Long-acting anti-IL31 antibody for veterinary use

Abstract

Various embodiments are provided relating to long-acting anti-IL31 antibodies that bind to canine IL31 and / or feline IL31. Such antibodies can be used in methods for treating IL31-induced conditions in companion animals, such as dogs, cats, and horses.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Application No. 63 / 014,033 filed on 22 April 2020 and U.S. Provisional Application No. 63 / 014,549 filed on 23 April 2020, each of which is incorporated herein by reference in whole for any purpose.

[0002] Sequence List This application is filed together with an electronic sequence listing. The sequence listing is provided as a file titled 2021-04-21_01157-0034-00PCT_ST25.txt, created on April 21, 2021, and its size is 225 KB. The electronic information of the sequence listing is incorporated herein by reference in its entirety.

[0003] The present invention relates to isolated, long-acting anti-IL31 antibodies, such as antibodies that bind to canine IL31 and / or feline IL31, and methods for using them, such as methods for treating IL31-induced conditions, or methods for reducing IL31 signaling function in the cells of companion animals such as dogs, cats, and horses. [Background technology]

[0004] Interleukin-31 (IL31) is a cytokine primarily produced by Th2 cells and is understood to be involved in promoting skin diseases such as pruritic and other forms of allergic diseases (e.g., atopic dermatitis). IL31 is thought to function by binding to its receptor and activating downstream activities such as JAK1 activation, leading to many of the clinical problems associated with dermatitis and other disorders.

[0005] Companion animals such as cats, dogs, and horses suffer from many skin diseases similar to those in humans, including atopic dermatitis. However, the IL31 sequence differs among humans, cats, dogs, and horses. Therefore, there is still a need for methods and compounds with increased serum half-lives that can be used to reduce IL31 signaling and treat IL31-induced conditions, and that can be used to specifically bind to companion animal IL31, and that can allow for longer dosing intervals and / or reduced doses. [Overview of the project]

[0006] In some embodiments, long-acting isolated antibodies that bind to canine IL-31 are provided. In some embodiments, the anti-IL-31 antibody has an increased serum half-life. In some embodiments, the anti-IL-31 antibody comprises a variant Fc polypeptide, in which case the anti-IL-31 antibody has an increased serum half-life compared to an antibody comprising a wild-type Fc polypeptide.

[0007] In some embodiments, there is provided an isolated antibody that binds to canine IL31 and comprises a variant IgG Fc polypeptide from a companion animal species that can bind to the neonatal Fc receptor (FcRn) with increased affinity compared to a wild-type Fc polypeptide. In some embodiments, the antibody binds to an epitope comprising amino acids 34 to 50 of SEQ ID NO:22. In some embodiments, the antibody binds to an epitope comprising the amino acid sequence of SEQ ID NO:23. In some embodiments, the antibody binds to an epitope comprising the amino acid sequence PSDX1X2KI (SEQ ID NO:45), wherein X is any amino acid residue. In some embodiments, X1 is a hydrophobic amino acid. In some embodiments, X1 is selected from A, V, I and L. In some embodiments, X1 is selected from V and I. In some embodiments, X2 is a hydrophilic amino acid. In some embodiments, X2 is selected from A, R, K, Q and N. In some embodiments, X2 is selected from R and Q. In some embodiments, X1 is V and X2 is R. In some embodiments, X1 is I and X2 is Q. In some embodiments, the antibody binds to an epitope comprising the amino acid sequence of SEQ ID NO:88.

[0008] In some embodiments, the antibody binds to canine IL31 with a dissociation constant (Kd) of less than 5×10, as measured by biolayer interferometry -6 M, less than 1×10 -6 M, less than 5×10 -7 M, less than 1×10 -7 M, less than 5×10 -8 M, less than 1×10 -8 M, less than 5×10 -9 M, less than 1×10 -9 M, less than 5×10 -10 M, less than 1×10 -10 M, less than 5×10 -11 M, less than 1×10 -11 M, less than 5×10 -12 M, or less than 1×10 -12 M.

[0009] In some embodiments, the antibody reduces IL31 signaling function in a companion animal species by reducing STAT-3 phosphorylation. In some embodiments, the companion animal species is a dog, cat, or horse.

[0010] In some embodiments, the antibody binds to feline IL31 or equine IL31, as determined by immunoblotting and / or biolayer interferometry. In some embodiments, the antibody competes with monoclonal M14 antibody for binding to canine IL31. In some embodiments, the antibody competes with monoclonal M14 antibody for binding to feline IL31. In some embodiments, the antibody does not bind to human IL31, as determined by immunoblotting and / or biolayer interferometry.

[0011] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a canine antibody, a feline antibody, a horse antibody, or a chimeric antibody. In some embodiments, the antibody is a chimeric antibody containing a mouse variable heavy chain framework region or a mouse variable light chain framework region.

[0012] In some embodiments, the antibody comprises a heavy chain and a light chain. a. The heavy chain comprises a CDR-H1 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1; a CDR-H2 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity to the amino acid sequences of SEQ ID NO: 2, 62, 89, or 87; and a CDR-H3 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 3. b. The light chain includes a CDR-L1 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 63; a CDR-L2 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 9; and a CDR-L3 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0013] In some embodiments, the antibody comprises a heavy chain including (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 2 or 89, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 3.

[0014] In some embodiments, the antibody comprises a heavy chain including (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 62 or 87, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 3.

[0015] In some embodiments, the antibody comprises a light chain comprising (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 8, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 9, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 10.

[0016] In some embodiments, the antibody comprises a light chain comprising (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 63, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 9, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 10.

[0017] In some embodiments, the antibody comprises one or more of the following: (a) the variable region heavy chain framework 1 (HC-FR1) sequence of SEQ ID NO: 4, 70, or 79; (b) the HC-FR2 sequence of SEQ ID NO: 5, 71, or 80; (c) the HC-FR3 sequence of SEQ ID NO: 6, 72, 73, or 81; (d) the HC-FR4 sequence of SEQ ID NO: 7, 74, 124, or 82; (e) the variable region light chain framework 1 (LC-FR1) sequence of SEQ ID NO: 11, 75, or 83; (f) the LC-FR2 sequence of SEQ ID NO: 12, 76, or 84; (g) the LC-FR3 sequence of SEQ ID NO: 13, 77, or 85; or (h) the LC-FR4 sequence of SEQ ID NO: 14, 78, or 86.

[0018] In some embodiments, the antibody a. (i) A variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with respect to the amino acid sequence of SEQ ID NO: 24; (ii) A variable heavy chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with respect to the amino acid sequence of SEQ ID NO: 25; or (iii) A variable light chain sequence like (i) and a variable heavy chain sequence like (ii); or b. (i) A variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with respect to the amino acid sequence of SEQ ID NO: 16; (ii) A variable heavy chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with respect to the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 123; or (iii) A variable light chain sequence like (i) and a variable heavy chain sequence like (ii); or c. (i) A variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 32; (ii) A variable heavy chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 33; or (iii) A variable light chain sequence like (i) and a variable heavy chain sequence like (ii) Includes.

[0019] In some embodiments, the antibody includes the variable light chain sequence of SEQ ID NO: 24, SEQ ID NO: 16, or SEQ ID NO: 32. In some embodiments, the antibody includes the variable heavy chain sequence of SEQ ID NO: 25, SEQ ID NO: 15, SEQ ID NO: 123, or SEQ ID NO: 33. In some embodiments, the antibody includes the variable light chain sequence of SEQ ID NO: 24 and the variable heavy chain sequence of SEQ ID NO: 25; the variable light chain sequence of SEQ ID NO: 16 and the variable heavy chain sequence of SEQ ID NO: 15 or SEQ ID NO: 123; or the variable light chain sequence of SEQ ID NO: 32 and the variable heavy chain sequence of SEQ ID NO: 33.

[0020] In some embodiments, the antibody is a chimeric antibody containing a constant heavy chain region or a constant light chain region derived from a companion animal.

[0021] In some embodiments, the variant IgG Fc polypeptide binds to FcRn with higher affinity than the wild-type IgG Fc polypeptide, as measured by biolayer interferometry, surface plasmon resonance, or any protein-protein interaction tool, in a pH range of about 5.0 to about 6.5, for example, about 5.0, about 5.2, about 5.5, about 6.0, about 6.2, or about 6.5.

[0022] In some embodiments, the variant IgG Fc polypeptide is measured at a pH in the range of about 5.0 to about 6.5, for example, about 5.0, about 5.5, about 6.0, or about 6.5, by biolayer interferometry, surface plasmon resonance, or any protein-protein interaction tool, to a pH of 5 × 10⁻¹⁶. -6 Less than M, 1 x 10 -6 Less than M, 5 x 10 -7 Less than M, 1 x 10 -7 Less than M, 5 x 10 -8 Less than M, 1 x 10 -8 Less than M, 5 x 10 -9 Less than M, 1 x 10 -9 Less than M, 5 x 10 -10 Less than M, 1 x 10 -10 Less than M, 5 x 10 11 Less than M, 1 x 10 11Less than M, 5 x 10 -12 Less than M, or 1 × 10 -12 It binds to FcRn with a dissociation constant (Kd) less than M.

[0023] In some embodiments, the variant IgG Fc polypeptide binds to FcRn with increased affinity compared to the wild-type Fc polypeptide, for example at low pH, and the antibody has an increased serum half-life compared to the antibody containing the wild-type Fc polypeptide.

[0024] In some embodiments, the variant IgG Fc polypeptide contains the amino acid sequence of SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, or SEQ ID NO: 107.

[0025] In some embodiments, the variant IgG Fc polypeptide is a) Tyrosine or phenylalanine at position 23 of SEQ ID NO: 90 b) Tyrosine at position 82 of sequence number 90, c) Tyrosine at position 82 and histidine at position 207 of sequence number 90. d) A tyrosine at position 82 and a tyrosine at position 207 of sequence number 90. e) Tyrosine at position 207 of sequence number 90, f) Tyrosine at position 82 and histidine at position 207 of sequence number 90. g) Tyrosine at the position corresponding to position 82 of sequence number 90 and tyrosine at the position corresponding to position 207, or h) Tyrosine at position 207 of sequence number 90 Includes.

[0026] In some embodiments, the variant IgG Fc polypeptide is a) Tyrosine or phenylalanine at position 23 of sequence number 90, b) Tyrosine at position 82 of sequence number 90, c) Tyrosine at position 82 and histidine at position 207 of sequence number 90, d) Tyrosine at position 82 and tyrosine at position 207 of sequence number 90, e) Tyrosine at position 207 of sequence number 90, f) Tyrosine at position 82 and histidine at position 207 of sequence number 90, g) Tyrosine at position 82 and tyrosine at position 207 of sequence number 90; or h) Tyrosine at position 207 of sequence number 90 Includes.

[0027] In some embodiments, the antibody a) Heavy chain amino acid sequence of SEQ ID NO: 108, SEQ ID NO: 109, or SEQ ID NO: 110, b) Heavy chain amino acid sequence of SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 125, SEQ ID NO: 126, or SEQ ID NO: 127, c) Heavy chain amino acid sequence of SEQ ID NO: 114, SEQ ID NO: 115, or SEQ ID NO: 116, d) The heavy chain amino acid sequence of SEQ ID NO: 117, SEQ ID NO: 118, or SEQ ID NO: 119, or e) Heavy chain amino acid sequence of SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122 Includes.

[0028] In some embodiments, the antibody a. (i) the light chain amino acid sequence of SEQ ID NO: 26; (ii) the heavy chain amino acid sequence of SEQ ID NO: 108, SEQ ID NO: 109, or SEQ ID NO: 110; or (iii) a light chain amino acid sequence like (i) and a heavy chain amino acid sequence like (ii); b. (i) the light chain amino acid sequence of SEQ ID NO: 21; (ii) the heavy chain amino acid sequences of SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 125, SEQ ID NO: 126 or SEQ ID NO: 127; or (iii) a light chain amino acid sequence like (i) and a heavy chain amino acid sequence like (ii); c. (i) the light chain amino acid sequence of SEQ ID NO: 37; (ii) the heavy chain amino acid sequence of SEQ ID NO: 114, SEQ ID NO: 115, or SEQ ID NO: 116; or (iii) a light chain amino acid sequence like (i) and a heavy chain amino acid sequence like (ii); d. (i) the light chain amino acid sequence of SEQ ID NO: 38; (ii) the heavy chain amino acid sequences of SEQ ID NO: 117, SEQ ID NO: 118, and SEQ ID NO: 119; or (iii) a light chain amino acid sequence like (i) and a heavy chain amino acid sequence like (ii); or e. (i) the light chain amino acid sequence of SEQ ID NO: 39; (ii) the heavy chain amino acid sequence of SEQ ID NO: 120, SEQ ID NO: 121 or SEQ ID NO: 122; or (iii) a light chain amino acid sequence like (i) and a heavy chain amino acid sequence like (ii) Includes.

[0029] In some embodiments, the antibody contains the light chain amino acid sequence of SEQ ID NO: 21. In some embodiments, the antibody contains the heavy chain amino acid sequence of SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 125, SEQ ID NO: 126, or SEQ ID NO: 127.

[0030] In some embodiments, the antibody is an antibody fragment selected from Fv, scFv, Fab, Fab', F(ab')2, and Fab'-SH.

[0031] In some embodiments, the antibody is bispecific and binds to IL31 and one or more antigens selected from IL4R, IL17, TNFα, CD20, CD19, CD25, IL4, IL13, IL23, IgE, CD11α, IL6R, α4 integrin, IL12, IL1β, IL5, IL5R, IL22, IL22R, IL33, IL33R, TSLP, TSLPR, or BlyS.

[0032] In some embodiments, an isolated nucleic acid encoding the above-described anti-IL31 antibody is provided. In some embodiments, a host cell containing the nucleic acid encoding the above-described anti-IL31 antibody is provided. In some embodiments, a method for producing an anti-IL31 antibody is provided, comprising culturing such a host cell containing the nucleic acid encoding the above-described anti-IL31 antibody and isolating the antibody.

[0033] In some embodiments, a pharmaceutical composition is provided comprising the anti-IL31 antibody described herein and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition is provided comprising the anti-IL31 antibody described herein and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises L-histidine, sodium chloride, and polysorbate 80.

[0034] In some embodiments, the pharmaceutical composition has a pH of 5.0 to 6.2. In some embodiments, the pharmaceutical composition has a pH of 5.0 to 6.0, or 5.3 to 5.7, or 5.5.

[0035] In some embodiments, the pharmaceutical composition has an L-histidine concentration of 5 mM to 100 mM, 10 mM to 50 mM, 20 mM to 30 mM, 10 mM to 30 mM, or 20 mM.

[0036] In some embodiments, the pharmaceutical composition has a sodium chloride concentration of 80–200 mM, 100–175 mM, 120–150 mM, or 140 mM.

[0037] In some embodiments, the pharmaceutical composition has a polysorbate 80 concentration of 0.005 mg / mL to 0.5 mg / mL, 0.01 mg / mL to 0.1 mg / mL, or 0.05 mg / mL.

[0038] In some embodiments, the pharmaceutically acceptable carrier comprises at least one sugar. In some embodiments, the pharmaceutical composition has a concentration of at least one sugar of 0.5% to 20%, 1% to 10%, 1% to 5%, or 1% to 3%. In some embodiments, the pharmaceutically acceptable carrier comprises sucrose, trehalose, D-mannitol, maltose, and / or sorbitol.

[0039] In some embodiments, the pharmaceutically acceptable carrier includes an antimicrobial agent. In some embodiments, the pharmaceutical composition includes m-cresol or methylparaben. In some embodiments, the pharmaceutical composition includes 0.2% m-cresol and / or 0.9% methylparaben.

[0040] Use of antibodies and pharmaceutical compositions In some embodiments, a method is provided for treating a companion animal species having an IL31-induced state, comprising administering to the companion animal species a therapeutically effective amount of the anti-IL31 antibody described herein or a pharmaceutical composition comprising the antibody described herein.

[0041] In some embodiments, a method is provided for treating a companion animal species having an IL31-induced state, comprising administering the companion animal species a long-acting anti-IL31 antibody. In some embodiments, a therapeutically effective amount of anti-IL31 antibody is administered to the companion animal species every 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, or 24 weeks.

[0042] In some embodiments, the companion animal species is a dog, cat, or horse. In some embodiments, the IL31-induced state is a pruritic or allergic state. In some embodiments, the IL31-induced state is selected from atopic dermatitis, pruritus, asthma, psoriasis, scleroderma, and eczema.

[0043] In some embodiments, the anti-IL31 antibody or pharmaceutical composition is administered parenterally. In some embodiments, the anti-IL31 antibody or pharmaceutical composition is administered via intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarterial, intrasynovial, intrathecal, or inhalation routes. In some embodiments, the anti-IL31 antibody or pharmaceutical composition is administered in amounts ranging from 0.01 mg / kg to 100 mg / kg body weight per dose.

[0044] 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. In some embodiments, the method includes administering one or more antibodies selected from anti-IL4R antibody, 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, anti-IL5 antibody, anti-IL5R antibody, anti-IL22 antibody, anti-IL22R antibody, anti-IL33 antibody, anti-IL33R antibody, anti-TSLP antibody, anti-TSLPR antibody, and anti-BlyS antibody in combination with an anti-IL31 antibody or a pharmaceutical composition.

[0045] In some embodiments, methods are provided for reducing intracellular IL31 signaling function, comprising exposing cells to an anti-IL31 antibody, a pharmaceutical composition described herein, under conditions that allow the antibody to bind to extracellular IL31, thereby reducing its binding to the IL31 receptor and / or reducing the IL31 signaling function by the cells. In some embodiments, the cells are exposed to the antibody or pharmaceutical composition ex vivo. In some embodiments, the cells are exposed to the antibody or pharmaceutical composition in vivo. In some embodiments, the cells are canine, feline, or equine cells.

[0046] In some embodiments, a method is provided for detecting IL31 in a sample derived from a companion animal species, comprising contacting the sample with an anti-IL31 antibody or a pharmaceutical composition described herein under conditions that allow the antibody to bind to IL31, and detecting whether a complex is formed between the antibody and IL31 in the sample. In some embodiments, the sample is a biological sample obtained from a dog, cat, or horse. [Brief explanation of the drawing]

[0047] [Figure 1A] Figure 1A shows the alignment of variable light chain sequences of M14, M18, M19, and M87 mouse monoclonal antibody clones. [Figure 1B] Figure 1B shows the alignment of variable heavy chain sequences of M14, M18, M19, and M87 mouse monoclonal antibody clones. [Figure 2A] This graph shows the results of canine IL31 binding analysis using chimeric M14 antibodies at various concentrations. [Figure 2B] This is another graph showing canine IL31 binding analysis using chimeric M14 antibodies at various concentrations. [Figure 3A] This graph shows the results of canine IL31 binding analysis using various concentrations of canine-modified M14 antibody. [Figure 3B] This is another graph showing canine IL31 binding analysis using various concentrations of canine-modified M14 antibody. [Figure 4] This is an immunoblot demonstrating inhibition of canine IL31 signaling with various concentrations of canine-modified M14 antibody. [Figure 5A] This is an immunoblot of GST-canine IL31 deletion probed with M14 antibody. [Figure 5B] This is an immunoblot of GST-canine IL31 deletion probed with an anti-GST antibody. [Figure 6A] This is an immunoblot of GST-canine IL31 deletion probed with M14 antibody. [Figure 6B] This is an immunoblot of GST-canine IL31 deletion probed with an anti-GST antibody. [Figure 7A] This is an immunoblot of feline and equine IL31 proteins fused to human Fc probes using the M14 antibody. [Figure 7B] This is an immunoblot of feline and equine IL31 proteins fused to human Fc, probed with anti-FC antibodies. [Figure 8] This image shows precise epitope mapping and alanine scanning immunoblot analysis of mature canine IL31 epitopes using anti-canine IL31 antibodies (upper panel) and anti-GST antibodies (lower panel). [Figure 9] This image shows precise epitope mapping and alanine scanning immunoblot analysis of mature canine IL31 epitopes using anti-canine IL31 antibodies (upper panel) and anti-GST antibodies (lower panel). [Figure 10] This image shows precise epitope mapping and alanine scanning immunoblot analysis of mature canine IL31 epitopes using anti-canine IL31 antibodies (upper panel) and anti-GST antibodies (lower panel). [Figure 11] This image shows precise epitope mapping and alanine scanning immunoblot analysis of mature canine IL31 epitopes using anti-canine IL31 antibodies (upper panel) and anti-GST antibodies (lower panel). [Figure 12] This image shows precise epitope mapping and alanine scanning immunoblot analysis of mature canine IL31 epitopes using anti-canine IL31 antibodies (upper panel) and anti-GST antibodies (lower panel). [Figure 13] This is the immunoblot cross-reactivity of the anti-canine IL31 antibody M14 against walrus IL31. [Figure 14] This shows Biacore sensorgrams of various concentrations of canine FcRn (12.5, 25, 50, 100, and 200 nM) that bind to wild-type canine IgG-B Fc polypeptide. [Figure 15]Biacore sensorgrams of various concentrations of canine FcRn (12.5, 25, 50, 100, and 200 nM) that bind to variant canine IgG-B Fc polypeptide L(23)Y are shown. [Figure 16] Biacore sensorgrams of various concentrations of canine FcRn (12.5, 25, 50, 100, and 200 nM) bound to variant canine IgG-B Fc polypeptide L(23)F are shown. [Figure 17] Biacore sensorgrams of various concentrations of canine FcRn (12.5, 25, 50, 100, and 200 nM) that bind to variant canine IgG-B Fc polypeptide L(23)M are shown. [Figure 18] Biacore sensorgrams of various concentrations of canine FcRn (12.5, 25, 50, 100, and 200 nM) that bind to variant canine IgG-B Fc polypeptide YTE are shown. [Figure 19] This figure compares the OctetRed sensorgrams of chimeric variant canine IgG-A Fc F00 antibody (A) and IgG-D Fc F00 antibody (B) that bind to canine FcRn with the sensorgrams of chimeric variant canine IgG-A Fc (C) and IgG-D Fc (D) that do not have the Phe mutation. [Figure 20] The serum pharmacokinetic profiles of chimeric variant canine IgG-A F00 antibody ("IgG-A F00"; n=2) and chimeric variant canine IgG-A without the Phe mutation ("IgG-A"; n=2) after subcutaneous administration to rats at a dose of 2 mg / kg are shown. [Figure 21] This figure compares the OctetRed sensorgram of a chimeric antibody having a variant canine IgG-B Fc (0Y0, 0YH, 0YY, or 00Y) that binds to canine FcRn with the sensorgram of a chimeric antibody having wild-type canine IgG-B. [Figure 22] This chart shows the time-normalized percentage of antibodies obtained from the in vivo pharmacokinetic study in dogs described in Example 18. [Modes for carrying out the invention]

[0048] Describe a specific sequence TIFF0007943816000001.tif255170TIFF0007943816000002.tif255170TIFF000 7943816000003.tif255170TIFF0007943816000004.tif255170TIFF00079438160 00005.tif255170TIFF0007943816000006.tif255170TIFF0007943816000007.t if255170TIFF0007943816000008.tif255170TIFF0007943816000009.tif255170 TIFF0007943816000010.tif255170TIFF0007943816000011.tif255170TIFF000 7943816000012.tif255170TIFF0007943816000013.tif255170TIFF00079438160 00014.tif255170TIFF0007943816000015.tif255170TIFF0007943816000016.t if255170TIFF0007943816000017.tif255170TIFF0007943816000018.tif126170

[0049] Description of a specific embodiment Antibodies that bind to canine IL31 and / or feline IL31, such as long-acting anti-IL31 antibodies, are provided. Heavy and light chains of antibodies capable of forming antibodies that bind to IL31 are also provided. Furthermore, antibodies, heavy chains, and light chains comprising one or more specific complementarity-determining regions (CDRs) are provided. Polynucleotides encoding antibodies against canine IL31 are provided. Methods for producing or purifying antibodies against canine IL31 are also provided. Therapeutic methods using antibodies against canine IL31 are provided. Such methods include, but are not limited to, methods for treating IL31-induced conditions in companion animal species. Methods for detecting IL31 in samples from companion animal species are provided.

[0050] Also provided herein are variant IgG Fc polypeptides from companion animals having increased binding to protein A, decreased binding to C1q, decreased binding to CD16, and increased binding to FcRn, which may be used in conjunction with the IL31 antibody provided herein.

[0051] For the convenience of the reader, the following definitions of terms used herein are provided.

[0052] Where used herein, numerical terms such as Kd are calculated based on scientific measurements and are therefore subject to appropriate measurement errors. In some cases, numerical terms may include numbers rounded to the nearest significant figure.

[0053] As used herein, “a” or “an” means “at least one” or “one or more” unless otherwise specified. As used herein, the term “or” means “and / or” unless otherwise specified. In the context of multiple dependent claims, the use of “or” when referring to other claims refers only to the alternative claim.

[0054] Anti-IL31 antibody Novel antibodies against IL31, such as antibodies that conjugate canine IL31, feline IL31, and / or equine IL31, are provided. Anti-IL31 antibodies provided herein include, but are not limited to, monoclonal antibodies, mouse antibodies, chimeric antibodies, canine antibodies, feline antibodies, and equine antibodies. In some embodiments, the anti-IL31 antibody is an isolated mouse monoclonal antibody such as M14, M18, M19, and M87.

[0055] Monoclonal antibodies M14, M18, M19, and M87 were isolated as follows: Briefly, mice were immunized with canine IL31, and mouse monoclonal antibody clones were obtained by standard hybridoma technology. Enzyme-linked immunosorbent assay (ELISA) was used to screen for hybridoma clones producing IL31-binding antibodies. Based on binding affinity and cell-based functional assays described herein, hybridoma clones producing monoclonal antibodies M14, M18, M19, and M87 were selected for further investigation. The variable heavy chain (VH) and variable light chain (VL) of each of the four clones were sequenced and analyzed by sequence alignment (Figure 1).

[0056] The amino acid sequences of the monoclonal antibody M14 are also provided herein. For example, the variable heavy chain CDRs (SEQ ID NOs. 1-3; SEQ ID NO. 89 is an alternative definition of CDR-H2), variable light chain CDRs (SEQ ID NOs. 8-10), variable region heavy chain framework sequences (SEQ ID NOs. 4-7), and variable region light chain framework sequences (SEQ ID NOs. 11-14) of the monoclonal antibody M14 are provided. The amino acid sequences of the variable light chain, light chain, variable heavy chain, and variable and hinged heavy chains of the monoclonal antibody M14 are also provided (SEQ ID NOs. 24, 36, 25, and 40, respectively).

[0057] Furthermore, the CDRs, framework sequences, variable light chains, and variable heavy chain amino acid sequences for monoclonal antibodies M18, M19, and M87 are provided (see, for example, Figure 1). The variable heavy chain CDRs (SEQ ID NOs. 1-3; SEQ ID NOs. 89 is an alternative definition for CDR-H2), variable light chain CDRs (SEQ ID NOs. 8-10), variable light chain (SEQ ID NOs. 65), light chain (SEQ ID NOs. 38), variable and hinged heavy chain (SEQ ID NOs. 42), and variable heavy chain (SEQ ID NOs. 68) for monoclonal antibody M19 are provided. The variable heavy chain CDRs (SEQ ID NOs. 1, 62, and 3; SEQ ID NOs. 87 is an alternative definition for CDR-H2), variable light chain CDRs (SEQ ID NOs. 63, 9, and 10), variable light chain (SEQ ID NOs. 64), light chain (SEQ ID NOs. 37), variable and hinged heavy chain (SEQ ID NOs. 41), and variable heavy chain (SEQ ID NOs. 67) for monoclonal antibody M18 are provided. The monoclonal antibody M87 is provided with a variable light chain (SEQ ID NO: 66), a light chain (SEQ ID NO: 39), a variable and hinged heavy chain (SEQ ID NO: 43), and a variable heavy chain (SEQ ID NO: 69).

[0058] Chimeric antibodies, canine antibodies, feline antibodies, and equine antibodies derived from monoclonal antibodies M14, M18, M19, and M87 are also provided herein. In some embodiments, amino acid sequences of canine monoclonal antibody M14, e.g., SEQ ID NOs. 15-21, 70-78, and 123-124 are provided. In some embodiments, amino acid sequences of feline antibodies derived from monoclonal antibody M14, e.g., SEQ ID NOs. 32-35 and 79-86 are provided. In some embodiments, amino acid sequences of chimeric antibodies derived from monoclonal antibody M14, e.g., SEQ ID NOs. 26, 27, 30, and 31 are provided.

[0059] In this specification, the term “antibody” is used in its broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies (such as bispecific T cell engagers) and tripspecific antibodies), and antibody fragments (Fab, F(ab')2, ScFv, minibody, diabody, triabody, tetrabody, etc.), as long as they exhibit the desired antigen-binding activity. Dogs, cats, and horses possess different types (classes) of antibodies common to many mammals.

[0060] The term antibody includes, but is not limited to, fragments capable of binding to antigens, such as Fv, single-chain Fv(scFv), Fab, Fab', di-scFv, sdAb (single-domain antibodies), and (Fab')2 (including chemically bound F(ab')2). Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments, each having a single antigen-binding site, and a residual "Fc" fragment whose name reflects its ability to readily crystallize. Pepsin treatment yields an F(ab')2 fragment having two antigen-binding sites and still capable of crosslinking antigens. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies from various species such as mouse, human, cynomolgus monkey, dog, cat, and horse. Furthermore, for all antibody constructs provided herein, variants having sequences derived from other organisms are also intended. Thus, where a mouse version of an antibody is disclosed, those skilled in the art will understand how to convert a mouse sequence-based antibody to sequences from cat, dog, horse, etc. Antibody fragments also include orientations such as single-chain scFv, tandem-scFv, diabody, tandem-tri-sdcFv, and minibody. Antibody fragments also include nanobody (sdAb, antibodies having a single monomer domain such as a pair of variable domains of a heavy chain, without a light chain). In some embodiments, antibody fragments may be referred to as being of a specific species (e.g., mouse scFv or canine scFv). This indicates a sequence of at least a portion of the non-CDR region, not the source of the construct. In some embodiments, the antibody includes a label or is conjugated to a second portion.

[0061] The terms “label” and “detectable label” refer to a portion bound to an antibody or its analyte in order to make the reaction (e.g., binding) between members of a specific binding pair detectable. A labeled member of a specific binding pair is referred to as “detectable label.” Thus, the term “labeled binding protein” refers to a protein into which a label has been incorporated that provides identification of the binding protein. In some embodiments, the label is a detectable marker that can produce a detectable signal by visual or instrumental means, e.g., the incorporation of a radiolabeled amino acid or the binding of a biotinyl moiety to a polypeptide that can be detected by a labeled avidin (e.g., a fluorescent marker or streptavidin containing enzymatic activity that can be detected by optical or colorimetric methods). Examples of polypeptide labeling 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); pigments, fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorescent), enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal-binding domains, epitope tags); and magnetic agents, e.g., gadolinium chelates. Typical examples of labels commonly used in immunoassays include light-producing moieties, e.g., acridinium compounds, and fluorescence-producing moieties, e.g., fluorescein. In this regard, the moieties themselves do not need to be detectably labeled, but may become detectable upon reaction with other moieties.

[0062] The term "monoclonal antibody" refers to an antibody from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical except for naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and target a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody targets a single determinant on an antigen. Therefore, samples of monoclonal antibodies can bind to the same epitope on an antigen. The modifier "monoclonal" indicates a characteristic of antibodies obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring antibody production by a specific method. For example, monoclonal antibodies can be produced by the hybridoma method first described in Kohler and Milstein, 1975, Nature 256:495, or by recombinant DNA methods such as those described in U.S. Patent No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries prepared using techniques such as those described in McCafferty et al., 1990, Nature 348:552-554.

[0063] In some embodiments, the monoclonal antibody is an isolated mouse antibody selected from clones M14, M18, M19, and M87.

[0064] "Amino acid sequence" means the sequence of amino acid residues in a peptide or protein. The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to minimum length. Such polymers of amino acid residues may contain, but are not limited to, native or non-native amino acid residues, and include peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and their fragments are encompassed in the definition. The term also includes post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, and phosphorylation. Furthermore, for the purposes of this disclosure, "polypeptide" refers to a protein that includes modifications such as deletions, additions, and substitutions (generally inherently conserved) to the native sequence, as long as the protein maintains the desired activity. These modifications may be intentional, such as site-directed mutagenesis, or accidental, such as mutations in the host producing the protein or errors due to PCR amplification.

[0065] As used herein, “IL31” refers to any native IL31 resulting from the expression and processing of IL31 in cells. Unless otherwise indicated, this term includes IL31 from any vertebrate origin, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), and companion animals (e.g., dogs, cats, and horses). This term also includes naturally occurring variants of IL31, such as splice variants or allele variants.

[0066] In some embodiments, canine IL31 includes the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 44. In some embodiments, cat IL31 includes the amino acid sequence of SEQ ID NO: 28. In some embodiments, horse IL31 includes the amino acid sequence of SEQ ID NO: 29. In some embodiments, human IL31 includes the amino acid sequence of SEQ ID NO: 46. In some embodiments, walrus IL31 includes the amino acid sequence of SEQ ID NO: 47. In some embodiments, mouse IL31 includes the amino acid sequence of SEQ ID NO: 61. In other embodiments, IL31 includes the amino acid sequence of SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60.

[0067] The term "IL31 binding domain" of an antibody refers to the binding domain formed by the light and heavy chains of an anti-IL31 antibody that binds to IL31.

[0068] In some embodiments, the IL31-binding domain binds to canine IL31 with greater affinity than it binds to human IL31. In some embodiments, the IL31-binding domain binds to IL31 of other companion animals, such as feline IL31 or horse IL31. In some embodiments, the IL31-binding domain does not bind to human IL31.

[0069] As used herein, the term “epitope” refers to a site on a target molecule (e.g., an antigen such as a protein, nucleic acid, carbohydrate, or lipid) to which an antigen-binding molecule (e.g., an antibody, antibody fragment, or scaffold protein containing an antibody-binding domain) binds. Epitopes often contain chemically active surface groups of molecules such as amino acids, polypeptides, or sugar side chains and have specific three-dimensional structural and specific charge characteristics. Epitopes can be formed from both adjacent residues or juxtaposed, non-adjacent residues (e.g., amino acids, nucleotides, sugars, lipid moieties) of the target molecule. Epitopes formed from adjacent residues (e.g., amino acids, nucleotides, sugars, lipid moieties) are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon processing with denaturing solvents. Epitopes may, but are not limited to, consist of at least 3, at least 5, or 8–10 residues (e.g., amino acids or nucleotides). In some examples, an epitope is less than 20 residues (e.g., an amino acid or nucleotide), less than 15 residues, or less than 12 residues in length. Two antibodies may bind to the same epitope within an antigen if they exhibit competitive binding to the antigen. In some embodiments, an epitope can be identified by a certain minimum distance to a CDR residue on the antigen-binding molecule. In some embodiments, an epitope can be identified by the above distance and further limited to residues involved in the binding (e.g., hydrogen bonding) between antibody residues and antigen residues. Epitopes can also be identified by various scans; for example, an alanine or arginine scan may indicate one or more residues with which the antigen-binding molecule can interact. Unless explicitly stated, a set of residues as an epitope does not preclude other residues from being part of the epitopes of a particular antibody. Rather, the presence of such a set indicates a minimal series (or set of species) of epitopes. Thus, in some embodiments, a set of residues identified as an epitope indicates the minimal epitope associated with the antigen, rather than an exclusive list of residues for epitopes on the antigen.

[0070] In some embodiments, the epitope comprises the amino acid sequence PSDX1X2KI (SEQ ID NO: 45), where X is any amino acid residue. In some embodiments, X1 is a hydrophobic amino acid. In some embodiments, X1 is selected from A, V, I, and L. In some embodiments, X1 is selected from V and I. In some embodiments, X2 is a hydrophilic amino acid. In some embodiments, X2 is selected from A, R, K, Q, and N. In some embodiments, X2 is selected from R and Q. In some embodiments, X1 is V and X2 is R. In some embodiments, X1 is I and X2 is Q. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO: 88. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the epitope is located within amino acids 34-50 of SEQ ID NO: 22. In some embodiments, the epitope comprises amino acids 34-50 of SEQ ID NO: 22.

[0071] The term "CDR" means a complementarity-determining region defined by at least one identification method to those skilled in the art. In some embodiments, a CDR can be defined according to any of the following: 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 can be indicated by their appropriate number and chain type, including, but are not limited to, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. The term "CDR" is also used herein to encompass "hypervariable regions" or HVRs, which include hypervariable loops.

[0072] In some embodiments, the anti-IL31 antibody comprises a heavy chain comprising (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 62, SEQ ID NO: 89, or SEQ ID NO: 87, or (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-IL31 antibody comprises a light chain comprising (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 63, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 9, or (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 10.

[0073] In some embodiments, the anti-IL31 antibody comprises a heavy chain comprising (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 2 or 89, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 3, and a light chain comprising (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 8 or 63, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 9, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 10.

[0074] In some embodiments, the anti-IL31 antibody comprises a heavy chain comprising (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 62 or 87, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 3, and a light chain comprising (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 8 or 63, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 9, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 10.

[0075] 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 framework region ("FR"). The terms “heavy chain variable region” or “variable heavy chain” are used interchangeably to refer to a region containing at least three heavy chain CDRs. The terms “light chain variable region” or “variable light chain” are used interchangeably to refer to a region containing at least three light chain CDRs. In some embodiments, the variable heavy chain or variable light chain includes at least one framework region. In some embodiments, the antibody includes at least one heavy chain framework region selected from HC-FR1, HC-FR2, HC-FR3, and HC-FR4. In some embodiments, the antibody includes at least one light chain framework region selected from LC-FR1, LC-FR2, LC-FR3, and LC-FR4. Framework 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).

[0076] 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: 4, (b) the HC-FR2 sequence of SEQ ID NO: 5, (c) the HC-FR3 sequence of SEQ ID NO: 6, (d) the HC-FR4 sequence of SEQ ID NO: 7, (e) the variable region light chain framework 1 (LC-FR1) sequence of SEQ ID NO: 11, (f) the LC-FR2 sequence of SEQ ID NO: 12, (g) the LC-FR3 sequence of SEQ ID NO: 13, or (h) the LC-FR4 sequence of SEQ ID NO: 14. In some embodiments, the anti-IL31 antibody comprises the variable light chain sequence of (a) SEQ ID NO: 16, (b) SEQ ID NO: 24, or (c) SEQ ID NO: 32. In some embodiments, the anti-IL31 antibody comprises the variable heavy chain sequence of (a) SEQ ID NO: 15 or SEQ ID NO: 123, (b) SEQ ID NO: 25, or (c) SEQ ID NO: 33. In some embodiments, the anti-IL31 antibody comprises (a) the variable light chain sequence of SEQ ID NO: 16 and the variable heavy chain sequence of SEQ ID NO: 15 or SEQ ID NO: 123, (b) the variable light chain sequence of SEQ ID NO: 24 and the variable heavy chain sequence of SEQ ID NO: 25, or (c) the variable light chain sequence of SEQ ID NO: 32 and the variable heavy chain sequence of SEQ ID NO: 33.

[0077] 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 to 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 (including the γ1 constant region), IgG2 (including the γ2 constant region), IgG3 (including the γ3 constant region), and IgG4 (including the γ4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (including the α1 constant region) and IgA2 (including 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 to refer to the region containing the light chain constant domain CL. Non-exclusive exemplary light chain constant regions include λ and κ. Deletions and changes within a domain that do not alter its function are included within the scope of the term “constant region” unless otherwise specified. Dogs, cats, and horses have antibody classes such as IgG, IgA, IgD, IgE, and IgM. Within the canine IgG antibody class, there are IgG-A, IgG-B, IgG-C, and IgG-D. The feline IgG antibody class includes IgG1a, IgG1b, and IgG2. The equine IgG antibody class includes IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, and IgG7.

[0078] The terms "chimeric antibody" or "chimera" refer to an antibody in which a portion of the heavy or light chain originates from a particular source or species, and at least a portion of the remaining heavy or light chain originates from a different source or species. In some embodiments, a chimeric antibody refers to an antibody that includes at least one variable region from a first species (e.g., mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (e.g., human, dog, cat, horse, etc.). In some embodiments, a chimeric antibody includes at least one mouse variable region and at least one canine constant region. In some embodiments, a chimeric antibody includes at least one mouse variable region and at least one feline constant region. In some embodiments, all variable regions of the chimeric antibody originate from the first species, and all constant regions of the chimeric antibody originate from the second species. In some embodiments, a chimeric antibody includes a constant heavy chain region or constant light chain region derived from a companion animal. In some embodiments, a chimeric antibody includes mouse variable heavy and light chains as well as companion animal constant heavy and light chains. For example, a chimeric antibody may contain mouse variable heavy and light chains as well as canine constant heavy and light chains; a chimeric antibody may contain mouse variable heavy and light chains as well as feline constant heavy and light chains; or a chimeric antibody may contain mouse variable heavy and light chains as well as equine constant heavy and light chains.

[0079] In some embodiments, the anti-IL31 antibody is a. (i) the light chain amino acid sequence of SEQ ID NO: 26, (ii) the heavy chain amino acid sequence of SEQ ID NO: 27, or (iii) a light chain amino acid sequence like (i) and a heavy chain sequence like (ii), b. (i) the light chain amino acid sequence of SEQ ID NO: 30, (ii) the heavy chain amino acid sequence of SEQ ID NO: 31, or (iii) a light chain amino acid sequence like (i) and a heavy chain sequence like (ii). Contains chimeric antibodies.

[0080] "Dog chimera" or "dog chimeric antibody" refers to a chimeric antibody having at least a portion of the heavy chain or at least a portion of the light chain derived from a dog. "Cat chimera" or "cat chimeric antibody" refers to a chimeric antibody having at least a portion of the heavy chain or at least a portion of the light chain derived from a cat. "Horse chimera" or "horse chimeric antibody" refers to a chimeric antibody having at least a portion of the heavy chain or at least a portion of the light chain derived from a horse. In some embodiments, the dog chimeric antibody includes mouse variable heavy and light chains as well as dog constant heavy and light chains. In some embodiments, the cat chimeric antibody includes mouse variable heavy and light chains as well as cat constant heavy and light chains. In some embodiments, the horse chimeric antibody includes mouse variable heavy and light chains as well as horse constant heavy and light chains. In some embodiments, the antibody is a chimeric antibody that includes a mouse variable heavy chain framework region or a mouse variable light chain framework region.

[0081] As used herein, "canine antibody" includes antibodies produced in dogs; antibodies produced in non-canine animals containing canine immunoglobulin genes or canine immunoglobulin peptides; or antibodies selected using in vitro methods such as phage display, whose antibody repertoire is based on canine immunoglobulin sequences. The term "canine antibody" indicates the genus of sequences that are canine sequences. Therefore, this term indicates the genus of the relevant sequences, not the process by which the antibody was created.

[0082] In some embodiments, the anti-IL31 antibody comprises a canine heavy chain constant region selected from the IgG-A, IgG-B, IgG-C, and IgG-D constant regions. In some embodiments, the anti-IL31 antibody is a canine IgG-A, IgG-B, IgG-C, or IgG-D antibody. In some embodiments, the anti-IL31 antibody comprises (a) the heavy chain amino acid sequence of SEQ ID NO: 17, (b) the heavy chain amino acid sequence of SEQ ID NO: 18, (c) the heavy chain amino acid sequence of SEQ ID NO: 19, or (d) the heavy chain amino acid sequence of SEQ ID NO: 20.

[0083] As used herein, "feline antibody" includes antibodies produced in cats; antibodies produced in non-feline animals containing feline immunoglobulin genes or feline immunoglobulin peptides; or antibodies selected using in vitro methods such as phage display, whose antibody repertoire is based on feline immunoglobulin sequences. The term "feline antibody" indicates the genus of sequences that are feline sequences. Therefore, this term indicates the genus of the relevant sequences, not the process by which the antibody was created.

[0084] In some embodiments, the anti-IL31 antibody comprises a feline heavy chain constant region selected from the IgG1, IgG2a, and IgG2b constant regions. In some embodiments, the anti-IL31 antibody is a feline IgG1, IgG2a, or IgG2b antibody.

[0085] As used herein, “equine antibody” includes antibodies produced in horses; antibodies produced in non-equine animals containing equine immunoglobulin genes or equine immunoglobulin peptides; or antibodies selected using in vitro methods such as phage display, whose antibody repertoire is based on equine immunoglobulin sequences. The term “equine antibody” indicates the genus of sequences that are equine sequences. Therefore, this term indicates the genus of sequences to which the antibody is related, not the process by which the antibody was created.

[0086] In some embodiments, the anti-IL31 antibody comprises a horse heavy chain constant region selected from the IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, and IgG7 constant regions. In some embodiments, the anti-IL31 antibody is a horse IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, and IgG7 antibody.

[0087] A "canine antibody" refers to an antibody in which at least one amino acid in a portion of the non-canine variable region is replaced with a corresponding amino acid from the canine variable region. In some embodiments, a canine antibody includes at least one canine constant region (e.g., the γ constant region, α constant region, δ constant region, ε constant region, μ constant region, etc.) or a fragment thereof. In some embodiments, a canine antibody is an antibody fragment such as Fab, scFv, (Fab')2. The term "canine" also refers to a form of non-canine (e.g., mouse) antibody that is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding sequence of the antibody) containing the minimal sequence of a non-canine immunoglobulin. A canine antibody may include canine immunoglobulin (recipient antibody) in which residues from the recipient's CDR are replaced with residues from a CDR of a non-canine species such as mouse, rat, or rabbit (donor antibody) having desired specificity, affinity, and ability. In some cases, Fv framework region (FR) residues of canine immunoglobulins are replaced by corresponding non-canine residues. Furthermore, canine antibodies may include residues that are not found in the recipient antibody or the imported CDR or framework sequence, but which can be included to further improve and optimize antibody performance.

[0088] In some embodiments, at least one amino acid residue of a portion of the mouse variable heavy chain or mouse variable light chain is substituted with a corresponding amino acid from the canine variable region. In some embodiments, the modified chain is fused to the canine constant heavy chain or canine constant light chain. In some embodiments, the anti-IL31 antibody is a canine antibody comprising (a) the heavy chain sequence of SEQ ID NO: 15 or SEQ ID NO: 123, (b) the heavy chain sequence of SEQ ID NO: 17, (c) the heavy chain sequence of SEQ ID NO: 18, (d) the heavy chain sequence of SEQ ID NO: 19, (e) the heavy chain sequence of SEQ ID NO: 20, (f) the light chain sequence of SEQ ID NO: 16, or (g) the light chain sequence of SEQ ID NO: 21.

[0089] A "feline antibody" refers to an antibody in which at least one amino acid in a portion of the non-feline variable region is replaced with a corresponding amino acid from the feline variable region. In some embodiments, a feline antibody includes at least one feline constant region (e.g., the γ constant region, α constant region, δ constant region, ε constant region, μ constant region, etc.) or a fragment thereof. In some embodiments, a feline antibody is an antibody fragment such as Fab, scFv, (Fab')2. The term "feline" also refers to a form of non-feline (e.g., mouse) antibody that is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of the antibody) containing the minimal sequence of a non-feline immunoglobulin. A feline antibody may include a feline immunoglobulin (recipient antibody) in which residues from the recipient's CDR are replaced with residues (donor antibody) from a CDR of a non-feline species such as mouse, rat, or rabbit, having the desired specificity, affinity, and ability. In some examples, Fv framework region (FR) residues of the feline immunoglobulin are replaced with corresponding non-feline residues. Furthermore, felineized antibodies may include residues that are not found in the recipient antibody or the imported CDR or framework sequence, but can be included to further improve and optimize antibody performance.

[0090] In some embodiments, at least one amino acid residue of a portion of the mouse variable heavy chain or mouse variable light chain is substituted with a corresponding amino acid from the feline variable region. In some embodiments, the modified chain is fused to a feline constant heavy chain or a canine constant light chain. In some embodiments, the anti-IL31 antibody is a felineized antibody comprising (a) the light chain sequence of SEQ ID NO: 32, (b) the light chain sequence of SEQ ID NO: 34, (c) the heavy chain sequence of SEQ ID NO: 33, or (d) the heavy chain sequence of SEQ ID NO: 35.

[0091] A “euthanized antibody” refers to an antibody in which at least one amino acid in a portion of the non-equine variable region is replaced with a corresponding amino acid from the equine variable region. In some embodiments, the euthanized antibody includes at least one equine constant region (e.g., the γ constant region, α constant region, δ constant region, ε constant region, μ constant region, etc.) or a fragment thereof. In some embodiments, the euthanized antibody is an antibody fragment such as Fab, scFv, (Fab')2. The term “euthanized” also refers to a form of non-equine (e.g., mouse) antibody that is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding sequence of the antibody) containing the minimal sequence of a non-equine immunoglobulin. An euthanized antibody may include an equine immunoglobulin (recipient antibody) in which residues from the recipient's CDR are replaced with residues from the CDR of a non-equine species such as mouse, rat, or rabbit (donor antibody) having desired specificity, affinity, and ability. In some cases, the Fv framework region (FR) residues of equine immunoglobulin are replaced by corresponding non-equine residues. Furthermore, eugenic antibodies may include residues that are not found in the recipient antibody or the imported CDR or framework sequence, but which can be included to further improve and optimize antibody performance.

[0092] In some embodiments, at least one amino acid residue of a portion of a mouse variable heavy chain or mouse variable light chain is substituted with a corresponding amino acid from a horse variable region. In some embodiments, the modified chain is fused to a horse constant heavy chain or a canine constant light chain.

[0093] A “fragment crystallizable polypeptide” or “Fc polypeptide” is a portion of an antibody molecule that interacts with an effector molecule and cells. This includes the C-terminal portion of an immunoglobulin heavy chain. As used herein, an Fc polypeptide comprises a fragment of one or more biologically active Fc domains of a whole Fc polypeptide. In some embodiments, the biological activity of an Fc polypeptide is its ability to bind to FcRn. In some embodiments, the biological activity of an Fc polypeptide is its ability to bind to C1q. In some embodiments, the biological activity of an Fc polypeptide is its ability to bind to CD16. In some embodiments, the biological activity of an Fc polypeptide is its ability to bind to protein A. The “effector function” of an Fc polypeptide is an action or activity performed whole or partially by any antibody in response to a stimulus, which may include complement fixation and / or ADCC (antibody-dependent cytotoxicity) induction.

[0094] The term "IgX Fc" means that the Fc region originates from a specific antibody isotype (e.g., IgG, IgA, IgD, IgE, IgM, etc.), where "X" indicates the antibody isotype. For example, "IgG Fc" indicates the Fc region of the γ chain, "IgA Fc" indicates the Fc region of the α chain, "IgD Fc" indicates the Fc region of the δ chain, "IgE Fc" indicates the Fc region of the ε chain, and "IgM Fc" indicates the Fc region of the μ chain. In some embodiments, the IgG Fc region includes CH1, hinge, CH2, CH3, and CL1. "IgG-N-Fc" indicates that the Fc region originates from a specific subclass of the antibody isotype (e.g., canine IgG subclass A, B, C, or D; feline IgG subclass 1, 2a, or 2b, etc.), where "N" indicates the subclass. In some embodiments, the IgX Fc or IgX-N-Fc region is derived from a companion animal such as a dog, cat, or horse. In some embodiments, the IgG Fc region is isolated from a canine γ heavy chain such as IgG-A, IgG-B, IgG-C, or IgG-D. In some examples, the IgG Fc region is isolated from a feline γ heavy chain such as IgG1, IgG2a, or IgG2b. Antibodies containing the Fc region of IgG-A, IgG-B, IgG-C, or IgG-D may provide higher expression levels in recombinant production systems.

[0095] The terms "IgX Fc" and "IgX Fc polypeptide" include wild-type IgX Fc polypeptide and variant IgX Fc polypeptide unless otherwise specified.

[0096] "Wild-type" refers to a naturally occurring polypeptide or a non-mutated version of a fragment thereof. Wild-type polypeptides may be produced recombinantly.

[0097] In some embodiments, the wild-type IgG Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 91.

[0098] A "variant" is a polypeptide that differs from a reference polypeptide by the substitution, deletion, and / or addition of one or more non-natural amino acids. In some embodiments, the variant retains at least one biological activity of the reference polypeptide (e.g., the wild-type polypeptide).

[0099] As used herein, “variant IgG Fc polypeptide” is an IgG Fc polypeptide that differs from the reference IgG Fc polypeptide by one or more amino acid substitutions, deletions, and / or additions, and substantially retains at least one biological activity of the reference IgG Fc polypeptide.

[0100] In some embodiments, the variant IgG Fc polypeptide comprises a variant IgG Fc polypeptide of a companion animal species. In some embodiments, the variant IgG Fc polypeptide comprises a variant canine IgG Fc polypeptide or a feline IgG Fc polypeptide. In some embodiments, the variant IgG Fc polypeptide (e.g., variant canine IgG-A Fc polypeptide, variant canine IgG-C Fc polypeptide, or variant canine IgG-D Fc polypeptide, variant feline IgG1a Fc polypeptide, variant feline IgG1b Fc polypeptide, or variant feline IgG2 Fc polypeptide) has activity substantially lacking in the reference (e.g., wild-type) polypeptide. For example, in some embodiments, the variant canine IgG-A Fc polypeptide, variant canine IgG-C Fc polypeptide, or variant canine IgG-D Fc polypeptide binds to protein A.

[0101] In some embodiments, the variant IgG Fc polypeptide has modified protein A binding affinity. In some embodiments, the variant IgG Fc polypeptide has increased binding affinity to protein A. In some embodiments, the variant IgG Fc polypeptide can be purified using protein A column chromatography. In some embodiments, the variant IgG Fc polypeptide has modified CD16 binding affinity. In some embodiments, the variant IgG Fc polypeptide has decreased binding affinity to CD16. In some embodiments, the variant IgG Fc may reduce the ADCC immune response. In some embodiments, the variant IgG Fc polypeptide has modified C1q binding affinity. In some embodiments, the variant IgG Fc polypeptide has reduced binding affinity to C1q. In some embodiments, the variant IgG Fc polypeptide may reduce complement binding. In some embodiments, the variant IgG Fc may reduce the complement-mediated immune response. In some embodiments, the variant IgG Fc polypeptide has modified FcRn binding affinity. In some embodiments, the variant IgG Fc polypeptide has increased binding affinity to FcRn.

[0102] In some embodiments, the variant IgG Fc polypeptide has a modified neonatal receptor (FcRn) binding affinity. In some embodiments, the variant IgG Fc polypeptide exhibits increased binding affinity to FcRn, for example, at low pH.

[0103] In some embodiments, the variant IgG Fc polypeptide binds to FcRn with higher affinity than the wild-type IgG Fc polypeptide, as measured by biolayer interferometry, surface plasmon resonance, or any protein-protein interaction tool, in a pH range of about 5.0 to about 6.5, for example, about 5.0, about 5.2, about 5.5, about 6.0, about 6.2, or about 6.5.

[0104] In some embodiments, the variant IgG Fc polypeptide is measured at a pH in the range of about 5.0 to about 6.5, for example, about 5.0, about 5.5, about 6.0, or about 6.5, by biolayer interferometry, surface plasmon resonance, or any protein-protein interaction tool, to a pH of 5 × 10⁻¹⁶. -6 Less than M, 1 x 10 -6 Less than M, 5 x 10 -7 Less than M, 1 x 10 -7 Less than M, 5 x 10 -8 Less than M, 1 x 10 -8 Less than M, 5 x 10 -9 Less than M, 1 x 10 -9 Less than M, 5 x 10 -10 Less than M, 1 x 10 -10 Less than M, 5 x 10 -11 Less than M, 1 x 10 -11 Less than M, 5 x 10 -12 Less than M, or 1 × 10 -12 It binds to FcRn with a dissociation constant (Kd) less than M.

[0105] In some embodiments, long-acting isolated antibodies that bind to canine IL-31 are provided. In some embodiments, the anti-IL-31 antibody has an increased serum half-life. In some embodiments, the anti-IL-31 antibody comprises a variant Fc polypeptide, in which case the anti-IL-31 antibody has an increased serum half-life compared to an antibody comprising a wild-type Fc polypeptide.

[0106] In some embodiments, the anti-IL31 antibody comprises a variant IgG Fc polypeptide that can bind to FcRn with increased affinity compared to the wild-type Fc polypeptide, and the antibody has an increased serum half-life compared to the anti-IL31 antibody containing the wild-type Fc polypeptide.

[0107] In some embodiments, the variant IgG Fc polypeptide contains tyrosine or phenylalanine at the position corresponding to position 23 of SEQ ID NO: 90. In some embodiments, the variant IgG Fc polypeptide contains tyrosine at the position corresponding to position 82 of SEQ ID NO: 90. In some embodiments, the variant IgG Fc polypeptide contains tyrosine at the position corresponding to position 82 of SEQ ID NO: 90 and histidine at the position corresponding to position 207. In some embodiments, the variant IgG Fc polypeptide contains tyrosine at the position corresponding to position 82 of SEQ ID NO: 90 and tyrosine at the position corresponding to position 207. In some embodiments, the variant IgG Fc polypeptide contains tyrosine at the position corresponding to position 207 of SEQ ID NO: 90. In some embodiments, the variant IgG Fc polypeptide contains tyrosine at the position corresponding to position 82 of SEQ ID NO: 90 and histidine at the position corresponding to position 207. In some embodiments, the variant IgG Fc polypeptide contains tyrosine at the position corresponding to position 82 of SEQ ID NO: 90 and tyrosine at the position corresponding to position 207. In some embodiments, the variant IgG Fc polypeptide contains tyrosine at the position corresponding to position 207 of SEQ ID NO: 90.

[0108] In some embodiments, the variant IgG Fc polypeptide contains tyrosine or phenylalanine at position 23 of SEQ ID NO: 90. In some embodiments, the variant IgG Fc polypeptide contains tyrosine at position 82 of SEQ ID NO: 90. In some embodiments, the variant IgG Fc polypeptide contains tyrosine at position 82 and histidine at position 207 of SEQ ID NO: 90. In some embodiments, the variant IgG Fc polypeptide contains tyrosine at position 82 and tyrosine at position 207 of SEQ ID NO: 90. In some embodiments, the variant IgG Fc polypeptide contains tyrosine at position 207 of SEQ ID NO: 90. In some embodiments, the variant IgG Fc polypeptide contains tyrosine at position 82 and histidine at position 207 of SEQ ID NO: 90. In some embodiments, the variant IgG Fc polypeptide contains tyrosine at position 82 and tyrosine at position 207 of SEQ ID NO: 90. In some embodiments, the variant IgG Fc polypeptide contains tyrosine at position 207 of SEQ ID NO: 90.

[0109] In some embodiments, the variant IgG Fc polypeptide contains the amino acid sequences of SEQ ID NOs: 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, and 107.

[0110] In some embodiments, the anti-IL31 antibody comprises a variant canine IgG-A, IgG-B, IgG-C, or IgG-D Fc polypeptide as described herein. In some embodiments, the anti-IL31 antibody comprises (a) a variant canine IgG-A Fc polypeptide comprising the amino acid sequence of SEQ ID NO: 96 or SEQ ID NO: 97; a variant canine IgG-B Fc polypeptide comprising the amino acid sequence of SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, or SEQ ID NO: 107; or (c) a variant canine IgG-D Fc polypeptide comprising the amino acid sequence of SEQ ID NO: 98 or SEQ ID NO: 99. In some embodiments, the anti-IL31 antibody comprises a variant canine IgG-B Fc polypeptide comprising the amino acid sequence of SEQ ID NO: 105. In some embodiments, the anti-IL31 antibody comprises a variant canine IgG-B Fc polypeptide comprising the amino acid sequence of SEQ ID NO: 106. In some embodiments, the anti-IL31 antibody comprises a variant canine IgG-B Fc polypeptide containing the amino acid sequence of SEQ ID NO: 107.

[0111] In some embodiments, the anti-IL31 antibody includes a heavy chain containing the amino acid sequence of SEQ ID NO: 108, SEQ ID NO: 109, or SEQ ID NO: 110. In some embodiments, the anti-IL31 antibody includes a heavy chain containing the amino acid sequence of SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 125, SEQ ID NO: 126, or SEQ ID NO: 127. In some embodiments, the anti-IL31 antibody includes a heavy chain containing the amino acid sequence of SEQ ID NO: 114, SEQ ID NO: 115, or SEQ ID NO: 116. In some embodiments, the anti-IL31 antibody includes a heavy chain containing the amino acid sequence of SEQ ID NO: 117, SEQ ID NO: 118, or SEQ ID NO: 119. In some embodiments, the anti-IL31 antibody includes a heavy chain containing the amino acid sequence of SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122.

[0112] In some embodiments, the anti-IL31 antibody comprises a) the heavy chain amino acid sequence of SEQ ID NO: 108, SEQ ID NO: 109, or SEQ ID NO: 110, b) the heavy chain amino acid sequence of SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 125, SEQ ID NO: 126, or SEQ ID NO: 127, c) the heavy chain amino acid sequence of SEQ ID NO: 114, SEQ ID NO: 115, or SEQ ID NO: 116, d) the heavy chain amino acid sequence of SEQ ID NO: 117, SEQ ID NO: 118, or SEQ ID NO: 119, or e) the heavy chain amino acid sequence of SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122.

[0113] In some embodiments, the anti-IL31 antibody comprises (i) the light chain amino acid sequence of SEQ ID NO: 26, (ii) the heavy chain amino acid sequence of SEQ ID NO: 108, SEQ ID NO: 109, or SEQ ID NO: 110, or (iii) a light chain amino acid sequence like (i) and a heavy chain amino acid sequence like (ii).

[0114] In some embodiments, the anti-IL31 antibody comprises (i) the light chain amino acid sequence of SEQ ID NO: 21, (ii) the heavy chain amino acid sequence of SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 125, SEQ ID NO: 126, or SEQ ID NO: 127, or (iii) a light chain amino acid sequence like (i) and a heavy chain amino acid sequence like (ii).

[0115] In some embodiments, the anti-IL31 antibody comprises (i) the light chain amino acid sequence of SEQ ID NO: 37, (ii) the heavy chain amino acid sequence of SEQ ID NO: 114, SEQ ID NO: 115, or SEQ ID NO: 116, or (iii) a light chain amino acid sequence like (i) and a heavy chain amino acid sequence like (ii).

[0116] In some embodiments, the anti-IL31 antibody comprises (i) the light chain amino acid sequence of SEQ ID NO: 38, (ii) the heavy chain amino acid sequences of SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, or (iii) a light chain amino acid sequence like (i) and a heavy chain amino acid sequence like (ii).

[0117] In some embodiments, the anti-IL31 antibody comprises (i) the light chain amino acid sequence of SEQ ID NO: 39, (ii) the heavy chain amino acid sequence of SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122, or (iii) a light chain amino acid sequence like (i) and a heavy chain amino acid sequence like (ii).

[0118] The term "affinity" refers to the sum of the strength 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 is generally expressed by the dissociation constant (K). D Affinity can be expressed by the following methods: Affinity can be measured by common methods known in the art, such as immunoblotting, ELISA KD, KinEx A, biolayer interferometry (BLI), or surface plasmon resonance devices.

[0119] "K D "K" d The terms "Kd" or "Kd value" are used interchangeably to refer to the equilibrium dissociation constant of the antibody-antigen interaction. In some embodiments, the K of the antibody d This is measured using a biolayer interferometry assay with a biosensor such as Octet® System (Pall ForteBio LLC, Fremont, CA), according to the supplier's instructions. Briefly, the biotinylated antigen is bound to the sensor tip, antibody association is monitored for 90 seconds, and dissociation is monitored for 600 seconds. The buffer for the dilution and binding steps is 20 mM phosphate, 150 mM NaCl, pH 7.2. A buffer-only blank curve is subtracted to correct for any drift. The data is 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 Determine the equilibrium dissociation constant (K d ) is k off / k onIt is calculated as a ratio. The term "kon" refers to the rate constant of association between the antibody and the antigen, and the term "koff" refers to the rate constant of dissociation of the antibody from the antibody / antigen complex.

[0120] The term “binding” to an antigen or epitope is a well-understood term in the art, and methods for determining such binding are also well-known in the art. A molecule is said to “bind” if it reacts with, associates with, or has affinity for a particular cell or substance, and the reaction, association, or affinity is detectable by one or more methods known in the art, such as immunoblotting, ELISA KD, KinEx A, biolayer interferometry (BLI), or surface plasmon resonance devices.

[0121] Surface plasmon resonance (SPL) refers to an optical phenomenon that enables real-time analysis of biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore® system (BIAcore International AB, GE Healthcare, Uppsala, Sweden and Piscataway, NJ). For further explanation, see Jonsson et al. (1993) Ann. Biol. Clin. 51:19-26.

[0122] "Biolayer interferometry" refers to an optical analysis technique that analyzes the interference patterns of light reflected from the biosensor tip and layers of immobilized proteins on the internal reference layer. Changes in the number of molecules bound to the biosensor tip cause shifts in the interference pattern that can be measured in real time. A non-exclusive exemplary device for biolayer interferometry is the Octet® system (Pall ForteBio LLC). See, for example, Abdiche et al., 2008, Anal. Biochem. 377:209-277.

[0123] In some embodiments, the anti-IL31 antibody has a value of 5×10 as measured by bio-layer interferometry -6 M or less, 1×10 -6 M or less, 5×10 -7 M or less, 1×10 -7 M or less, 5×10 -8 M or less, 1×10 -8 M or less, 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, 1×10 -10 M or less, 5×10 -11 M or less, 1×10 -11 M or less, 5×10 -12 M or less, or 1×10 -12 M or less dissociation constant (Kd), binds to canine IL31, feline IL31 or equine IL31. In some embodiments, the anti-IL31 antibody has, as measured by bio-layer interferometry, 5x10 -6 M~1x10 -6 M, 5x10 -6 M~5x10 -7 M, 5x10 -6 M~1x10 -7 M, 5x10 -6 M~5x10 -8 M, 5x10 -6 M~1x10 -8 M, 5x10 -6 M~5x10 -9 M, 5x10 -6 M~1x10 -9 M, 5x10 -6 M~5x10 -10 M, 5x10 -6 M~1x10 -10 M, 5x10 -6 M~5x10 -11 M, 5x10 -6 M~1x10 -11 M, 5x10 -6 M~5x10 -12 M, 5x10 -6 M~1x10 -12 M, 1x10 -6 M~5x10 -7 M, 1x10 -6 M~1x10 -7 M, 1x10 -6M~5x10 -8 M、1x10 -6 M~1x10 -8 M、1x10 -6 M~5x10 -9 M、1x10 -6 M~1x10 -9 M、1x10 -6 M~5x10 -10 M、1x10 -6 M~1x10 -10 M、1x10 -6 M~5x10 -11 M、1x10 -6 M~1x10 -11 M、1x10 -6 M~5x10 -12 M、1x10 -6 M~1x10 -12 M、5x10 -7 M~1x10 -7 M、5x10 -7 M~5x10 -8 M、5x10 -7 M~1x10 -8 M、5x10 -7 M~5x10 -9 M、5x10 -7 M~1x10 -9 M、5x10 -7 M~5x10 -10 M、5x10 -7 M~1x10 -10 M、5x10 -7 M~5x10 -11 M、5x10 -7 M~1x10 -11 M、5x10 -7 M~5x10 -12 M、5x10 -7 M~1x10 -12 M、1x10 -7 M~5x10 -8 M、1x10 -7 M~1x10 -8 M、1x10 -7 M~5x10 -9 M、1x10 -7 M~1x10 -9 M、1x10 -7 M~5x10 -10 M、1x10 -7 M~1x10-10 M、1x10 -7 M~5x10 -11 M、1x10 -7 M~1x10 -11 M、1x10 -7 M~5x10 -12 M、1x10 -7 M~1x10 -12 M、5x10 -8 M~1x10 -8 M、5x10 -8 M~5x10 -9 M、5x10 -8 M~1x10 -9 M、5x10 -8 M~5x10 -10 M、5x10 -8 M~1x10 -10 M、5x10 -8 M~5x10 -11 M、5x10 -8 M~1x10 -11 M、5x10 -8 M~5x10 -12 M、5x10 -8 M~1x10 -12 M、1x10 -8 M~5x10 -9 M、1x10 -8 M~1x10 -9 M、1x10 -8 M~5x10 -10 M、1x10 -8 M~1x10 -10 M、1x10 -8 M~5x10 -11 M、1x10 -8 M~1x10 -11 M、1x10 -8 M~5x10 -12 M、1x10 -8 M~1x10 -12 M、5x10 -9 M~1x10 -9 M、5x10 -9 M~5x10 -10 M、5x10 -9 M~1x10 -10 M、5x10 -9 M~5x10 -11 M、5x10 -9 M~1x10 -11M, 5x10 -9 M~5x10 -12 M, 5x10 -9 M~1x10 -12 M, 1x10 -9 M~5x10 -10 M, 1x10 -9 M~1x10 -10 M, 1x10 -9 M~5x10 -11 M, 1x10 -9 M~1x10 -11 M, 1x10 -9 M~5x10 -12 M, 1x10 -9 M~1x10 -12 M, 5x10 -10 M~1x10 -10 M, 5x10 -10 M~5x10 -11 M, 1x10 -10 M~5x10 -11 M, 1x10 -10 M~1x10 -11 M, 1x10 -10 M~5x10 -12 M, 1x10 -10 M~1x10 -12 M, 5x10 -11 M~1x10 -12 M, 5x10 -11 M~5x10 -12 M, 5x10 -11 M~1x10 -12 M, 1x10 -11 M~5x10 -12 M, or 1x10 -11 M~1x10 -12 The M-Kd antibody binds to canine IL31, feline IL31, or equine IL31. In some embodiments, the anti-IL31 antibody binds to canine IL31, feline IL31, or equine IL31 as determined by immunoblotting analysis.

[0124] In some embodiments, the anti-IL31 antibody does not bind to human IL31, as determined by immunoblotting and / or biolayer interferometry.

[0125] In some embodiments, anti-IL31 antibodies are provided that compete with the anti-IL31 antibodies described herein (e.g., M14, M18, M19, or M87) for binding to IL31. In some embodiments, antibodies can be produced or used that compete with any of the antibodies provided herein for binding. In some embodiments, anti-IL31 antibodies are provided that compete with monoclonal M14 antibodies for binding to canine IL31 or feline IL31.

[0126] "Increased" or "greater" means an increase relative to the reference. In some embodiments, "increased" or "greater" means the ability to cause an overall increase of about 5% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or more, about 125% or more, about 150% or more, about 200% or more, or about 300% or more relative to the reference value. In some embodiments, "increased" or "greater" means the ability to cause an overall increase of about 5% to about 50%, about 10% to about 20%, about 50% to about 100%, or about 25% to about 70% relative to the reference value.

[0127] In some embodiments, a variant Fc polypeptide, such as a variant IgG Fc polypeptide, can bind to FcRn or FcRn / B2M with an increased affinity of about 5% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or more, about 125% or more, about 150% or more, or about 200% or more compared to a reference Fc polypeptide. In some embodiments, a variant Fc polypeptide can bind to FcRn or FcRn / B2M with an increased affinity of about 5% to about 50%, about 10% to about 20%, about 50% to about 100%, or about 25% to about 70% compared to a reference Fc polypeptide. In some embodiments, the reference Fc polypeptide is a wild-type Fc polypeptide. In some embodiments, the Fc polypeptide is a different variant Fc polypeptide. In some embodiments, affinity is measured by biolayer interferometry at a pH in the range of approximately 5.0 to approximately 6.5.

[0128] In some embodiments, pharmacokinetic analysis is performed to determine any number of pharmacokinetic parameters, including half-life, Tmax, Cmax, and area under the curve (AUC). For example, animals may be administered the anti-IL31 antibody described herein and serum samples collected at different time intervals (e.g., before injection and / or 0.5, 1, 6, 24, 48, 72, 168, 216, and / or 336 hours after administration). The antibody concentration in the serum samples can be determined, for example, by ELISA.

[0129] In some embodiments, the anti-IL31 antibody has a serum half-life of approximately 5% or more, approximately 10% or more, approximately 20% or more, approximately 30% or more, approximately 40% or more, approximately 50% or more, approximately 60% or more, approximately 70% or more, approximately 80% or more, approximately 90% or more, approximately 100% or more, approximately 125% or more, approximately 150% or more, approximately 200% or more, approximately 250% or more, or approximately 300% or more compared to the reference anti-IL31 antibody. In some embodiments, the anti-IL31 antibody has a serum half-life of approximately 5% to approximately 50%, approximately 10% to approximately 20%, approximately 50% to approximately 100%, or approximately 25% to approximately 70% compared to the reference anti-IL31 antibody. In some embodiments, the anti-IL31 antibody has a serum half-life of approximately 1.5 times or more, approximately 2 times or more, or approximately 3 times or more compared to the reference anti-IL31 antibody. In some embodiments, the anti-IL31 antibody has a serum half-life that is approximately 5% to 50%, 10% to 20%, 50% to 100%, 25% to 70%, and 200% to 300% longer than the reference anti-IL31 antibody. In some embodiments, the reference anti-IL31 antibody contains a wild-type Fc polypeptide. In some embodiments, the Fc polypeptide is a different variant Fc polypeptide.

[0130] A "variant" refers to a biologically active polypeptide that has at least approximately 50% amino acid sequence identity with the native polypeptide, after the sequence has been aligned and gaps introduced as necessary to achieve maximum percent sequence identity, and no conservative substitutions have been considered as part of the sequence identity. Examples of such variants include polypeptides in which one or more amino acid residues have been added or deleted from the N-terminus or C-terminus of the polypeptide.

[0131] In some embodiments, the variant has at least about 50% amino acid sequence identity with the natural sequence polypeptide, 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, and at least about 95% amino acid sequence identity.

[0132] As used herein, “percent (%) amino acid sequence identity” and “homology” with respect to 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 particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the scope of the art of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALINE® (DNASTAR) software. A person skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm necessary to achieve maximum alignment over the full length of the sequences being compared.

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

[0134] TIFF0007943816000019.tif124170

[0135] Amino acids can be classified according to their general side-chain properties. (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln (3) Acidic: Asp, Glu (4) Basicity: His, Lys, Arg (5) Residues that affect chain orientation: Gly, Pro (6) Aromatic: Trp, Tyr, Phe

[0136] Non-conservative substitution involves swapping one member of one class with another.

[0137] In some embodiments, the anti-IL31 antibody comprises a heavy chain and a light chain. a. The heavy chain comprises a CDR-H1 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1; a CDR-H2 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity to the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 62, SEQ ID NO: 89, or SEQ ID NO: 87; and a CDR-H3 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 3. b. The light chain includes a CDR-L1 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 63; a CDR-L2 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 9; and a CDR-L3 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0138] In some embodiments, the anti-IL31 antibody comprises a heavy chain and a light chain. a. (i) A variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with respect to the amino acid sequence of SEQ ID NO: 24; (ii) A variable heavy chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with respect to the amino acid sequence of SEQ ID NO: 25; or (iii) A variable light chain sequence like (i) and a variable heavy chain sequence like (ii); or b. (i) A variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with respect to the amino acid sequence of SEQ ID NO: 16; (ii) A variable heavy chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with respect to the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 123; or (iii) A variable light chain sequence like (i) and a variable heavy chain sequence like (ii); or c. (i) A variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with respect to the amino acid sequence of SEQ ID NO: 32; (ii) A variable heavy chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with respect to the amino acid sequence of SEQ ID NO: 33; or (iii) A variable light chain sequence like (i) and a variable heavy chain sequence like (ii); or d. (i) A variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 64; (ii) A variable heavy chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 67; or (iii) A variable light chain sequence like (i) and a variable heavy chain sequence like (ii); or e. (i) A variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with respect to the amino acid sequence of SEQ ID NO: 65; (ii) A variable heavy chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; or (iii) A variable light chain sequence like (i) and a variable heavy chain sequence like (ii); or f. (i) A variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 66; (ii) A variable heavy chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 69; or (iii) A variable light chain sequence like (i) and a variable heavy chain sequence like (ii).

[0139] The term "vector" is used to describe a cloned polynucleotide or a polynucleotide that can be manipulated to contain a polynucleotide that can be grown in a host cell. A vector may contain one or more of the following elements: a replication origin, one or more regulatory sequences that regulate the expression of the polypeptide of interest (e.g., promoter or enhancer), or one or more select marker genes (e.g., antibiotic resistance genes and genes that can be used in colorimetric assays, e.g., β-galactosidase). The term "expression vector" refers to a vector used to express the polypeptide of interest in a host cell.

[0140] "Host cell" refers to a cell that may or may have been a recipient of a vector or isolated polynucleotide. A host cell may be a prokaryotic or eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, e.g., primate or non-primate animal cells; fungal cells, e.g., yeast; plant cells; and insect cells. Non-exclusive exemplary mammalian cells include, but are not limited to, NS0 cells, PER.C6® cells (Crucell), 293 cells, and CHO cells, as well as their derivatives such as 293-6E, DG44, CHO-S, and CHO-K cells. A host cell may include offspring of a single host cell, which may not necessarily be completely identical to the original parent cell (in morphology or genomic DNA complement) due to natural, accidental, or intentional mutations. A host cell may include cells transfected in vivo with polynucleotides encoding the amino acid sequences provided herein.

[0141] As used herein, the term “isolated” refers to a molecule separated from at least some of its components that are typically found or produced in nature. For example, a polypeptide is called “isolated” if it is separated from at least some of the components of the cell in which it was produced. If a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered “isolated” the polypeptide. Similarly, a polynucleotide is called “isolated” if it is not part of a larger polynucleotide typically found in nature (e.g., in the case of a DNA polynucleotide, such as genomic DNA or mitochondrial DNA), or, for example, in the case of an RNA polynucleotide, if it is separated from at least some of the components of the cell in which it was produced. Thus, a DNA polynucleotide contained in a vector within a host cell may be called “isolated.” In some embodiments, anti-IL31 antibodies are purified using chromatography, such as size exclusion chromatography, ion exchange chromatography, protein A column chromatography, hydrophobic interaction chromatography, and CHT chromatography.

[0142] The term "companion animal species" refers to animals suitable to serve as human companions. In some embodiments, companion animal species are small mammals such as dogs, cats, horses, rabbits, ferrets, guinea pigs, and rodents. In some embodiments, companion animal species are domesticated animals such as horses, cattle, and pigs.

[0143] The term "IL31 signaling function" refers to any one or combination of downstream activities that occur when IL31 binds to its receptor or receptor complex. In some embodiments, IL31 signaling function includes activation of Janus kinase (Jak) 1 or Jak 2 signaling molecules. In some embodiments, IL31 signaling function includes phosphorylation of STAT-3 or STAT-5 proteins. In some embodiments, IL31 signaling function includes activation of the ERK1 / 2 MAP kinase signaling pathway. In some embodiments, IL31 signaling function includes activation of the PI3K / AKT signaling pathway. In some embodiments, IL31 signaling function includes activation of the Jak1 / 2 signaling pathway.

[0144] "STAT phosphorylation" refers to post-expression modification of STAT proteins by phosphorylation. For example, "STAT-3 phosphorylation" refers to phosphorylation of STAT-3, and "STAT-5 phosphorylation" refers to phosphorylation of STAT-5. In some embodiments, STAT-3 phosphorylation is measured by immunoblotting. For example, cells (e.g., canine monocytes DH82 cells) are incubated at 37°C for 24 hours in the presence of the anti-IL31 antibody described herein in growth medium (e.g., MEM, Life Technologies®) containing 15% heat-inactivated fetal bovine serum, 2 mmol / L GlutaMax, 1 mmol / L sodium pyruvate, and 10 nm / mL canine interferon-c (R&D Systems, Minneapolis, MN, USA) at a rate of 1 × 10⁶ cells per well. 5Cells were placed in 96-well cell culture plates at the specified cell density. The concentrations of phosphorylated and unphosphorylated STAT-3 were detected relative to each other and compared to a β-actin control using immunoblotting analysis of cell lysates with anti-phospho STAT-3 antibodies and anti-STAT-3 antibodies (R&D Systems). Methods for qualitatively or quantitatively determining protein concentrations by immunoblotting are well understood by those skilled in the art. In some embodiments, relative concentrations are qualitatively determined by visual inspection of the immunoblots. In some embodiments, the concentrations of phosphorylated and unphosphorylated STAT-3 are quantitatively determined by digitally imaging the immunoblots, determining the intensity of the bands, and inversely calculating the concentration of phosphorylated or unphosphorylated STAT-3 in the sample using a linear standard curve of STAT-3 protein at known concentrations.

[0145] "To reduce" or "to inhibit" means to decrease, reduce, or halt activity, function, or amount compared to a reference. In some embodiments, "to reduce" or "to inhibit" means the ability to cause an overall reduction of 20% or more. In some embodiments, "to reduce" or "to inhibit" means the ability to cause an overall reduction of 50% or more. In some embodiments, "to reduce" or "to inhibit" means the ability to cause an overall reduction of 75%, 85%, 90%, 95% or more. In some embodiments, the above amounts are inhibited or reduced over a period of time compared to a control dose (such as a placebo) over the same period. As used herein, "reference" refers to any sample, standard, or level used for comparative purposes. References may be obtained from healthy or non-disease samples. In some examples, references may be obtained from non-disease or non-treatment samples of companion animals. In some examples, references may be obtained from one or more healthy animals of a particular species that are not being tested or treated.

[0146] As used herein, the term “substantially reduced” indicates 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 the value. In some embodiments, a substantially reduced numerical value is reduced by more than one of approximately 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the reference value.

[0147] In some embodiments, an IL31 antibody can reduce IL31 signaling function in companion animal species by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to IL31 signaling function in the absence of the antibody, by a reduction in STAT-3 phosphorylation.In some embodiments, the reduction of IL31 signaling function or STAT-3 phosphorylation is 10%~15%, 10%~20%, 10%~25%, 10%~30%, 10%~35%, 10%~40%, 10%~45%, 10%~50%, 10%~60%, 10%~70%, 10%~80%, 10%~90%, 10%~100%, 15%~20%, 15%~25%, 15%~30%, 15%~35%, 15%~40%, 15%~45%, 15%~50%, 15%~60%, 15%~70%, 15%~80%, 15%~90%, 15%~100%, 20%~25%, 20%~30%, 20%~35%, 20%~40%, 20%~45%, 20%~50%, 20%~60%, 20%~70%, 20%~80%, 20%~90%, 20%~100%, 25%~30%, 25%~35%, 25%~40%, 25%~45%, 25%~50%, 25%~60%, 25%~70%, 25%~ 80%, 25%~90%, 25%~100%, 30%~35%, 30%~40%, 30%~45%, 30%~50%, 30%~60%, 30%~70%, 30%~80%, 30%~90%, 30%~100%, 35%~40%, 35%~45%, 35%~50%, 35%~60%, 35%~70%, 35%~80%, 35%~90%, 35%~100%, 40%~45%, 40%~50%, 40%~60%, 40%~70%, 40%~80% The percentages are 40%~90%, 40%~100%, 45%~50%, 45%~60%, 45%~70%, 45%~80%, 45%~90%, 45%~100%, 50%~60%, 50%~70%, 50%~80%, 50%~90%, 50%~100%, 60%~70%, 60%~80%, 60%~90%, 60%~100%, 70%~80%, 70%~90%, 70%~100%, 80%~90%, 80%~100%, or 90%~100%.

[0148] Pharmaceutical composition The terms "pharmaceutical preparation" and "pharmaceutical composition" refer to preparations that are in a form that allows the biological activity of the active ingredient to be effective and that do not contain additional ingredients that are unacceptably toxic to the subject to which the preparation is administered.

[0149] A "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation adjuvant, or carrier that is conventional in the art for use with a therapeutic agent containing a "pharmaceutically acceptable composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dose and concentration used and is compatible with the other components of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation in which it is used. Examples of pharmaceutically acceptable carriers include alumina; aluminum stearate; lecithin; serum proteins, e.g., human serum albumin, canine or other animal albumin; buffers, e.g., phosphates, citrates, tromethamine or HEPES buffer; glycine; sorbic acid; potassium sorbate; partial glyceride mixtures of saturated vegetable fatty acids; water; salts or electrolytes, e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, or magnesium trisilicate; polyvinylpyrrolidone, cellulosic substances; polyethylene glycol; sucrose; mannitol; or amino acids, including but not limited to arginine.

[0150] Pharmaceutical compositions can be stored in lyophilized form. Therefore, in some embodiments, the preparation process includes a lyophilization step. The lyophilized composition can then typically be reformulated 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 against thermal and oxidative denaturation, the pharmaceutical composition can be stored as a liquid, i.e., an aqueous composition, which can then be administered directly to dogs, cats, or horses or with appropriate dilution. The lyophilized composition can be reconstituted with sterile water for injection (WFI). Antimicrobial agents (e.g., bacteriostatic reagents, e.g., benzyl alcohol) may be included. Thus, the present invention provides pharmaceutical compositions in solid or liquid form.

[0151] The pH of the pharmaceutical composition may be in the range of approximately pH 5 to approximately pH 8 when administered. The compositions of the present invention are sterile when used for therapeutic purposes. Sterility can be achieved by any of several means known in the art, including filtration with a sterile filtration membrane (e.g., a 0.2 micron membrane). Sterility can be maintained with or without antimicrobial agents.

[0152] In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition has a pH of 5.0–6.2, 5.0–6.0, or 5.3–5.7. In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition has a pH of 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, or 6.2.

[0153] In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition comprises L-histidine, sodium chloride, and polysorbate 80.

[0154] In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition contains sodium chloride at concentrations of 80 nM to 200 nM, 100 nM to 180 nM, 100 nM to 175 nM, 110 nM to 170 nM, 120 nM to 160 nM, 120 nM to 150 nM, 130 nM to 150 nM, 130 nM to 160 nM, 100 nM, 80 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, or 200 nM.

[0155] In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition includes polysorbate 80 at concentrations of 0.005 mg / mL to 0.5 mg / mL, 0.01 mg / mL to 0.1 mg / mL, 0.1 mg / mL to 0.5 mg / mL, 0.005 mg / mL to 0.01 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, or 0.1 mg / mL.

[0156] In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition contains L-histidine at concentrations of 5 mM to 100 mM, 10 mM to 50 mM, 20 mM to 30 mM, 10 mM to 30 mM, 20 mM to 80 mM, 30 mM to 70 mM, 40 mM to 60 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, or 50 mM.

[0157] In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition comprises m-cresol or benzyl alcohol. In some embodiments, the concentration of m-cresol is about 0.2%, about 0.1% to about 0.3%, about 0.08% to about 0.25%, or about 0.05% to about 0.25%. In some embodiments, the concentration of benzyl alcohol is about 1%, about 0.5% to about 2%, about 0.2% to about 2.5%, about 1% to about 5%, about 0.5% to about 5%, or about 1% to about 3%.

[0158] In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition contains sugars. In some embodiments, the sugars are sucrose, trehalose, D-mannitol, maltose, and / or sorbitol. In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition contains sugars at concentrations of 0.5% to 20%, 1% to 10%, 1% to 5%, 1% to 3%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%.

[0159] In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition contains an antimicrobial agent. In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition contains m-cresol or methylparaben. In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition contains 0.2% m-cresol. In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition contains 0.9% methylparaben.

[0160] Use of antibodies and pharmaceutical compositions An antibody or pharmaceutical composition comprising the antibody of the present invention may be useful for treating an IL-31-induced condition. 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 diseases or allergic diseases. In some embodiments, the IL-31-induced condition is atopic dermatitis, pruritus, asthma, psoriasis, scleroderma or eczema. The IL-31-induced condition may be present in companion animals including, but not limited to, dogs, cats or horses.

[0161] As used herein, "treatment" refers to a procedure for obtaining beneficial or desired clinical results. As used herein, "treatment" encompasses any administration or application of a therapeutic agent for a disease in mammals, including companion animals. For the purposes of the present disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, attenuation of the extent of the disease, prevention or delay of disease spread, prevention or delay of disease recurrence, delay or slowing of disease progression, amelioration of the disease state, inhibition of the disease or its progression, inhibition or slowing of the disease or its progression, arrest of its onset, and remission (whether partial or total), any one or more of these. Reduction of the pathological consequences of a proliferative disease is also encompassed by "treatment". The methods provided herein contemplate any one or more of these aspects of treatment. Consistent with the above, the term treatment does not require 100% elimination of all aspects of the disorder.

[0162] In some embodiments, an anti-IL-31 antibody or a pharmaceutical composition comprising the same can be utilized according to the methods herein to treat an IL-31-induced condition. In some embodiments, the anti-IL-31 antibody or pharmaceutical composition is administered to a companion animal such as a dog, cat or horse to treat an IL-31-induced condition.

[0163] The "therapeutic effective dose" of a substance / molecule, agonist, or antagonist may vary depending on factors such as the type of disease being treated, the condition, the severity and course of the disease, the type of therapeutic objective, any previous treatments, medical history, response to previous treatments, the discretion of the attending veterinarian, the animal's age, sex, and weight, and the ability of the substance / molecule, agonist, or antagonist to induce the desired response in the animal. The therapeutic effective dose is also the amount in which the therapeutically beneficial effects outweigh any toxic or adverse effects of the substance / molecule, agonist, or antagonist. The therapeutic effective dose may be delivered in one or more doses. The therapeutic effective dose refers to the amount effective in the required dosage and duration to achieve the desired therapeutic or prophylactic outcome.

[0164] In some embodiments, the anti-IL31 antibody or the pharmaceutical composition containing the anti-IL31 antibody is administered by parenteral administration, subcutaneous administration, intravenous infusion, or intramuscular injection. In some embodiments, the anti-IL31 antibody or the pharmaceutical composition containing the anti-IL31 antibody is administered as a bolus injection or by continuous infusion over a period of time. In some embodiments, the anti-IL31 antibody or the pharmaceutical composition containing the anti-IL31 antibody is administered by an intramuscular route, an intraperitoneal route, an intracerebrospinal route, a subcutaneous route, an intraarterial route, an intrasynovial route, an intrathecal route, or an inhalation route.

[0165] The anti-IL31 antibodies described herein can be administered in an amount ranging from 0.01 mg / kg body weight to 100 mg / kg body weight per administration. In some embodiments, the anti-IL31 antibodies can be administered in an amount ranging from 0.5 mg / kg body weight to 50 mg / kg body weight per administration. In some embodiments, the anti-IL31 antibodies can be administered in an amount ranging from 0.1 mg / kg body weight to 10 mg / kg body weight per administration. In some embodiments, the anti-IL31 antibodies can be administered in an amount ranging from 0.1 mg / kg body weight to 100 mg / kg body weight per administration. In some embodiments, the anti-IL31 antibodies can be administered in an amount ranging from 1 mg / kg body weight to 10 mg / kg body weight per administration. In some embodiments, the anti-IL31 antibodies can be administered in an amount in the range of 0.5 mg / kg body weight to 100 mg / kg body weight, 1 mg / kg body weight to 100 mg / kg body weight, 5 mg / kg body weight to 100 mg / kg body weight, 10 mg / kg body weight to 100 mg / kg body weight, 20 mg / kg body weight to 100 mg / kg body weight, 50 mg / kg body weight to 100 mg / kg body weight, 1 mg / kg body weight to 10 mg / kg body weight, 5 mg / kg body weight to 10 mg / kg body weight, 0.5 mg / kg body weight to 10 mg / kg body weight, 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.

[0166] Anti-IL31 antibodies or pharmaceutical compositions containing anti-IL31 antibodies can be administered to companion animals as a single dose or over a series of treatments. For example, anti-IL31 antibodies or pharmaceutical compositions containing anti-IL31 antibodies may be administered at least once, twice or more times, at least twice, at least three times, at least four times, or at least five times. In some embodiments, anti-IL31 antibodies or pharmaceutical compositions containing anti-IL31 antibodies may be administered to companion animals once a month, for example, up to six months. In some embodiments, long-acting anti-IL31 antibodies or pharmaceutical compositions containing long-acting anti-IL31 antibodies may be administered to companion animals every two months, every three months, every four months, every five months, or every six months, as needed. In some embodiments, anti-IL31 antibodies or pharmaceutical compositions containing anti-IL31 antibodies may be administered to companion animals every five weeks, for example, up to six months. In some embodiments, an anti-IL31 antibody or a pharmaceutical composition containing an anti-IL31 antibody may be administered to a companion animal every six weeks, for example, up to six months. In some embodiments, a long-acting anti-IL31 antibody or a pharmaceutical composition containing a long-acting anti-IL31 antibody may be administered to a companion animal every four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks, twelve weeks, thirteen weeks, fourteen weeks, fifteen weeks, sixteen weeks, eighteen weeks, twenty weeks, twenty-two weeks, or twenty-four weeks.

[0167] 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, with optional intervals of one week or more without treatment. In other embodiments, the therapeutically effective dose is administered once daily for two to five consecutive days, and in some embodiments, this treatment cycle is repeated two or more times, with optional intervals of one day or more, or one week or more without treatment.

[0168] In some embodiments, the long-acting anti-IL31 antibody is administered at a reduced dose and / or an increased dosing interval compared to the reference anti-IL31 antibody.

[0169] Administering one or more additional therapeutic agents “in combination” includes concurrent and sequential or sequential administration in any order. The term “concurrently” is used herein to refer to the administration of two or more therapeutic agents where at least a portion of the administrations overlaps in time, or where the administration of one therapeutic agent occurs within a shorter period compared to the administration of another therapeutic agent. For example, two or more therapeutic agents are administered at time intervals of approximately no more than a specified number of minutes. The term “sequentially” is used herein to refer to the administration of two or more therapeutic agents where the administration of one or more agents follows the interruption of the administration of one or more other agents, or where the administration of one or more agents begins before the administration of one or more other agents. For example, the administration of two or more therapeutic agents is administered at time intervals exceeding approximately a specified number of minutes. As used herein, “in combination with” means administering one therapeutic agent in addition to another therapeutic agent. Thus, “in combination with” means administering one therapeutic agent before, during, or after the administration of other therapeutic agents to an animal.

[0170] In some embodiments, the method includes administering an anti-IL31 antibody or a pharmaceutical composition containing an anti-IL31 antibody in combination with a Jak inhibitor, a PI3K inhibitor, an AKT inhibitor, or a MAPK inhibitor. In some embodiments, the method includes administering an anti-IL31 antibody or a pharmaceutical composition containing an anti-IL31 antibody, an anti-IL4R antibody, an anti-IL17 antibody, an anti-TNFα antibody, an anti-CD20 antibody, an anti-CD19 antibody, an anti-CD25 antibody, an anti-IL4 antibody, an anti-IL13 antibody, an anti-IL23 antibody, an anti-IgE antibody, an anti-CD11α antibody, an anti-IL6R antibody, an anti-α4-integrin antibody, an anti-IL12 antibody, an anti-IL1β antibody, an anti-IL5 antibody, an anti-IL5R antibody, an anti-IL22 antibody, an anti-IL22R antibody, an anti-IL33 antibody, an anti-IL33R antibody, an anti-TSLP antibody, an anti-TSLPR antibody, or an anti-BlyS antibody in combination with a Jak inhibitor, a PI3K inhibitor, an anti-IL31 antibody, an anti-IL17 antibody, an anti-IL17 antibody, an anti-TNFα antibody, an anti-CD20 antibody, an anti-CD19 antibody, an anti-CD25 antibody, an anti-IL4 antibody, an anti-IL13 antibody, an anti-IL23 antibody, an anti-IL33 antibody, an anti-IL33R antibody, an anti-TSLP antibody, an anti-TSLPR antibody, or an anti-BlyS antibody.

[0171] Methods 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 are provided herein. In some embodiments, cells are exposed to the antibody or pharmaceutical composition ex vivo. In some embodiments, cells are exposed to the antibody or pharmaceutical composition in vivo. In some embodiments, cells are exposed to the anti-IL31 antibody or pharmaceutical composition under conditions that allow the antibody to bind to intracellular IL31. In some embodiments, cells are exposed to the anti-IL31 antibody or pharmaceutical composition under conditions that allow the antibody to bind to extracellular IL31. In some embodiments, cells may be exposed in vivo to the anti-IL31 antibody or pharmaceutical composition by one or more of the administration methods described herein, including, but not limited to, intraperitoneal injection, intramuscular injection, or intravenous injection into a subject. In some embodiments, cells may be exposed to the anti-IL31 antibody or pharmaceutical composition ex vivo by exposing the cells to a culture medium containing the antibody or pharmaceutical composition. In some embodiments, the permeability of the cell membrane can be influenced by the use of several methods known to those skilled in the art, such as electroporating the cells or exposing the cells to a solution containing calcium chloride, before exposing the cells to a culture medium containing an antibody or pharmaceutical composition.

[0172] In some embodiments, binding results in a reduction of IL31 signaling function by cells. In some embodiments, an 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 IL31 signaling function in the absence of the antibody, as measured by a reduction in STAT-3 phosphorylation.In some embodiments, the reduction of IL31 signaling function or STAT-3 phosphorylation is 10%~15%, 10%~20%, 10%~25%, 10%~30%, 10%~35%, 10%~40%, 10%~45%, 10%~50%, 10%~60%, 10%~70%, 10%~80%, 10%~90%, 10%~100%, 15%~20%, 15%~25%, 15%~30%, 15%~35%, 15%~40%, 15%~45%, 15%~50%, 15%~60%, 15%~70%, 15%~80%, 15%~90%, 15%~100%, 20%~25%, 20%~30%, 20%~35%, 20%~40%, 20%~45%, 20%~50%, 20%~60%, 20%~70%, 20%~80%, 20%~90%, 20%~100%, 25%~30%, 25%~35%, 25%~40%, 25%~45%, 25%~50%, 25%~60%, 25%~70%, 25%~ 80%, 25%~90%, 25%~100%, 30%~35%, 30%~40%, 30%~45%, 30%~50%, 30%~60%, 30%~70%, 30%~80%, 30%~90%, 30%~100%, 35%~40%, 35%~45%, 35%~50%, 35%~60%, 35%~70%, 35%~80%, 35%~90%, 35%~100%, 40%~45%, 40%~50%, 40%~60%, 40%~70%, 40%~80% The percentages are 40%~90%, 40%~100%, 45%~50%, 45%~60%, 45%~70%, 45%~80%, 45%~90%, 45%~100%, 50%~60%, 50%~70%, 50%~80%, 50%~90%, 50%~100%, 60%~70%, 60%~80%, 60%~90%, 60%~100%, 70%~80%, 70%~90%, 70%~100%, 80%~90%, 80%~100%, or 90%~100%.

[0173] Methods using anti-IL31 antibodies, polypeptides, and polynucleotides for the detection, diagnosis, and monitoring of IL31 induction status are provided herein. Methods for determining whether a companion animal responds to anti-IL31 antibody therapy are also provided herein. In some embodiments, the method includes detecting whether an animal has cells expressing IL31 using an anti-IL31 antibody. In some embodiments, the detection method includes contacting a sample with an antibody, polypeptide, or polynucleotide and determining whether the binding level differs from that of a reference or comparative sample (e.g., control). In some embodiments, the method may be useful for determining whether the antibody or polypeptide described herein is an appropriate treatment for an animal of interest.

[0174] In some embodiments, the sample is a biological sample. The term “biological sample” means a quantity of material from something that is alive or has been alive in the past. In some embodiments, the biological sample is cells or cell / tissue lysates. In some embodiments, biological samples include, but are not limited to, blood (e.g., whole blood), plasma, serum, urine, synovial fluid, and epithelial cells.

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

[0176] Various methods known in the art can be used to detect specific antibody-antigen binding. Exemplary immunoassays that can be performed include fluorescence-polarized immunoassays (FPIA), fluorescence immunoassays (FIA), enzyme immunoassays (EIA), turbidimetry-inhibited immunoassays (NIA), enzyme-linked immunosorbent assays (ELISA), and radioimmunoassays (RIA). The indicator moiety or labeling group can be bound to the target antibody and is selected to meet the needs of various methods used, often determined by the availability of assay equipment and suitable immunoassay procedures. Suitable labels include, but are not limited to, radionuclides (e.g., 125 I, 131 I, 35 S, 3 H, or 32 Examples include P), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or p-galactosidase), fluorescent moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g., Qdot® nanoparticles supplied by Quantum Dot Corporation (Palo Alto, Calif.)). Common techniques used in carrying out the various immunoassays described above are known to those skilled in the art.

[0177] For diagnostic purposes, antibody-containing polypeptides can be labeled with detectable portions, including, but not limited to, radioisotopes, fluorescent labels, and various enzyme-substrate labels known in the art. Methods for conjugating labels to antibodies are known in the art. In some embodiments, the anti-IL31 antibody does not need to be labeled, and its presence can be detected using a second labeled antibody conjugated to a first anti-IL31 antibody. In some embodiments, the anti-IL31 antibody can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. (Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987)). Anti-IL31 antibodies and polypeptides can also be used in in vivo diagnostic assays, such as in vivo imaging. Generally, the antibody or polypeptide can be conjugated with radionuclides (e.g., radioscintigraphy) so that the target cells or tissues can be localized using immunoscintigraphy. 111 In, 99 Tc, 14 C, 131 I, 125 I, 3 The antibodies are labeled with 1H or any other radionuclide (including those outlined herein). Antibodies may also be used as staining reagents in pathology using techniques known in the art.

[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 and second antibodies are different. In some embodiments, both the first and second antibodies can bind to the antigen simultaneously by binding to separate epitopes.

[0179] The following embodiments are illustrative of specific aspects of the Disclosure and are not intended to limit the Disclosure in any way. [Examples]

[0180] Example 1 Identification of mouse monoclonal antibodies that bind to canine IL31 The canine IL31 gene encoding the IL31 protein (SEQ ID NO: 22) was synthesized with a poly-His tag at its C-terminus and cloned into a mammalian expression vector. Plasmids containing the canine IL31 gene were transfected into 293 cells.

[0181] The supernatant containing the canine IL31 protein was collected and filtered. Canine IL31 was purified using a Ni-NTA column (CaptivA® Protein A Affinity Resin, Repligen).

[0182] Mouse monoclonal antibodies were identified using standard immunization with canine IL-31 produced by 293 cells as the immunogen. Two different adjuvants were used during immunization (Antibody Solutions, Sunnyvale, CA), and monoclonal antibodies were obtained by standard hybridoma technology. An enzyme-linked immunosorbent assay (ELISA) was developed to screen clones producing IL-31-binding antibodies. Canine IL-31 was biotinylated and then introduced into streptavidin-coated wells. Immunized serum was then added to the wells, washed, and detected with HRP-conjugated anti-mouse antibody. A positive signal was expressed in the presence of canine IL-31-binding antibodies. Of the thousands of clones tested by ELISA, 170 clones with the highest binding affinity based on signal intensity were selected for further testing by a biosensor assay (Forte Bio Octet). Biotinylated canine IL31 was bound to the sensor tip, and hybridoma clone supernatants containing anti-canine IL31 antibodies were selected based on their slow off-rate (dissociation rate between antibody and ligand). Binding affinity was measured at single concentrations for the top 19 candidates, and antibody concentrations were measured by a protein A titer assay using Biosensor Octet, after which the equilibrium dissociation constant (Kd) was reported. The Kd values ​​for the top 19 candidates were all less than 10 nM.

[0183] Furthermore, each of the 170 clones with high ELISA-based binding activity was tested for neutralizing activity. The cell-based functional assay described in Example 4 below was performed to evaluate the activity of top candidates in reducing canine IL31-mediated pSTAT signaling using canine DH82 cells. Four top clones (M14, M18, M19 and M87) were selected for further investigation. Notably, the majority of high-affinity binders identified by ELISA were not functional.

[0184] Example 2 Identification of DNA sequences encoding VH and VL of monoclonal antibodies Hybridoma cells producing M14, M18, M19 and M87 were pelleted. RNA was extracted, and cDNA was obtained using standard techniques with oligonucleotide primers for amplifying mouse immunoglobulin (Ig) variable domains. The variable heavy chain (VH) and variable light chain (VL) of each of the four clones were sequenced and analyzed by sequence alignment (Figure 1, SEQ ID NO: 36 to 43). Notably, three of the four active antibodies (M14, M18 and M19) share the same six CDR sequences, except that the CDR-L1 of M18 has isoleucine at position 14 (SEQ ID NO: 63), M14 and M19 have methionine at position 14 (SEQ ID NO: 8), the CDR-H2 of M18 has tyrosine at position 9 (SEQ ID NO: 62 and SEQ ID NO: 87), and M14 and M19 have aspartic acid at position 14 (SEQ ID NO: 2 and SEQ ID NO: 89). The similarity of CDR sequences suggests that M14, M18 and M19 share a common epitope.

[0185] Example 3 Expression and purification of mouse-canine chimeric and caninized IL31-mAb M14 from CHO cells DNA sequences encoding chimeric antibodies were designed to fuse mouse M14 VH (SEQ ID NO: 25) and mouse VL (SEQ ID NO: 24) with canine constant heavy and light chains. The nucleotide sequences were chemically synthesized and inserted into expression vectors suitable for transfection into CHO host cells. After transfection into CHO cells, the light chain, heavy chain, or both were secreted from the cells. For example, chimeric M14 using canine IgG-B was purified by one-step protein A column chromatography.

[0186] Mice M14 VH and VL were canineized by searching for and selecting suitable canine germline antibody sequences as templates for CDR transplantation, followed by protein modeling. Canineized M14 IgG-B (SEQ ID NO: 18 and 21) were readily expressed and purified in one step using Protein A columns or other chromatography methods, such as ion exchange column chromatography, hydrophobic interaction column chromatography, mixed-mode column chromatography, such as CHT, or multimodal-mode column chromatography, such as CaptoMMC. Low pH or other viral inactivation and removal steps can be applied. The purified protein was mixed with excipients and sterilized by filtration to prepare the pharmaceutical composition of the present invention. The pharmaceutical composition was administered to dogs with atopic dermatitis in an amount sufficient to bind to inhibit IL31.

[0187] Next, the vector was used to perform pilot-scale transfection in CHO-S cells using FreestyleMax® transfection reagent (Life Technologies). The supernatant was collected by clarifying the conditioned medium. The protein was purified by single-pass protein A chromatography and used for further investigation.

[0188] Example 4 Demonstration of IL31 binding activity This example demonstrates that the antibodies of the present invention, exemplified using chimeric M14 (SEQ ID NOs. 26 and 27) and canine M14 (SEQ ID NOs. 18 and 21), bind to canine IL31 in the kinetics necessary for therapeutic activity.

[0189] Binding analysis was performed using the Octet biosensor as follows: Briefly, canine IL31 was biotinylated with primary amine groups using EZ-Link® NHS-LC-Biotin (Thermo Scientific, catalog no. 21336) or with glycan groups using EZ-Link® Biotin-LC-Hydrazide (Thermo Fisher Scientific, catalog no. 21340), according to the manufacturer's instructions. Free, unreacted biotin was removed from the biotinylated IL31 by extensive dialysis. The biotinylated canine IL31 was captured at the tip of the streptavidin sensor. The association of chimeric and canine-modified M14 antibodies with human and canine IL31 (amine-conjugated biotin) at four different concentrations (400, 200, 66.6, and 33 nM), as well as the association of 100 nM canine-modified M14 antibody with canine IL31 (glycan-conjugated biotin), was monitored for 90 seconds. Dissociation was monitored for 600 seconds. A buffer-only blank curve was subtracted to correct for any drift. The data were fitted to a 2:1 coupling model using ForteBio® data analysis software. on , k off The phosphate group and Kd were determined. The buffer for dilution and all binding steps was 20 mM phosphate, 150 mM NaCl, pH 7.2.

[0190] Canine IL31 with a C-terminal polyHis tag was expressed from CHO-S cells and purified. Human IL31 was obtained from Sino Biologicaland, and the Streptavidin biosensor was obtained from ForteBio (catalog no. 18-509). The binding kinetics were as follows: For the ligand canine IL31 (amine conjugate biotin), the Kd(M) of chimeric M14 was <1.0 × 10⁻¹⁴ for canine M14.-11 (Figure 2) and <1.0 × 10 -11 (Figure 3) In the case of ligand canine IL31 (glycan conjugate biotin), the Kd(M) of canine M14 was <1.0 × 10⁻¹⁴ -12 and k off (1 / s) is <1.0 × 10 -7 That was the case.

[0191] Chimeric M14 and canine-modified M14 did not exhibit a clear binding signal to human IL-31. Therefore, Kd could not be measured.

[0192] Example 5 Demonstration that M14 inhibits canine IL31 signaling. After binding to the IL-31 receptor, IL-31 activates Janus kinase (Jak) 1 and Jak 2 signaling molecules. Activated Jak then stimulates the phosphorylation of downstream signaling molecules STAT-3 and STAT-5. Anti-IL-31 activity was detected from the purified protein A fraction in cell-free medium using anti-phospho-Stat3 immunoblot analysis (Gonzales et al. Vet Dermatol 2013;24:48-e12). Briefly, canine monocyte DH82 cells (American type culture collection, Manassas, VA, USA) were incubated at 37°C for 24 hours in MEM growth medium (Life Technologies) containing 15% heat-inactivated fetal bovine serum, 2 mMol / L GlutaMax, 1 mmol / L sodium pyruvate, and 10 ng / mL canine interferon-c (R&D Systems, Minneapolis, MN, USA), at a rate of 1 × 10⁶ cells per well. 5Cells were placed in 96-well flat-bottom cell culture plates at the specified cell density. In this experiment, the concentration of canine IL31-Fc was 5 ng / mL (8 nM). Anti-phospho-STAT-3 and anti-STAT-3 antibodies were purchased from R&D Systems. The anti-beta-actin antibody was from Sigma-Aldrich. As shown in Figure 4, canine IL31 signaling decreased as the concentration of canine-modified M14 exposed to the cells increased (as evidenced by the reduction in STAT-3 phosphorylation) (lane 1: no anti-IL31 antibody, lane 2: 3.3 nM, lane 3: 6.6 nM, lane 4: 9.9 nM, lane 5: 13.2 nM).

[0193] Example 6 Identification of the M14 canine IL31-binding epitope To identify the canine IL31 epitope recognized by M14, multiple GST canine IL31 fragment fusion molecules were constructed, and the proteins were expressed intracellularly in Escherichia coli (E. coli). After transcribing the GST fusion proteins to the membrane, the membrane was probed using chimeric M14. A positive signal was obtained when the IL31 fragment contained the epitope.

[0194] Combining Figure 5 with Figure 6 demonstrates that M14 can recognize the smallest fragment (sequence number 23).

[0195] Example 7 Demonstration of cross-reactivity between M14 and cat IL31. To investigate whether the M14 antibody recognizes feline IL31 (SEQ ID NO: 28) or equine IL31 (SEQ ID NO: 29), each protein was fused to human Fc cells and expressed in 293 mammalian cells. Partially purified proteins were blotted to membranes and probed with the M14 antibody. The immunoblot in Figure 1 demonstrates that M14 binds to feline IL31. The immunoblot assay did not detect binding between M14 and equine IL31. However, biolayer interferometry analysis revealed that the M14 antibody does bind to equine IL31, but with lower affinity. Preliminary Kd measurements using biotinylated equine IL31 immobilized on a sensor revealed an affinity (Kd) of approximately 10–50 nM.

[0196] Example 8 Feline transformation M14 The M14 variable light chain was felineized as (SEQ ID NO: 32), and the M14 variable heavy chain was felineized as (SEQ ID NO: 33). First, appropriate variants of feline VH and VL were searched using mouse heavy chain variable sequences and light chain variable sequences. Appropriate feline frames were selected for CDR transplantation. These were further optimized using structural modeling. Felineized VH and VL were fused to the feline IgG heavy chain constant domains (CH1, CH2, and CH3) and the feline light chain constant domain (CL1).

[0197] Feline M14 chimeric antibodies (SEQ ID NOs. 30 and 31) or feline M14 antibodies (SEQ ID NOs. 34 and 35) may be administered to cats for the treatment of IL31-induced conditions.

[0198] Example 9 Identification of the M14 canine IL31-binding epitope To further identify the amino acid residues of the canine IL31 epitope recognized by M14, multiple GST canine IL31 epitope fragment fusion molecules containing alanine mutations were expressed in Escherichia coli (E. coli). Figures 8–12 show immunoblots of precise epitope mapping of canine IL31-GST fusion proteins probed with anti-canine IL31-mAb (M14) or canine M14 (upper panel) and anti-GST antibody control (lower panel). The epitope fragments tested in each lane are listed below the immunoblot. The fragment name indicates the amino acid range of the tested mature canine IL31 (SEQ ID NO: 44) and, if present, the location of the alanine mutation. A positive signal was obtained if the IL31 fragment contained the wild-type epitope, and a negative signal was obtained if the IL31 fragment contained alanine substitutions in residues important for antibody-ligand interaction.

[0199] The results of the epitope mapping study are summarized in Table 3 below. The results suggest that P12, S13, D14, and K17 of mature canine IL31 (SEQ ID NO: 44) are involved in the binding of antibody M14, and that R16 and I18 are partially involved in M14 recognition. In short, the results of this study suggest that the motif of the IL31 polypeptide that binds to the CDR of antibody M14 is PSDX1X2KI, where X1 and X2 are variable (SEQ ID NO: 45).

[0200] TIFF0007943816000020.tif69170

[0201] Example 10 M14 specifically binds to IL31 of other species that possess the PSDX1X2KI epitope motif. The IL31 epitope motif recognized by M14 (PSDX1X2KI; SEQ ID NO: 45) is not present in the IL31 amino acid sequence of humans (SEQ ID NO: 46) or mice (SEQ ID NO: 61). However, this motif has been identified in several other species, including cats. Felis catus(XP_011286140.1; Sequence ID 28) Odobenus rosmarus divergens(XP_004395998.1; Sequence ID 47) Papio Anubis(XP_003907358.1; Sequence ID 49) Ursus maritimus (XP_008687166.1; Sequence ID 51) Leptonychotes weddellii(XP_006746595.1; Sequence ID 52) Panthera tigris altaica (XP_007079636.1; SEQ ID NO: 53) Acinonyx jubatus(XP_014919275.1; Sequence ID 54) Macaca fascicularis (EHH66805.1; Sequence ID 55) Macaca mulatta (EHH21279.1; Sequence ID 56) Mandrillus leucophaeus (XP_011819882.1; Sequence ID 57) Chlorocebus sabaeus(XP_008003211.1; Sequence ID 58) Cercocebus atys(XP_011926625.1; Sequence ID 59) Rhinopithecus roxellana (XP_010366647.1; Sequence ID 60)

[0202] Walrus IL31 (SEQ ID NO: 47) and olive baboon IL31 (SEQ ID NO: 49) possess the PSDX1X2KI epitope (SEQ ID NO: 45), which can be recognized by the antibody M14. To facilitate protein purification, C-terminal His tags were added to walrus IL31 (SEQ ID NO: 48) and olive baboon IL31 (SEQ ID NO: 50). Western blot analysis confirmed that M14 binds to walrus IL31 (Figure 13). The M14 antibody or its derivatives can be used as a therapeutic and diagnostic agent for IL31-induced diseases in any of the above species.

[0203] Example 11 Thermal stability of canine-modified M14 antibody The thermal stability of canine-modified M14 antibody was compared with Zoetis CYTOPOINT® and a commercially available anti-IL31 antibody over a wide pH range and analyzed using differential scanning fluorescence (DSF). The melting temperatures (Tm) of each antibody at different pH levels are listed in Table 4 below. Both CYTOPOINT® and canine-modified M14 antibody were buffer-exchanged using a PD Minitrap® G-25 column (GE Healthcare) with the assay buffers listed in Table 4. The Tm of each antibody was evaluated using the same buffer and protein concentration. The canine-modified M14 antibody showed improved thermal stability over a wide pH range compared to CYTOPOINT®. Furthermore, precipitation occurred with the 0.22 mg / ml antibody and CYTOPOINT® under stress conditions at 55°C for 2 days, while no precipitate was observed with the canine-modified M14 antibody.

[0204] TIFF0007943816000021.tif76170

[0205] Example 12 Canine M14 Antibody Buffer Preparation The thermal stability of canine-modified M14 antibody in various buffer formulations was analyzed. Buffers containing sodium phosphate, sodium acetate, or L-histidine were examined. Other formulation variables included different pH levels (5.2, 5.5, 6.0, 6.5, and 7.0), different concentrations of sodium chloride (50 mM and 140 mM), different polysorbates (polysorbate 20 and polysorbate 80), and different antimicrobial agents (m-cresol and methylparaben). The melting temperature (Tm) of canine-modified M14 antibody in each buffer was measured at 20°C to 95°C using differential scanning fluorescence (DSF) technology. Table 5 lists the Tm values ​​of canine-modified M14 antibody in the various buffers tested.

[0206] Table 5 TIFF0007943816000022.tif255170TIFF0007943816000023.tif255170TIFF0007943816000024.tif255170TIFF0007943816000025.tif157170* No peak indicates that a clear melting point was not observed.

[0207] The Tm of formulations D1, D2, D3, D4, D5, and D6 was further tested using DSF in the presence of one of each of the following sugars (1%): sucrose, trehalose, D-mannitol, maltose, and sorbitol.

[0208] Formulations containing and without sugars were tested under stress conditions of 40°C for 1 day, followed by 45°C for 1 day, and then 55°C for 4 days. Size exclusion HPLC analysis was used to detect and quantify the monomeric and aggregated forms of antibodies in the various formulations after exposure to stress conditions. Samples were loaded onto SHODEX® KW803 columns (8 mm × 300 mm) and mounted on KW-G guard columns. An Agilent 1100 chromatography system was used at a constant flow rate of 0.5 mL / min, with 2 × PBS (270 mM NaCl, 5.4 mM KCl, 8.6 mM Na2PO4), pH 7.2, as the running buffer. The columns were calibrated with BIORAD gel filtration standards (catalog no. 151-1901) consisting of thyroglobulin, bovine γ-globulin, chicken oocyte albumin, horse myoglobin, and vitamin B12 (molecular weight 1,350–670,000). The amount of monomer antibody remaining in the solution was determined by measuring UV absorbance at 214 nm or 280 nm and calculating the peak area under the curve.

[0209] Based on DSF and HPLC analysis, formulations containing L-histidine, sodium chloride, and polysorbate 80, with a pH of 5.0–6.2, such as formulations D1, D2, D3, D4, D6, D7, D10, and D12, were considered more desirable. For example, the formulation preferred for single administration is as follows: 20 mM L-histidine; 140 mM sodium chloride; polysorbate 80 (0.05 mg / mL); pH 5.5. The formulation preferred for multiple administrations (in the presence of preservatives) is as follows: 1.20 mM L-histidine; 140 mM sodium chloride; polysorbate 80 (0.05 mg / mL); sucrose (1-3%); m-cresol (0.2%); pH 5.5; and 2.20 mM L-histidine; 140 mM sodium chloride; polysorbate 80 (0.05 mg / mL); trehalose (1-3%); methylparaben (0.9%); pH 5.5.

[0210] Example 13 Screening of variant canine IgG-B polypeptides with enhanced canine FcRn / B2M binding. Poly-His tagged canine FcRn (SEQ ID NO: 92) and canine B2M (SEQ ID NO: 93) heterodimer complexes were transiently expressed in HEK cells and purified using Ni-NTA chromatography.

[0211] Fast Screening for Expression, Biophysical Properties and Affinity (FASEBA) was performed on a canine IgG-B Fc phage library. Briefly, reading frames of canine IgG-B Fc polypeptides were subcloned into plasmid pFASEBA. Based on a three-dimensional protein modeling of the canine IgG-B / canine FcRn / canine B2M complex, 12 amino acid positions of canine IgG-B were identified as potentially involved in the binding of IgG-B to FcRn / B2M. The 12 identified positions of canine IgG-B were Thr(21), Leu(22), Leu(23), Ile(24), Ala(25), Thr(27), Gly(80), His(81), Gln(82), Leu(85), Met(201), and Asn(207) in Sequence ID No. 90.

[0212] Twelve single-site NNK mutation libraries of canine IgG-B Fc were prepared, with each library containing a variant IgG-B Fc polypeptide in which each of the 20 possible amino acids was substituted at each of the 12 sites. Each phage library was panned against the canine FcRn / B2M complex at pH 6.0. After three panning cycles, a total of 53 Fc phage clones were identified as potentially exhibiting enhanced FcRn / B2M binding, and mutations were identified by sequencing.

[0213] Single Escherichia coli (E. coli) colonies expressing each of 53 variant canine IgG-B Fc polypeptides with SASA tags were cultured and induced to express the Fc polypeptide. Cell media containing the variant canine IgG-B Fc polypeptides were exposed to immobilized BSA on plates or Biacore chips. Plates or chips conjugated with the variant canine IgG-B Fc polypeptides were exposed to soluble canine FcRn / B2M complexes and screened for slow off-rate (koff) at pH 6. Each variant canine IgG-B Fc polypeptide showing a slower koff with the canine FcRn / B2M complex compared to the wild-type IgG-B Fc polypeptide was identified. Four lead variant canine IgG-B polypeptides were identified: L(23)Y (SEQ ID NO: 95; "Y00"); L(23)F (SEQ ID NO: 94; "F00"); L(23)M; and L(23)S.

[0214] The koff values ​​of each lead variant canine IgG-B polypeptide were further investigated. Biotinylated canine FcRn / B2M complexes were immobilized on Biacore chips and exposed to each variant canine IgG-B polypeptide as the analyte using Biacore T200 at pH 6.0. The koff (1 / s) of wild-type canine IgG-B Fc polypeptide was 1.22 × 10⁻⁶. -1 The koff (1 / s) of variant canine IgG-B Fc polypeptide L(23)Y ("Y00") is 1.38 × 10⁻¹⁰. -2 The koff(1 / s) of variant IgG-B Fc polypeptide L(23)F ("F00") is 6.31 × 10⁻¹⁰. -2 and 8.47 × 10 -2 The koff (1 / s) of variant canine IgG-B polypeptide L(23)M is 1.26 × 10⁻⁶. -1 ; and the koff(1 / s) of variant canine IgG-B polypeptide L(23)S is 2.41 × 10⁻⁶. -1 That was the case.

[0215] Binding analysis was performed using Biacore T200. Briefly, lead variant canine IgG-B Fc polypeptides with SASA tags were immobilized on Series S Sensor Chips CM5. The association of each variant IgG-B Fc polypeptide with various concentrations of canine FcRn / B2M complexes (12.5, 25, 50, 100, and 200 nM) was monitored at 25°C until a steady state was reached. A running buffer of 10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.005% Tween-20, pH 6.0 was used. A blank curve with buffer only was used as a control. The results are shown in Figures 10-14. The steady-state Kd of wild-type canine IgG-B Fc polypeptide was 1.25 × 10⁻¹⁶. -6 (Figure 14); The steady-state Kd of variant canine IgG-B Fc polypeptide L(23)Y ("Y00") is 1.13 × 10⁻¹⁰. -7 (Figure 15); The steady-state Kd of variant canine IgG-B Fc polypeptide L(23)F ("F00") is 3.67 × 10⁻¹⁴. -7 (Figure 16); The steady-state Kd of variant canine IgG-B Fc polypeptide L(23)M is 4.06 × 10⁻⁶ -7 (Figure 17); and the steady-state Kd of variant canine IgG-B Fc polypeptide YTE is 8.62 × 10⁻⁶. -8 (Figure 18)

[0216] Example 14 Phe mutations in canine IgG enhance canine FcRn interaction. The affinity of variant canine Fc polypeptides for FcRn was evaluated in association with chimeric antibodies. Variable light chains of antibodies fused to the canine kappa light chain, as well as variable heavy chains fused to variant canine IgG-A Fc polypeptides containing SEQ ID NO: 96 (F00; Protein A+; C1q-; CD16-) or SEQ ID NO: 97 (Protein A+; C1q+; CD16+), and variant canine IgG-D Fc polypeptides containing SEQ ID NO: 98 (F00; Protein A+; C1q-; CD16-) or SEQ ID NO: 99 (Protein A+; C1q+; CD16+), were expressed.

[0217] Binding analysis was performed using the OctetRed biosensor as follows: Briefly, biotinylated TNFα was captured at the tip of the streptavidin sensor. An aggregate of 20 μg / mL antibody was conjugated to the TNFα. The complex was then conjugated to canine FcRn (50 μg / mL) at pH 6.0. Dissociation was performed at pH 7.2.

[0218] As shown by the binding profiles of chimeric variant canine IgG-A "F00" antibody (Figure 19, A) and IgG-D "F00" antibody (Figure 19, B), the Phe mutation enhanced canine FcRn binding at low pH (pH 6.0, 20 mM sodium citrate, 140 mM NaCl) compared to chimeric variant canine IgG-A (Figure 19, C) and IgG-D (Figure 19, D) without the Phe mutation. Chimeric variant canine IgG-A and IgG-D antibodies with the Phe mutation (Figure 19, A and B) showed enhanced association with canine FcRn at low pH (pH 6.0) and rapid dissociation at neutral pH (PBS pH 7.2). Similar enhancement of the binding profile was observed with the chimeric variant canine IgG-B "F00" antibody.

[0219] Example 15 Pharmacokinetics of Phe mutations in canine IgG Pharmacokinetic analyses were performed using Sprague Dawley rats. Rats were subcutaneously administered 2 mg / kg of chimeric variant canine IgG-A "F00" antibody and chimeric variant canine IgG-A without the Phe mutation (2 rats per group). Serum samples were collected from the rats before injection and at 0.5, 1, 6, 24, 48, 72, 168, 216, and 336 hours after injection. The concentration of canine chimeric antibody in the serum samples was determined by ELISA as follows.

[0220] Capture antibody (1 μg / mL in PBS) was coated onto a 96-well Maxisorp plate with 100 μl per well. The plate was incubated overnight at 4°C and washed five times with PBST (PBS containing 0.05% Tween-20). Each well was blocked with 200 μl of 5% BSA in PBST, and the plate was incubated at room temperature for 1 hour. The plate was washed five times with PBST. Dilutions of the control antibody (1,000 ng / mL to 0.1 ng / mL) were added to the plate in double decans and used with blank wells without the control antibody to create a standard curve. Serum samples were prepared by 10-fold, 20-fold, and 40-fold dilutions in 5% BSA-PBST and added to the plate. The plate was incubated at room temperature for 1 hour and washed five times with PBST. 100 μl of HRP conjugate antibody (Bio-Rad, catalog number HCA204P) was added to each well in 5% BSA-PBST at a concentration of 0.25 μg / mL. The plate was incubated at room temperature for 1 hour and washed 5 times with PBST. 100 μl of QuantaBlu (Thermo Scientific, catalog number 15169) was added to each well. Fluorescence was measured at 325 nm / 420 nm after 10–15 minutes of incubation (luminescence / excitation). The titer of anti-TNFα in serum samples was calculated against a standard curve.

[0221] AUC of IgG-A 0~336h The pharmacokinetic value for rats was 150970, while that for IgG-A "F00" was 848924 ng / mL*hr (Figure 20). The final half-lives were estimated to be 33 hours and 152 hours, respectively. Therefore, a single Phe mutation significantly improved the pharmacokinetic profile of the antibody in rats.

[0222] Example 16 Phe mutations in canine, feline, and equine IgG Fc The interaction between Phe mutations in canine IgG-A, IgG-B, IgG-C, and IgG-D Fc and FcRn was modeled using three-dimensional protein structure analysis. The aromatic side chain of Phe appears to have a hydrophobic interaction with canine FcRn at the hydrophobic ring (π-CH) of the Pro in the "WPE" motif. Furthermore, the hydrophobic side chain of Phe may be in direct contact with the adjacent Glu side chain of the same "WPE" motif. This interaction may have an energy penalty if the Glu side chain is deprotonated and becomes negatively charged, such as at neutral pH. Therefore, some protonation of the Glu residue may be required to minimize the interaction from aromatic to Glu-H. This may explain why the interaction between variant IgG with Phe mutations and FcRn is reduced at neutral pH. Based on the protein structure analysis, the interaction appears to be conserved among canine IgG-A, IgG-B, IgG-C, and IgG-D Fc.

[0223] Furthermore, the interaction between Phe mutations in feline IgG1a and IgG2 Fc was modeled when complexed with feline FcRn. The same interaction observed in canine IgG Fc appeared to be conserved in feline IgG Fc.

[0224] We also modeled the interactions between Phe mutations in equine IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, and IgG7 Fc in their complex with equine FcRn. The same interactions appeared to be maintained in equine IgG Fc.

[0225] Example 17 Other exemplary variants of canine IgG Fc enhance canine FcRn interaction. The affinity of further variant canine Fc polypeptides to FcRn was evaluated in association with chimeric antibodies. Antibody variable light chains fused to the canine kappa light chain, as well as variable heavy chain sequences fused to wild-type IgG-B Fc polypeptide (including SEQ ID NO: 90), variant canine IgG-B Fc polypeptide 0Y0 (including SEQ ID NO: 100), variant canine IgG-B Fc polypeptide 0YH (including SEQ ID NO: 101), variant canine IgG-B Fc polypeptide 0YY (including SEQ ID NO: 102), and variant canine IgG-B Fc polypeptide 00Y (including SEQ ID NO: 103) were expressed.

[0226] Binding analysis was performed using the OctetRed biosensor as follows: Briefly, the biotinylated target was captured at the tip of the streptavidin sensor. An aggregate of 20 μg / mL antibody was conjugated to the biotinylated target. The complex was then used to conjugate canine FcRn (50 μg / mL) at pH 6.0. Dissociation was performed at pH 7.2.

[0227] Each of the chimeric variant canine IgG-B antibodies showed enhanced binding to canine FcRn at pH 6.0 compared to the chimeric wild-type canine IgG-B antibody, and each exhibited a considerable dissociation rate at neutral pH (Figure 21).

[0228] Example 18 Variant canine IgG Fc extends the half-life of antibodies in vivo in dogs. The in vivo half-life of variant canine Fc polypeptides against FcRn was evaluated in relation to chimeric antibodies. Variable light chains of antibodies fused to the canine kappa light chain, as well as variable heavy chains fused to wild-type IgG-B Fc polypeptide (including SEQ ID NO: 90), variant canine IgG-B Fc polypeptide YTE (including SEQ ID NO: 104), variant canine IgG-B Fc polypeptide 0Y0 (including SEQ ID NO: 100), variant canine IgG-B Fc polypeptide F00 (including SEQ ID NO: 94), variant canine IgG-B Fc polypeptide 0YH (including SEQ ID NO: 101), and variant canine IgG-B Fc polypeptide Y00 (including SEQ ID NO: 95), were expressed and purified to 40 mg / mL in PBS, pH 7.2.

[0229] Canine pharmacokinetics were performed at Absorption Systems California, LLC. Male beagles (approximately 8-14 kg) were obtained from Marshall Bioresources, North Rose, New York. A total of 12 dogs were used in the study, with n=2 dogs per group. Six antibodies were administered subcutaneously to the dogs at a dose of 4 mg / kg. Serum samples were collected before injection and at 6, 24, 48, 72, 96, 120, 144, 168, 216, 264, 336, 504, and 672 hours after injection. Canine chimeric antibody concentrations were determined by ELISA as described. Cp between 144 and 336 hours was converted to Ln[Cp], and then Ln[Cp] t =-k*t+Ln[Cp] 144h The equation was fitted to a linear equation of the form . Then, the final half-life was calculated from the gradient k, as listed in Table 6 below. The 0Y0, F00, 0YH, and Y00 mutations in canine IgG-B Fc significantly improved the half-life of the antibody in vivo in dogs. The time-normalized antibody percentages obtained from the study are shown in Figure 22.

[0230] Table 6: Effect of variant canine IgG Fc on antibody half-life in dogs TIFF0007943816000026.tif97170

Claims

1. (a) Below, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 63; (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 9; and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 10, Light chains including, (b) Below, (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 1; (ii) CDR-H2 containing the amino acid sequence of SEQ ID NOs: 2, 62, 89 or 87; and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 3, Heavy chains including An antibody that binds to canine interleukin 31 (IL31), comprising: The antibody comprises a variant canine IgG Fc polypeptide, and the variant canine IgG Fc polypeptide is a) A sequence that is 100% identical to sequence number 95, or b) Sequences that have 100% identity with sequence number 100 Antibodies containing antibodies.

2. 5×10, as measured by biolayer interferometry －６ less than M, 1×10 －６ less than M, 5×10 －７ less than M, 1×10 －７ less than M, 5×10 －８ less than M, 1×10 －８ less than M, 5×10 －９ less than M, 1×10 －９ less than M, 5×10 －１０ less than M, 1×10 －１０ less than M, 5×10 －１１ less than M, 1×10 －１１ less than M, 5×10 －１２ less than M, or 1×10 －１２ less than M, that binds to canine IL31 with a dissociation constant (Kd) of less than M, The antibody according to claim 1.

3. The antibody according to claim 1 or 2, wherein the antibody is a chimeric antibody and / or a monoclonal antibody.

4. The antibody according to any one of claims 1 to 3, wherein the antibody comprises a variant IgG Fc polypeptide derived from a companion animal species that can bind to the neonatal Fc receptor (FcRn) with increased affinity compared to the wild-type Fc polypeptide, and the antibody protein has an increased serum half-life compared to the antibody comprising the wild-type Fc polypeptide.

5. The antibody according to any one of claims 1 to 4, comprising SEQ ID NO: 112 or SEQ ID NO:

127.

6. The antibody according to any one of claims 1 to 5, comprising the light chain amino acid sequence of SEQ ID NO: 21 and the heavy chain amino acid sequence of SEQ ID NO: 112 or SEQ ID NO:

127.

7. The antibody according to any one of claims 1 to 6, wherein the antibody is bispecific and binds to IL31 and one or more antigens selected from IL4R, IL17, TNFα, CD20, CD19, CD25, IL4, IL13, IL23, IgE, CD11α, IL6R, α4-integrin, IL12, IL1β, IL5, IL5R, IL22, IL22R, IL33, IL33R, TSLP, TSLPR, or BlyS.

8. An isolated nucleic acid encoding an antibody according to any one of claims 1 to 7.

9. A host cell containing the nucleic acid described in claim 8.

10. A method for producing an antibody, comprising culturing the host cells described in claim 9 and isolating the antibody.

11. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

12. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier; a) The pharmaceutically acceptable carrier comprises L-histidine, sodium chloride, and polysorbate 80, and the pharmaceutical composition has a pH of 5.0 to 6.2, and / or b) The pharmaceutical composition has an L-histidine concentration of 5 mM to 100 mM, 10 mM to 50 mM, 20 mM to 30 mM, 10 mM to 30 mM, or 20 mM, and / or c) The pharmaceutical composition has a sodium chloride concentration of 80–200 mM, 100–175 mM, 120–150 mM, or 140 mM, and / or d) The pharmaceutical composition has a polysorbate 80 concentration of 0.005 mg / mL to 0.5 mg / mL, 0.01 mg / mL to 0.1 mg / mL, or 0.05 mg / mL, and / or e) The pharmaceutically acceptable carrier contains at least one sugar, and / or f) The pharmaceutical composition has a sugar concentration of 0.5% to 20%, 1% to 10%, 1% to 5%, or 1% to 3%, and / or g) The pharmaceutically acceptable carrier includes sucrose, trehalose, D-mannitol, maltose and / or sorbitol, and / or h) The pharmaceutical composition contains an antimicrobial agent and / or i) The pharmaceutical composition contains m-cresol or methylparaben, and / or j) The pharmaceutical composition comprises 0.2% m-cresol and / or 0.9% methylparaben, Pharmaceutical composition.

13. A method for treating a companion animal species having an IL31-induced state, comprising administering to the companion animal species a therapeutically effective amount of the antibody according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 11 or 12; wherein the companion animal species is a dog, a cat or a horse.

14. A method for reducing IL-31 signaling function in cells, comprising exposing cells to an antibody according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 11 or 12 under conditions that allow the antibody to bind to extracellular IL-31, thereby reducing binding to the IL-31 receptor and / or reducing the IL-31 signaling function by the cells; wherein the cells are canine, feline, or equine cells.

15. A method for detecting IL-31 in a sample derived from a companion animal species, comprising: contacting the sample with an antibody according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 11 or 12 under conditions that allow the antibody to bind to IL-31; and detecting whether a complex is formed between the antibody and IL-31 in the sample; wherein the sample is a biological sample obtained from a dog, cat or horse.

16. The antibody according to any one of claims 1 to 7, wherein the light chain sequence is at least 90% identical to SEQ ID NO: 24 and the heavy chain sequence is at least 90% identical to SEQ ID NO:

25.

17. The antibody according to any one of claims 1 to 7 and 16, wherein the light chain sequence is at least 95% identical to SEQ ID NO: 24 and the heavy chain sequence is at least 95% identical to SEQ ID NO:

25.

18. The antibody according to any one of claims 1 to 7, 16, and 17, wherein the light chain sequence is at least 98% identical to SEQ ID NO: 24 and the heavy chain sequence is at least 98% identical to SEQ ID NO:

25.

19. The antibody according to any one of claims 1 to 7, wherein the light chain sequence comprises SEQ ID NO: 24 having any one, two, three, four, or five amino acid substitutions, and the heavy chain sequence comprises SEQ ID NO: 25 having any one, two, three, four, or five amino acid substitutions.

20. The antibody according to claim 19, wherein the amino acid substitution is a conservative substitution.

21. The antibody according to any one of claims 1 to 3, wherein the antibody binds to IL-31 of a dog, cat, or horse.

22. Antibodies, (i) binding to feline IL31 or horse IL31 as determined by immunoblotting and / or biolayer interferometry; (ii) Competes with monoclonal M14 antibody in binding to canine IL31; (iii) Competes with monoclonal M14 antibody in binding to feline IL31; and / or (iv) Non-binding to human IL31, as determined by immunoblotting and / or biolayer interferometry. The antibody according to any one of claims 1 to 7 and 16 to 21.

23. The antibody according to claim 4, wherein the variant IgG Fc polypeptide comprises SEQ ID NO:

95.

24. The antibody according to claim 4, wherein the variant IgG Fc polypeptide comprises SEQ ID NO:

100.

25. (a) Below, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 63; (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 9; and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 10, Light chains including, (b) Below, (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 1; (ii) CDR-H2 containing the amino acid sequence of SEQ ID NOs: 2, 62, 89 or 87; and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 3, Heavy chains including Includes, The antibody according to claim 1, wherein the antibody comprises a variant canine IgG Fc polypeptide, and the variant canine IgG Fc polypeptide comprises a sequence having 100% identity with SEQ ID NO:

95.

26. (a) Below, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 8; (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 9; and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 10, Light chains including, (b) Below, (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 1; (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 2; and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 3, Heavy chains including Includes, The antibody according to claim 1, wherein the antibody comprises a variant canine IgG Fc polypeptide, and the variant canine IgG Fc polypeptide comprises a sequence having 100% identity with SEQ ID NO:

95.

27. The antibody according to any one of claims 1, 23, 25, and 26, wherein the antibody half-life in dogs is at least 33 days.

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