Veterinary anti-il31 antibodies

Abstract

Various embodiments involving anti-IL31 antibodies having enhanced binding to canine IL31 and feline IL31 are provided. Such antibodies can be used in methods of treating IL31-induced disorders in companion animals such as canids and felines. Antibodies having enhanced binding to canine IL31 and / or feline IL31 are provided. Antibody heavy and light chains capable of forming antibodies that bind canine and feline IL31 are also provided.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 893,799, filed August 29, 2019, and U.S. Provisional Application No. 62 / 894,526, filed August 30, 2019, which are each incorporated herein by reference in their entirety for any purpose. Technical Field

[0003] This invention relates to isolated anti-IL31 antibodies, for example, having enhanced binding to canine and feline IL31; and methods of using said isolated anti-IL31 antibodies, for example, to treat IL31-induced conditions in companion animals (such as canines and felines) or to reduce IL31 signaling function in cells. Background Technology

[0004] Interleukin-31 (IL31) is a cytokine primarily produced by Th2 cells and is believed to be involved in promoting skin conditions such as pruritus and other forms of allergic disorders (e.g., atopic dermatitis). IL31 functions by binding to a receptor complex (a complex of the IL31 receptor A (IL-31Ra) and the OSMR receptor subunit) and activating downstream activities such as activation of JAK kinase and subsequent phosphorylation and activation of STAT1, STAT3, and STAT5. Activation of this pathway is thought to lead to many clinical problems associated with dermatitis and other disorders.

[0005] Companion animals (such as cats, dogs, and horses) suffer from many skin diseases similar to human skin conditions, including atopic dermatitis. However, the IL31 sequence differs among humans, cats, dogs, and horses. Therefore, there remains a need for methods and compounds that can specifically bind to IL31 in companion animals to treat IL31-induced conditions and reduce IL31 signaling. Summary of the Invention

[0006] Implementation Scheme 1. An isolated antibody that binds to canine IL31 or feline IL31, wherein the antibody comprises:

[0007] a) A heavy chain comprising a CDR-H3 sequence having the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:15; and / or

[0008] b) a light chain comprising a CDR-L1 sequence having the amino acid sequence of SEQ ID NO:20; and / or c) a light chain comprising a CDR-L3 sequence having the amino acid sequence of SEQ ID NO:23 or SEQ ID NO:24.

[0009] Implementation Scheme 2. An isolated antibody that binds to canine IL31 or feline IL31, wherein the antibody comprises:

[0010] a) a heavy chain comprising a CDR-H1 sequence having the amino acid sequence of SEQ ID NO:11, a CDR-H2 sequence having the amino acid sequence of SEQ ID NO:12, and a CDR-H3 sequence having the amino acid sequence of SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15; and / or b) a light chain comprising a CDR-L1 sequence having the amino acid sequence of SEQ ID NO:20, a CDR-L2 sequence having the amino acid sequence of SEQ ID NO:21, and a CDR-L3 sequence having the amino acid sequence of SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24.

[0011] Implementation Scheme 3. The antibody according to any one of the foregoing implementation schemes, wherein, as measured by biolayer interferometry, the antibody has a value of less than 5 x 10⁻⁶. -8 M, less than 1x10 -8 M, less than 5x10 -9 M, less than 1x10 -9 M, less than 5x10 -10 M, less than 1x10 -10 M, less than 5x10 -11 M, less than 1x10 -11 M, less than 5x10 -12 M or less than 1x10 -12 The dissociation constant (Kd) of M binds to canine IL31 or cat IL31.

[0012] Implementation Scheme 4. The antibody according to any one of the preceding implementation schemes, wherein, as measured by a reduction in STAT-3 phosphorylation, the antibody reduces IL31 signaling function in the associated animal species.

[0013] Implementation Scheme 5. The antibody according to Implementation Scheme 4, wherein the associated animal species is a canine or a feline.

[0014] Implementation Scheme 6. The antibody according to any one of the preceding implementation schemes, wherein, as determined by immunoblotting analysis and / or biolayer interference, the antibody binds to canine IL31 or feline IL31.

[0015] Implementation Scheme 7. The antibody according to any one of the preceding implementation schemes, wherein the antibody competes with the monoclonal M14 antibody for binding to canine IL31.

[0016] Implementation Scheme 8. The antibody according to any one of the foregoing implementation schemes, wherein the antibody competes with the monoclonal M14 antibody for binding to feline IL31.

[0017] Implementation Scheme 9. The antibody according to any one of the preceding implementation schemes, wherein, as determined by immunoblotting analysis and / or biolayer interferometry, the antibody does not bind to human IL31.

[0018] Implementation Scheme 10. The antibody according to any one of the foregoing implementation schemes, wherein the antibody is a monoclonal antibody.

[0019] Implementation Scheme 11. The antibody according to any one of the foregoing implementation schemes, wherein the antibody is a canine-derived, feline-derived, or chimeric antibody.

[0020] Implementation Scheme 12. The antibody according to any one of the preceding embodiments, wherein the antibody further comprises one or more of the following: (a) the variable region heavy chain framework 1 (HC-FR1) sequence of SEQ ID NO:16; (b) the HC-FR2 sequence of SEQ ID NO:17; (c) the HC-FR3 sequence of SEQ ID NO:18; (d) the HC-FR4 sequence of SEQ ID NO:19; (e) the variable region light chain framework 1 (LC-FR1) sequence of SEQ ID NO:25; (f) the LC-FR2 sequence of SEQ ID NO:26; (g) the LC-FR3 sequence of SEQ ID NO:27; or (h) the LC-FR4 sequence of SEQ ID NO:28.

[0021] Implementation Scheme 13. The antibody according to any one of Implementation Schemes 1 to 11, wherein the antibody further comprises one or more of the following: (a) a variable region heavy chain framework 1 (HC-FR1) sequence of SEQ ID NO:55 or SEQ ID NO:56; (b) an HC-FR2 sequence of SEQ ID NO:57, SEQ ID NO:58, or SEQ ID NO:59; (c) an HC-FR3 sequence of SEQ ID NO:60 or SEQ ID NO:61; (d) an HC-FR4 sequence of SEQ ID NO:62 or SEQ ID NO:63; (e) a variable region light chain framework 1 (LC-FR1) sequence of SEQ ID NO:64 or SEQ ID NO:65; (f) an LC-FR2 sequence of SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68; (g) an LC-FR3 sequence of SEQ ID NO:69 or SEQ ID NO:70; or (h) an LC-FR4 sequence of SEQ ID NO:71 or SEQ ID NO:72.

[0022] Implementation Scheme 14. The antibody according to any one of the foregoing implementation schemes, wherein the antibody comprises:

[0023] a)(i) a variable heavy chain sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; (ii) a variable light chain sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; or (iii) a variable heavy chain sequence as in (i) and a variable light chain sequence as in (ii); or

[0024] b)(i) a variable heavy chain sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequences of SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:90; (ii) a variable light chain sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the amino acid sequences of SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54; or (iii) the variable heavy chain sequence as in (i) and the variable light chain sequence as in (ii).

[0025] Implementation Scheme 15. The antibody according to any one of the preceding embodiments, wherein the antibody comprises a variable heavy chain sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 or SEQ ID NO:90.

[0026] Implementation Scheme 16. An antibody according to any one of the preceding embodiments, wherein the antibody comprises a variable light chain sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54.

[0027] Implementation Scheme 17. The antibody according to any one of the foregoing embodiments, wherein the antibody comprises:

[0028] a) SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:8 of a variable heavy chain; and SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11 of a variable light chain; or b) SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 or SEQ ID NO:90 of a variable heavy chain; and SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54 of a variable light chain.

[0029] Implementation Scheme 18. The antibody according to any one of the preceding embodiments, wherein the antibody comprises a canine or cat constant heavy chain region and / or a canine or cat constant light chain region.

[0030] Implementation Scheme 19. An antibody according to any one of the preceding embodiments, wherein the antibody comprises (a) a canine heavy chain constant region selected from the constant regions of IgG-A, IgG-B, IgG-C and IgG-D; or (b) a feline heavy chain constant region selected from the constant regions of IgG1, IgG2a and IgG2b.

[0031] Implementation Scheme 20. The antibody according to any one of the foregoing embodiments, wherein the antibody comprises:

[0032] a)(i) the heavy chain amino acid sequence of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 or SEQ ID NO:45; (ii) the light chain amino acid sequence of SEQ ID NO:46, SEQ ID NO:47 or SEQ ID NO:48; or (iii) the heavy chain amino acid sequence as in (i) and the light chain amino acid sequence as in (ii); or

[0033] b)(i) the heavy chain amino acid sequence of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75 or SEQ ID NO:91; (ii) the light chain amino acid sequence of SEQ ID NO:76, SEQ ID NO:77 or SEQ ID NO:78; or (iii) the heavy chain amino acid sequence as in (i) and the light chain amino acid sequence as in (ii).

[0034] Implementation Scheme 21. An isolated antibody comprising the variable heavy chain amino acid sequence of SEQ ID NO:3 and / or the variable light chain amino acid sequence of SEQ ID NO:4.

[0035] Implementation Scheme 22. An isolated antibody comprising a variable heavy chain amino acid sequence of SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.

[0036] Implementation Scheme 23. An isolated antibody comprising a variable light chain amino acid sequence of SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10.

[0037] Implementation Scheme 24. The isolated antibody according to Implementation Scheme 21, wherein the antibody comprises a variable light chain amino acid sequence of SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10.

[0038] Implementation Scheme 25. An isolated antibody comprising a variable heavy chain amino acid sequence of SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 or SEQ ID NO:90.

[0039] Implementation Scheme 26. An isolated antibody comprising a variable light chain amino acid sequence of SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54.

[0040] Implementation Scheme 27. An isolated antibody according to any one of Implementation Schemes 1 to 25, wherein the antibody comprises a variable light chain amino acid sequence of SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54.

[0041] Implementation Scheme 28. The antibody according to any one of the preceding implementation schemes, wherein the antibody is an antibody fragment selected from Fv, scFv, Fab, Fab', F(ab')2 and Fab'-SH.

[0042] Implementation Scheme 29. An antibody according to any one of the preceding implementation schemes, wherein the antibody is a bispecific antibody, wherein 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.

[0043] Implementation Scheme 30. A nucleic acid encoding an antibody isolated according to any one of Implementation Schemes 1 to 29.

[0044] Implementation Scheme 31. A host cell comprising the nucleic acid described in Implementation Scheme 30.

[0045] Implementation Scheme 32. A method for generating antibodies, the method comprising culturing host cells according to Implementation Scheme 31 and isolating the antibodies.

[0046] Implementation Scheme 33. A pharmaceutical composition comprising an antibody according to any one of Implementation Schemes 1 to 29 and a pharmaceutically acceptable carrier.

[0047] Implementation Scheme 34. A method for treating a companion animal species suffering from IL31-induced disease, the method comprising administering to the companion animal species a therapeutically effective amount of an antibody according to any one of Implementation Schemes 1 to 29 or a pharmaceutical composition according to Implementation Scheme 33.

[0048] Implementation Scheme 35. The method according to Implementation Scheme 33, wherein the associated animal species is a canine or a feline.

[0049] Implementation Scheme 36. The method according to Implementation Scheme 34 or Implementation Scheme 35, wherein the IL31-induced condition is an pruritus condition or an allergic condition.

[0050] Implementation Scheme 37. The method according to any one of Implementation Schemes 34 to 36, wherein the IL31-induced condition is selected from atopic dermatitis, allergic dermatitis, pruritus, asthma, psoriasis, scleroderma, and eczema.

[0051] Implementation Scheme 38. The method according to any one of Implementation Schemes 34 to 37, wherein the antibody or the pharmaceutical composition is administered parenterally.

[0052] Implementation Scheme 39. The method according to any one of Implementation Schemes 34 to 38, wherein the antibody or the pharmaceutical composition is administered via an intramuscular, intraperitoneal, intraspinal, subcutaneous, intraarterial, intrasynovial, intrathecal, or inhalation route.

[0053] Implementation Scheme 40. The method according to any one of Implementation Schemes 34 to 39, wherein the method comprises administering a Jak inhibitor, PI3K inhibitor, AKT inhibitor, or MAPK inhibitor in combination with the antibody or the pharmaceutical composition.

[0054] Implementation Scheme 41. The method according to any one of Implementation Schemes 34 to 40, wherein the method comprises administering, in combination with the antibody or the pharmaceutical composition, one or more antibodies selected from the group consisting of: 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.

[0055] Implementation Scheme 42. A method for reducing IL31 signaling function in cells, the method comprising exposing the cells to an antibody according to any one of Implementation Schemes 1 to 29 or a pharmaceutical composition according to Implementation Scheme 33, under conditions allowing the antibody to bind to extracellular IL31, thereby reducing the binding of the cells to the IL31 receptor and / or reducing the IL31 signaling function of the cells.

[0056] Implementation Scheme 43. The method according to Implementation Scheme 42, wherein the cells are exposed in vitro to the antibody or the pharmaceutical composition.

[0057] Implementation Scheme 44. The method according to Implementation Scheme 42, wherein the cells are exposed in vivo to the antibody or the pharmaceutical composition.

[0058] Implementation Scheme 45. The method according to any one of Implementation Schemes 42 to 44, wherein the cell is a canine cell or a cat cell.

[0059] Implementation Scheme 46. A method for detecting IL31 in a sample from a companion animal species, the method comprising contacting the sample with an antibody according to any one of Implementation Schemes 1 to 29 or a pharmaceutical composition according to Implementation Scheme 33 under conditions that allow the antibody to bind to IL31, and detecting in the sample whether a complex is formed between the antibody and IL31.

[0060] Implementation Scheme 47. The method according to Implementation Scheme 46, wherein the sample is a biological sample obtained from a canine or feline animal.

[0061] Implementation Scheme 48. A method for identifying an IL31 antagonist, the method comprising contacting an engineered cell line with an IL31 antagonist candidate, wherein the engineered cell line is a mammalian cell line not derived from canines or felines, and wherein the engineered cell line expresses canine IL31Ra and / or feline IL31Ra.

[0062] Implementation Scheme 49. The method according to Implementation Scheme 48, wherein the engineered cell line is the HeLa cell line.

[0063] Implementation Scheme 50. The method according to Implementation Scheme 48 or 49, wherein the engineered cell line expresses canine IL31Ra or feline IL31Ra.

[0064] Implementation Scheme 51. The method according to any one of Implementation Schemes 48 to 50, wherein the engineered cell line expresses a polypeptide having an amino acid sequence having SEQ ID NO:92 or SEQ ID NO:93.

[0065] Implementation Scheme 52. The method according to any one of Implementation Schemes 48 to 51, wherein the engineered cell line does not express canine or feline oncogene M receptor (OSMR).

[0066] Implementation Scheme 53. The method according to any one of Implementation Schemes 48 to 52, wherein the IL31 antagonist candidate is an IL31 antibody, a soluble IL31 receptor, an IL31Ra antibody, or a small molecule, aptamer, or peptide.

[0067] Implementation Scheme 54. The method according to any one of Implementation Schemes 48 to 53, wherein the method includes measuring the signal conduction function of IL31.

[0068] Implementation Scheme 55. The method according to Implementation Scheme 54, wherein the IL31 signal transduction function is measured by the phosphorylation levels of STAT-1, STAT-3 and / or STAT-5.

[0069] Implementation Scheme 56. The method according to any one of Implementation Schemes 48 to 55, wherein the IL31 antagonist candidate is identified by detecting a decrease in STAT-1, STAT-3 and / or STAT-5 phosphorylation. Attached Figure Description

[0070] Figure 1 The analysis was based on the binding of six chimeric antibodies to feline IL-31 according to Example 2.

[0071] Sequence Description

[0072] Table 1 provides a list of some of the sequences mentioned in this article.

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082] Detailed Implementation

[0083] Antibodies with enhanced binding to canine and / or feline IL31 are provided. Antibody heavy and light chains capable of forming antibodies binding to canine and feline IL31 are also provided. Furthermore, antibodies, heavy and light chains containing one or more specific complementarity-determining regions (CDRs) are provided. Polynucleotides encoding antibodies against canine and feline IL31 are provided. Methods for producing or purifying antibodies against canine and feline IL31 are also provided. Treatment methods using antibodies against canine and feline IL31 are provided. Such methods include, but are not limited to, methods for treating IL31-induced symptoms in companion animal species. Methods for detecting IL31 in samples from companion animal species are provided.

[0084] For the convenience of readers, the following definitions of the terms used in this article are provided.

[0085] As used herein, numerical terms (such as Kd) are based on scientific measurements and are therefore subject to appropriate measurement errors. In some cases, numerical terms may include values ​​rounded to the nearest significant digit.

[0086] As used herein, “a / an” means “at least one / an” or “one / an or more / an”, unless otherwise stated. As used herein, the term “or” means “and / or”, unless otherwise stated. In the context of multiple dependent claims, when referring to other claims, the use of “or” refers only to those claims in the alternatives.

[0087] Example anti-IL31 antibody

[0088] Antibodies with enhanced affinity for canine and feline IL31 are provided. The anti-IL31 antibodies provided herein include, but are not limited to, monoclonal antibodies, chimeric antibodies, canine-derived antibodies, and feline-derived antibodies.

[0089] This document also provides amino acid sequences of mature antibodies with enhanced affinity for canine and feline IL-31 binding. For example, variable heavy chain CDRs (SEQ ID NO: 11-15), variable light chain CDRs (SEQ ID NO: 20-24), variable region heavy chain framework sequences (SEQ ID NO: 16-19, 25-28, 55-72), and variable region light chain framework sequences (SEQ ID NO: 11-14) of exemplary mature antibodies (canine and feline). Exemplary amino acid sequences (e.g., SEQ ID NO: 5-10, 29-54, 73-78) of the variable light chain, light chain, variable heavy chain, and heavy chain of exemplary mature antibodies (canine and feline) are also provided.

[0090] The term "antibody" is used in the broadest sense and includes a wide variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies such as bispecific T-cell adaptors and trispecific antibodies), and antibody fragments (e.g., Fab, F(ab')2, ScFv, microantibodies, biantibodies, triantibodies, and tetraantibodies), as long as they exhibit the desired antigen-binding activity. Dogs, cats, and equines possess different types (categories) of antibodies common to many mammals.

[0091] The term antibody includes, but is not limited to, fragments capable of binding antigens, such as Fv, single-chain Fv (scFv), Fab, Fab', di-scFv, sdAb (single-domain antibody), and F(ab')2 (including chemically linked F(ab')2). Papain digestion of an antibody yields two identical antigen-binding fragments, referred to as "Fab" fragments, each having a single antigen-binding site; and a residual "Fc" fragment, the name reflecting its ease of crystallization. Pepsin treatment yields an F(ab')2 fragment having two antigen-binding sites and still capable of cross-linking the antigen. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies from various species, such as mice, humans, cynomolgus monkeys, canines, felines, horses, etc. Furthermore, for all antibody constructs provided herein, variants with sequences from other organisms are also considered. Therefore, if a mouse-form antibody is disclosed, those skilled in the art will understand how to convert mouse-sequence-based antibodies into cat, dog, horse, etc. sequences. Antibody fragments also include orientations of single-chain scFvs, tandem di-scFvs, double-chain antibodies, tandem tri-sdcFvs, microantibodies, etc. Antibody fragments also include nanobodies (sdAbs, antibodies having a single monomeric domain, such as a pair of heavy chain variable domains without a light chain). In some embodiments, the antibody fragment may be referred to as species-specific (e.g., mouse scFv or canine scFv). This indicates a sequence of at least a portion of the non-CDR region, rather than the source of the construct. In some embodiments, the antibody comprises a label or is conjugated to a second portion.

[0092] The terms "label" and "detectable label" refer to a portion attached to an antibody or its analyte to make the reaction (e.g., binding) between members of a specific binding pair detectable. The labeled member of the specific binding pair is referred to as "detectably labeled." Therefore, the term "labeled binding protein" refers to a protein incorporating a label provided for identifying the binding protein. In some embodiments, the label is a detectable label capable of generating a signal detectable by visual or instrumental means, such as a polypeptide incorporating a radiolabeled amino acid or attached to a biotinylated moiety, the biotinylated portion of which can be detected by labeled avidin (e.g., streptomycin containing a fluorescent label or enzyme activity detectable by optical or colorimetric methods). Examples of labeling polypeptides include, but are not limited to, 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); chromogen; fluorescent label (e.g., FITC, rhodamine, lanthanide phosphor); enzyme label (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent label; biotinylate group; predetermined polypeptide epitope recognized by a second reporter (e.g., leucine zipper pair sequence, binding site of secondary antibody, metal-binding domain, epitope tag); and magnetic agent (e.g., gadolinium chelate). Representative examples of labels commonly used in immunoassays include light-generating portions, such as acridinium compounds; and fluorescence-generating portions, such as fluorescein. In this respect, the portion itself may not be detectably labeled, but may become detectable upon reaction with another portion.

[0093] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, a single antibody that is identical to the rest of the population except for a small number of potentially naturally occurring mutations. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, unlike polyclonal antibody formulations, which typically consist of different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. Therefore, a sample of a monoclonal antibody can bind to the same epitope on the antigen. The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies can be prepared by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or by recombinant DNA methods (as described in U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage libraries, for example, using techniques described in McCafferty et al., 1990, Nature 348:552-554.

[0094] "Amino acid sequence" refers to the sequence of amino acid residues in a peptide or protein. The terms "peptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may contain native or non-native amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and polymers of amino acid residues. The definition covers both full-length proteins and fragments thereof. The term also includes post-expression modifications of peptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. Furthermore, for the purposes of this disclosure, "peptide" refers to a protein that includes modifications to the native sequence (such as deletions, additions, and substitutions, which are generally conserved in nature) as long as the protein retains the desired activity. These modifications may be intentional (e.g., by site-directed mutagenesis) or accidental (e.g., by mutations in the host that produces the protein or by errors due to PCR amplification).

[0095] As used herein, “IL31” refers to any naturally occurring IL31 produced by the expression and processing of IL31 in cells. Unless otherwise indicated, the term includes IL31 from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats) and symbiotic animals (e.g., dogs, cats, and horses). The term also includes naturally occurring IL31 variants, such as splice variants or allelic variants.

[0096] In some embodiments, canine IL31 comprises the amino acid sequence of SEQ ID NO:79 or SEQ ID NO:80. In some embodiments, feline IL31 comprises the amino acid sequence of SEQ ID NO:81. In some embodiments, mouse IL31 comprises the amino acid sequence of SEQ ID NO:82.

[0097] The term "IL31 binding domain" in antibody refers to the IL31 binding domain formed by the light and heavy chains of an anti-IL31 antibody.

[0098] In some embodiments, the IL31 binding domain binds canine IL31 with a greater affinity than it binds to human or cat IL31. In some embodiments, the IL31 binding domain binds to IL31 from other associated animals (such as horse IL31). In some embodiments, the IL31 binding domain does not bind to human IL31.

[0099] As used herein, the term "epitope" refers to a site on which an antigen-binding molecule (e.g., an antibody, antibody fragment, or scaffold protein containing an antibody-binding region) binds to a target molecule (e.g., an antigen, such as a protein, nucleic acid, carbohydrate, or lipid). Epitopes typically comprise chemically active surface groups of a molecule (e.g., amino acids, polypeptides, or sugar side chains) and possess specific three-dimensional structural features and specific charge characteristics. Epitopes can be formed from consecutive or juxtaposed discontinuous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) of the target molecule. Epitopes formed from consecutive residues (e.g., amino acids, nucleotides, sugars, lipid moieties) are generally retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents. Epitopes can include, but are not limited to, at least 3, at least 5, or 8-10 residues (e.g., amino acids or nucleotides). In some examples, epitopes are less than 20 residues (e.g., amino acids or nucleotides), less than 15 residues, or less than 12 residues in length. If two antibodies exhibit competitive binding to a certain antigen, they can bind to the same epitope within the antigen. In some embodiments, epitopes can be identified by a certain minimum distance from CDR residues on an antigen-binding molecule. In some embodiments, epitopes can be identified by the aforementioned distance and are further limited to those residues involved in the bonding (e.g., hydrogen bonding) between antibody residues and antigen residues. Epitopes can also be identified by various scans, such as alanine or arginine scans, which can indicate one or more residues that the antigen-binding molecule can interact with. Unless explicitly stated otherwise, a set of residues that constitutes an epitope does not exclude other residues that are part of an epitope for a particular antibody. Rather, the presence of such a set represents a minimal series (or species group) of epitopes. Thus, in some embodiments, a set of residues identified as an epitope represents a minimal epitope associated with the antigen, rather than an exclusive list of residues on the antigen that constitute an epitope.

[0100] The term "CDR" refers to a complementary determination region defined by at least one identification method known to those skilled in the art. In some embodiments, a CDR may be defined according to a Chothia numbering scheme, a Kabat numbering scheme, a combination of Kabat and Chothia, an AbM definition, a contact definition, or a combination of Kabat, Chothia, AbM, or a contact definition. Various CDRs within an antibody may be represented by their appropriate number and chain type, including but not limited to CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. As used herein, the term "CDR" also encompasses "hypervariant region" or HVR, including hypervariant loops.

[0101] In some implementations, the anti-IL31 antibody comprises:

[0102] a) A heavy chain comprising a CDR-H3 sequence having the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:15; and / or

[0103] b) A light chain comprising a CDR-L1 sequence having the amino acid sequence of SEQ ID NO:20; and / or

[0104] c) A light chain comprising a CDR-L3 sequence having an amino acid sequence of SEQ ID NO:23 or SEQ ID NO:24.

[0105] In some implementations, the anti-IL31 antibody comprises:

[0106] a) A heavy chain comprising a CDR-H1 sequence having the amino acid sequence of SEQ ID NO:11, a CDR-H2 sequence having the amino acid sequence of SEQ ID NO:12, and a CDR-H3 sequence having the amino acid sequence of SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15; and / or

[0107] b) A light chain comprising a CDR-L1 sequence having the amino acid sequence of SEQ ID NO:20, a CDR-L2 sequence having the amino acid sequence of SEQ ID NO:21, and a CDR-L3 sequence having the amino acid sequences of SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24.

[0108] As used herein, the term "variable region" refers to a region containing at least three CDRs. In some embodiments, the variable region includes three CDRs and at least one frame region ("FR"). The terms "heavy chain variable region" or "variable heavy chain" are used interchangeably and refer to a region containing at least three heavy chain CDRs. The terms "light chain variable region" or "variable light chain" are used interchangeably and refer to a region containing at least three light chain CDRs. In some embodiments, the variable heavy chain or variable light chain contains at least one frame region. In some embodiments, the antibody contains at least one heavy chain frame region selected from HC-FR1, HC-FR2, HC-FR3, and HC-FR4. In some embodiments, the antibody contains at least one light chain frame region selected from LC-FR1, LC-FR2, LC-FR3, and LC-FR4. The frame regions may be juxtaposed between light chain CDRs or between heavy chain CDRs. For example, an antibody may contain a variable heavy chain having the following structure: (HC-FR1)-(CDR-H1)-(HC-FR2)-(CDR-H2)-(HC-FR3)-(CDR-H3)-(HC-FR4). An antibody may contain a variable heavy chain having the following structure: (CDR-H1)-(HC-FR2)-(CDR-H2)-(HC-FR3)-(CDR-H3). An antibody may also contain a variable light chain having the following structure: (LC-FR1)-(CDR-L1)-(LC-FR2)-(CDR-L2)-(LC-FR3)-(CDR-L3)-(LC-FR4). An antibody may also contain a variable light chain having the following structure: (CDR-L1)-(LC-FR2)-(CDR-L2)-(LC-FR3)-(CDR-L3).

[0109] In some embodiments, the anti-IL31 antibody comprises one or more of the following: (a) the variable region heavy chain framework 1 (HC-FR1) sequence of SEQ ID NO:16; (b) the HC-FR2 sequence of SEQ ID NO:17; (c) the HC-FR3 sequence of SEQ ID NO:18; (d) the HC-FR4 sequence of SEQ ID NO:19; (e) the variable region light chain framework 1 (LC-FR1) sequence of SEQ ID NO:25; (f) the LC-FR2 sequence of SEQ ID NO:26; (g) the LC-FR3 sequence of SEQ ID NO:27; or (h) the LC-FR4 sequence of SEQ ID NO:28.

[0110] In some embodiments, the anti-IL31 antibody comprises one or more of the following: (a) the variable region heavy chain framework 1 (HC-FR1) sequence of SEQ ID NO:55 or SEQ ID NO:56; (b) the HC-FR2 sequence of SEQ ID NO:57, SEQ ID NO:58, or SEQ ID NO:59; (c) the HC-FR3 sequence of SEQ ID NO:60 or SEQ ID NO:61; (d) the HC-FR4 sequence of SEQ ID NO:62 or SEQ ID NO:63; (e) the variable region light chain framework 1 (LC-FR1) sequence of SEQ ID NO:64 or SEQ ID NO:65; (f) the LC-FR2 sequence of SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68; (g) the LC-FR3 sequence of SEQ ID NO:69 or SEQ ID NO:70; or (h) the LC-FR4 sequence of SEQ ID NO:71 or SEQ ID NO:72.

[0111] As used herein, the term "constant region" refers to a region containing at least three constant domains. The terms "heavy chain constant region" or "constant heavy chain" are used interchangeably and refer to 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 antibody isotype. 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. Certain isotypes 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 refer to the region containing the light chain constant domain CL. Non-limiting exemplary light chain constant regions include λ and κ. Unless otherwise indicated, the absence and alteration of non-functional changes within the domain are included within the scope of the term "constant region." Dogs, cats, and horses have antibody classes such as IgG, IgA, IgD, IgE, and IgM. Among the canine IgG antibody classes are IgG-A, IgG-B, IgG-C, and IgG-D. Among the feline IgG antibody classes are IgG1a, IgG1b, and IgG2.

[0112] The term "chimeric antibody" or "chimerism" refers to an antibody in which a portion of the heavy or light chain originates from a specific source or species, while at least a portion of the remaining portion of the heavy or light chain originates from a different source or species. In some embodiments, a chimeric antibody refers to an antibody comprising at least one variable region from a first species (such as a mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (such as a human, dog, cat, horse, etc.).

[0113] In some embodiments, the anti-IL31 antibody comprises a canine heavy chain constant region selected from the constant regions of IgG-A, IgG-B, IgG-C, and IgG-D. In some embodiments, the anti-IL31 antibody is (a) a canine IgG-A antibody comprising the heavy chain amino acid sequence of SEQ ID NO:83; (b) a canine IgG-B antibody comprising the heavy chain amino acid sequence of SEQ ID NO:84; (c) a canine IgG-C antibody comprising the heavy chain amino acid sequence of SEQ ID NO:85; or (d) a canine IgG-D antibody comprising the heavy chain amino acid sequence of SEQ ID NO:86.

[0114] In some implementations, the anti-IL31 antibody contains a cat heavy chain constant region selected from the constant regions of IgG1, IgG2a, and IgG2b.

[0115] "Canine-derived antibody" refers to an antibody in which at least one amino acid in a portion of a non-canine variable region has been replaced by a corresponding amino acid from a canine variable region. In some embodiments, the canine-derived 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, etc.) or a fragment thereof. In some embodiments, the canine-derived antibody is an antibody fragment such as Fab, scFv, (Fab')2, etc. The term "canine-derived" also refers to the form of a non-canine (e.g., mouse) antibody as a chimeric immunoglobulin, an immunoglobulin chain, or a fragment thereof containing the minimal sequence of a non-canine immunoglobulin (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of an antibody). Canine-derived antibodies may include canine immunoglobulins (recipient antibodies) in which residues of the recipient's CDR are replaced by residues of a CDR from a non-canine species (donor antibody) (e.g., mouse, rat, or rabbit) having the desired specificity, affinity, and ability. In some cases, the Fv framework region (FR) residues of the canine immunoglobulin are replaced by corresponding non-canine residues. In addition, the canine-derived antibody may contain residues that are not found in the recipient antibody or in the introduced CDR or frame sequence, but are included to further improve and optimize antibody performance.

[0116] In some implementations, the canine-derived variable chain is fused with a canine constant heavy chain or a canine constant light chain.

[0117] "Cat-derived antibody" refers to an antibody in which at least one amino acid in a portion of a non-cat variable region has been replaced by a corresponding amino acid from a cat variable region. In some embodiments, the cat-derived antibody comprises at least one cat constant region (e.g., γ constant region, α constant region, δ constant region, ε constant region, μ constant region, etc.) or a fragment thereof. In some embodiments, the cat-derived antibody is an antibody fragment such as Fab, scFv, F(ab')2. The term "cat-derived" also refers to the form of a non-cat (e.g., mouse) antibody as a chimeric immunoglobulin, an immunoglobulin chain, or a fragment thereof containing the minimal sequence of a non-cat immunoglobulin (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding sequence of an antibody). Cat-derived antibodies may comprise cat immunoglobulins (recipient antibodies) in which residues of the CDR from the recipient are replaced by residues of the CDR from a non-cat species (donor antibody) (such as mouse, rat, or rabbit) having the desired specificity, affinity, and capability. In some cases, the Fv framework region (FR) residues of the cat immunoglobulin are replaced by corresponding non-cat residues. In addition, the cat-derived antibody may contain residues that are not found in the recipient antibody or in the imported CDR or frame sequence, but are included to further improve and optimize antibody performance.

[0118] In some embodiments, the feline-derived variable chain is fused with a feline constant heavy chain or a feline constant light chain. The term "IgX Fc" indicates that the Fc region is derived from a specific antibody isotype (e.g., IgG, IgA, IgD, IgE, IgM, etc.), where "X" represents the antibody isotype. Therefore, "IgG Fc" represents the Fc region of the γ chain, "IgA Fc" represents the Fc region of the α chain, "IgD Fc" represents the Fc region of the δ chain, "IgE Fc" represents the Fc region of the ε chain, "IgM Fc" represents the Fc region of the μ chain, and so on. In some embodiments, the IgG Fc region comprises CH1, a hinge, CH2, CH3, and CL1. "IgX-N-Fc" indicates that the Fc region is derived from a specific subtype of the antibody isotype (e.g., canine IgG subtypes A, B, C, or D; or feline IgG subtypes 1, 2a, or 2b, etc.), where "N" represents the subtype. In some implementations, the IgXFc or IgX-N-Fc region is derived from a companion animal (such as a dog, cat, or horse). In some implementations, the IgG Fc region is isolated from canine γ-heavy chains (such as IgG-A, IgG-B, IgG-C, or IgG-D). In some cases, the IgG Fc region is isolated from feline γ-heavy chains (such as IgG1, IgG2a, or IgG2b). Antibodies containing Fc regions of IgG-A, IgG-B, IgG-C, or IgG-D can provide higher expression levels in recombinant production systems.

[0119] The term "affinity" refers to the strength of the sum of 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 can generally be determined by the dissociation constant (K). D Affinity can be measured using conventional methods known in the art, such as immunoblotting, ELISAKD, KinEx A, biolayer interferometry (BLI), or surface plasmon resonance devices.

[0120] Term "K" D “K” d "Kd" or "Kd value" are used interchangeably and refer to the equilibrium dissociation constant of antibody-antigen interactions. In some implementations, according to the supplier's instructions, biosensors (such as...) are used. The system (Pall Forte Bio LLC, Fremont, California) measures the K-value of antibodies using biolayer interferometry. d In short, biotinylated antigens were bound to the sensor tip, and antibody association was monitored for 90 seconds, followed by dissociation for 600 seconds. The buffer used for dilution and binding steps was 20 mM phosphate, 150 mM NaCl, pH 7.2. A blank curve containing only buffer was subtracted to correct for any drift. Data were fitted to a 2:1 binding model using ForteBio data analysis software to determine the association rate constant (k). on ), dissociation rate constant (k off ) and K d The equilibrium dissociation constant (K) d ) is calculated as k off / k on The ratio. The term "kon" refers to the rate constant of antibody-antigen association, while the term "koff" refers to the rate constant of antibody-antigen dissociation.

[0121] The term "binding" of an antigen or epitope is well known in the art, as are the methods for determining such binding. A molecule is said to "bind" if it reacts, associates, or has an affinity for a particular cell or substance, and said reaction, association, or affinity can be detected by one or more methods known in the art (e.g., like Western blotting, ELISA KD, KinEx A, biolayer interferometry (BLI), surface plasmon resonance devices, etc.).

[0122] "Surface plasmon resonance" refers to an optical phenomenon that allows for the analysis of real-time, biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using BIAcore. TMThe system (BIAcore International AB, GE Healthcare, Uppsala, Sweden and Piscataway, New Jersey). For further description, see Jonsson et al. (1993) Ann. Biol. Clin. 51:19-26.

[0123] "Biofilm interferometry" refers to an optical analysis technique that analyzes the interference pattern of light reflected from an immobilized protein layer on a biosensor tip and an internal reference layer. Changes in the number of molecules bound to the biosensor tip cause a shift in the interference pattern that can be measured in real time. A non-limiting exemplary device for biofilm interferometry is... The system (Pall ForteBio LLC). See, for example, Abdiche et al., 2008, Anal. Biochem. 377:209-277.

[0124] In some implementations, such as those measured by biolayer interferometry, anti-IL31 antibodies bind to canine or feline IL31 with a dissociation constant (Kd) of less than 5 x 10⁻⁶. -6 M, less than 1x10 -6 M, less than 5x10 -7 M, less than 1x10 -7 M, less than 5x10 -8 M, less than 1x10 -8 M, less than 5x10 -9 M, less than 1x10 -9 M, less than 5x10 -10 M, less than 1x10 -10 M, less than 5x10 -11 M, less than 1x10 -11 M, less than 5x10 -12 M or less than 1x10 -12 M. In some implementations, such as those measured by biolayer interferometry, the anti-IL31 antibody binds to canine IL31, feline IL31, or equine IL31 at the following Kd: 5 x 10 -6 M and 1x10 -6 Between M, 5x10 -6 M and 5x10 -7 Between M, 5x10 -6 M and 1x10 -7 Between M, 5x10 -6 M and 5x10 -8 Between M, 5x10 -6 M and 1x10 -8 Between M, 5x10 -6 M and 5x10 -9Between M, 5x10 -6 M and 1x10 -9 Between M, 5x10 -6 M and 5x10 -10 Between M, 5x10 -6 M and 1x10 -10 Between M, 5x10 -6 M and 5x10 -11 Between M, 5x10 -6 M and 1x10 -11 Between M, 5x10 -6 M and 5x10 -12 Between M, 5x10 -6 M and 1x10 -12 Between M, 1x10 -6 M and 5x10 -7 Between M, 1x10 -6 M and 1x10 -7 Between M, 1x10 -6 M and 5x10 -8 Between M, 1x10 -6 M and 1x10 -8 Between M, 1x10 -6 M and 5x10 -9 Between M, 1x10 -6 M and 1x10 -9 Between M, 1x10 -6 M and 5x10 -10 Between M, 1x10 -6 M and 1x10 -10 Between M, 1x10 -6 M and 5x10 -11 Between M, 1x10 -6 M and 1x10 -11 Between M, 1x10 -6 M and 5x10 -12 Between M, 1x10 -6 M and 1x10 -12 Between M, 5x10 -7 M and 1x10 -7 Between M, 5x10 -7 M and 5x10 -8 Between M, 5x10 -7 M and 1x10 -8 Between M, 5x10 -7 M and 5x10 -9 Between M, 5x10 -7 M and 1x10 -9 Between M, 5x10 -7 M and 5x10 -10Between M, 5x10 -7 M and 1x10 - 10 Between M, 5x10 -7 M and 5x10 -11 Between M, 5x10 -7 M and 1x10 -11 Between M, 5x10 -7 M and 5x10 -12 Between M, 5x10 -7 M and 1x10 -12 Between M, 1x10 -7 M and 5x10 -8 Between M, 1x10 -7 M and 1x10 -8 Between M, 1x10 -7 M and 5x10 -9 Between M, 1x10 -7 M and 1x10 -9 Between M, 1x10 -7 M and 5x10 -10 Between M, 1x10 -7 M and 1x10 -10 Between M, 1x10 -7 M and 5x10 -11 Between M, 1x10 -7 M and 1x10 -11 Between M, 1x10 -7 M and 5x10 -12 Between M, 1x10 -7 M and 1x10 -12 Between M, 5x10 -8 M and 1x10 -8 Between M, 5x10 -8 M and 5x10 -9 Between M, 5x10 -8 M and 1x10 -9 Between M, 5x10 -8 M and 5x10 -10 Between M, 5x10 -8 M and 1x10 -10 Between M, 5x10 -8 M and 5x10 -11 Between M, 5x10 -8 M and 1x10 -11 Between M, 5x10 -8 M and 5x10 -12 Between M, 5x10 -8 M and 1x10 -12 Between M, 1x10 -8 M and 5x10-9 Between M, 1x10 -8 M and 1x10 -9 Between M, 1x10 -8 M and 5x10 -10 Between M, 1x10 -8 M and 1x10 -10 Between M, 1x10 -8 M and 5x10 -11 Between M, 1x10 -8 M and 1x10 -11 Between M, 1x10 -8 M and 5x10 -12 Between M, 1x10 -8 M and 1x10 -12 Between M, 5x10 -9 M and 1x10 -9 Between M, 5x10 -9 M and 5x10 -10 Between M, 5x10 -9 M and 1x10 -10 Between M, 5x10 -9 M and 5x10 -11 Between M, 5x10 -9 M and 1x10 -11 Between M, 5x10 -9 M and 5x10 -12 Between M, 5x10 -9 M and 1x10 -12 Between M, 1x10 -9 M and 5x10 -10 Between M, 1x10 -9 M and 1x10 -10 Between M, 1x10 -9 M and 5x10 -11 Between M, 1x10 -9 M and 1x10 -11 Between M, 1x10 -9 M and 5x10 -12 Between M, 1x10 -9 M and 1x10 -12 Between M, 5x10 -10 M and 1x10 -10 Between M, 5x10 -10 M and 5x10 -11 M, 1x10 -10 M and 5x10 -11 Between M, 1x10 -10 M and 1x10 -11 Between M, 1x10 - 10M and 5x10 -12 Between M, 1x10 -10 M and 1x10 -12 Between M, 5x10 -11 M and 1x10 -12 Between M, 5x10 -11 M and 5x10 -12 Between M, 5x10 -11 M and 1x10 -12 Between M, 1x10 -11 M and 5x10 -12 M or 1x10 -11 M and 1x10 -12 Between M. In some implementations, such as those determined by immunoblotting analysis, anti-IL31 antibodies bind to canine or feline IL31.

[0125] In some implementations, such as those determined by immunoblotting and / or biolayer interference, anti-IL31 antibodies do not bind to human IL31.

[0126] In some implementations, an anti-IL31 antibody is provided that competes with M14 for binding to IL31.

[0127] "Variant" means a bioactive polypeptide that, after sequence alignment and cleavage (if desired) to achieve the maximum percentage of sequence identity and without treating any conserved substitutions as part of the sequence identity, has at least about 50% amino acid sequence identity with the native sequence polypeptide. 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.

[0128] 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, at least about 97% amino acid sequence identity, at least about 98% amino acid sequence identity, and at least about 99% amino acid sequence identity with the natural sequence polypeptide.

[0129] In some embodiments, the anti-IL31 antibody comprises:

[0130] a)(i) a variable heavy chain sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; (ii) a variable light chain sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; or (iii) a variable heavy chain sequence as in (i) and a variable light chain sequence as in (ii); or

[0131] b)(i) a variable heavy chain sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequences of SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:90; (ii) a variable light chain sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the amino acid sequences of SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54; or (iii) the variable heavy chain sequence as in (i) and the variable light chain sequence as in (ii).

[0132] As used herein, “percentage of amino acid sequence identity (%)” and “homology” for peptide, polypeptide, or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific peptide or polypeptide sequence after sequence alignment and cleavage (if necessary) to achieve the maximum percentage of sequence identity and without treating any conserved substitutions as part of sequence identity. Alignments used to determine the percentage of amino acid sequence identity can be performed in a variety of ways well known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALINE. TM (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment across the full length of the sequences being compared.

[0133] Amino acid substitutions can include, but are not limited to, replacing one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 2. Amino acid substitutions can be introduced into target antibodies, and the desired activity of the product can be screened, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0134] Table 2

[0135]

[0136]

[0137] Amino acids can be grouped according to common side-chain characteristics:

[0138] (1) Hydrophobicity: Leucine, Met, Ala, Val, Leu, Ile;

[0139] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0140] (3) Acidity: Asp, Glu;

[0141] (4) Alkaline: His, Lys, Arg;

[0142] (5) Residues that affect chain orientation: Gly, Pro;

[0143] (6) Aromatics: Trp, Tyr, Phe.

[0144] Non-conservative substitution would require swapping members of one of these categories with another.

[0145] The term "vector" is used to describe a polynucleotide that is engineered to contain one or more clones that can replicate in a host cell. A vector may include one or more of the following elements: an origin of replication, one or more regulatory sequences (e.g., promoters or enhancers) that regulate the expression of a target polypeptide, or one or more selective marker genes (e.g., antibiotic resistance genes and genes that can be used for colorimetric assays, such as β-galactosidase). The term "expression vector" refers to a vector used to express a target polypeptide in a host cell.

[0146] "Host cell" refers to a cell that can be, or already is, a recipient of a vector or isolated polynucleotide. Host cells can be prokaryotic or eukaryotic cells. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells. Crucell, 293 cells, and CHO cells, and their derivatives such as 293-6E, DG44, CHO-S, and CHO-K cells. Host cells include the progeny of a single host cell, and said progeny may not necessarily be identical to the original parent cell (in morphology or complementary genomic DNA sequences) due to natural, accidental, or intentional mutations. Host cells include cells transfected in vivo with one or more polynucleotides encoding one or more amino acid sequences provided herein.

[0147] As used herein, the term "isolated" refers to a molecule that has been separated from at least some of its components, which are normally found or produced in nature. For example, a polypeptide is called "isolated" when it is separated from at least some of the components of the cell that produced it. In the case of a polypeptide being secreted by a cell after expression, the physical separation of the supernatant containing the polypeptide from the cell that produced it is considered "isolated" of the polypeptide. Similarly, a polynucleotide is called "isolated" when it is not part of a larger polynucleotide normally found in nature (e.g., genomic DNA or mitochondrial DNA in the case of DNA polynucleotides), or, for example, in the case of RNA polynucleotides, when it is separated from at least some of the components of the cell that produced it. Thus, DNA polynucleotides contained in a vector within a host cell can be referred to as "isolated." In some embodiments, anti-IL31 antibodies are purified using chromatographic methods (e.g., size exclusion chromatography, ion exchange chromatography, protein A column chromatography, hydrophobic interaction chromatography, and CHT chromatography).

[0148] The term "companion animal species" refers to animals suitable for living alongside humans. In some implementations, companion animal species are small mammals such as canines, felines, dogs, cats, horses, rabbits, ferrets, guinea pigs, rodents, etc. In other implementations, companion animal species are farm animals such as horses, cattle, pigs, etc.

[0149] The term "IL31 signaling function" refers to any one or a combination of downstream activities that occur when IL31 binds to its receptor or receptor complex. In some embodiments, 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 activation of the ERK1 / 2 MAP kinase signaling pathway. In some embodiments, the IL31 signaling function includes activation of the PI3K / AKT signaling pathway. In some embodiments, the IL31 signaling function includes activation of the Jak1 / 2 signaling pathway.

[0150] "STAT phosphorylation" refers to post-expression modification of STAT proteins via phosphorylation. For example, "STAT-3 phosphorylation" refers to the phosphorylation of STAT-3, and "STAT-5 phosphorylation" refers to the phosphorylation of STAT-5. In some implementations, STAT-3 phosphorylation is measured by immunoblotting analysis.

[0151] For example, in the presence of an anti-IL31 antibody as described herein, cells (e.g., canine DH82 cells transfected with canine and / or feline IL31Ra or mammalian cells (e.g., HeLa cells)) are incubated at 37°C at a concentration of 1 x 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 10 cells / well into 96-well cell culture plates containing 15% heat-inactivated fetal bovine serum, 2 mmol / L GlutaMax, 1 mmol / L sodium pyruvate, and 10 nm / mL canine interferon-c (R&D Systems, Minneapolis, Minnesota, USA) in growth medium (e.g., MEM, Life). The immunoblotting of cell lysates for 24 hours can be used to detect the concentrations of phosphorylated and unphosphorylated STAT proteins relative to each other and compared to a β-actin control. Those skilled in the art understand methods for qualitatively or quantitatively determining protein concentrations via immunoblotting. In some embodiments, relative concentrations are qualitatively determined by visual inspection of the immunoblot. In some embodiments, band intensity is determined by digital imaging immunoblotting, and the concentrations of phosphorylated or unphosphorylated STAT proteins in the sample are inversely calculated using a linear standard curve of known concentrations of STAT proteins to quantitatively determine the concentrations of phosphorylated and unphosphorylated STAT proteins.

[0152] "Reduction" or "inhibition" means a reduction, decrease, or cessation of activity, function, or quantity compared to a reference. In some embodiments, "reduction" or "inhibition" means the ability to result in an overall reduction of 20% or more. In some embodiments, "reduction" or "inhibition" means the ability to result in an overall reduction of 50% or more. In some embodiments, "reduction" or "inhibition" means the ability to cause an overall reduction of 75%, 85%, 90%, 95%, or higher. In some embodiments, the aforementioned quantities are inhibited or reduced over a period of time relative to a control dose (such as a placebo). As used herein, "reference" means any sample, standard, or level used for comparative purposes. A reference may be obtained from healthy or non-disease-prone samples. In some examples, a reference is obtained from unaffected or untreated samples of concomitant animals. In some examples, a reference is obtained from one or more healthy animals of a particular species, rather than from the tested or treated animal.

[0153] As used herein, the term "significant reduction" means a sufficiently high degree of reduction between a numerical value and a reference value, such that a person skilled in the art would consider the difference between the two values ​​to be statistically significant in the context of the biological characteristic measured by said value. In some embodiments, a significant reduction is a numerical reduction greater than any one of about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the reference value.

[0154] In some embodiments, the IL31 antibody can reduce IL31 signaling function in the associated 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 a reduction in STAT-3 phosphorylation, compared to IL31 signaling function in the absence of the antibody.In some implementations, the reduction in IL31 signaling function or STAT-3 phosphorylation is between 10% and 15%, 10% and 20%, 10% and 25%, 10% and 30%, 10% and 35%, 10% and 40%, 10% and 45%, 10% and 50%, 10% and 60%, 10% and 70%, 10% and 80%, 10% and 90%, 10% and 100%, 15% and 20%, 15% and 25%, 15% and 30%, 15% and 35%, 15% and 40%, 15% and 45%, 15% and 50%, or 15%. Between 60% and 15%; between 70% and 15%; between 80% and 15%; between 90% and 15%; between 100% and 20%; between 25% and 20%; between 30% and 20%; between 35% and 20%; between 40% and 20%; between 45% and 20%; between 50% and 20%; between 60% and 25%; between 70% and 25%; between 80% and 25%; between 90% and 20%; between 100% and 25%; between 30% and 25%; between 40% and 25%; between 45% and 25%; between 50% and 25%; between 60% and 25%; between 70% and 25%; between 80% and 25% Between 5% and 90%, between 25% and 100%, between 30% and 35%, between 30% and 40%, between 30% and 45%, between 30% and 50%, between 30% and 60%, between 30% and 70%, between 30% and 80%, between 30% and 90%, between 30% and 100%, between 35% and 40%, between 35% and 45%, between 35% and 50%, between 35% and 60%, between 35% and 70%, between 35% and 80%, between 35% and 90%, between 35% and 100%, between 40% and 45%, between 40% and 50%, between 40% and 60%, between 40% and 70%, between 40% and 80%. Between, 40% and 90%, 40% and 100%, 45% and 50%, 45% and 60%, 45% and 70%, 45% and 80%, 45% and 90%, 45% and 100%, 50% and 60%, 50% and 70%, 50% and 80%, 50% and 90%, 50% and 100%, 60% and 70%, 60% and 80%, 60% and 90%, 60% and 100%, 70% and 80%, 70% and 90%, 70% and 100%, 80% and 90%, 80% and 100%, or 90% and 100%.

[0155] Exemplary pharmaceutical compositions

[0156] The terms “pharmaceutical formulation” and “pharmaceutical composition” refer to a formulation which is in a form that enables the biological activity of one or more active ingredients and does not contain any other components that would have unacceptable toxicity to a subject to which the formulation is administered.

[0157] "Pharmaceutically acceptable carriers" refer to non-toxic solid, semi-solid, or liquid fillers, diluents, encapsulation materials, formulation aids, or carriers conventional in the art used in conjunction with therapeutic agents, which together constitute a "pharmaceutical composition" for administration to a subject. Pharmaceutically acceptable carriers are non-toxic to the recipient at the dosage and concentration used and are compatible with other components of the formulation. Pharmaceutically acceptable carriers are suitable for the formulation used. Examples of pharmaceutically acceptable carriers include alumina; aluminum stearate; lecithin; serum proteins such as human serum albumin, canine or other animal albumin; buffers such as phosphate, citrate, tromethamine, or HEPES buffer; glycine; sorbic acid; potassium sorbate; mixtures of metaglycerides of saturated vegetable fatty acids; water; salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, or magnesium trisilicate; polyvinylpyrrolidone; cellulose-based substances; polyethylene glycol; sucrose; mannitol; or amino acids including, but not limited to, arginine.

[0158] The pharmaceutical composition can be stored in lyophilized form. Therefore, in some embodiments, the preparation method includes a lyophilization step. The lyophilized composition can then be reformed, typically as an aqueous composition suitable for parenteral administration, before administration to dogs, cats, or horses. In other embodiments, particularly when the antibody is highly stable to heat and oxidative denaturation, the pharmaceutical composition can be stored as a liquid, i.e., as an aqueous composition, which can be administered directly to dogs, cats, or horses, or with appropriate dilution. The lyophilized composition can be reconstituted with sterile water for injection (WFI). Antibacterial agents (e.g., bacteriostatic agents such as benzyl alcohol) may be included. Therefore, the present invention provides pharmaceutical compositions in solid or liquid form.

[0159] When applied, the pH of the pharmaceutical composition may be in the range of about pH 5 to about pH 8. If the compositions of the present invention are used for therapeutic purposes, they are sterile. Sterility can be achieved by any of several means known in the art, including filtration through a sterile filter membrane (e.g., a 0.2-micron membrane). Sterility may or may not be maintained using antibacterial agents.

[0160] Exemplary uses of antibody and pharmaceutical compositions

[0161] The antibodies of the present invention, or pharmaceutical compositions comprising said antibodies, can be used to treat IL-31-induced conditions. As used herein, "IL-31-induced condition" means a disease associated with, caused by, or characterized by an elevated level or altered gradient of IL-31 concentration. Such IL-31-induced conditions include, but are not limited to, pruritic or allergic diseases. In some embodiments, said IL-31-induced conditions are atopic dermatitis, allergic dermatitis, pruritus, asthma, psoriasis, scleroderma, and eczema. IL-31-induced conditions may be manifested in companion animals, including, but not limited to, canines or felines.

[0162] As used herein, “treatment” is a means of obtaining a beneficial or desired clinical outcome. As used herein, “treatment” includes any administration or application of a disease-treating agent in mammals (including companion animals). For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, any one or more of the following: relief of one or more symptoms, reduction of disease severity, prevention or delay of disease spread, prevention or delay of disease recurrence, delay or slowing of disease progression, improvement of disease status, inhibition of disease or disease progression, inhibition or slowing of disease or disease progression, cessation of disease development, and remission (whether partial or complete). “Treatment” also encompasses the reduction of the pathological consequences of proliferative diseases. The methods provided herein take into account any one or more of these therapeutic aspects. Consistent with the foregoing, the term “treatment” does not require the complete removal of all aspects of the obstruction.

[0163] In some embodiments, anti-IL31 antibodies or pharmaceutical compositions containing them may be used to treat IL31-induced symptoms according to the methods described herein. In some embodiments, anti-IL31 antibodies or pharmaceutical compositions are administered to companion animals (such as canines or felines) to treat IL31-induced symptoms.

[0164] The "therapeutic effective dose" of a substance / molecule, agonist, or antagonist can vary depending on factors such as the type of disease to be treated, disease state, severity and course of the disease, type of treatment objective, any prior treatments, clinical history, response to previous treatments, the attending veterinarian's judgment, the animal's age, sex, and weight, and the ability of the substance / molecule, agonist, or antagonist to elicit the desired response in the animal. Therapeutic effective dose is also the amount at which the beneficial therapeutic effect outweighs any toxic or harmful effects of the substance / molecule, agonist, or antagonist. Therapeutic effective doses can be delivered in single or multiple administrations. "Therapeutic effective dose" refers to the amount that effectively achieves the desired therapeutic or preventative outcome at the necessary dose and for the necessary duration.

[0165] In some embodiments, the anti-IL31 antibody or a pharmaceutical composition containing an anti-IL31 antibody is administered parenterally via subcutaneous, intravenous, or intramuscular injection. In some embodiments, the anti-IL31 antibody or a pharmaceutical composition containing an anti-IL31 antibody is administered as a bolus or via continuous infusion for a period of time. In some embodiments, the anti-IL31 antibody or a pharmaceutical composition containing an anti-IL31 antibody is administered via intramuscular, intraperitoneal, intraspinal, subcutaneous, intraarterial, intrasynovial, intrathecal, or inhalation routes.

[0166] The anti-IL31 antibody described herein can be administered in doses ranging from 0.01 mg / kg body weight to 100 mg / kg body weight. In some embodiments, the anti-IL31 antibody can be administered in doses ranging from 0.5 mg / kg body weight to 50 mg / kg body weight. In some embodiments, the anti-IL31 antibody can be administered in doses ranging from 0.1 mg / kg body weight to 10 mg / kg body weight. In some embodiments, the anti-IL31 antibody can be administered in doses ranging from 0.1 mg / kg body weight to 100 mg / kg body weight. In some embodiments, the anti-IL31 antibody can be administered in doses ranging from 1 mg / kg body weight to 10 mg / kg body weight. In some implementations, the anti-IL31 antibody can be administered in doses ranging from 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, 0.01 mg / kg body weight to 0.5 mg / kg body weight, 0.01 mg / kg body weight to 0.1 mg / kg body weight, or 5 mg / kg body weight to 50 mg / kg body weight.

[0167] Anti-IL31 antibodies or pharmaceutical compositions containing anti-IL31 antibodies can be administered to companion animals once or in a series of treatments. For example, anti-IL31 antibodies or pharmaceutical compositions containing anti-IL31 antibodies can be administered at least once, more than once, at least twice, at least three times, at least four times, or at least five times.

[0168] In some embodiments, the dose is administered once a week for at least two or three consecutive weeks, and in some embodiments, this treatment cycle is repeated two or more times, optionally interspersed with one or more weeks of no treatment. In other embodiments, the effective therapeutic dose is administered once daily for two to five consecutive days, and in some embodiments, this treatment cycle is repeated two or more times, optionally interspersed with one or more days or one or more weeks of no treatment.

[0169] Administering in combination with one or more other therapeutic agents includes simultaneous (concurrent) and sequential or continuous administration in any order. The term "concurrent" is used herein to refer to the administration of two or more therapeutic agents, wherein at least part of the administration overlaps in time or the administration of one therapeutic agent occurs within a very short time relative to the administration of another. For example, the two or more therapeutic agents are administered at time intervals not exceeding approximately a specified number of minutes. The term "sequentially" is used herein to refer to the administration of two or more therapeutic agents, wherein one or more agents are administered after an interruption of administration of one or more other agents, or wherein one or more agents are administered before administration of one or more other agents. For example, the two or more therapeutic agents are administered at time intervals exceeding approximately a specified number of minutes. As used herein, the term "in combination with" means administering a treatment modality in addition to another treatment modality. Thus, "in combination with" means administering another treatment modality before, during, or after administration of a treatment modality to the animal.

[0170] In some embodiments, the method includes administering a Jak inhibitor, PI3K inhibitor, AKT inhibitor, or MAPK inhibitor in combination with an anti-IL31 antibody or a pharmaceutical composition containing an anti-IL31 antibody. In some embodiments, the method includes administering an 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 an anti-IL31 antibody or a pharmaceutical composition containing an anti-IL31 antibody.

[0171] This document provides a method for exposing cells to an anti-IL31 antibody or a pharmaceutical composition containing an anti-IL31 antibody, under conditions that allow the antibody to bind to IL31. In some embodiments, the cells are exposed to the antibody or pharmaceutical composition in vitro. In some embodiments, the cells are exposed to the antibody or pharmaceutical composition in vivo. In some embodiments, the cells are exposed to the anti-IL31 antibody or the pharmaceutical composition under conditions that allow the antibody to bind to intracellular IL31. In some embodiments, the cells are exposed to the anti-IL31 antibody or the pharmaceutical composition under conditions that allow the antibody to bind to extracellular IL31. In some embodiments, the cells may be exposed in vivo to the anti-IL31 antibody or the pharmaceutical composition via any one or more of the administration methods described herein, including but not limited to intraperitoneal, intramuscular, or intravenous injection into the subject. In some embodiments, the cells may be exposed to the anti-IL31 antibody or the pharmaceutical composition in vitro by exposing the cells to a culture medium containing the antibody or the pharmaceutical composition. In some embodiments, prior to exposing the cells to a culture medium containing the antibody or the pharmaceutical composition, any number of methods understood by those skilled in the art (such as electroporating the cells or exposing the cells to a solution containing calcium chloride) may be used to influence the permeability of the cell membrane.

[0172] In some embodiments, the binding results in a reduction in the IL31 signaling function of the cells. In some embodiments, as measured by a reduction in STAT-3 phosphorylation, the IL31 antibody can reduce IL31 signaling function in cells by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to the absence of the antibody.In some implementations, the reduction in IL31 signaling function or STAT-3 phosphorylation is between 10% and 15%, 10% and 20%, 10% and 25%, 10% and 30%, 10% and 35%, 10% and 40%, 10% and 45%, 10% and 50%, 10% and 60%, 10% and 70%, 10% and 80%, 10% and 90%, 10% and 100%, 15% and 20%, 15% and 25%, 15% and 30%, 15% and 35%, 15% and 40%, 15% and 45%, 15% and 50%, or 15%. Between 60% and 15%; between 70% and 15%; between 80% and 15%; between 90% and 15%; between 100% and 20%; between 25% and 20%; between 30% and 20%; between 35% and 20%; between 40% and 20%; between 45% and 20%; between 50% and 20%; between 60% and 25%; between 70% and 25%; between 80% and 25%; between 90% and 20%; between 100% and 25%; between 30% and 25%; between 40% and 25%; between 45% and 25%; between 50% and 25%; between 60% and 25%; between 70% and 25%; between 80% and 25% Between 5% and 90%, between 25% and 100%, between 30% and 35%, between 30% and 40%, between 30% and 45%, between 30% and 50%, between 30% and 60%, between 30% and 70%, between 30% and 80%, between 30% and 90%, between 30% and 100%, between 35% and 40%, between 35% and 45%, between 35% and 50%, between 35% and 60%, between 35% and 70%, between 35% and 80%, between 35% and 90%, between 35% and 100%, between 40% and 45%, between 40% and 50%, between 40% and 60%, between 40% and 70%, between 40% and 80%. Between, 40% and 90%, 40% and 100%, 45% and 50%, 45% and 60%, 45% and 70%, 45% and 80%, 45% and 90%, 45% and 100%, 50% and 60%, 50% and 70%, 50% and 80%, 50% and 90%, 50% and 100%, 60% and 70%, 60% and 80%, 60% and 90%, 60% and 100%, 70% and 80%, 70% and 90%, 70% and 100%, 80% and 90%, 80% and 100%, or 90% and 100%.

[0173] This document provides methods for detecting, diagnosing, and monitoring IL31-induced conditions using the described anti-IL31 antibodies, peptides, and polynucleotides. This document provides methods for determining whether a companion animal responds to anti-IL31 antibody therapy. In some embodiments, the method includes detecting whether the animal has cells expressing IL31 using an anti-IL31 antibody. In some embodiments, the detection method includes contacting the sample with the antibody, peptide, or polynucleotide and determining whether the binding level differs from the binding level of a reference or control sample (e.g., a control). In some embodiments, the method can be used to determine whether the antibody or peptide described herein is a suitable treatment for the test animal.

[0174] In some embodiments, the sample is a biological sample. The term "biological sample" means an amount of substance derived from or formerly from a living organism. In some embodiments, the biological sample is cells or cell / tissue lysates. 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.

[0175] In some embodiments, the cells or cell / tissue lysate are contacted with an anti-IL31 antibody, and the binding between the antibody and the cells is measured. When the test cells show binding activity compared to reference cells of the same tissue type, it can indicate that the subject will benefit from treatment with the anti-IL31 antibody. In some embodiments, the test cells are derived from tissue of a companion animal.

[0176] Various methods known in the art for detecting specific antibody-antigen binding can be used. Exemplary immunoassays that can be performed include fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), turbidimetric inhibition immunoassay (NIA), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA). Indicator portions or labeling groups can be attached to the subject antibody and selected to meet the needs of various applications of the method, typically determined by the availability of the assay equipment and compatible immunoassay procedures. Suitable labeling includes, but is not limited to, radionuclides (e.g., radioisotopes). 125 I, 131 I, 35 S, 3 H or 32 P), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or p-lactosidase), fluorescent moieties or proteins (e.g., luciferin, rhodamine, phycoerythrin, GFP, or BFP) or luminescent moieties (e.g., Qdot supplied by Quantum Dot Corporation, Palo Alto, Calif). TM(Nanoparticles). The general techniques used to perform the various immunoassays described above are known to those skilled in the art.

[0177] For diagnostic purposes, the polypeptide (including antibodies) can be labeled with a detectable portion, 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, labeling of the anti-IL31 antibody is not required, and its presence can be detected using a second labeled antibody bound to a first anti-IL31 antibody. In some embodiments, the anti-IL31 antibody can be used with any known assay, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. (Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987)). The anti-IL31 antibody and polypeptide can also be used for in vivo diagnostic assays (e.g., in vivo imaging). Typically, a radionuclide (e.g.) is used. 111 In、 99 Tc, 14 C 131 I, 125 I, 3 Antibodies or peptides (H or any other radionuclide labeling as outlined herein) can be labeled so that target cells or tissues can be located using immunoscanning. Using techniques well known in the art, the antibodies can also be used as staining reagents in pathology.

[0178] In some embodiments, the first antibody is used for diagnosis, and the second antibody is used as a therapeutic agent. In some embodiments, the first antibody and the second antibody are different. In some embodiments, the first antibody and the second antibody can simultaneously bind the antigen by binding to separate epitopes.

[0179] The following examples illustrate specific aspects of this disclosure and are not intended to limit this disclosure in any way.

[0180] Example

[0181] Example 1: In vitro affinity maturation for enhancing binding to IL31

[0182] The variable heavy chain (VH) and variable light chain (VL) sequences of the mouse monoclonal antibody M14 against IL-31 were identified (SEQ ID NO: 1 and 2). See WO 2018 / 156367, which is incorporated herein by reference in its entirety for any purpose. The M14 VH and VL sequences were canine-derived (SEQ ID NO: 3 and 4) by searching for and selecting suitable canine antibody sequences as templates for CDR transplantation, followed by protein modeling.

[0183] Phage libraries containing mutant Fab peptides with mutations within and around the CDRs of canine-derived M14 VH and VL sequences (SEQ ID NO 3 and 4) were prepared, and slower-release Fab peptides were screened by canine IL31 dissociation assay. off Rate. After three rounds of panning against canine IL31 phage libraries, phage colonies expressing variant Fab peptides that may have enhanced canine IL31 binding were identified and peptide sequencing was performed.

[0184] Single *E. coli* colonies expressing each of the identified variant Fab peptides with the SASA tag were cultured and induced to express the peptide. Cell culture medium containing the variant Fab peptide was exposed to immobilized BSA on a plate or Biacore chip. Plates or chips containing the bound variant Fab peptide were exposed to soluble canine IL31 to screen for slow-release peptides. off rate.

[0185] Three canine-derived, mature variable heavy chain polypeptides (cmVH1 (SEQ ID NO:5), cmVH2 (SEQ ID NO:6), and cmVH3 (SEQ ID NO:7)) and three canine-derived, mature light chain polypeptides (cmVL1 (SEQ ID NO:8), cmVL2 (SEQ ID NO:9), and cmVL3 (SEQ ID NO:10)) were selected. Exemplary CDR sequences of the canine-derived, mature variable chains are represented by SEQ ID NO:11, 12, 13, 14, 15, 20, 21, 22, 23, and 24, and frame region sequences are represented by SEQ ID NO:16, 17, 18, 19, 25, 26, 27, and 28.

[0186] Chimeric antibodies were generated consisting of different combinations of cmVH1, cmVH2, or cmVH3 fused to the human IgG1 heavy chain and cmVL1, cmVL2, and cmVL3 fused to the human κ constant light chain. Canine VH (SEQ ID NO:3) fused to the human IgG1 heavy chain and canine VL (SEQ ID NO:4) fused to the human κ constant light chain were also generated. The affinity of canine IL31 for the different chimeric antibodies was measured using a BIAcore 8K, as shown in Table 3 below:

[0187] Table 3.

[0188]

[0189] The affinity and kinetics of each chimeric antibody tested for canine IL31 are summarized in Table 4 below. Compared to the canine VH-VLM14 antibody control (analyte 1), the mature antibodies (analytes 2-6) tested for each affinity exhibited higher affinity (as evidenced by lower Kd values) and slower dissociation rates (as evidenced by lower k... off (Proof of value).

[0190] Table 4.

[0191] 1. Canine-derived VH-VL M14 <![CDATA[2.40x10 5 ]]> <![CDATA[5.23x10 -4 ]]> <![CDATA[2.18x10 -9 ]]> 49.2 <![CDATA[6.99x10 -2 ]]> 2.cmVH1+cmVL1 <![CDATA[2.28x10 5 ]]> <![CDATA[1.82x10 -4 ]]> <![CDATA[8.00x10 -10 ]]> 32 <![CDATA[2.93x10 -2 ]]> 3.cmVH2+cmVL2 <![CDATA[1.74x10 5 ]]> <![CDATA[1.59x10 -4 ]]> <![CDATA[9.14x10 -10 ]]> 34.7 <![CDATA[2.36x10 -2 ]]> 4.cmVH2+cmVL3 <![CDATA[1.86x10 5 ]]> <![CDATA[1.62x10 -4 ]]> <![CDATA[8.68x10 -10 ]]> 33.8 <![CDATA[1.45x10 -2 ]]> 5.cmVH3+cmVL2 <![CDATA[1.87x10 5 ]]> <![CDATA[1.68x10 -4 ]]> <![CDATA[9.00x10 -10 ]]> 35.9 <![CDATA[3.59x10 -2 ]]> 6.cmVH3+cmVL3 <![CDATA[2.16x10 5 ]]> <![CDATA[1.85x10 -4 ]]> <![CDATA[8.58x10 -10 ]]> 32.1 <![CDATA[3.05x10 -2 ]]>

[0192] Example 2: A mutant with enhanced binding to feline IL31

[0193] The affinity of six chimeric antibodies for canine IL-31, tested in Tables 3 and 4 (above), for feline IL-31 was also examined. Binding analysis was performed using the OctetRed biosensor as follows. In short, feline IL-31 is biotinylated. Free unreacted biotin was removed from the biotinylated IL-31 by extensive dialysis. Biotinylated feline IL-31 was captured at the tip of a strepto-avidin sensor. Association of the antibody (20 μg / mL) with feline IL-31 was monitored for 300 seconds. Dissociation was monitored for 300 seconds. The buffer used for dilution and all binding steps was: 20 mM phosphate, 150 mM NaCl (pH 7.2). The results of the binding analysis are shown below. Figure 1 The affinity ranking of the tested antibodies was: (cmVH3+cmVL2) or (cmVH3+cmVL3)>(cmVH2+cmVL3)>(cmVH2+cmVL2)>(cmVH1+cmVL1)>(canine-derived VH-VL M14).

[0194] Example 3: Canine-derived, mature variable chain with a canine constant structural domain

[0195] Canine-derived, mature VH (e.g., SEQ ID NO: 5, 6, and 7) and VL (e.g., SEQ ID NO: 8, 9, and 10) can be fused with canine IgG-A, IgG-B, IgG-C, or IgG-D heavy chain constant domains (e.g., SEQ ID NO: 83, 84, 85, and 86) and canine κ light chain constant domains (e.g., SEQ ID NO: 87), respectively. Exemplary canine-derived, mature heavy chain, and light chain sequences having canine constant domains include SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, and 48.

[0196] Example 4: Cat-derived and expressed variable heavy and light chains with enhanced affinity

[0197] The affinity-enhanced variable heavy and light chains (e.g., cmVH1-3 and cmVL1-3) can be cat-derived using methods understood in the art. For example, cmVH2 (SEQ ID NO:6) can be cat-derived as exemplary SEQ ID NO:90; cmVH3 (SEQ ID NO:7) can be cat-derived as exemplary SEQ ID NO:49, 50, and 51; and cmVL3 (SEQ ID NO:10) can be cat-derived as any one of SEQ ID NO:52, 53, and 54. The cat-derived VH and VL can be expressed using a cat IgG heavy chain constant domain (e.g., SEQ ID NO:88) and a cat κ light chain constant domain (e.g., SEQ ID NO:89), respectively. Exemplary cat-derived, mature heavy and light chain sequences having cat constant regions include SEQ ID NO:73, 74, 75, 91, 76, 77, and 78.

[0198] The DNA sequence encoding a feline-derived IL-31 antibody having the heavy chain of SEQ ID NO:91 (VH2) and the light chain of SEQ ID NO:78 (VL3c) was expressed and purified in mammalian cells. Using the cell-based assay of Example 5, the feline-derived VH2-VL3 IL31 antibody quantitatively blocked feline IL31-induced STAT3-phosphorylation in HeLa cells transfected with feline IL31R and canine IL31R.

[0199] Example 5: Development of Signal Transduction Assay Based on Canine IL31 Cells

[0200] Purchase HeLa cell lines, a human epithelial cell line (American Type Culture Collection (ATCC), catalog number CCL-2), and culture them in Eagle's Limit Essential Medium (ATCC, catalog number 30-2033) prepared by ATCC with 10% fetal bovine serum (FBS) supplemented (ATCC, catalog number 30-2020). Use the liposome method (Thermo Fisher, catalog number 11668027) to generate HeLa cells stably transfected with canine or feline IL31Ra-FLAG expression plasmids (pcDNA3.1_canine IL31Ra_FLAG (SEQ ID NO:92) or pcDNA3.1_feline IL31Ra-FLAG (SEQ ID NO:93)), and select G418-resistant transfectants using G418 at a final concentration of 400 μg / mL (Thermo Fisher, catalog number 10131035). G418 resistant clones were screened using Western blotting targeting STAT1, STAT3, and STAT5 phosphorylation induced by canine or feline IL31. HeLa clones stably transfected with IL31Ra in response to IL31 were used for subsequent studies.

[0201] IL31-mediated STAT protein phosphorylation in dogs or cats was performed by seeding HeLa / IL31Ra cells at 10e5 cells / well in 96-well plates and incubating overnight at 37°C, 5% CO2 (in 10% FBSD-MEM as recommended by ATCC). Cell starvation was achieved by replacing the medium in each well with medium without FBS supplementation for 1 hour at 37°C, 5% CO2. Serially diluted anti-IL31 antibody was pre-incubated with IL31 cytokines for 1 hour and then added to each well of the serum-starved cells at room temperature for 5 minutes. Then, 20 μL of stop solution (M-PER from Thermo Fisher, catalog 78501) was added to each well to lyse the cells. Cell lysates were separated by SDS-PAGE (4%–12% Bis-Tris gel, Invitrogen, catalog NP0329). IL31-induced STAT phosphorylation was determined by Western blotting using an antiphosphorylated STAT3 antibody (RnD, catalog number AF 4607), an antiphosphorylated STAT1 antibody (Cell Signaling, catalog number 7649), or an antiphosphorylated STAT5 antibody (Cell Signaling, catalog number 9359).

[0202] Surprisingly, the expression of the canine or feline co-receptor OSMR is not essential for IL31 signaling.

Claims

1. An antibody that binds to canine IL31 or feline IL31, wherein the antibody has: a) A heavy chain, wherein the CDR-H1 sequence is composed of the amino acid sequence of SEQ ID NO: 11, the CDR-H2 sequence is composed of the amino acid sequence of SEQ ID NO: 12, and the CDR-H3 sequence is composed of the amino acid sequence of SEQ ID NO: 13; and b) Light chains, wherein the CDR-L1 sequence is composed of the amino acid sequence of SEQ ID NO: 20, the CDR-L2 sequence is composed of the amino acid sequence of SEQ ID NO: 21, and the CDR-L3 sequence is composed of the amino acid sequence of SEQ ID NO:

22.

2. The antibody according to claim 1, wherein the antibody has a variable heavy chain sequence of SEQ ID NO: 8 and a variable light chain sequence of SEQ ID NO:

5.

3. An antibody that binds to canine IL31 or feline IL31, wherein the antibody has: a) A heavy chain, wherein the CDR-H1 sequence is composed of the amino acid sequence of SEQ ID NO: 11, the CDR-H2 sequence is composed of the amino acid sequence of SEQ ID NO: 12, and the CDR-H3 sequence is composed of the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15; and b) Light chains, wherein the CDR-L1 sequence consists of the amino acid sequence of SEQ ID NO: 20, the CDR-L2 sequence consists of the amino acid sequence of SEQ ID NO: 21, and the CDR-L3 sequence consists of the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO:

24.

4. The antibody according to claim 3, wherein the antibody has a variable heavy chain sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 or SEQ ID NO: 90 and a variable light chain sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 52, SEQ ID NO: 53 or SEQ ID NO:

54.

5. The antibody according to any one of claims 1-4, wherein the antibody is in a fraction of less than 1 x 10⁻⁶. -9 The dissociation constant (Kd) of M binds to canine IL31.

6. The antibody according to claim 3, wherein the antibody reduces IL31 signaling function in isolated cultured cell lines in vitro.

7. The antibody according to any one of claims 1-4, wherein the antibody binds to feline IL31.

8. The antibody according to any one of claims 1-4, wherein the antibody is a monoclonal antibody.

9. The antibody according to any one of claims 1-4, wherein the antibody is a canine-derived, feline-derived, or chimeric antibody.

10. The antibody according to any one of claims 1-4, wherein the antibody comprises (a) a canine heavy chain constant region selected from the constant regions of IgG-A, IgG-B, IgG-C and IgG-D; or (b) a feline heavy chain constant region selected from the constant regions of IgG1, IgG2a and IgG2b.

11. The antibody according to claim 1 or 2, wherein the antibody comprises: (i) a heavy chain amino acid sequence selected from SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37; and (ii) a light chain amino acid sequence of SEQ ID NO:

46.

12. The antibody according to claim 3 or 4, wherein the antibody comprises: (i) a heavy chain amino acid sequence selected from SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 or SEQ ID NO: 91; and (ii) The light chain amino acid sequence of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 76, SEQ ID NO: 77 or SEQ ID NO:

78.

13. The antibody according to any one of claims 1-4, wherein the antibody is an antibody fragment selected from Fv, scFv, Fab, Fab', F(ab')2 and Fab'-SH.

14. The antibody according to any one of claims 1-4, wherein the antibody is bispecific, and wherein 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.

15. An isolated nucleic acid encoding an antibody according to any one of claims 1 to 14.

16. A host cell comprising the nucleic acid according to claim 15.

17. A method for producing an antibody, the method comprising culturing a host cell according to claim 16 and isolating the antibody.

18. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

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