Veterinary anti-IL-31 antibody

Abstract

To provide methods for treating IL31-induced conditions in companion animals, such as canines, felines, and equines and for reducing IL31 signaling functions in cells.SOLUTION: The present invention provides an isolated antibody that binds to canine IL31, wherein the antibody binds to an epitope comprising an amino acid sequence having a specific sequence.SELECTED DRAWING: None

Description

Technical Field

[0001] This application claims the benefit of International Application No. PCT / US2017 / 023788, filed Mar. 23, 2017, and U.S. Provisional Patent Application No. 62 / 463,543, filed Feb. 24, 2017, each of which is hereby incorporated by reference in its entirety for all purposes.

[0002] The present invention relates to, for example, isolated anti-IL31 antibodies that bind to canine IL31, and methods of using the same, such as methods of treating IL31-induced conditions in companion animals, such as dogs, cats, and horses, or reducing IL31 signaling function in cells.

Background Art

[0003] Interleukin 31 (IL31) is a cytokine produced mainly by Th2 cells and is understood to be involved in the promotion of skin diseases such as pruritus and other forms of allergic diseases (e.g., atopic dermatitis). IL31 binds to its receptor and has a function of activating downstream activities such as the activation of JAK1, and is thought to cause many of the clinical problems associated with dermatitis and other disorders.

[0004] Companion animals such as cats, dogs, and horses are affected by many of the same skin diseases as human skin diseases such as atopic dermatitis. However, the sequence of IL31 differs among humans, cats, dogs, and horses. Therefore, there is a need for methods and compounds that can be specifically used to bind to IL31 in companion animals in order to treat IL31-induced conditions and reduce IL31 signaling.

Summary of the Invention

[0005] In some embodiments, an isolated antibody that binds to canine IL31 is provided. In some embodiments, the antibody binds to an epitope comprising amino acids 34-50 of SEQ ID NO: 22. In some embodiments, the antibody binds to an epitope comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the antibody binds to an epitope comprising the amino acid sequence of PSDX1X2KI (SEQ ID NO: 45), where X is any amino acid residue. In some embodiments, X1 is a hydrophobic amino acid. In some embodiments, X1 is selected from A, V, I, and L. In some embodiments, X1 is selected from V and I. In some embodiments, X2 is a hydrophilic amino acid. In some embodiments, X2 is selected from A, R, K, Q, and N. In some embodiments, X2 is selected from R and Q. In some embodiments, X1 is V and X2 is R. In some embodiments, X1 is I and X2 is Q. In some embodiments, the antibody binds to an epitope comprising the amino acid sequence of SEQ ID NO: 88.

[0006] In some embodiments, the antibody has a density 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 It binds to canine IL31 with a dissociation constant (Kd) of less than M.

[0007] In some embodiments, the antibody reduces IL31 signaling function in the companion animal species as measured by reduced phosphorylation of STAT-3, hi some embodiments, the companion animal species is a dog, cat, or horse.

[0008] In some embodiments, the antibody binds to feline IL31 or equine IL31 as determined by immunoblot analysis and / or biolayer interferometry. In some embodiments, the antibody competes with monoclonal M14 antibody for binding to canine IL31. In some embodiments, the antibody competes with monoclonal M14 antibody for binding to feline IL31. In some embodiments, the antibody does not bind to human IL31 as determined by immunoblot analysis and / or biolayer interferometry.

[0009] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a canine, caninized, feline, felineized, equine, equine, or chimeric antibody. In some embodiments, the antibody is a chimeric antibody comprising a murine variable heavy chain framework region or a murine variable light chain framework region.

[0010] In some embodiments, the antibody comprises a heavy chain and a light chain: a. the heavy chain comprises a CDR-H1 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1, a CDR-H2 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 2, 62, 89, or 87, and a CDR-H3 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 3; b. The light chain comprises a CDR-L1 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 63, a CDR-L2 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 9, and a CDR-L3 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 10.

[0011] In some embodiments, the antibody comprises a heavy chain comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2 or 89, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3.

[0012] In some embodiments, the antibody comprises a heavy chain comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 62 or 87, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3.

[0013] In some embodiments, the antibody comprises a light chain comprising (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

[0014] In some embodiments, the antibody comprises a light chain comprising (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 63, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

[0015] In some embodiments, the antibody comprises one or more of (a) a variable region heavy chain framework 1 (HC-FR1) sequence of SEQ ID NO: 4, 70, or 79, (b) an HC-FR2 sequence of SEQ ID NO: 5, 71, or 80, (c) an HC-FR3 sequence of SEQ ID NO: 6, 72, 73, or 81, (d) an HC-FR4 sequence of SEQ ID NO: 7, 74, or 82, (e) a variable region light chain framework 1 (LC-FR1) sequence of SEQ ID NO: 11, 75, or 83, (f) an LC-FR2 sequence of SEQ ID NO: 12, 76, or 84, (g) an LC-FR3 sequence of SEQ ID NO: 13, 77, or 85, or (h) an LC-FR4 sequence of SEQ ID NO: 14, 78, or 86.

[0016] In some embodiments, the antibody is a. (i) a variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 24, (ii) a variable heavy chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 25, or (iii) the variable light chain sequence of (i) and the variable heavy chain sequence of (ii), or b. (i) a variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 16, (ii) a variable heavy chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 15, or (iii) the variable light chain sequence of (i) and the variable heavy chain sequence of (ii), or c. (i) a variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 32, (ii) a variable heavy chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 33, or (iii) the variable light chain sequence of (i) and the variable heavy chain sequence of (ii) and comprises.

[0017] In some embodiments, the antibody comprises a variable light chain sequence of SEQ ID NO: 24, SEQ ID NO: 16, or SEQ ID NO: 32. In some embodiments, the antibody comprises a variable heavy chain sequence of SEQ ID NO: 25, SEQ ID NO: 15, or SEQ ID NO: 33. In some embodiments, the antibody comprises a variable light chain sequence of SEQ ID NO: 24 and a variable heavy chain sequence of SEQ ID NO: 25, a variable light chain sequence of SEQ ID NO: 16 and a variable heavy chain sequence of SEQ ID NO: 15, or a variable light chain sequence of SEQ ID NO: 32 and a variable heavy chain sequence of SEQ ID NO: 33.

[0018] In some embodiments, the antibody is a chimeric antibody comprising a constant heavy chain region or a constant light chain region derived from a companion animal.

[0019] In some embodiments, the antibody comprises (a) a canine heavy chain constant region selected from the IgG-A, IgG-B, IgG-C, and IgG-D constant regions, (b) a feline heavy chain constant region selected from the IgG1, IgG2a, and IgG2b constant regions, or (c) a equine heavy chain constant region selected from the IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, and IgG7 constant regions.

[0020] In some embodiments, the antibody is a. (i) the light chain amino acid sequence of SEQ ID NO: 26, (ii) the heavy chain amino acid sequence of SEQ ID NO: 27, or (iii) the light chain amino acid sequence of (i) and the heavy chain amino acid sequence of (ii), or b. (i) the light chain amino acid sequence of SEQ ID NO: 30, (ii) the heavy chain amino acid sequence of SEQ ID NO: 31, or (iii) the light chain amino acid sequence of (i) and the heavy chain amino acid sequence of (ii), or c. (i) the light chain amino acid sequence of SEQ ID NO: 34, (ii) the heavy chain amino acid sequence of SEQ ID NO: 35, or (iii) the light chain amino acid sequence of (i) and the heavy chain amino acid sequence of (ii) and comprises.

[0021] In some embodiments, the antibody comprises the light chain amino acid sequence of SEQ ID NO: 21. In some embodiments, the antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.

[0022] In some embodiments, the antibody is an antibody fragment selected from Fv, scFv, Fab, Fab’, F(ab’)2, and Fab’-SH.

[0023] In some embodiments, 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-integrin, IL12, IL1β, or BlyS.

[0024] In some embodiments, there is provided an isolated nucleic acid encoding an anti-IL31 antibody as described above herein. In some embodiments, there is provided a host cell comprising a nucleic acid encoding an anti-IL31 antibody as described above herein. In some embodiments, there is provided a method for producing an anti-IL31 antibody, comprising culturing such a host cell comprising a nucleic acid encoding an anti-IL31 antibody as described above herein, and isolating the antibody.

[0025] In some embodiments, there is provided a pharmaceutical composition comprising an anti-IL31 antibody as described herein and a pharmaceutically acceptable carrier. In some embodiments, there is provided a pharmaceutical composition comprising an anti-IL31 antibody as described herein and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises L-histidine, sodium chloride, and polysorbate 80.

[0026] In some embodiments, the pharmaceutical composition has a pH of 5.0 to 6.2. In some embodiments, the pharmaceutical composition has a pH of 5.0 to 6.0, or 5.3 to 5.7, or 5.5.

[0027] In some embodiments, the pharmaceutical composition has an L-histidine concentration of 5 mM to 100 mM, 10 mM to 50 mM, 20 mM to 30 mM, 10 to 30 mM, or 20 mM.

[0028] In some embodiments, the pharmaceutical composition has a sodium chloride concentration of 80 - 200 mM, 100 - 175 mM, 120 - 150 mM, or 140 mM.

[0029] In some embodiments, the pharmaceutical composition has a concentration of polysorbate 80 of 0.005 mg / mL - 0.5 mg / mL, 0.01 mg / mL - 0.1 mg / mL, or 0.05 mg / mL.

[0030] In some embodiments, the pharmaceutically acceptable carrier comprises at least one sugar. In some embodiments, the pharmaceutical composition has a concentration of at least one sugar of 0.5% - 20%, 1% - 10%, 1% - 5%, or 1% - 3%. In some embodiments, the pharmaceutically acceptable carrier comprises sucrose, trehalose, D - mannitol, maltose, and / or sorbitol.

[0031] In some embodiments, the pharmaceutically acceptable carrier comprises an antibacterial agent. In some embodiments, the pharmaceutical composition comprises m - cresol or methylparaben. In some embodiments, the pharmaceutical composition comprises 0.2% m - cresol and / or 0.9% methylparaben.

[0032] Use of the antibody and the pharmaceutical composition In some embodiments, there is provided a method of treating a companion animal species having a condition induced by IL31, the method comprising administering to the companion animal species a therapeutically effective amount of the anti - IL31 antibody described herein or a pharmaceutical composition comprising the antibody described herein. In some embodiments, the companion animal species is a dog, a cat, or a horse. In some embodiments, the condition induced by IL31 is a pruritus or an allergic condition. In some embodiments, the condition induced by IL31 is selected from atopic dermatitis, pruritus, asthma, psoriasis, scleroderma, and eczema.

[0033] In some embodiments, the anti-IL31 antibody or pharmaceutical composition is administered parenterally. In some embodiments, the anti-IL31 antibody or pharmaceutical composition is administered by intramuscular, intraperitoneal, intrathecal, subcutaneous, intraarterial, intraarticular, intrathecal, or inhalation routes.

[0034] In some embodiments, the method comprises administering a Jak inhibitor, a PI3K inhibitor, an AKT inhibitor, or a MAPK inhibitor in combination with the anti-IL31 antibody or pharmaceutical composition. In some embodiments, the method comprises administering one or more antibodies selected from anti-IL17 antibody, anti-TNFα antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CD25 antibody, anti-IL4 antibody, anti-IL13 antibody, anti-IL23 antibody, anti-IgE antibody, anti-CD11α antibody, anti-IL6R antibody, anti-α4-integrin antibody, anti-IL12 antibody, anti-IL1β antibody, and anti-BlyS antibody in combination with the anti-IL31 antibody or pharmaceutical composition.

[0035] In some embodiments, provided is a method of reducing IL31 signaling function in a cell, the method comprising exposing the cell to the anti-IL31 antibody or pharmaceutical composition described herein under conditions that permit binding of the antibody to extracellular IL31, thereby reducing binding to the IL31 receptor and / or reducing the IL31 signaling function by the cell. In some embodiments, the cell is exposed to the antibody or pharmaceutical composition ex vivo. In some embodiments, the cell is exposed to the antibody or pharmaceutical composition in vivo. In some embodiments, the cell is a canine cell, a feline cell, or a equine cell.

[0036] In some embodiments, provided is a method of detecting IL31 in a sample from a companion animal species, the method comprising contacting the sample with the anti-IL31 antibody or pharmaceutical composition described herein under conditions that permit binding of the antibody to IL31 and detecting whether a complex is formed between the antibody and IL31 in the sample. In some embodiments, the sample is a biological sample obtained from a dog, a cat, or a horse. BRIEF DESCRIPTION OF THE DRAWINGS

[0037]

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Mode for Carrying Out the Invention

[0038] Description of Specific Sequences Table 1 provides a list of specific sequences referred to in this specification. TIFF0007716441000001.tif249170TIFF0007716441000002.tif253170TIFF0007716441000003.tif254170TIFF0007716441000004.tif254170TIFF0007716441000005.tif254170TIFF0007716441000006.tif254170TIFF0007716441000007.tif254170TIFF0007716441000008.tif254170TIFF0007716441000009.tif254170TIFF0007716441000010.tif254170TIFF0007716441000011.tif242170

[0039] Description of Specific Embodiments Antibodies that bind to canine IL31, feline IL31, or equine IL31 are provided. Also provided are antibody heavy chains and antibody light chains capable of forming antibodies that bind to IL31. Further provided are antibodies, heavy chains, and light chains that include one or more specific complementarity determining regions (CDRs). Polynucleotides encoding antibodies to canine IL31 are provided. Methods of producing or purifying antibodies to canine IL31 are also provided. Methods of treating with antibodies to canine IL31 are provided. Such methods include, but are not limited to, methods of treating IL31-induced conditions in companion animal species. Methods of detecting IL31 in samples from companion animal species are provided.

[0040] For the convenience of the reader, the following definitions of terms used herein are provided.

[0041] As used herein, numerical terms such as Kd are calculated based on scientific measurements and thus are not immune to appropriate measurement error. In some cases, numerical terms may include values rounded to the nearest significant figure.

[0042] 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 otherwise specified. In the context of multiple dependent claims, the use of "or" when referring back to other claims means only the claims in the alternatives.

[0043] Anti-IL31 antibody Novel antibodies to IL31 are provided, such as antibodies that bind to canine IL31, feline IL31, and / or equine IL31. The anti-IL31 antibodies provided herein include, but are not limited to, monoclonal antibodies, mouse antibodies, chimeric antibodies, canineized antibodies, felineized antibodies, and equinized antibodies. In some embodiments, the anti-IL31 antibodies are isolated mouse monoclonal antibodies such as M14, M18, M19, and M87.

[0044] Monoclonal antibodies M14, M18, M19, and M87 were isolated as follows. Briefly, mice were immunized with canine IL31, and mouse monoclonal antibody clones were obtained by standard hybridoma technology. Enzyme-linked immunosorbent assay (ELISA) was used to screen for hybridoma clones that produce IL31-binding antibodies. Based on the binding affinity and cell-based functional assays described herein, hybridoma clones that produce monoclonal antibodies M14, M18, M19, and M87 were selected for further study. The variable heavy chain (VH) and variable light chain (VL) of each of the four clones were sequenced and analyzed by sequence alignment (Figure 1).

[0045] The amino acid sequence of monoclonal antibody M14 is also provided herein. For example, for monoclonal antibody M14, variable heavy chain CDRs (SEQ ID NOs: 1-3; SEQ ID NO: 89 is an alternative definition of CDR-H2), variable light chain CDRs (SEQ ID NOs: 8-10), variable region heavy chain framework sequences (SEQ ID NOs: 4-7), and variable region light chain framework sequences (SEQ ID NOs: 11-14) are provided. The amino acid sequences of the variable light chain, light chain, variable heavy chain, and variable and hinge heavy chains of monoclonal antibody M14 are provided (SEQ ID NOs: 24, 36, 25, and 40, respectively).

[0046] Furthermore, the amino acid sequences of the CDRs, framework regions, variable light chains, and variable heavy chains of monoclonal antibodies M18, M19, and M87 are provided (see, for example, FIG. 1). For monoclonal antibody M19, the variable heavy chain CDRs (SEQ ID NOs: 1-3; SEQ ID NO: 89 is an alternative definition of CDR-H2), variable light chain CDRs (SEQ ID NOs: 8-10), variable light chain (SEQ ID NO: 65), light chain (SEQ ID NO: 38), variable and hinge heavy chain (SEQ ID NO: 42), and variable heavy chain (SEQ ID NO: 68) are provided. For monoclonal antibody M18, the variable heavy chain CDRs (SEQ ID NOs: 1, 62, and 3; SEQ ID NO: 87 is an alternative definition of CDR-H2), variable light chain CDRs (SEQ ID NOs: 63, 9, and 10), variable light chain (SEQ ID NO: 64), light chain (SEQ ID NO: 37), variable and hinge heavy chain (SEQ ID NO: 41), and variable heavy chain (SEQ ID NO: 67) are provided. For monoclonal antibody M87, the variable light chain (SEQ ID NO: 66), light chain (SEQ ID NO: 39), variable and hinge heavy chain (SEQ ID NO: 43), and variable heavy chain (SEQ ID NO: 69) are provided.

[0047] Chimeric, canineized, felineized, and equinized antibodies derived from monoclonal antibodies M14, M18, M19, and M87 are also provided herein. In some embodiments, the amino acid sequences of the canineized monoclonal antibody M14, such as SEQ ID NOs: 15-21 and 70-78, are provided. In some embodiments, the amino acid sequences of the felineized antibodies derived from monoclonal antibody M14, such as SEQ ID NOs: 32-35 and 79-86, are provided. In some embodiments, the amino acid sequences of the chimeric antibodies derived from monoclonal antibody M14, such as SEQ ID NOs: 26, 27, 30, and 31, are provided.

[0048] The term "antibody" is used in the broadest sense herein and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific (bispecific T cell engagers, etc.) and trispecific antibodies), and various antibody structures including antibody fragments (Fab, F(ab’)2, ScFv, minibodies, diabodies, triabodies, and tetra-bodies, etc.), as long as they exhibit the desired antigen-binding activity. The species of dog, cat, and horse have different diversities (classes) of antibodies that are shared by many mammals.

[0049] The term "antibody" includes, but is not limited to, fragments capable of binding to an antigen, such as Fv, single-chain Fv (scFv), Fab, Fab’, di-scFv, sdAb (single domain antibody), and (Fab’)2 (including chemically linked F(ab’)2). Papain digestion of an antibody produces two identical antigen-binding fragments, termed "Fab" fragments, each having a single antigen-binding site, and the remaining "Fc" fragment, whose name reflects its ability to readily crystallize. Pepsin treatment yields an F(ab’)2 fragment having two antigen-binding sites and still having the ability to cross-link antigens. The term "antibody" includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species such as mouse, human, cynomolgus monkey, dog, cat, horse, and others. Further, for all antibody constructs provided herein, variants having sequences from other organisms are also contemplated. Thus, if a mouse antibody version is disclosed, one of ordinary skill in the art should understand how to convert an antibody based on a mouse sequence to a cat, dog, horse, or other sequence. Antibody fragments include single-chain scFv, tandem di-scFv, diabody, tandem tri-sdcFv, minibody, and any other orientation. Antibody fragments also include nanobodies (sdAb, antibodies having a single monomeric domain without a light chain, such as pairs of variable domains of the heavy chain). Antibody fragments can, in some embodiments, also be referred to as being of a particular species (e.g., mouse scFv or dog scFv). This refers to at least a portion of the sequence of the non-CDR regions rather than the source of the construct. In some embodiments, the antibody includes a label or is conjugated to a second moiety.

[0050] The terms "label" and "detectable label" mean a moiety attached to an antibody or its analyte such that a reaction (e.g., binding) between members of a particular binding pair becomes detectable. A labeled member of a particular binding pair is referred to as "detectably labeled." Thus, the term "labeled binding protein" means a protein into which a label that provides for the identification of the binding protein has been incorporated. In some embodiments, the label is a detectable marker capable of producing a signal detectable by visual or mechanical means, such as incorporation of a radiolabeled amino acid, or binding of a biotinylated moiety detectable by a marked avidin (e.g., streptavidin containing a fluorescent marker detectable by optical or colorimetric methods or enzyme activity) to a polypeptide. 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 employed for immunoassays include moieties that generate light, such as acridinium compounds, and moieties that generate fluorescence, such as fluorescein. In this regard, the moiety itself need not be detectably labeled, but may become detectable by reaction with yet another moiety.

[0051] The term "monoclonal antibody" means an antibody of 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 directed 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 directed against a single determinant on the antigen. Thus, a sample of monoclonal antibodies can bind to the same epitope on an antigen. The modifier "monoclonal" indicates the property of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be made by the hybridoma method first described by Kohler and Milstein, Nature 256:495, 1975, or by recombinant DNA methods such as those described in U.S. Patent No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries generated using, for example, the techniques described in McCafferty et al., Nature 348:552-554, 1990.

[0052] In some embodiments, the monoclonal antibody is an isolated mouse antibody selected from clone M14, M18, M19, and M87.

[0053] "Amino acid sequence" means the sequence of amino acid residues in a peptide or protein. The terms "polypeptide" and "protein" are used interchangeably and mean a polymer of amino acid residues, without limitation as to minimum length. Such a polymer of amino acid residues may contain natural or non-natural amino acid residues and includes, but is not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. This definition includes both full-length proteins and fragments thereof. The term includes post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, and others. Further, for the purposes of the present disclosure, "polypeptide" means a protein that includes modifications to the native sequence, such as deletions, additions, and substitutions (generally being essentially conservative), as long as the protein maintains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or may be incidental, such as mutations in the host producing the protein or errors due to PCR amplification.

[0054] "IL31", as used herein, means any native IL31 resulting from the expression and processing of IL31 within a cell. The term includes IL31 from any vertebrate source, including, but not limited to, mammals such as primates (e.g., humans and cynomolgus monkeys), rodents (e.g., mice and rats), and companion animals (e.g., dogs, cats, and horses), unless otherwise indicated. The term also includes naturally occurring variants of IL31, such as splice variants or allelic variants.

[0055] In some embodiments, canine IL31 comprises the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 44. In some embodiments, feline IL31 comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, equine IL31 comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, human IL31 comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, sea urchin IL31 comprises the amino acid sequence of SEQ ID NO: 47. In some embodiments, mouse IL31 comprises the amino acid sequence of SEQ ID NO: 61. In other embodiments, IL31 comprises the amino acid sequence of SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60.

[0056] 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.

[0057] In some embodiments, the IL31 binding domain binds to canine IL31 with greater affinity than it binds to human IL31. In some embodiments, the IL31 binding domain binds to IL31 of other companion animals, such as feline IL31 or equine IL31. In some embodiments, the IL31 binding domain does not bind to human IL31.

[0058] As used herein, the term "epitope" means a site on a target molecule (e.g., an antigen such as a protein, nucleic acid, carbohydrate, or lipid) to which an antigen-binding molecule (e.g., an antibody, antibody fragment, or scaffold protein containing an antibody-binding region) binds. Epitopes often contain chemically active surface groups of molecules such as amino acids, polypeptides, or sugar side chains, and have specific three-dimensional structural features as well as specific charge features. Epitopes can be formed from both contiguous or juxtaposed and non-contiguous residues (e.g., amino acids, nucleotides, saccharides, lipid moieties) of the target molecule. Epitopes formed from contiguous residues (e.g., amino acids, nucleotides, saccharides, 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 contain, 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 length of the epitope is less than 20 residues (e.g., amino acids or nucleotides), less than 15 residues, or less than 12 residues. If two antibodies exhibit competitive binding to an antigen, they can bind to the same epitope in the antigen. In some embodiments, an epitope can be identified by a certain minimum distance to the CDR residues of the antigen-binding molecule. In some embodiments, an epitope is identified by the above distance and is further limited to those residues involved in the binding (e.g., hydrogen bonding) between antibody residues and antigen residues. Epitopes can also be identified by various scans, for example, alanine or arginine scans can indicate one or more residues with which the antigen-binding molecule can interact. Unless explicitly noted otherwise, a set of residues as an epitope does not exclude other residues from being part of the epitope for a particular antibody. Rather, the presence of such a set represents the minimal series (or set of species) of epitopes. Thus, in some embodiments, a set of residues identified as an epitope represents the minimal epitope associated with the antigen rather than an exclusive list of the residues of the epitope on the antigen.

[0059] In some embodiments, the epitope comprises the amino acid sequence PSDX1X2KI (SEQ ID NO: 45), where X is any amino acid residue. In some embodiments, X1 is a hydrophobic amino acid. In some embodiments, X1 is selected from A, V, I, and L. In some embodiments, X1 is selected from V and I. In some embodiments, X2 is a hydrophilic amino acid. In some embodiments, X2 is selected from A, R, K, Q, and N. In some embodiments, X2 is selected from R and Q. In some embodiments, X1 is V and X2 is R. In some embodiments, X1 is I and X2 is Q. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO: 88. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the epitope is within amino acids 34 - 50 of SEQ ID NO: 22. In some embodiments, the epitope comprises amino acids 34 - 50 of SEQ ID NO: 22.

[0060] The term "CDR" means a complementarity-determining region defined by at least one mode of identification to those skilled in the art. In some embodiments, the CDR can be defined according to any of the Chothia numbering scheme, Kabat numbering scheme, combination of Kabat and Chothia, AbM definition, contact definition, or combination of Kabat, Chothia, AbM, or contact definition. The various CDRs in an antibody can be represented by appropriate numbers and chain types 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 that include hypervariable loops.

[0061] In some embodiments, the anti-IL31 antibody comprises a heavy chain comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 62, SEQ ID NO: 89, or SEQ ID NO: 87, or (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-IL31 antibody comprises a light chain comprising (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 63, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, or (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

[0062] In some embodiments, the anti-IL31 antibody comprises a heavy chain comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2 or 89, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain comprising (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8 or 63, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

[0063] In some embodiments, the anti-IL31 antibody comprises a heavy chain comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 62 or 87, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain comprising (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8 or 63, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

[0064] The term "variable region," as used herein, 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 to refer to a region comprising at least three heavy chain CDRs. The terms "light chain variable region" or "variable light chain" are used interchangeably to 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 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 comprise a variable light chain having the following structure: (CDR-L1)-(LC-FR2)-(CDR-L2)-(LC-FR3)-(CDR-L3).

[0065] 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: 4, (b) the HC-FR2 sequence of SEQ ID NO: 5, (c) the HC-FR3 sequence of SEQ ID NO: 6, (d) the HC-FR4 sequence of SEQ ID NO: 7, (e) the variable region light chain framework 1 (LC-FR1) sequence of SEQ ID NO: 11, (f) the LC-FR2 sequence of SEQ ID NO: 12, (g) the LC-FR3 sequence of SEQ ID NO: 13, or (h) the LC-FR4 sequence of SEQ ID NO: 14. In some embodiments, the anti-IL31 antibody comprises a variable light chain sequence of: (a) SEQ ID NO: 16, (b) SEQ ID NO: 24, or (c) SEQ ID NO: 32. In some embodiments, the anti-IL31 antibody comprises a variable heavy chain sequence of: (a) SEQ ID NO: 15, (b) SEQ ID NO: 25, or (c) SEQ ID NO: 33. In some embodiments, the anti-IL31 antibody comprises: (a) the variable light chain sequence of SEQ ID NO: 16 and the variable heavy chain sequence of SEQ ID NO: 15, (b) the variable light chain sequence of SEQ ID NO: 24 and the variable heavy chain sequence of SEQ ID NO: 25, or (c) the variable light chain sequence of SEQ ID NO: 32 and the variable heavy chain sequence of SEQ ID NO: 33.

[0066] As used herein, the term "constant region" means a region containing at least three constant domains. The terms "heavy chain constant region" or "constant heavy chain" are used interchangeably and mean a region containing at least three heavy chain constant domains, CH1, CH2, and CH3. Non-limiting exemplary heavy chain constant regions include γ, δ, α, ε, and μ. Each heavy chain constant region corresponds to an isotype of the antibody. For example, an antibody containing a γ constant region is an IgG antibody, an antibody containing a δ constant region is an IgD antibody, an antibody containing an α constant region is an IgA antibody, an antibody containing a μ constant region is an IgM antibody, and an antibody containing an ε constant region is an IgE antibody. A particular isotype can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (containing the γ1 constant region), IgG2 (containing the γ2 constant region), IgG3 (containing the γ3 constant region), and IgG4 (containing the γ4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (containing the α1 constant region) and IgA2 (containing the 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 mean a 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 the function are included within the scope of the term "constant region" unless otherwise specified. Dogs, cats, and horses have antibody classes such as IgG, IgA, IgD, IgE, and IgM. Among dog IgG antibody classes are IgG-A, IgG-B, IgG-C, and IgG-D. Among cat IgG antibody classes are IgG1a, IgG1b, and IgG2. Among horse IgG antibody classes are IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, and IgG7.

[0067] The term "chimeric antibody" or "chimeric" means 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 remaining heavy or light chain is derived from a different source or species. In some embodiments, a chimeric antibody means an antibody comprising 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.). In some embodiments, a chimeric antibody comprises at least one mouse variable region and at least one dog constant region. In some embodiments, a chimeric antibody comprises at least one mouse variable region and at least one cat constant region. In some embodiments, all of the variable regions of the chimeric antibody are from the first species and all of the constant regions of the chimeric antibody are from the second species. In some embodiments, a chimeric antibody comprises a constant heavy chain region or a constant light chain region from a companion animal. In some embodiments, a chimeric antibody comprises a mouse variable heavy and light chain, and a constant heavy and light chain from a companion animal. For example, a chimeric antibody may comprise a mouse variable heavy and light chain, and a dog constant heavy and light chain, a chimeric antibody may comprise a mouse variable heavy and light chain, and a cat constant heavy and light chain, or a chimeric antibody may comprise a mouse variable heavy and light chain, and a horse constant heavy and light chain.

[0068] In some embodiments, the anti-IL31 antibody a. (i) the light chain amino acid sequence of SEQ ID NO: 26, (ii) the heavy chain amino acid sequence of SEQ ID NO: 27, or (iii) the light chain amino acid sequence of (i) and the heavy chain sequence of (ii), or b. (i) the light chain amino acid sequence of SEQ ID NO: 30, (ii) the heavy chain amino acid sequence of SEQ ID NO: 31, or (iii) the light chain amino acid sequence of (i) and the heavy chain sequence of (ii), comprising a chimeric antibody.

[0069] "Canine chimeric" or "canine chimeric antibody" means a chimeric antibody having at least a portion of a heavy chain or a portion of a light chain derived from a dog. "Feline chimeric" or "feline chimeric antibody" means a chimeric antibody having at least a portion of a heavy chain or a portion of a light chain derived from a cat. "Equine chimeric" or "equine chimeric antibody" means a chimeric antibody having at least a portion of a heavy chain or a portion of a light chain derived from a horse. In some embodiments, the canine chimeric antibody comprises a murine variable heavy chain and light chain, and a canine constant heavy chain and light chain. In some embodiments, the feline chimeric antibody comprises a murine variable heavy chain and light chain, and a feline constant heavy chain and light chain. In some embodiments, the equine chimeric antibody comprises a murine variable heavy chain and light chain, and an equine constant heavy chain and light chain. In some embodiments, the antibody is a chimeric antibody comprising a murine variable heavy chain framework region or a murine variable light chain framework region.

[0070] "Canine antibody", as used herein, includes an antibody produced in a dog, an antibody produced in a non-dog animal that contains a canine immunoglobulin gene or contains a canine immunoglobulin peptide, or an antibody selected using an in vitro method such as phage display in which the antibody repertoire is based on a canine immunoglobulin sequence. The term "canine antibody" means a genus of sequences that are canine sequences. Thus, this term represents the genus of related sequences, rather than the process of creating the antibody.

[0071] In some embodiments, the anti-IL31 antibody comprises a canine heavy chain constant region selected from the IgG-A, IgG-B, IgG-C, and IgG-D constant regions. In some embodiments, the anti-IL31 antibody is a canine IgG-A, IgG-B, IgG-C, or IgG-D antibody. 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: 17, (b) a canine IgG-B antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 18, (c) a canine IgG-C antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 19, or (d) a canine IgG-D antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 20.

[0072] As used herein, the term "feline antibody" encompasses antibodies produced in cats, antibodies produced in non-cat animals that contain feline immunoglobulin genes or contain feline immunoglobulin peptides, or antibodies selected using in vitro methods such as phage display in which the antibody repertoire is based on feline immunoglobulin sequences. The term "feline antibody" means the genus of sequences that are feline sequences. Thus, this term represents the genus of related sequences rather than the process of creating the antibody.

[0073] In some embodiments, the anti-IL31 antibody comprises a feline heavy chain constant region selected from the IgG1, IgG2a, and IgG2b constant regions. In some embodiments, the anti-IL31 antibody is a feline IgG1, IgG2a, or IgG2b antibody.

[0074] As used herein, the term "equine antibody" encompasses antibodies produced in horses, antibodies produced in non-horse animals that contain equine immunoglobulin genes or contain equine immunoglobulin peptides, or antibodies selected using in vitro methods such as phage display in which the antibody repertoire is based on equine immunoglobulin sequences. The term "equine antibody" means the genus of sequences that are equine sequences. Thus, this term represents the genus of related sequences rather than the process of creating the antibody.

[0075] In some embodiments, the anti-IL31 antibody comprises an equine heavy chain constant region selected from the IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, and IgG7 constant regions. In some embodiments, the anti-IL31 antibody is an equine IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, or IgG7 antibody.

[0076] "Caninized antibody" means an antibody in which at least one amino acid in a portion of the non-canine variable region is replaced with the corresponding amino acid from the canine variable region. In some embodiments, the caninized antibody comprises at least one canine constant region (e.g., γ constant region, α constant region, δ constant region, ε constant region, μ constant region, or others) or a fragment thereof. In some embodiments, the caninized antibody is a Fab, scFv, (Fab')2, or other antibody fragment. The term "caninized" also means a form of a non-canine (e.g., mouse) antibody that comprises a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of an antibody, etc.) that contains a minimal sequence of a non-canine antibody. A caninized antibody may comprise a canine immunoglobulin (recipient antibody) in which residues from the CDR of the recipient are replaced by residues from the CDR of a non-canine species such as a mouse, rat, or rabbit that have the desired specificity, affinity, and potency (donor antibody). In some examples, the Fv framework region (FR) residues of the canine immunoglobulin are replaced by the corresponding non-canine residues. Further, the caninized antibody may comprise residues that are not found in the CDR or framework sequences introduced into the recipient antibody, but are included to further improve and optimize the performance of the antibody.

[0077] In some embodiments, at least one amino acid residue in a portion of the mouse variable heavy chain or mouse variable light chain is replaced with the corresponding amino acid from the canine variable region. In some embodiments, the modified chain is fused to a canine constant heavy chain or canine constant light chain. In some embodiments, the anti-IL31 antibody is a caninized antibody comprising (a) the heavy chain sequence of SEQ ID NO: 15, (b) the heavy chain sequence of SEQ ID NO: 17, (c) the heavy chain sequence of SEQ ID NO: 18, (d) the heavy chain sequence of SEQ ID NO: 19, (e) the heavy chain sequence of SEQ ID NO: 20, (f) the light chain sequence of SEQ ID NO: 16, or (g) the light chain sequence of SEQ ID NO: 21.

[0078] "Catized antibody" means an antibody in which at least one amino acid in a part of the non-cat variable region is replaced with the corresponding amino acid from the cat variable region. In some embodiments, the catized antibody comprises at least one cat constant region (e.g., gamma constant region, alpha constant region, delta constant region, epsilon constant region, mu constant region, or others) or a fragment thereof. In some embodiments, the catized antibody is a Fab, scFv, (Fab')2, or other antibody fragment. The term "catized" also means a form of a non-cat (e.g., mouse) antibody that includes a chimeric immunoglobulin, immunoglobulin chain, or a fragment thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of an antibody, etc.) that contains the minimal sequence of a non-cat immunoglobulin. A catized antibody may include a cat immunoglobulin (recipient antibody) in which residues from the CDR of the recipient are replaced by residues from the CDR of a non-cat species such as a mouse, rat, or rabbit that have the desired specificity, affinity, and potency (donor antibody). In some examples, the Fv framework region (FR) residues of the cat immunoglobulin are replaced by the corresponding non-cat residues. Further, a catized antibody may include residues that are not found in the CDR or framework sequences introduced into the recipient antibody but are included to further improve and optimize the performance of the antibody.

[0079] In some embodiments, at least one amino acid residue in a part of the mouse variable heavy chain or mouse variable light chain is replaced with the corresponding amino acid from the cat variable region. In some embodiments, the modified chain is fused to a cat constant heavy chain or a dog constant light chain. In some embodiments, the anti-IL31 antibody is a catized antibody comprising (a) the light chain sequence of SEQ ID NO: 32, (b) the light chain sequence of SEQ ID NO: 34, (c) the heavy chain sequence of SEQ ID NO: 33, or (d) the heavy chain sequence of SEQ ID NO: 35.

[0080] "Humanized antibody" means an antibody in which at least one amino acid in a part of the non-human variable region is replaced with the corresponding amino acid from the human variable region. In some embodiments, the humanized antibody comprises at least one human constant region (e.g., γ constant region, α constant region, δ constant region, ε constant region, μ constant region, or others) or a fragment thereof. In some embodiments, the humanized antibody is a Fab, scFv, (Fab')2, or other antibody fragment. The term "humanized" also means a form of a non-human (e.g., mouse) antibody that comprises a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of an antibody, etc.) that contains the minimal sequence of a non-human immunoglobulin. A humanized antibody may comprise a human immunoglobulin (recipient antibody) in which residues from the CDRs of the recipient are replaced by residues from the CDRs of a non-human species such as a mouse, rat, or rabbit that have the desired specificity, affinity, and capacity (donor antibody). In some examples, the Fv framework region (FR) residues of the human immunoglobulin are replaced by the corresponding non-human residues. Further, the humanized antibody may contain residues that are not found in the CDRs or framework sequences introduced into the recipient antibody, but are included to further improve and optimize the performance of the antibody.

[0081] In some embodiments, at least one amino acid residue in a part of the mouse variable heavy chain or mouse variable light chain is replaced with the corresponding amino acid from the human variable region. In some embodiments, the modified chain is fused to a human constant heavy chain or a canine constant light chain.

[0082] The term "IgX Fc" means that the Fc region is derived from a specific antibody isotype (e.g., IgG, IgA, IgD, IgE, IgM, etc.), where "X" means the antibody isotype. Thus, "IgG Fc" means the Fc region of the γ chain, "IgA Fc" means the Fc region of the α chain, "IgD Fc" means the Fc region of the δ chain, "IgE Fc" means the Fc region of the ε chain, "IgM Fc" means the Fc region of the μ chain, and so on. In some embodiments, the IgG Fc region includes CH1, the hinge, CH2, CH3, and CL1. "IgX-N-Fc" means that the Fc region is derived from a specific subclass of an antibody isotype (e.g., canine IgG subclass A, B, C, or D, feline IgG subclass 1, 2a, or 2b, or equine IgG subclass IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, or IgG7, etc.), where "N" means the subclass. In some embodiments, the IgX Fc or IgX-N-Fc region is derived from a companion animal such as a dog, a cat, or a horse. In some embodiments, the IgG Fc region is isolated from a canine γ heavy chain such as IgG-A, IgG-B, IgG-C, or IgG-D. In some examples, the IgG Fc region is isolated from a feline γ heavy chain such as IgG1, IgG2a, or IgG2b. Antibodies containing the Fc region of IgG-A, IgG-B, IgG-C, or IgG-D can provide high expression levels in recombinant production systems.

[0083] The term "affinity" means the overall strength of the 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 molecule X for its partner Y is generally represented by the dissociation constant (K D ). Affinity can be measured by common methods known in the art, such as immunoblotting, ELISA KD, KinEx A, biolayer interferometry (BLI), or a surface plasmon resonance device.

[0084] The term "K D ", "K d", "Kd", or "Kd value" are used interchangeably and mean the equilibrium dissociation constant of an antibody-antigen interaction. In some embodiments, the K of the antibody d is measured using biolayer interferometry with a biosensor such as the Octet® system (Pall ForteBio LLC, Fremont (CA)) according to the supplier's instructions. Briefly, biotinylated antigen is bound to the sensor chip and the association of the antibody is monitored for 90 seconds and the 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 blank curve of buffer alone is subtracted to correct for drift. The data is fit to a 2:1 binding model using ForteBio data analysis software to determine the association rate constant (k on ), the dissociation rate constant (k off ), and K d . The equilibrium dissociation constant (K d ) is calculated as the ratio of k off / k on . The term "k on " means the rate constant of association of the antibody to the antigen and the term "k off " means the rate constant of dissociation of the antibody from the antibody / antigen complex.

[0085] 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 "bind" if it reacts with, associates with, or has an affinity for a particular cell or substance and that reaction, association, or affinity is detectable by one or more methods known in the art, such as immunoblot, ELISA KD, KinEx A, biolayer interferometry (BLI), surface plasmon resonance devices, among others.

[0086] "Surface plasmon resonance" refers to an optical phenomenon that enables the analysis of real-time dual-specific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using a BIAcore (trademark) system (BIACore International AB, GE Healthcare Company, Uppsala, Sweden and Piscataway, N.J.). For further explanation, see Jonsson et al., (1993), Ann. Biol. Clin. 51:19-26.

[0087] "Biolayer interferometry" means an optical analysis technique that analyzes the interference pattern of light reflected from a layer of immobilized protein and an internal reference layer on a biosensor chip. A change in the number of molecules bound to the biosensor chip changes the interference pattern, which can be measured in real time. A non-limiting exemplary device for biolayer interferometry is the Octet (registered trademark) system (Pall ForteBio LLC). See, for example, Abdiche et al., 2008, Anal. Biochem. 377:209-277.

[0088] In some embodiments, the anti-IL31 antibody binds to canine IL31, feline IL31, or equine IL31 with a dissociation constant (Kd) of less than 5×10 -6 M, less than 1×10 -6 M, less than 5×10 -7 M, less than 1×10 -7 M, less than 5×10 -8 M, less than 1×10 -8 M, less than 5×10 -9 M, less than 1×10 -9 M, less than 5×10 -10 M, less than 1×10 -10 M, less than 5×10 -11 M, less than 1×10 -11 M, less than 5×10 -12 M, or less than 1×10 -12 M. In some embodiments, the anti-IL31 antibody binds to canine IL31, feline IL31, or equine IL31 with a dissociation constant (Kd) of 5×10 -6 M to 1×10-6 M, 5×10 -6 M to 5×10 -7 M, 5×10 -6 M to 1×10 -7 M, 5×10 -6 M to 5×10 -8 M, 5×10 -6 M to 1×10 -8 M, 5×10 -6 M to 5×10 -9 M, 5×10 -6 M to 1×10 -9 M, 5×10 -6 M to 5×10 -10 M, 5×10 -6 M to 1×10 -10 M, 5×10 -6 M to 5×10 -11 M, 5×10 -6 M to 1×10 -11 M, 5×10 -6 M to 5×10 -12 M, 5×10 -6 M to 1×10 -12 M, 1×10 -6 M to 5×10 -7 M, 1×10 -6 M to 1×10 -7 M, 1×10 -6 M to 5×10 -8 M, 1×10 -6 M to 1×10 -8 M, 1×10 -6 M to 5×10 -9 M, 1×10 -6 M to 1×10 -9 M, 1×10 -6 M to 5×10 -10 M, 1×10 -6 M to 1×10 -10 M, 1×10 -6 M to 5×10 -11 M, 1×10 -6 M to 1×10 -11 M, 1×10 -6 M to 5×10 -12 M, 1×10 -6 M to 1×10 -12 M, 5×10 -7 M to 1×10 -7 M, 5×10 -7 M to 5×10 -8M, 5×10 -7 M to 1×10 -8 M, 5×10 -7 M to 5×10 -9 M, 5×10 -7 M to 1×10 -9 M, 5×10 -7 M to 5×10 -10 M, 5×10 -7 M to 1×10 -10 M, 5×10 -7 M to 5×10 -11 M, 5×10 -7 M to 1×10 -11 M, 5×10 -7 M to 5×10 -12 M, 5×10 -7 M to 1×10 -12 M, 1×10 -7 M to 5×10 -8 M, 1×10 -7 M to 1×10 -8 M, 1×10 -7 M to 5×10 -9 M, 1×10 -7 M to 1×10 -9 M, 1×10 -7 M to 5×10 -10 M, 1×10 -7 M to 1×10 -10 M, 1×10 -7 M to 5×10 -11 M, 1×10 -7 M to 1×10 -11 M, 1×10 -7 M to 5×10 -12 M, 1×10 -7 M to 1×10 -12 M, 5×10 -8 M to 1×10 -8 M, 5×10 -8 M to 5×10 -9 M, 5×10 -8 M to 1×10 -9 M, 5×10 -8 M to 5×10 -10 M, 5×10 -8 M to 1×10 -10 M, 5×10 -8 M to 5×10 -11 M, 5×10 -8 M to 1×10 -11 M, 5×10-8 M ~ 5×10 -12 M, 5×10 -8 M ~ 1×10 -12 M, 1×10 -8 M ~ 5×10 -9 M, 1×10 -8 M ~ 1×10 -9 M, 1×10 -8 M ~ 5×10 -10 M, 1×10 -8 M ~ 1×10 -10 M, 1×10 -8 M ~ 5×10 -11 M, 1×10 -8 M ~ 1×10 -11 M, 1×10 -8 M ~ 5×10 -12 M, 1×10 -8 M ~ 1×10 -12 M, 5×10 -9 M ~ 1×10 -9 M, 5×10 -9 M ~ 5×10 -10 M, 5×10 -9 M ~ 1×10 -10 M, 5×10 -9 M ~ 5×10 -11 M, 5×10 -9 M ~ 1×10 -11 M, 5×10 -9 M ~ 5×10 -12 M, 5×10 -9 M ~ 1×10 -12 M, 1×10 -9 M ~ 5×10 -10 M, 1×10 -9 M ~ 1×10 -10 M, 1×10 -9 M ~ 5×10 -11 M, 1×10 -9 M ~ 1×10 -11 M, 1×10 -9 M ~ 5×10 -12 M, 1×10 -9 M ~ 1×10 -12 M, 5×10 -10 M ~ 1×10 -10 M, 5×10 -10 M ~ 5×10 -11 M, 1×10 -10 M ~ 5×10 -11 M, 1×10 -10M ~ 1×10 -11 M, 1×10 -10 M ~ 5×10 -12 M, 1×10 -10 M ~ 1×10 -12 M, 5×10 -11 M ~ 1×10 -12 M, 5×10 -11 M ~ 5×10 -12 M, 5×10 -11 M ~ 1×10 -12 M, 1×10 -11 M ~ 5×10 -12 M, or 1×10 -11 M ~ 1×10 -12 Binds to canine IL31, feline IL31, or equine IL31 with a Kd of M. In some embodiments, the anti-IL31 antibody binds to canine IL31, feline IL31, or equine IL31 as determined by immunoblot analysis.

[0089] In some embodiments, the anti-IL31 antibody does not bind to human IL31 as determined by immunoblot analysis and / or biolayer interferometry.

[0090] In some embodiments, provided are anti-IL31 antibodies that compete with the anti-IL31 antibodies described herein for binding to IL31 (e.g., M14, M18, M19, or M87). In some embodiments, antibodies can be made or used that compete for binding with any of the antibodies provided herein. In some embodiments, provided are anti-IL31 antibodies that compete with the monoclonal M14 antibody for binding to canine IL31 or feline IL31.

[0091] "Variant" means a biologically active polypeptide having at least about 50% amino acid sequence identity with the native sequence polypeptide without consideration of conservative substitutions even if there are such conservative substitutions after aligning the sequences and introducing gaps if necessary to maximize the percent sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N-terminus or C-terminus of the polypeptide.

[0092] In some embodiments, the variant has 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 with the polypeptide of the unmodified sequence.

[0093] As used herein, "percent amino acid sequence identity (%)" and "homology" with respect to the sequence of a peptide, polypeptide, or antibody are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps if necessary to maximize the percent sequence identity, without considering conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved by various methods in the art using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN, or MEGALINE™ (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve the maximum alignment over the full length of the sequences being compared.

[0094] Amino acid substitutions can include, but are not limited to, replacement of one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 2. Amino acid substitutions may be introduced into the antibody of interest, and the product is screened for retention / improvement of the desired activity, such as antigen binding, reduction of immunogenicity, or improvement of ADCC or CDC. Table 2 TIFF0007716441000012.tif208170

[0095] Amino acids can be classified according to common side-chain characteristics. (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 affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0096] Non-conservative substitutions will involve the exchange of one member of these classes with another.

[0097] In some embodiments, the anti-IL31 antibody comprises a heavy chain and a light chain, a. The heavy chain comprises a CDR-H1 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 1, a CDR-H2 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 62, SEQ ID NO: 89, or SEQ ID NO: 87, and a CDR-H3 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 3, b. The light chain comprises a CDR-L1 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 63, a CDR-L2 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 9, and a CDR-L3 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 10.

[0098] In some embodiments, the anti-IL31 antibody comprises a heavy chain and a light chain, a. (i) a variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 24, (ii) a variable heavy chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 25, or (iii) the variable light chain sequence of (i) and the variable heavy chain sequence of (ii), or b. (i) a variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 16, (ii) a variable heavy chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 15, or (iii) the variable light chain sequence of (i) and the variable heavy chain sequence of (ii), or c. (i) a variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 32, (ii) a variable heavy chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 33, or (iii) the variable light chain sequence of (i) and the variable heavy chain sequence of (ii), or d. (i) a variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 64, (ii) a variable heavy chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 67, or (iii) the variable light chain sequence of (i) and the variable heavy chain sequence of (ii), or e. (i) A variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 65, (ii) a variable heavy chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 68, or (iii) the variable light chain sequence of (i) and the variable heavy chain sequence of (ii), or f. (i) A variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 66, (ii) a variable heavy chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 69, or (iii) the variable light chain sequence of (i) and the variable heavy chain sequence of (ii).

[0099] The term "vector" is used to describe a polynucleotide that can be manipulated to include a cloned polynucleotide or a polynucleotide that can be increased in a host cell. A vector can include one or more of the following elements: an origin of replication, one or more regulatory sequences (such as a promoter or enhancer, etc.) that regulate the expression of the polypeptide of interest, or one or more selectable marker genes (such as an antibiotic resistance gene and a gene that can be used in a colorimetric assay, such as β - galactosidase). The term "expression vector" means a vector used to express the polypeptide of interest in a host cell.

[0100] "Host cell" means a cell that may be or has been the recipient of a vector or isolated polynucleotide. Host cells may be prokaryotic or eukaryotic cells. Exemplary eukaryotic cells include mammalian cells such as primate or non-primate animal cells, fungal cells such as yeast, plant cells, and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NS0 cells, PER.C6® cells (Crucell), 293 cells, and CHO cells, and derivatives thereof, such as 293-6E, DG44, CHO-S, and CHO-K cells. Host cells include the progeny of a single host cell, and due to natural, accidental, or intentional mutations, the progeny need not be exactly identical to the original parent cell (in terms of morphology or genomic DNA complementarity). Host cells include cells transfected in vivo with a polynucleotide encoding the amino acid sequences provided herein.

[0101] The term "isolated", as used herein, typically means a molecule that has been separated from at least some of the components with which it is naturally found or produced. For example, a polypeptide is referred to as "isolated" if it has been separated from at least some of the components of the cell that produced it. When a polypeptide is secreted from a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to "isolate" the polypeptide. Similarly, a polynucleotide is "isolated" if it is not part of a larger polynucleotide (such as genomic DNA or mitochondrial DNA in the case of a DNA polynucleotide) in which it is typically found naturally, or, in the case of an RNA polynucleotide, if it has been separated from at least some of the components of the cell that produced it. 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.

[0102] The term "companion animal species" means an animal suitable as a companion to humans. In some embodiments, the companion animal species are small mammals such as canines, felines, dogs, cats, horses, rabbits, ferrets, guinea pigs, rodents, and others. In some embodiments, the companion animal species are horses, cows, pigs, and other livestock.

[0103] The term "IL31 signaling function" means any one or combination of downstream activities that occur when IL31 binds to its receptor or receptor complex. In some embodiments, the IL31 signaling function includes activation of Janus kinase (Jak) 1 or Jak2 signaling molecules. In some embodiments, the IL31 signaling function includes phosphorylation of STAT-3 or STAT-5 proteins. In some embodiments, the IL31 signaling function includes activating the ERK1 / 2 MAP kinase signaling pathway. In some embodiments, the IL31 signaling function includes activating the PI3K / AKT signaling pathway. In some embodiments, the IL31 signaling function includes activating the Jak1 / 2 signaling pathway.

[0104] "STAT phosphorylation" means the modification of the STAT protein by phosphorylation after its expression. For example, "STAT-3 phosphorylation" means the phosphorylation of STAT-3, and "STAT-5 phosphorylation" means the phosphorylation of STAT-5. In some embodiments, the phosphorylation of STAT-3 is measured by immunoblot analysis. For example, cells (such as canine monocyte DH82 cells) are cultured in a growth medium (such as MEM, Life Technologies (registered trademark)) containing 15% heat-inactivated fetal bovine serum, 2 mmol / L GlutaMax, 1 mmol / L sodium pyruvate, and 10 ng / mL canine interferon-c (R&D Systems, Minneapolis, MN, USA) at 1 × 10 5Seed a 96-well cell culture plate at a cell / well density in the presence of the anti-IL31 antibody described herein at 37° C. for 24 hours. Use immunoblot analysis of cell lysates with anti-phospho STAT-3 antibody and anti-STAT-3 antibody (R&D Systems) to detect the relative concentrations of phosphorylated STAT-3 and non-phosphorylated STAT-3 compared to a control of β-actin. Methods for determining the qualitative or quantitative concentration of a protein by immunoblot are understood by those of ordinary skill in the art. In some embodiments, the relative concentration is determined qualitatively by visual inspection of the immunoblot. In some embodiments, the concentrations of phosphorylated STAT-3 and non-phosphorylated STAT-3 are determined quantitatively by digital imaging of the immunoblot, determining the intensity of the bands, and back-calculating the concentration of phosphorylated or non-phosphorylated STAT-3 in the sample using a linear standard curve of STAT-3 protein of known concentration.

[0105] "Reduce" or "inhibit" means to cause a decrease, reduction, or cessation in activity, function, or amount as compared to a reference. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall decrease of 20% or more. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall decrease of 50% or more. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or more. In some embodiments, the above amounts are inhibited or reduced over a period of time as compared to a control dose (such as a placebo) over the same period of time. "Reference" as used herein means any sample, standard, or level used for purposes of comparison. A reference may be obtained from a healthy or non-diseased sample. In some examples, the reference is obtained from a non-diseased or untreated sample of a companion animal. In some examples, the reference is obtained from one or more healthy animals of a particular species that are not the animals being tested or treated.

[0106] As used herein, the term "substantially reduced" refers to a degree of reduction between a numerical value and a reference value that is high enough such that a person of ordinary skill in the art would consider the difference between the two values to be statistically significant within the context of a biological property measured by the numerical value and the reference value. In some embodiments, the numerically substantially reduced value is greater than any of about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or is reduced by 100% compared to the reference value.

[0107] In some embodiments, the IL31 antibody can reduce the IL31 signaling function in 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% as measured by reduction of STAT-3 phosphorylation compared to the IL31 signaling function in the absence of the antibody.In some embodiments, the reduction of IL31 signaling function or the reduction of STAT-3 phosphorylation is 10% - 15%, 10% - 20%, 10% - 25%, 10% - 30%, 10% - 35%, 10% - 40%, 10% - 45%, 10% - 50%, 10% - 60%, 10% - 70%, 10% - 80%, 10% - 90%, 10% - 100%, 15% - 20%, 15% - 25%, 15% - 30%, 15% - 35%, 15% - 40%, 15% - 45%, 15% - 50%, 15% - 60%, 15% - 70%, 15% - 80%, 15% - 90%, 15% - 100%, 20% - 25%, 20% - 30%, 20% - 35%, 20% - 40%, 20% - 45%, 20% - 50%, 20% - 60%, 20% - 70%, 20% - 80%, 20% - 90%, 20% - 100%, 25% - 30%, 25% - 35%, 25% - 40%, 25% - 45%, 25% - 50%, 25% - 60%, 25% - 70%, 25% - 80%, 25% - 90%, 25% - 100%, 30% - 35%, 30% - 40%, 30% - 45%, 30% - 50%, 30% - 60%, 30% - 70%, 30% - 80%, 30% - 90%, 30% - 100%, 35% - 40%, 35% - 45%, 35% - 50%, 35% - 60%, 35% - 70%, 35% - 80%, 35% - 90%, 35% - 100%, 40% - 45%, 40% - 50%, 40% - 60%, 40% - 70%, 40% - 80%, 40% - 90%, 40% - 100%, 45% - 50%, 45% - 60%, 45% - 70%, 45% - 80%, 45% - 90%, 45% - 100%, 50% - 60%, 50% - 70%, 50% - 80%, 50% - 90%, 50% - 100%, 60% - 70%, 60% - 80%, 60% - 90%, 60% - 100%, 70% - 80%, 70% - 90%, 70% - 100%, 80% - 90%, 80% - 100%, or 90% - 100%.

[0108] Pharmaceutical composition The terms "pharmaceutical formulation" and "pharmaceutical composition" mean a preparation in a form that enables the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is to be administered.

[0109] "Pharmaceutically acceptable carrier" means a conventional non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier in a technique for use with a therapeutic agent that together includes a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosages and concentrations employed, is compatible with the other ingredients of the formulation, and is suitable for the formulation employed. Examples of pharmaceutically acceptable carriers include alumina, aluminum stearate, lecithin, human serum albumin, serum proteins such as albumin of dog or other animals, phosphates, citrates, tromethamine, or buffers such as HEPES buffer, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, or salts or electrolytes such as magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sucrose, mannitol, or amino acids including, but not limited to, arginine.

[0110] The pharmaceutical composition can be stored in a lyophilized form. Thus, in some embodiments, the preparation process includes a lyophilization step. The lyophilized composition can then be reformulated as an aqueous composition typically suitable for parenteral administration prior to administration to a dog, cat, or horse. In other embodiments, particularly when the antibody is highly stable to denaturation by heat and oxidation, the pharmaceutical composition can be stored as a liquid, i.e., as an aqueous composition that can be administered directly or appropriately diluted to a dog, cat, or horse. The lyophilized composition can be reconstituted with water for injection (WFI). An antibacterial agent (e.g., a bacteriostatic agent such as benzyl alcohol) may be included. Thus, the present invention provides a pharmaceutical composition in solid or liquid form.

[0111] The pH of the pharmaceutical composition may be in the range of about pH 5 to about pH 8 upon administration. The composition of the present invention is sterile when used for therapeutic purposes. The sterile state 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). The sterile state may be maintained in the presence or absence of an antibacterial agent.

[0112] In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition has a pH of 5.0 to 6.2, 5.0 to 6.0, or 5.3 to 5.7. In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition has a pH of 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, or 6.2.

[0113] In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition contains L-histidine, sodium chloride, and polysorbate 80.

[0114] In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition contains sodium chloride at a concentration of 80 nM to 200 nM, 100 nM to 180 nM, 100 nM to 175 nM, 110 nM to 170 nM, 120 nM to 160 nM, 120 nM to 150 nM, 130 nM to 150 nM, 130 nM to 160 nM, 100 nM, 80 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, or 200 nM.

[0115] In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition contains polysorbate 80 at a concentration of 0.005 mg / mL to 0.5 mg / mL, 0.01 mg / mL to 0.1 mg / mL, 0.1 mg / mL to 0.5 mg / mL, 0.005 mg / mL to 0.01 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, or 0.1 mg / mL.

[0116] In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition contains L-histidine at a concentration of 5 mM to 100 mM, 10 mM to 50 mM, 20 mM to 30 mM, 10 mM to 30 mM, 20 mM to 80 mM, 30 mM to 70 mM, 40 mM to 60 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, or 50 mM.

[0117] In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition contains m-cresol or benzyl alcohol. In some embodiments, the concentration of m-cresol is about 0.2%, about 0.1% to about 0.3%, about 0.08% to about 0.25%, or about 0.05% to about 0.25%. In some embodiments, the concentration of benzyl alcohol is about 1%, about 0.5% to about 2%, about 0.2% to about 2.5%, about 1% to about 5%, about 0.5% to about 5%, or about 1% to about 3%.

[0118] In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition contains sugar. In some embodiments, the sugar is sucrose, trehalose, D-mannitol, maltose, and / or sorbitol. In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition contains sugar at a concentration of 0.5% to 20%, 1% to 10%, 1% to 5%, 1% to 3%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%.

[0119] In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition contains an antibacterial agent. In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition contains m-cresol or methylparaben. In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition contains 0.2% m-cresol. In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition contains 0.9% methylparaben.

[0120] Use of Antibodies and Pharmaceutical Compositions The antibody or pharmaceutical composition comprising the antibody of the present invention may be useful for treating an IL31-induced condition. As used herein, "IL31-induced condition" means a disease associated with, caused by, or characterized by an elevated level or altered gradient 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, pruritus, asthma, psoriasis, scleroderma, or eczema. IL31-induced conditions may occur in companion animals including, but not limited to, dogs, cats, or horses.

[0121] As used herein, "treatment" is a procedure for obtaining a beneficial or desired clinical outcome. "Treatment" as used herein encompasses any administration or application for the treatment of a disease in a mammal, including companion animals. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, reduction in the degree of the disease, prevention or delay of disease spread, prevention or delay of disease recurrence, delay or retardation of disease progression, improvement of the medical condition, arrest of the disease or its progression, prevention or retardation of the disease or its progression, stopping its advancement, and any one or more of remission (partial or complete). Reduction of the pathological consequences of a proliferative disease is also encompassed by "treatment". The methods provided herein are intended for any one or more of these aspects of treatment. Consistent with the above, the term treatment does not require complete elimination of all aspects of the disorder.

[0122] In some embodiments, the anti-IL31 antibody or pharmaceutical composition comprising the same can be utilized according to the methods herein for treating an IL31-induced condition. In some embodiments, the anti-IL31 antibody or pharmaceutical composition is administered to a companion animal such as a dog, cat, or horse for treating an IL31-induced condition.

[0123] The "therapeutically effective amount" of a substance / molecule, agonist, or antagonist can vary depending on factors such as the type of disease to be treated, the condition, the severity and course of the disease, the type of treatment objective, past treatments if any, the medical history, the response to previous treatments, the discretion of the attending veterinarian, the age, sex, and body weight of the animal, and the ability of the substance / molecule, agonist, or antagonist to elicit the desired response from the animal. The therapeutically effective amount is also an amount at which the therapeutically beneficial effects of the substance / molecule, agonist, or antagonist exceed any toxic or harmful effects. The therapeutically effective amount may be delivered in one or more administrations. The therapeutically effective amount means an amount effective to achieve the desired therapeutic or prophylactic result at the required dosage and for the required period of time.

[0124] In some embodiments, the anti-IL31 antibody or pharmaceutical composition comprising the anti-IL31 antibody is administered parenterally, by subcutaneous administration, by intravenous infusion, or by intramuscular injection. In some embodiments, the anti-IL31 antibody or pharmaceutical composition comprising the anti-IL31 antibody is administered as a bolus injection or by continuous infusion over a period of time. In some embodiments, the anti-IL31 antibody or pharmaceutical composition comprising the anti-IL31 antibody is administered intramuscularly, intraperitoneally, intrathecally, subcutaneously, intraarterially, intraarticularly, intramedullary, or by the inhalation route.

[0125] The anti-IL31 antibodies described herein may be administered in an amount in the range of 0.1 mg / kg body weight to 100 mg / kg body weight per dose. In some embodiments, the anti-IL31 antibody may be administered in an amount in the range of 0.5 mg / kg body weight to 50 mg / kg body weight per dose. In some embodiments, the anti-IL31 antibody may be administered in an amount in the range of 1 mg / kg body weight to 10 mg / kg body weight per dose. In some embodiments, the anti-IL31 antibody may be administered in an amount in the range of 0.5 mg / kg body weight to 100 mg / kg body weight, 1 mg / kg body weight to 100 mg / kg body weight, 5 mg / kg body weight to 100 mg / kg body weight, 10 mg / kg body weight to 100 mg / kg body weight, 20 mg / kg body weight to 100 mg / kg body weight, 50 mg / kg body weight to 100 mg / kg body weight, 1 mg / kg body weight to 10 mg / kg body weight, 5 mg / kg body weight to 10 mg / kg body weight, 0.5 mg / kg body weight to 10 mg / kg body weight, or 5 mg / kg body weight to 50 mg / kg body weight per dose.

[0126] The anti-IL31 antibody or a pharmaceutical composition comprising the anti-IL31 antibody can be administered to a companion animal either once or over a series of treatments. For example, the anti-IL31 antibody or a pharmaceutical composition comprising the anti-IL31 antibody may be administered at least once, two or more times, at least twice, at least three times, at least four times, or at least five times.

[0127] In some embodiments, the dose is administered once a week for at least 2 or 3 consecutive weeks, and in some embodiments, the cycle of this treatment is repeated two or more times, optionally with one or more non-treatment weeks in between. In other embodiments, a therapeutically effective dose is administered once a day for 2 to 5 consecutive days, and in some embodiments, the cycle of this treatment is repeated two or more times, optionally with one or more non-treatment weeks in between.

[0128] Administration “in combination with” one or more additional therapeutic agents includes simultaneous (parallel) administration and sequential or successive administration in any order. The term “in parallel” as used herein means the administration of two or more therapeutic agents where at least a portion of the administrations overlap in time or where the administration of one therapeutic agent occurs within a short period of time relative to the administration of another therapeutic agent. For example, two or more therapeutic agents are administered with a time separation of approximately a specified number of minutes or less. The term “sequentially” as used herein means 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 has been interrupted or where the administration of one or more agents begins prior to the administration of one or more other agents. For example, the administration of two or more therapeutic agents is separated by a time interval of approximately more than a specified number of minutes. As used herein, “in combination with” means the administration of one mode of treatment in addition to another mode of treatment. Thus, “in combination with” means the administration of one mode of treatment before, during, or after the administration of another mode of treatment to an animal.

[0129] In some embodiments, the method includes 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 includes 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.

[0130] The present specification provides a method of exposing a cell to an anti-IL31 antibody or a pharmaceutical composition comprising the anti-IL31 antibody under conditions that permit binding of the antibody to IL31. In some embodiments, the cell is exposed to the antibody or the pharmaceutical composition ex vivo. In some embodiments, the cell is exposed to the antibody or the pharmaceutical composition in vivo. In some embodiments, the cell is exposed to the anti-IL31 antibody or the pharmaceutical composition under conditions that permit binding of the antibody to intracellular IL31. In some embodiments, the cell is exposed to the anti-IL31 antibody or the pharmaceutical composition under conditions that permit binding of the antibody to extracellular IL31. In some embodiments, the cell may be exposed in vivo to the anti-IL31 antibody or the pharmaceutical composition by any one or more of the administration methods described herein including, but not limited to, intraperitoneal, intramuscular, intravenous injection to a subject. In some embodiments, the cell may be exposed ex vivo to the anti-IL31 antibody or the pharmaceutical composition by exposing the cell to a medium comprising the antibody or the pharmaceutical composition. In some embodiments, the cell membrane permeability can be affected by the use of any number of methods understood by those skilled in the art (e.g., subjecting the cell to electroporation, or exposing the cell in a solution containing calcium chloride) prior to exposing the cell to a medium comprising the antibody or the pharmaceutical composition.

[0131] In some embodiments, the binding reduces the IL31 signaling function of the cell. In some embodiments, the IL31 antibody reduces the STAT-3 phosphorylation and can reduce the 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% as measured compared to the IL31 signaling function in the absence of the antibody.In some embodiments, the reduction of IL31 signaling function or the reduction of STAT-3 phosphorylation is 10% - 15%, 10% - 20%, 10% - 25%, 10% - 30%, 10% - 35%, 10% - 40%, 10% - 45%, 10% - 50%, 10% - 60%, 10% - 70%, 10% - 80%, 10% - 90%, 10% - 100%, 15% - 20%, 15% - 25%, 15% - 30%, 15% - 35%, 15% - 40%, 15% - 45%, 15% - 50%, 15% - 60%, 15% - 70%, 15% - 80%, 15% - 90%, 15% - 100%, 20% - 25%, 20% - 30%, 20% - 35%, 20% - 40%, 20% - 45%, 20% - 50%, 20% - 60%, 20% - 70%, 20% - 80%, 20% - 90%, 20% - 100%, 25% - 30%, 25% - 35%, 25% - 40%, 25% - 45%, 25% - 50%, 25% - 60%, 25% - 70%, 25% - 80%, 25% - 90%, 25% - 100%, 30% - 35%, 30% - 40%, 30% - 45%, 30% - 50%, 30% - 60%, 30% - 70%, 30% - 80%, 30% - 90%, 30% - 100%, 35% - 40%, 35% - 45%, 35% - 50%, 35% - 60%, 35% - 70%, 35% - 80%, 35% - 90%, 35% - 100%, 40% - 45%, 40% - 50%, 40% - 60%, 40% - 70%, 40% - 80%, 40% - 90%, 40% - 100%, 45% - 50%, 45% - 60%, 45% - 70%, 45% - 80%, 45% - 90%, 45% - 100%, 50% - 60%, 50% - 70%, 50% - 80%, 50% - 90%, 50% - 100%, 60% - 70%, 60% - 80%, 60% - 90%, 60% - 100%, 70% - 80%, 70% - 90%, 70% - 100%, 80% - 90%, 80% - 100%, or 90% - 100%.

[0132] This specification provides methods for detecting, diagnosing, and monitoring a state induced by IL31 using anti-IL31 antibodies, polypeptides, and polynucleotides. This specification provides methods for determining whether a companion animal will respond to anti-IL31 antibody treatment. In some embodiments, the method includes detecting, using an anti-IL31 antibody, whether an animal has cells that express IL31. In some embodiments, the detection method includes contacting a sample with an antibody, polypeptide, or polynucleotide and determining whether the degree 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 treatment for a subject animal.

[0133] In some embodiments, the sample is a biological sample. The term "biological sample" means an amount of substance from a living or formerly living body. In some embodiments, the biological sample is a cell or a cell / tissue lysate. In some embodiments, the biological sample includes, but is not limited to, blood (e.g., whole blood), plasma, serum, urine, synovial fluid, and epithelial cells.

[0134] In some embodiments, a cell or cell / tissue lysate is contacted with an anti-IL31 antibody and the binding between the antibody and the cell is determined. If the test cells exhibit binding activity compared to reference cells of the same tissue type, it is shown that the subject will benefit from treatment with an anti-IL31 antibody. In some embodiments, the test cells are from the tissue of a companion animal.

[0135] A variety of 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), nephelometric inhibition immunoassay (NIA), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA). The indicator portion, or label group, can be attached to the antibody of interest and is selected to match the availability of assay equipment and the need for the use of a variety of methods, which are often determined by the compatible immunoassay procedure. 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-glucosidase), fluorescent moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g., Qdot™ nanoparticles supplied by Quantum Dot Corporation, Palo Alto, Calif.). The general techniques used in performing the various immunoassays described above are known to those of ordinary skill in the art.

[0136] For diagnostic purposes, the polypeptide containing the antibody can be labeled with detectable moieties 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 employed 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, pages 147-158 (CRC Press, Inc., 1987). The anti-IL31 antibody and polypeptide can also be used in in vivo diagnostic assays such as in vivo imaging. Generally, the antibody or polypeptide is labeled with a radionuclide (e.g., 111 In, 99 Tc, 14 C, 131 I, 125 I, 3 H, or any other radionuclide label including those outlined herein) such that the location of the target cell or tissue is identified using immunoscintigraphy. Antibodies are also used as staining agents in pathology using techniques well known in the art.

[0137] In some embodiments, the first antibody is used for diagnosis and the second antibody is used for treatment. In some embodiments, the first antibody and the second antibody are different. In some embodiments, the first antibody and the second antibody can both bind to the antigen simultaneously by binding to different epitopes.

[0138] The following examples illustrate specific aspects of the disclosure and are not intended to limit the disclosure in any way.

Examples

[0139] Example 1: Identification of a Mouse Monoclonal Antibody that Binds to Canine IL31 The canine IL31 gene encoding the IL31 protein (SEQ ID NO: 22) was synthesized with a C-terminal poly-His tag and cloned into a mammalian expression vector. A plasmid having the canine IL31 gene was transfected into 293 cells.

[0140] The supernatant containing the canine IL31 protein was collected and filtered. Canine IL31 was affinity purified using a Ni-NTA column (CaptivA® Protein A Affinity Resin, Repligen).

[0141] Mouse monoclonal antibodies were identified using standard immunization with canine IL31 produced by 293 cells as an immunogen. Two different adjuvants were used during immunization (Antibody Solutions, Sunnyvale, CA), and monoclonal antibodies were obtained by standard hybridoma technology. To screen for clones producing IL31-binding antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed. First, canine IL31 was biotinylated and then introduced into streptavidin-coated wells. Then, the immunized serum was added to the wells, washed, and detected with an HRP-conjugated anti-mouse antibody. The presence of a canine IL31-binding antibody resulted in a positive signal. Among thousands of clones tested by ELISA, 170 clones with the highest binding affinity based on signal intensity were selected and further tested by a biosensor assay (Forte Bio Octet). Biotinylated canine IL31 was bound to the sensor chip, and the supernatant of hybridoma clones containing anti-canine IL31 antibodies was selected based on the slow-off rate (the rate of dissociation of the antibody and ligand). The binding affinities of the top 19 candidates were measured at a single concentration, and the antibody concentration was measured by a protein A titer assay using a Biosensor Octet and then reported as the equilibrium dissociation constant (Kd). The Kd of the top 19 candidates was less than 10 nM each.

[0142] Furthermore, each of the 170 clones having high binding activity based on ELISA was tested for neutralizing activity. To evaluate the activity of top candidates in reducing canine IL31-mediated pSTAT signaling using canine DH82 cells, a functional assay based on the cells described below in Example 4 was performed. The top four clones (M14, M18, M19, and M87) were selected for further investigation. In particular, the majority of the high-affinity binders identified by ELISA were non-functional.

[0143] Example 2: Identification of DNA Sequences Encoding VH and VL of Monoclonal Antibodies The hybridoma cells producing M14, M18, M19, and M87 were pelleted. RNA was extracted and cDNA was obtained using standard techniques with oligonucleotide primers for amplifying the mouse immunoglobulin (Ig) variable domains. The variable heavy (VH) and variable light (VL) chains of each of the four clones were sequenced and analyzed by sequence alignment (Figure 1, SEQ ID NOs: 36-43). In particular, three of the four active antibodies (M14, M18, and M19) share the same six CDR sequences, with the exception that the CDR-L1 of M18 has isoleucine at position 14 (SEQ ID NO: 63) while M14 and M19 have methionine at position 14 (SEQ ID NO: 8), and the CDR-H2 of M18 has tyrosine at position 9 (SEQ ID NOs: 62 and 87) while M14 and M19 have aspartic acid at position 14 (SEQ ID NOs: 2 and 89). The similarity of the CDR sequences suggests that M14, M18, and M19 share a common epitope.

[0144] Example 3: Expression and Purification of Mouse-Canine Chimeric and Caninized IL31 mAb M14 from CHO Cells To fuse mouse M14VH (SEQ ID NO: 25) and mouse VL (SEQ ID NO: 24) to canine constant heavy chain and canine constant light chain, a DNA sequence encoding a chimeric antibody was designed. The nucleotide sequence was chemically synthesized and inserted into an expression vector suitable for transfection into CHO host cells. After transfection into CHO cells, the light chain or heavy chain protein or both were secreted from the cells. For example, chimeric M14 using canine IgG-B was purified by single-step protein A column chromatography.

[0145] The correct canine germline antibody sequences were searched for and selected as templates for CDR grafting, and by performing protein modeling, the VH and VL of mouse M14 were caninized. Caninized M14IgG-B (SEQ ID NO: 18 and SEQ ID NO: 21) was easily expressed and was purified in a single step by a protein A column or other chromatography methods, such as ion exchange column chromatography, hydrophobic interaction column chromatography, mixed mode column chromatography such as CHT, or multimodal mode column chromatography such as CaptoMMC. Low pH or other virus inactivation and virus removal steps can be applied. The purified protein was mixed with excipients and filter sterilized to prepare the pharmaceutical composition of the present invention. The pharmaceutical composition was administered to dogs with atopic dermatitis in an amount sufficient to bind to and inhibit IL31.

[0146] Then, using the vector, a pilot scale transfection was performed in CHO-S cells using FreestyleMax (trademark) transfection reagent (Life Technologies). The supernatant was harvested by clarifying the conditioned medium. The protein was purified by a single pass protein A chromatography step and used for further study.

[0147] Example 4: Demonstration of IL31 Binding Activity This example demonstrates that the antibodies of the present invention represented by chimeric M14 (SEQ ID NO: 26 and SEQ ID NO: 27) and canineized M14 (SEQ ID NO: 18 and SEQ ID NO: 21) bind to canine IL31 with the kinetics essential for therapeutic activity.

[0148] Binding analysis was performed as follows using a Biacore Octet. Briefly, canine IL31 was biotinylated using EZ-Link™ NHS-LC-Biotin (Thermo Scientific, Catalog No. 21336) at primary amine groups or EZ-Link™ Biotin-LC-Hydrazide (Thermo Fisher Scientific, Catalog No. 21340) at glycan groups according to the manufacturer's instructions. Free unreacted biotin was removed from biotinylated IL31 by extensive dialysis. Biotinylated canine IL31 was captured on a streptavidin sensor chip. Association of chimeric and canineized M14 antibodies at four different concentrations (400, 200, 66.6, and 33 nM) with human and canine IL31 (amine-conjugate-biotin), and association of 100 nM canineized M14 antibody with canine IL31 (glycan-conjugate-biotin) were monitored for 90 seconds. Dissociation was monitored for 600 seconds. A blank curve of buffer only was subtracted to correct for drift. The data was fit to a 2:1 binding model using ForteBio™ data analysis software to determine k on 、k off 、and kd. The buffer for dilution and all binding steps was 20 mM phosphate, 150 mM NaCl, pH 7.2.

[0149] Canine IL31 having a polyHis tag at the C-terminus was expressed and purified from CHO-S cells. Human IL31 was obtained from Sino Biological and the streptavidin biosensor was obtained from ForteBio (Cat.#18-509). The binding kinetics were as follows. For canine IL31 (amine-conjugate-biotin) as the ligand, the Kd (M) of chimeric M14 was <1.0×10 -11(Figure 2), in Canine M14, <1.0×10 -11 (Figure 3). For the ligand Canine IL31 (glycan-conjugate-biotin), the Kd (M) of Canine M14 was <1.0×10 -12 , k off (1 / s) was <1.0×10 -7 .

[0150] Chimeric M14 and Canine M14 did not have an obvious binding signal to human IL31. Therefore, the Kd could not be measured.

[0151] Example 5: Demonstration that M14 Inhibits the Signaling of Canine IL31 After binding to its IL31 receptor, IL31 activates the Janus kinase (Jak) 1 and Jak2 signaling molecules. Subsequently, the activated Jak stimulates the phosphorylation of the downstream signaling STAT-3 and STAT-5. To detect the anti-IL31 activity from the purified fraction by protein A in cell-free medium, anti-phospho-Stat3 immunoblot analysis was used (Gonzales et al., Vet Dermatol, 2013, 24, 48 - e12). Briefly, Canine monocyte DH82 cells (American Type Culture Collection, Manassas, VA, USA) were seeded at 1×10 per well in MEM growth medium (Life Technologies) containing 15% heat-inactivated fetal bovine serum, 2 mmol / L GlutaMax, 1 mmol / L sodium pyruvate, and 10 ng / mL Canine interferon-c (R&D Systems, Minneapolis, MN, USA). 5Cells were seeded at a density of 37°C for 24 hours in a 96-well flat-bottom cell culture plate. In this experiment, the concentration of canine IL31-Fc was 5 ng / mL (8 nM). Anti-phospho STAT-3 antibody and anti-STAT-3 antibody were purchased from R&D Systems. Anti-β-actin antibody was obtained from Sigma-Aldrich. As shown in Figure 4, as the concentration of canine M14 exposed to the cells increased, the signal transduction of canine IL31 (as demonstrated by the reduction of phosphorylation of STAT-3) decreased (lane 1: without anti-IL31 antibody, lane 2: 3.3 nM, lane 3: 6.6 nM, lane 4: 9.9 nM, and lane 5: 13.2 nM).

[0152] Example 6: Identification of the M14 Canine IL31 Binding Epitope To identify the canine IL31 epitope recognized by M14, multiple GST canine IL31 fragment fusion molecules were generated and the proteins were expressed intracellularly in Escherichia coli (E. coli). After transferring the GST fusion proteins to the membrane, the membrane was probed with chimeric M14. A positive signal was obtained if the IL31 fragment contained the epitope.

[0153] By combining Figure 5 and Figure 6, it was demonstrated that M14 could recognize the smallest fragment (SEQ ID NO: 23).

[0154] Example 7: Demonstration that M14 Cross-Reacts with Feline IL31 To examine whether the M14 antibody recognizes feline IL31 (SEQ ID NO: 28) or equine IL31 (SEQ ID NO: 29), each protein was fused with human Fc and expressed in mammalian 293 cells. The partially purified protein was blotted onto a membrane and probed with the M14 antibody. The immunoblot in Figure 1 demonstrates that M14 binds to feline IL31. The immunoblot assay did not detect binding between M14 and equine IL31. However, by biolayer interferometry analysis, it was revealed that the M14 antibody binds to equine IL31 with low affinity. Preliminary Kd measurements using biotinylated equine IL31 immobilized on the sensor showed that the affinity (Kd) was approximately 10 - 50 nM.

[0155] Example 8: Felineized M14 The M14 variable light chain was felineized as (SEQ ID NO: 32), and the M14 variable heavy chain was felineized as (SEQ ID NO: 33). First, the correct variants of feline VH and VL were searched using mouse heavy chain variable and light chain variable sequences. The correct feline framework was selected for grafting the CDRs. These were further optimized using structural modeling. The felineized VH and VL were fused to feline IgG heavy chain constant domains (CH1, CH2, and CH3) and feline light chain constant domain (CL1).

[0156] The feline M14 chimeric antibody (SEQ ID NO: 30 and SEQ ID NO: 31) or felineized M14 antibody (SEQ ID NO: 34 and SEQ ID NO: 35) can be administered to cats for the treatment of conditions induced by IL31.

[0157] Example 9: Identification of the M14 Canine IL31 Binding Epitope To further identify the amino acid residues of the canine IL31 epitope recognized by M14, a multiple GST canine IL31 epitope fragment fusion molecule with alanine mutations was expressed in E. coli. Figures 8 - 12 show immunoblots of fine epitope mapping of canine IL31 - GST fusion proteins probed with anti - canine IL31 - mAb (M14) or canine - ized M14 (upper panel) and anti - GST antibody control (lower panel). The epitope fragments tested in each lane are listed below the immunoblot. The fragment names indicate the range of amino acids of mature canine IL31 (SEQ ID NO: 44) tested and the position of the alanine mutation if included. A positive signal occurred when the IL31 fragment contained the wild - type epitope, and a negative signal occurred when the IL31 fragment contained an alanine substitution at residues important for antibody - ligand interaction.

[0158] The results of the epitope mapping study are summarized in Table 3 below. The results suggest that P12, S13, D14, and K17 of mature canine IL31 (SEQ ID NO: 44) are involved in the binding of antibody M14, and that R16 and I18 are partially involved in the recognition by M14. Overall, the results of this study suggest that the motif of the IL31 polypeptide that binds to the CDR of antibody M14 is PSDX1X2KI, where X1 and X2 are variable (SEQ ID NO: 45). Table 3. TIFF0007716441000013.tif68170

[0159] Example 10: M14 specifically binds to IL31 of other species having the PSDX1X2KI epitope motif. The epitope motif of IL31 recognized by M14 (PSDX1X2KI, SEQ ID NO: 45) does not exist in the amino acid sequences of human (SEQ ID NO: 46) or mouse (SEQ ID NO: 61) IL31. However, this motif is present in the domestic cat (Felis catus) (XP_011286140.1; SEQ ID NO: 28) Walrus (Odobenus rosmarus divergens) (XP_004395998.1; SEQ ID NO: 47) Anubis baboon (Papio Anubis) (XP_003907358.1; SEQ ID NO: 49) Polar bear (Ursus maritimus) (XP_008687166.1; SEQ ID NO: 51) Weddell seal (Leptonychotes weddellii) (XP_006746595.1; SEQ ID NO: 52) Amur tiger (Panthera tigris altaica) (XP_007079636.1; SEQ ID NO: 53) Cheetah (Acinonyx jubatus) (XP_014919275.1; SEQ ID NO: 54) Cynomolgus macaque (Macaca fascicularis) (EHH66805.1; SEQ ID NO: 55) Rhesus macaque (Macaca mulatta) (EHH21279.1; SEQ ID NO: 56) Drill (Mandrillus leucophaeus) (XP_011819882.1; SEQ ID NO: 57) Green monkey (Chlorocebus sabaeus) (XP_008003211.1; SEQ ID NO: 58) Sooty mangabey (Cercocebus atys) (XP_011926625.1; SEQ ID NO: 59) Golden snub-nosed monkey (Rhinopithecus roxellana) (XP_010366647.1; SEQ ID NO: 60) Identified in several other species including

[0160] Seiuchi IL31 (SEQ ID NO: 47) and Anubis baboon IL31 (SEQ ID NO: 49) carry the PSDX1X2KI epitope (SEQ ID NO: 45) recognizable by antibody M14. To facilitate protein purification, a C-terminal His tag was added to Seiuchi IL31 (SEQ ID NO: 48) and Anubis baboon IL31 (SEQ ID NO: 50). Western blot analysis confirmed that M14 binds to Seiuchi IL31 (Figure 13). The M14 antibody or its derivatives can be used as therapeutic agents and diagnostic agents for diseases induced by IL31 in any of the species listed above.

[0161] Example 11: Thermal stability of the canineized M14 antibody The thermal stability of the canineized M14 antibody was analyzed over a wide range of pH using differential scanning fluorimetry (DSF) and compared with the commercially available anti-IL31 antibody, Zoetis' CYTOPOINT™. The melting points (Tm) of each antibody at various pH values are listed in Table 4 below. Both CYTOPOINT™ and the canineized M14 antibody were buffer-exchanged into the assay buffer listed in Table 4 using a PD Minitrap™ G-25 column (GE Healthcare). The Tm of each antibody was evaluated using the same buffer and protein concentration. The canineized M14 antibody showed improved thermal stability over a wide range of pH compared to CYTOPOINT™. Furthermore, CYTOPOINT™ precipitated under stress conditions of 0.22 mg / ml antibody, 55°C, for 2 days, while no precipitation was observed with the canineized M14 antibody. Table 4. TIFF0007716441000014.tif95170

[0162] Example 12: Canineized M14 antibody buffer formulation The thermal stability of the canine M14 antibody in various buffer formulations was analyzed. Buffers containing sodium phosphate, sodium acetate, or L-histidine were considered. Other formulation variables included different pH values (pH 5.2, 5.5, 6.0, 6.5, and 7.0), different sodium chloride concentrations (50 mM and 140 mM), different polysorbates (polysorbate 20 and polysorbate 80), and different antibacterial agents (m-cresol and methylparaben). The melting point (Tm) of the canine M14 antibody in each buffer was measured by differential scanning fluorimetry (DSF) in the range of 20°C to 95°C. Table 5 lists the Tm values of the canine M14 antibody in the various buffers tested. Table 5 TIFF0007716441000015.tif224170TIFF0007716441000016.tif254170TIFF0007716441000017.tif254170TIFF0007716441000018.tif138170

[0163] The Tm of formulations D1, D2, D3, D4, D5, and D6 was further tested using DSF in the presence of each of the following sugars (1%): sucrose, trehalose, D-mannitol, maltose, and sorbitol.

[0164] Formulations were tested under stress conditions of 40 °C for 1 day, then 45 °C for 1 day, and then 55 °C for 4 days, both in the presence and absence of saccharides. Size-exclusion HPLC analysis was used to detect and quantify monomeric and aggregated forms of the antibody in various formulations after being subjected to the stress conditions. Samples were loaded onto a SHODEX™ KW803 column (8 mm × 300 mm) and attached to a KW-G guard column. Using an Agilient 1100 chromatography system, 2-fold PBS (270 mM NaCl, 5.4 mM KCl, 8.6 mM Na2PO4), pH 7.2, with a constant flow rate of 0.5 mL / min was used as the running buffer. The column was calibrated using a BIORAD gel filtration standard (Catalog No. 151-1901) (molecular weight 1,350 - 670,000) consisting of thyroglobulin, bovine γ-globulin, chicken ovalbumin, horse myoglobin, and vitamin B12. The amount of monomeric antibody remaining in the solution was determined by measuring the UV absorbance at 214 nm or 280 nm and calculating the peak area under the curve.

[0165] Based on DSF and HPLC analysis, formulations such as D1, D2, D3, D4, D6, D7, D10, and D12, which contain L-histidine, sodium chloride, polysorbate 80, and have a pH of 5.0 - 6.2, were considered more desirable. For example, a formulation desirable for single-dose is 20 mM L-histidine, 140 mM sodium chloride, polysorbate 80 (0.05 mg / mL), pH 5.5, and a formulation desirable for multiple-dose (in the presence of a preservative) is 1. 20 mM L-histidine, 140 mM sodium chloride, polysorbate 80 (0.05 mg / mL), sucrose (1 - 3%), m-cresol (0.2%), pH 5.5 and 2. 20 mM L-histidine, 140 mM sodium chloride, polysorbate 80 (0.05 mg / mL), trehalose (1 - 3%), methylparaben (0.9%), pH 5.5 is.

Claims

【Claim 1】 An isolated antibody that binds to canine IL-31, which binds to an epitope consisting of the amino acid sequence of SEQ ID NO: 23.

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