0051] Amino acids can be grouped according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) Residues that influence chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0052] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0053] The term "vector" is used to describe a polynucleotide that can be manipulated to contain a cloned polynucleotide or a polynucleotide that can be propagated in a host cell. A vector may contain one or more of the following elements: an origin of replication, one or more control sequences (such as a promoter or enhancer) that control the expression of a polypeptide of interest, or one or more selectable marker genes (such as antibiotic resistance genes and genes that can be used in colorimetric assays, e.g., β-galactosidase). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0054] A "host cell" refers to a cell that can be or has been the recipient of a vector or isolated polynucleotide. Host cells can be prokaryotic or eukaryotic. Exemplary eukaryotic cells include mammalian cells, e.g., primate or non-primate animal cells; fungal cells, e.g., yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), 293 cells, and CHO cells, as well as their derivatives, e.g., 293-6E, DG44, CHO-S, and CHO-K cells. A host cell includes the progeny of a single host cell; the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide encoding the amino acid sequences provided herein.

[0055] As used herein, the term "isolated" refers to a molecule that has been separated from at least some of the components in which it is typically found or produced in nature. For example, a polypeptide is referred to as "isolated" if it is separated from at least some of the components of the cell in which it is produced. If a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell in which it was produced is considered to "isolate" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" if it is not part of a larger polynucleotide in which it is typically found in nature (e.g., genomic DNA or mitochondrial DNA in the case of a DNA polynucleotide) or, for example, if it is an RNA polynucleotide, if it is separated from at least some of the components of the cell in which it was produced. Thus, a DNA polynucleotide contained in a vector within a host cell may be referred to as "isolated." In some embodiments, the anti-IL31 antibody is purified using chromatography, such as size exclusion chromatography, ion exchange chromatography, Protein A column chromatography, hydrophobic interaction chromatography, and CHT chromatography.

[0056] The term "companion animal species" refers to animals suitable for serving as companions to humans. In some embodiments, companion animal species are small mammals such as canines, felines, dogs, cats, horses, rabbits, ferrets, guinea pigs, rodents, etc. In some embodiments, companion animal species are domestic animals such as horses, cows, pigs, etc.

[0057] The term "IL31 signaling function" refers to any one or a combination of downstream activities that occur when IL31 binds to its receptor or receptor complex. In some embodiments, IL31 signaling function includes activation of Janus kinase (Jak) 1 or Jak2 signaling molecules. In some embodiments, IL31 signaling function includes phosphorylation of STAT-3 or STAT-5 proteins. In some embodiments, IL31 signaling function includes activation of the ERK1 / 2 MAP kinase signaling pathway. In some embodiments, IL31 signaling function includes activation of the PI3K / AKT signaling pathway. In some embodiments, IL31 signaling function includes activation of the Jak1 / 2 signaling pathway.

[0058] "STAT phosphorylation" refers to the post-expression modification of STAT proteins by phosphorylation. For example, "STAT-3 phosphorylation" refers to the phosphorylation of STAT-3, and "STAT-5 phosphorylation" refers to the phosphorylation of STAT-5. In some embodiments, STAT-3 phosphorylation is measured by immunoblot analysis.

[0059] For example, cells (e.g., canine monocytic DH82 cells, or mammalian cells (e.g., HeLa cells) transfected with canine and / or feline IL31Ra) are plated at 1 x 10 cells per well in growth medium (e.g., MEM, Life Technologies®) containing 15% heat-inactivated fetal bovine serum, 2 mmol / L GlutaMax, 1 mmol / L sodium pyruvate, and 10 nM canine interferon-c (R&D Systems, Minneapolis, MN, USA) at 37°C for 24 hours in the presence of an anti-IL31 antibody described herein. 5Cells are plated into 96-well cell culture plates at a density of 1 / 3. Immunoblot analysis of cell lysates using anti-phospho-STAT-3, anti-STAT-3, anti-phospho-STAT-1, anti-STAT-1, anti-phospho-STAT-5, or anti-STAT-5 antibodies (R&D Systems) can be used to detect the concentrations of phosphorylated and non-phosphorylated STAT proteins relative to each other and compared with a beta-actin control. Methods for qualitatively or quantitatively determining protein concentrations by immunoblot are understood by those of skill in the art. In some embodiments, relative concentrations are determined qualitatively by visual inspection of the immunoblot. In some embodiments, the concentrations of phosphorylated and non-phosphorylated STAT proteins are determined quantitatively by digitally imaging the immunoblot, determining the intensity of the bands, and back-calculating the concentration of phosphorylated or non-phosphorylated STAT protein in the sample using a linear standard curve of known concentrations of STAT protein.

[0060] "Decrease" or "inhibit" means to decrease, reduce, or stop an activity, function, or amount compared to a reference. In some embodiments, "decrease" or "inhibit" refers to the ability to cause an overall decrease of 20% or more. In some embodiments, "decrease" or "inhibit" refers to the ability to cause an overall decrease of 50% or more. In some embodiments, "decrease" or "inhibit" refers to the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or more. In some embodiments, the amount is inhibited or reduced over a period of time compared to a control dose (e.g., a placebo) over the same period of time. As used herein, "reference" refers to any sample, standard, or level used for comparison purposes. A reference can be obtained from a healthy or non-diseased sample. In some instances, a reference is obtained from a non-diseased or untreated sample of a companion animal. In some instances, a reference is obtained from one or more healthy animals of a particular species that are not the animal being tested or treated.

[0061] As used herein, the term "substantially reduced" refers to a sufficiently high degree of reduction between a numerical value and a reference numerical value such that one of skill in the art would consider the difference between the two values ​​to be statistically significant in the context of the biological characteristic measured by the values. In some embodiments, a substantially reduced numerical value is reduced by more than approximately any one of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the reference value.

[0062] In some embodiments, the IL31 antibody can reduce IL31 signaling function in a companion animal species by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to IL31 signaling function in the absence of the antibody, as measured by a reduction in STAT-3 phosphorylation.In some embodiments, the reduction in IL31 signaling function or reduction in STAT-3 phosphorylation is between 10% and 15%, between 10% and 20%, between 10% and 25%, between 10% and 30%, between 10% and 35%, between 10% and 40%, between 10% and 45%, between 10% and 50%, between 10% and 60%, between 10% and 70%, between 10% and 80%, between 10% and 90%, between 10% and 100%, between 15% and 20%, between 15% and 25%, between 15% and 30%, between 15% and 35%, between 15% and 40%, between 15% and 45%, or between 15% and 50%. Between 15% and 60%, Between 15% and 70%, Between 15% and 80%, Between 15% and 90%, Between 15% and 100%, Between 20% and 25%, Between 20% and 30%, Between 20% and 35%, Between 20% and 40%, Between 20% and 45%, Between 20% and 50%, Between 20% and 60%, Between 20% and 70%, Between 20% and 80%, Between 20% and 90%, Between 20% and 100%, Between 25% and 30%, Between 25% and 35%, Between 25% and 40%, Between 25% and 45%, Between 25% and 50%, Between 25% and 60%, Between 25% and 70%, Between 25% and 80% , between 25% and 90%, between 25% and 100%, between 30% and 35%, between 30% and 40%, between 30% and 45%, between 30% and 50%, between 30% and 60%, between 30% and 70%, between 30% and 80%, between 30% and 90%, between 30% and 100%, between 35% and 40%, between 35% and 45%, between 35% and 50%, between 35% and 60%, between 35% and 70%, between 35% and 80%, between 35% and 90%, between 35% and 100%, between 40% and 45%, between 40% and 50%, between 40% and 60%, between 40% and 70%, between 40% and 80% , between 40% and 90%, between 40% and 100%, between 45% and 50%, between 45% and 60%, between 45% and 70%, between 45% and 80%, between 45% and 90%, between 45% and 100%, between 50% and 60%, between 50% and 70%, between 50% and 80%, between 50% and 90%, between 50% and 100%, between 60% and 70%, between 60% and 80%, between 60% and 90%, between 60% and 100%, between 70% and 80%, between 70% and 90%, between 70% and 100%, between 80% and 90%, between 80% and 100%, or between 90% and 100%.

[0063] Exemplary Pharmaceutical Compositions The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0064] A "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier common in the art for use with therapeutic agents that together comprise a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation with which it is used. Examples of pharmaceutically acceptable carriers include alumina; aluminum stearate; lecithin; serum proteins such as human serum albumin, canine or other animal albumin; buffers such as phosphate, citrate, tromethamine or HEPES buffers; glycine; sorbic acid; potassium sorbate; partial glyceride mixtures of saturated vegetable fatty acids; water; salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, or magnesium trisilicate; polyvinylpyrrolidone, cellulose-based materials; polyethylene glycol; sucrose; mannitol; or amino acids including, but not limited to, arginine.

[0065] The pharmaceutical composition can be stored in lyophilized form. Thus, in some embodiments, the preparation method includes a lyophilization step. The lyophilized composition can then be re-formulated prior to administration to dogs, cats, or horses, typically as an aqueous composition suitable for parenteral administration. In other embodiments, particularly when the antibody is highly stable to thermal and oxidative denaturation, the pharmaceutical composition can be stored as a liquid, i.e., an aqueous composition, that can be administered directly or after appropriate dilution to dogs, cats, or horses. The lyophilized composition can be reconstituted with sterile water for injection (WFI). An antimicrobial agent (e.g., a bacteriostatic agent such as benzyl alcohol) may be included. Thus, the present invention provides pharmaceutical compositions in solid or liquid form.

[0066] The pH of the pharmaceutical composition when administered can range from about pH 5 to about pH 8. The compositions of the present invention are sterile when used for therapeutic purposes. Sterility can be achieved by any of several means known in the art, including filtration through a sterile filtration membrane (e.g., a 0.2 micron membrane). Sterility can be maintained with or without antibacterial agents.

[0067] Exemplary Uses of Antibodies and Pharmaceutical Compositions The antibodies of the present invention or pharmaceutical compositions comprising the antibodies may be useful for treating IL31-induced conditions. As used herein, "IL31-induced conditions" refers to diseases associated with, caused by, or characterized by elevated levels or altered gradients of IL31 concentration. Such IL31-induced conditions include, but are not limited to, pruritic or allergic diseases. In some embodiments, the IL31-induced condition is atopic dermatitis, allergic dermatitis, pruritus, asthma, psoriasis, scleroderma, or eczema. The IL31-induced conditions may be observed in companion animals, including, but not limited to, dogs or cats.

[0068] As used herein, "treatment" is an approach to obtain beneficial or desired clinical results. As used herein, "treatment" includes any administration or application of a therapeutic agent for a disease in a mammal, including a companion animal. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, reduction in the extent of the disease, prevention or slowing of disease spread, prevention or slowing of disease recurrence, slowing or slowing of disease progression, amelioration of the condition, inhibition of the disease or disease progression, inhibition or slowing of the disease or its progression, arrest of its occurrence, and remission (partial or complete). Also encompassed by "treatment" is the reduction of the pathological consequences of a proliferative disease. The methods provided herein contemplate any one or more of these aspects of treatment. Consistent with the above, the term treatment does not require 100% elimination of all aspects of the disorder.

[0069] In some embodiments, an anti-IL31 antibody or a pharmaceutical composition comprising the same can be used in accordance with the methods herein to treat an IL31-induced condition, hi some embodiments, an anti-IL31 antibody or pharmaceutical composition is administered to a companion animal, such as a dog or cat, to treat an IL31-induced condition.

[0070] A "therapeutically effective amount" of a substance / molecule, agonist, or antagonist can vary depending on factors such as the type of disease being treated, the disease state, the severity and course of the disease, the type of therapeutic objective, previous treatments, clinical history, response to previous treatments, the discretion of the attending physician, the age, sex, and weight of the animal, and the ability of the substance / molecule, agonist, or antagonist to elicit a desired response in the animal. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the substance / molecule, agonist, or antagonist are outweighed by the therapeutically beneficial effects. A therapeutically effective amount can be delivered in one or more administrations. A therapeutically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0071] In some embodiments, the anti-IL31 antibody or pharmaceutical composition comprising an anti-IL31 antibody is administered parenterally by subcutaneous administration, intravenous infusion, or intramuscular injection. In some embodiments, the anti-IL31 antibody or pharmaceutical composition comprising an anti-IL31 antibody is administered as a bolus injection or by continuous infusion over a predetermined period of time. In some embodiments, the anti-IL31 antibody or pharmaceutical composition comprising an anti-IL31 antibody is administered intramuscularly, intraperitoneally, intracerebrospinally, subcutaneously, intraarterially, intrasynovially, intrathecally, or by inhalation.

[0072] The anti-IL31 antibodies described herein may be administered in an amount ranging from 0.01 mg / kg to 100 mg / kg of body weight per dose. In some embodiments, the anti-IL31 antibodies may be administered in an amount ranging from 0.5 mg / kg to 50 mg / kg of body weight per dose. In some embodiments, the anti-IL31 antibodies may be administered in an amount ranging from 0.1 mg / kg to 10 mg / kg of body weight per dose. In some embodiments, the anti-IL31 antibodies may be administered in an amount ranging from 0.1 mg / kg to 100 mg / kg of body weight per dose. In some embodiments, the anti-IL31 antibodies may be administered in an amount ranging from 1 mg / kg to 10 mg / kg of body weight per dose. In some embodiments, the anti-IL31 antibody may be administered in an amount per dose in the range of 0.5 mg / kg to 100 mg / kg body weight, 1 mg / kg to 100 mg / kg body weight, 5 mg / kg to 100 mg / kg body weight, 10 mg / kg to 100 mg / kg body weight, 20 mg / kg to 100 mg / kg body weight, 50 mg / kg to 100 mg / kg body weight, 1 mg / kg to 10 mg / kg body weight, 5 mg / kg to 10 mg / kg body weight, 0.5 mg / kg to 10 mg / kg body weight, 0.01 mg / kg to 0.5 mg / kg body weight, 0.01 mg / kg to 0.1 mg / kg body weight, or 5 mg / kg to 50 mg / kg body weight.

[0073] The anti-IL31 antibody or pharmaceutical composition comprising an anti-IL31 antibody can be administered to the companion animal at one time or over a series of treatments. For example, the anti-IL31 antibody or pharmaceutical composition comprising an anti-IL31 antibody can be administered at least once, more than once, at least two times, at least three times, at least four times, or at least five times.

[0074] In some embodiments, the dose is administered once per week for at least two or three consecutive weeks, and in some embodiments, this treatment cycle is repeated two or more times, optionally interspersed with one or more week-long treatment-free periods. In other embodiments, the therapeutically effective dose is administered once per day for two to five consecutive days, and in some embodiments, this treatment cycle is repeated two or more times, optionally interspersed with one or more day- or week-long treatment-free periods.

[0075] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) and sequential or sequential administration in any order. The term "in combination" is used herein to refer to the administration of two or more therapeutic agents, where at least a portion of the administration overlaps in time, or where the administration of one therapeutic agent is within a short time period relative to the administration of another therapeutic agent. For example, two or more therapeutic agents are administered at a time interval of about a specified number of minutes or less. The term "sequentially" is used herein to refer to the administration of two or more therapeutic agents, where the administration of one or more agents continues after the administration of one or more other agents is discontinued, or where the administration of one or more agents is initiated before the administration of one or more other agents. For example, the administration of two or more therapeutic agents is separated in time by more than about a specified number of minutes. As used herein, "in conjunction with" refers to the administration of one therapeutic modality in addition to another therapeutic modality. Thus, "in conjunction with" refers to the administration of one therapeutic modality before, during, or after the administration of another therapeutic modality to an animal.

[0076] In some embodiments, the method comprises administering a Jak inhibitor, a PI3K inhibitor, an AKT inhibitor, or a MAPK inhibitor in combination with an anti-IL31 antibody or a pharmaceutical composition comprising an anti-IL31 antibody. In some embodiments, the method comprises administering an anti-IL17 antibody, an anti-TNFα antibody, an anti-CD20 antibody, an anti-CD19 antibody, an anti-CD25 antibody, an anti-IL4 antibody, an anti-IL13 antibody, an anti-IL23 antibody, an anti-IgE antibody, an anti-CD11α antibody, an anti-IL6R antibody, an anti-α4-integrin antibody, an anti-IL12 antibody, an anti-IL1β antibody, or an anti-BlyS antibody in combination with an anti-IL31 antibody or a pharmaceutical composition comprising an anti-IL31 antibody.

[0077] Provided herein are methods of exposing cells to an anti-IL31 antibody or a pharmaceutical composition comprising an anti-IL31 antibody under conditions that allow binding of the antibody to IL31. In some embodiments, the cells are exposed to the antibody or pharmaceutical composition ex vivo. In some embodiments, the cells are exposed to the antibody or pharmaceutical composition in vivo. In some embodiments, the cells are exposed to the anti-IL31 antibody or pharmaceutical composition under conditions that allow binding of the antibody to intracellular IL31. In some embodiments, the cells are exposed to the anti-IL31 antibody or pharmaceutical composition under conditions that allow binding of the antibody to extracellular IL31. In some embodiments, the cells can be exposed to the anti-IL31 antibody or pharmaceutical composition in vivo by any one or more of the administration methods described herein, including, but not limited to, intraperitoneal, intramuscular, or intravenous injection into a subject. In some embodiments, the cells can be exposed to the anti-IL31 antibody or pharmaceutical composition ex vivo by exposing the cells to culture medium containing the antibody or pharmaceutical composition. In some embodiments, the permeability of the cell membrane can be affected by using any number of methods understood by one of skill in the art (e.g., electroporating the cells or exposing the cells to a solution containing calcium chloride) prior to exposing the cells to culture medium containing the antibody or pharmaceutical composition.

[0078] In some embodiments, binding results in a reduction in IL31 signaling function by the cell. In some embodiments, the anti-IL31 antibody can reduce IL31 signaling function in the cell by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to IL31 signaling function in the absence of the antibody, as measured by a reduction in STAT-3 phosphorylation.In some embodiments, the reduction in IL31 signaling function or reduction in STAT-3 phosphorylation is between 10% and 15%, between 10% and 20%, between 10% and 25%, between 10% and 30%, between 10% and 35%, between 10% and 40%, between 10% and 45%, between 10% and 50%, between 10% and 60%, between 10% and 70%, between 10% and 80%, between 10% and 90%, between 10% and 100%, between 15% and 20%, between 15% and 25%, between 15% and 30%, between 15% and 35%, between 15% and 40%, between 15% and 45%, or between 15% and 50%. Between 15% and 60%, Between 15% and 70%, Between 15% and 80%, Between 15% and 90%, Between 15% and 100%, Between 20% and 25%, Between 20% and 30%, Between 20% and 35%, Between 20% and 40%, Between 20% and 45%, Between 20% and 50%, Between 20% and 60%, Between 20% and 70%, Between 20% and 80%, Between 20% and 90%, Between 20% and 100%, Between 25% and 30%, Between 25% and 35%, Between 25% and 40%, Between 25% and 45%, Between 25% and 50%, Between 25% and 60%, Between 25% and 70%, Between 25% and 80% , between 25% and 90%, between 25% and 100%, between 30% and 35%, between 30% and 40%, between 30% and 45%, between 30% and 50%, between 30% and 60%, between 30% and 70%, between 30% and 80%, between 30% and 90%, between 30% and 100%, between 35% and 40%, between 35% and 45%, between 35% and 50%, between 35% and 60%, between 35% and 70%, between 35% and 80%, between 35% and 90%, between 35% and 100%, between 40% and 45%, between 40% and 50%, between 40% and 60%, between 40% and 70%, between 40% and 80% , between 40% and 90%, between 40% and 100%, between 45% and 50%, between 45% and 60%, between 45% and 70%, between 45% and 80%, between 45% and 90%, between 45% and 100%, between 50% and 60%, between 50% and 70%, between 50% and 80%, between 50% and 90%, between 50% and 100%, between 60% and 70%, between 60% and 80%, between 60% and 90%, between 60% and 100%, between 70% and 80%, between 70% and 90%, between 70% and 100%, between 80% and 90%, between 80% and 100%, or between 90% and 100%.

[0079] Provided herein are methods of using anti-IL31 antibodies, polypeptides, and polynucleotides for the detection, diagnosis, and monitoring of anti-IL31-induced conditions. Provided herein are methods of determining whether a companion animal will respond to anti-IL31 antibody therapy. In some embodiments, the method comprises using an anti-IL31 antibody to detect whether the animal has cells that express IL31. In some embodiments, the method of detection comprises contacting a sample with the antibody, polypeptide, or polynucleotide and determining whether the level of binding differs from that of a reference or comparative sample (e.g., a control). In some embodiments, the method may be useful for determining whether an antibody or polypeptide described herein is an appropriate therapeutic agent for a subject animal.

[0080] In some embodiments, the sample is a biological sample. The term "biological sample" refers to a quantity of material from a living organism or formerly living organism. In some embodiments, the biological sample is a cell or cell / tissue lysate. In some embodiments, biological samples include, but are not limited to, blood (e.g., whole blood), plasma, serum, urine, synovial fluid, and epithelial cells.

[0081] In some embodiments, cells or cell / tissue lysates are contacted with an anti-IL31 antibody and binding between the antibody and the cells is determined. If the test cells exhibit binding activity compared to reference cells of the same tissue type, it may indicate that the subject would benefit from treatment with an anti-IL31 antibody. In some embodiments, the test cells are from a tissue of a companion animal.

[0082] Various methods known in the art for detecting specific antibody-antigen binding can be used. Exemplary immunoassays that can be performed include fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), turbidimetric inhibition immunoassay (NIA), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA). Indicator moieties, or label groups, can be attached to the antibody of interest and are selected to meet the needs of the various methods used, which are often determined by the availability of assay equipment and compatible immunoassay procedures. Suitable labels include, but are not limited to, radionuclides (e.g., 125 I, 131 I, 35 S, 3 H, or 32 P), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or p-lactosidase), fluorescent moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g., Qdot supplied by Quantum Dot Corporation, Palo Alto, Calif.). TM General techniques to be used in performing the various immunoassays noted above are known to those of skill in the art.

[0083] For diagnostic purposes, polypeptides, including antibodies, can be labeled with a detectable moiety, including but not limited to radioisotopes, fluorescent labels, and various enzyme-substrate labels known in the art. Methods for conjugating labels to antibodies are known in the art. In some embodiments, the anti-IL31 antibody need not be labeled, and its presence can be detected using a second, labeled antibody that binds to the first anti-IL31 antibody. In some embodiments, the anti-IL31 antibody can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987). Anti-IL31 antibodies and polypeptides can also be used in in vivo diagnostic assays, such as in vivo imaging. Generally, the antibody or polypeptide is conjugated to a radionuclide (e.g., a nucleotide sequence encoding a nucleotide sequence) so that the location of cells or tissues of interest can be identified using immunoscintigraphy. 111 In, 99 Tc, 14 C. 131 I, 125 I, 3 H, or any other radionuclide label, including those outlined herein. The antibodies may also be used as staining reagents in pathology using techniques well known in the art.

[0084] In some embodiments, the first antibody is used for diagnosis and the second antibody is used as a therapeutic agent. In some embodiments, the first antibody and the second antibody are different. In some embodiments, the first antibody and the second antibody bind to different epitopes, thereby allowing both to bind to the antigen simultaneously.

[0085] The following examples illustrate certain aspects of the disclosure and are not intended to limit the disclosure in any way. [Example]

[0086] Example 1: In vitro affinity maturation to enhance binding to IL31 The variable heavy (VH) and variable light (VL) chain sequences of the murine monoclonal antibody M14, raised against IL-31, were identified (SEQ ID NOs: 1 and 2). See WO 2018 / 156367, incorporated herein by reference in its entirety for all purposes. The M14 VH and VL sequences were caninized (SEQ ID NOs: 3 and 4) by searching and selecting suitable canine germline antibody sequences as templates for CDR grafting, followed by protein modeling.

[0087] A phage library of variant Fab polypeptides with mutations in and around the CDRs of the caninized M14 VH and VL sequences (SEQ ID NOs: 3 and 4) was prepared and demonstrated to have slower kDa expression by canine IL31 dissociation assay. off After three rounds of panning the phage library against canine IL31, phage colonies expressing variant Fab polypeptides with potentially enhanced canine IL31 binding were identified and the polypeptides were sequenced.

[0088] A single E. coli colony expressing each of the identified variant Fab polypeptides containing a SASA tag was cultured and induced to express the polypeptide. Cell culture medium containing the variant Fab polypeptide was exposed to BSA immobilized on either a plate or a Biacore chip. The plate or chip to which the variant Fab polypeptide was bound was exposed to soluble canine IL-31 to measure the slow kappa activity. off Screened for speed.

[0089] Three lead caninized mature variable heavy chain polypeptides (cmVH1 (SEQ ID NO: 5), cmVH2 (SEQ ID NO: 6), and cmVH3 (SEQ ID NO: 7)) and three caninized mature light chain polypeptides (cmVL1 (SEQ ID NO: 8), cmVL2 (SEQ ID NO: 9), and cmVL3 (SEQ ID NO: 10)) were selected. Exemplary CDR sequences of the lead caninized mature variable chains are represented by SEQ ID NOs: 11, 12, 13, 14, 15, 20, 21, 22, 23, and 24, and framework region sequences are represented by SEQ ID NOs: 16, 17, 18, 19, 25, 26, 27, and 28.

[0090] Chimeric antibodies were generated consisting of different combinations of cmVH1, cmVH2, or cmVH3 fused to a human IgG1 heavy chain and cmVL1, cmVL2, and cmVL3 fused to a human kappa constant light chain. A caninized VH of M14 (SEQ ID NO: 3) fused to a human IgG1 heavy chain and a caninized VL of M14 (SEQ ID NO: 4) fused to a human kappa constant light chain were also generated. The affinity of canine IL31 to the different chimeric antibodies was measured using BIAcore 8K, as described in Table 3 below:

[0091] JPEG0007671275000012.jpg99168

[0092] The affinity and kinetics of each chimeric antibody tested for canine IL31 are summarized in Table 4 below. Each of the affinity matured antibodies tested (analytes 2-6) exhibited higher affinity (as evidenced by lower Kd values) and lower k (as evidenced by lower k) values ​​compared to the caninized VH-VL M14 antibody control (analyte 1). off The results showed slow off-rates (as evidenced by the .times. ...

[0093] JPEG0007671275000013.jpg46170

[0094] Example 2: Mutations with enhanced binding to feline IL31 The six chimeric antibodies tested for affinity to canine IL-31 in Tables 3 and 4 (above) were also tested for affinity to feline IL-31. Binding assays were performed using the biosensor OctetRed as follows. Briefly, feline IL-31 was biotinylated. Free, unreacted biotin was removed from the biotinylated IL-31 by extensive dialysis. Biotinylated feline IL-31 was captured onto a streptavidin sensor tip. Binding of the antibody (20 μg / mL) to feline IL-31 was monitored for 300 seconds. Dissociation was monitored for 300 seconds. The buffer for dilution and all binding steps was 20 mM phosphate, 150 mM NaCl, pH 7.2. The results of the binding assay are shown in Figure 2. The affinities of the antibodies tested were ranked as follows: (cmVH3 + cmVL2) or (cmVH3 + cmVL3) > (cmVH2 + cmVL3) > (cmVH2 + cmVL2) > (cmVH1 + cmVL1) > (caninized VH-VL M14).

[0095] Example 3: Caninized mature variable chain containing canine constant domains The caninized mature VH (e.g., SEQ ID NOs:5, 6, and 7) and VL (e.g., SEQ ID NOs:8, 9, and 10) can be fused to a canine IgG-A, IgG-B, IgG-C, or IgG-D heavy chain constant domain (e.g., SEQ ID NOs:83, 84, 85, and 86) and a canine kappa light chain constant domain (e.g., SEQ ID NO:87), respectively. Exemplary caninized mature heavy and light chain sequences with canine constant domains include SEQ ID NOs:34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, and 48.

[0096] Example 4: Felineization and expression of affinity-enhanced variable heavy and light chains Affinity-enhanced variable heavy and light chains (e.g., cmVH1-3 and cmVL1-3) can be felineized using methods understood in the art. For example, cmVH2 (SEQ ID NO: 6) was felineized to exemplary SEQ ID NO: 90; cmVH3 (SEQ ID NO: 7) was felineized to exemplary SEQ ID NOs: 49, 50, and 51; and cmVL3 (SEQ ID NO: 10) was felineized to any one of SEQ ID NOs: 52, 53, and 54. Felineized VH and VL with a feline IgG heavy chain constant domain (e.g., SEQ ID NO: 88) and a feline kappa light chain constant domain (e.g., SEQ ID NO: 89), respectively, can be expressed. Exemplary felineized mature heavy and light chain sequences with feline constant regions include SEQ ID NOs: 73, 74, 75, 91, 76, 77, and 78.

[0097] A DNA sequence encoding a felinized IL-31 antibody having a heavy chain (VH2) of SEQ ID NO: 91 and a light chain (VL3c) of SEQ ID NO: 78 was expressed in mammalian cells and purified. Using the cell-based assay of Example 5, the felinized VH2-VL3 IL31 antibody quantitatively blocked feline IL31-induced STAT3 phosphorylation in both feline IL31R- and canine IL31R-transfected HeLa cells.

[0098] Example 5: Development of a canine IL31 cell-based signaling assay The HeLa cell line, a human epithelial cell line (American Type Culture Collection (ATCC), catalog number CCL-2), was purchased and cultured in ATCC-formulated Eagle's minimum essential medium (ATCC, catalog number 30-2033) supplemented with 10% fetal bovine serum (FBS) (ATCC, catalog number 30-2020). HeLa cells stably transfected with canine or feline IL31Ra-FLAG expression plasmids (pcDNA3.1_canine IL31Ra_FLAG (SEQ ID NO: 92) or pcDNA3.1_feline IL31Ra-FLAG (SEQ ID NO: 93)) were generated using the Lipofectamine method (Thermo Fisher, catalog no. 11668027) and selected for G418-resistant transfectants (Thermo Fisher catalog no. 10131035) using G418 at a final concentration of 400 μg / mL. G418-resistant clones were screened for STAT1, STAT3, and STAT5 phosphorylation in response to canine IL31 or feline IL31 induction by Western blotting. HeLa clones stably transfected with IL31Ra in response to IL31 induction were used for subsequent studies.

[0099] Canine or feline IL31-mediated STAT protein phosphorylation was measured by seeding HeLa / IL31Ra cells at 10e5 cells per well in a 96-well plate and incubating overnight at 37°C and 5% CO2 (in 10% FBS D-MEM as recommended by ATCC). Serum starvation of cells was achieved by replacing the medium in each well with medium without FBS for 1 hour at 37°C and 5% CO2. Serially diluted anti-IL31 antibodies were preincubated with IL31 cytokine for 1 hour and then added to serum-starved cells in each well of the 96-well plate for 5 minutes at room temperature. Next, 20 μL of stop solution (Thermo Fisher M-PER, catalog no. 78501) was added to each well to lyse the cells. Cell lysates were resolved by SDS-PAGE (4-12% Bis-Tris gel, Invitrogen, catalog no. NP0329). IL31-induced STAT phosphorylation was assayed by Western blotting using either an anti-phospho-STAT3 antibody (RnD, Catalog No. AF4607), an anti-phospho-STAT1 antibody (Cell Signaling, Catalog No. 7649), or an anti-phospho-STAT5 antibody (Cell Signaling Catalog No. 9359).

[0100] Surprisingly, expression of the canine or feline co-receptor OSMR was not required for IL31 signaling.

Claims

1. An antibody that binds to canine IL31, a) a heavy chain comprising a CDR-H1 sequence having the amino acid sequence of SEQ ID NO: 11, a CDR-H2 sequence having the amino acid sequence of SEQ ID NO: 12, and a CDR-H3 sequence having the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15; and b) a light chain comprising a CDR-L1 sequence having the amino acid sequence of SEQ ID NO:20, a CDR-L2 sequence having the amino acid sequence of SEQ ID NO:21, and a CDR-L3 sequence having the amino acid sequence of SEQ ID NO:23 or SEQ ID NO:

24. An antibody comprising:

2. The antibody of claim 1, wherein the antibody comprises a variable light chain amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10 and a variable heavy chain amino acid sequence of SEQ ID NO:6 or SEQ ID NO:

7.

3. (a) The antibody was found to be 1×10 －９ binds to canine IL31 with a dissociation constant (Kd) of less than M; (b) the antibody is a monoclonal antibody; (c) the antibody is a caninized antibody; (d) the antibody comprises a canine heavy chain constant region selected from an IgG-A, IgG-B, IgG-C, and IgG-D constant region; (e) the antibody comprises: (i) a heavy chain amino acid sequence of SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, or SEQ ID NO:45; (ii) a light chain amino acid sequence of SEQ ID NO:47 or SEQ ID NO:

48. The antibody according to claim 1 or 2.

4. An isolated nucleic acid encoding an antibody according to any one of claims 1 to 3.

5. A host cell comprising the nucleic acid of claim 4.

6. A method for producing an antibody according to any one of claims 1 to 3, comprising culturing a host cell according to claim 5 and isolating the antibody.

7. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 3 and a pharma- ceutically acceptable carrier.

8. A composition for use in treating a dog having an IL31-induced condition, the treatment method comprising administering to the dog a therapeutically effective amount of an antibody described in any one of claims 1 to 3 or a pharmaceutical composition described in claim 7.

9. The species is dog, (a) the IL31-induced condition is a pruritic or allergic condition; (b) the IL31-induced condition is selected from atopic dermatitis, allergic dermatitis, pruritus, asthma, psoriasis, scleroderma and eczema; The composition of claim 8.

10. A method for reducing IL31 signaling function in a cell, comprising exposing the cell to an antibody described in any one of claims 1 to 3 or a pharmaceutical composition described in claim 7 under conditions that allow binding of the antibody to extracellular IL31, thereby reducing binding to the IL31 receptor and / or reducing IL31 signaling function by the cell, wherein the cell is exposed to the antibody or pharmaceutical composition ex vivo and the cell is a canine cell.

11. A composition for use in a method for reducing IL31 signaling function in a cell, the method comprising exposing the cell to an antibody described in any one of claims 1 to 3 or a pharmaceutical composition described in claim 7 under conditions that allow binding of the antibody to extracellular IL31, thereby reducing binding to the IL31 receptor and / or reducing IL31 signaling function by the cell, wherein the cell is exposed to the antibody or pharmaceutical composition in vivo and the cell is a canine cell.

12. A method for detecting IL31 in a sample from a dog, comprising contacting the sample with an antibody described in any one of claims 1 to 3 or a pharmaceutical composition described in claim 7 under conditions allowing binding of the antibody to IL31, and detecting whether a complex is formed between the antibody and IL31 in the sample.

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