Canine IL-31 specific antibody and application thereof

Abstract

The present disclosure relates to the field of antibodies, in particular to antibodies that bind to animal IL-31 and uses thereof. The invention provides an antibody specifically binding to animal IL-31 or an antigen binding fragment thereof, a coding nucleic acid comprising the antibody or the antigen binding fragment thereof, an expression vector, a cell, a composition, a method for preparing the antibody or the antigen binding fragment thereof, and an application of the antibody or the antigen binding fragment thereof. The antibody specifically binding to animal IL-31 disclosed by the invention can be used for effectively relieving IL-31 mediated diseases such as pruritus, allergy or asthma and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibodies, in particular to IL-31 specific antibodies and uses thereof. BACKGROUND

[0002] Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by infiltration of T cells, eosinophils, and mast cells into the skin. There are many potential factors involved in AD, components in food, environmental allergens (such as fleas, dust mites, etc.) can all trigger atopic dermatitis. It is estimated that the prevalence of AD is 10% of the total population of dogs, and the incidence has shown a significant increasing trend in the past decade. Pruritus is a fundamental feature and major symptom of AD, which seriously affects the quality of life of dogs and their owners. And the inhibition or reduction of pruritus has become the most important therapeutic goal for canine AD.

[0003] Cytokines are the most important factors that mediate pruritic stimuli and inflammation. Interleukin-31 (IL-31) has been identified as an important cytokine that triggers canine pruritus. Intravenous injection of IL-31 causes canine pruritus within 4 hours. IL-31 is a four-helix bundle cytokine belonging to the IL-6 cytokine family, which is preferentially produced by Th2 cells, and also produced in mast cells and macrophages. IL-31 signals through a heterodimeric receptor composed of oncostatin M receptor (OSMR) and IL-31 receptor alpha (IL-31RA) subunits. The expression of co-receptors has been shown in macrophages, keratinocytes and in dorsal root ganglia. The pro-pruritic and pro-inflammatory cytokine IL-31 binds to the receptor, activates intracellular JAK-STAT and PI3K-AKT signaling pathways, produces more cell active substances that mediate pruritus and inflammation, leading to a vicious cycle of "pruritus-scratching-inflammation".

[0004] Based on the existing research on the relationship between IL-31 signaling and AD pruritus, IL-31 signal blocking is considered as an effective target for treating atopic dermatitis. Therefore, it is necessary to provide IL-31 antibodies or functional fragments thereof to block, inhibit, reduce, limit or neutralize the activity of canine IL-31 and inhibit its cell transmission, so as to treat IL-31 mediated disorders. SUMMARY

[0005] The present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31, a composition comprising the antibody or antigen-binding fragment thereof, and a preparation method and application.

[0006] According to a first aspect of the present disclosure, an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 is provided, the antibody or antigen-binding fragment thereof comprising: a1) CDR-H1, CDR-H2, and CDR-H3 in a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 1; and, CDR-L1, CDR-L2, and CDR-L3 in a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 2; or a2) at least one CDR contains one or more amino acid substitutions, deletions, or additions compared to the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of a1); wherein the CDRs are defined by the IMGT, Kabat, Chothia, or Contact numbering system.

[0007] In some embodiments, the animal can be selected from canine, feline, or equine.

[0008] In some embodiments, the antibody or antigen-binding fragment thereof comprises: b1) a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequences set forth, in order, in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; and, a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequences set forth, in order, in SEQ ID NO: 14, YTS, and SEQ ID NO: 15; wherein the CDRs are defined by the IMGT numbering system, or a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequences set forth, in order, in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8; and, a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequences set forth, in order, in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 15; wherein the CDRs are defined by the Kabat numbering system, or a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequences set forth, in order, in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 8; and, a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequences set forth, in order, in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 15; wherein the CDRs are defined by the Chothia numbering system, or a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of amino acid sequences set forth, in order, in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13; and, a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of amino acid sequences set forth, in order, in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20; wherein the CDRs are defined by the Contact numbering system, or b2) a heavy chain variable region; and, a light chain variable region, wherein at least one CDR of the heavy chain variable region and / or light chain variable region contains one or several amino acid substitutions, deletions, or additions as compared to the heavy chain variable region and / or light chain variable region of b1).

[0009] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region that can comprise an amino acid sequence as set forth in SEQ ID NO: 1, or an amino acid sequence with at least 75% sequence identity thereto.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region that can comprise an amino acid sequence as set forth in SEQ ID NO: 2, or an amino acid sequence with at least 75% sequence identity thereto.

[0011] In some embodiments, the antibody can be a monoclonal antibody.

[0012] In some embodiments, the antibody can be a monospecific antibody, a bispecific antibody, or a multispecific antibody.

[0013] In some embodiments, the antibody can be a canine, caninized, feline, felinized, equine, equinized, or chimeric antibody.

[0014] In some embodiments, the antibody can be of the IgA, IgD, IgE, IgG, or IgM class. In some particular embodiments, the antibody can be of the IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2 class.

[0015] In some embodiments, the antibody can comprise a heavy chain constant region and / or a light chain constant region of an antibody derived from an animal. In some embodiments, the antibody can comprise a heavy chain constant region and / or a light chain constant region of an antibody derived from a canine.

[0016] According to a second aspect of the present disclosure, there is provided an antibody or an antigen binding fragment thereof that specifically binds to an animal IL-31, characterized in that the antibody or the antigen binding fragment thereof is caninized, the antibody or the antigen binding fragment thereof comprises: c1) a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 having the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively; and, a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 having the amino acid sequences of SEQ ID NO: 14, YTS and SEQ ID NO: 15, respectively; wherein the CDRs are defined by the IMGT numbering system, or c2) a heavy chain variable region; and, a light chain variable region, wherein at least one CDR of the heavy chain variable region and / or the light chain variable region contains one or several amino acid substitutions, deletions or additions compared to the heavy chain variable region and / or the light chain variable region of b1).

[0017] In some embodiments, the heavy chain constant region of the caninized antibody can comprise an amino acid sequence as set forth in SEQ ID NO: 52, or an amino acid sequence having at least 75% sequence identity thereto.

[0018] In some embodiments, the light chain constant region of the caninized antibody can comprise an amino acid sequence as set forth in SEQ ID NO: 53, or an amino acid sequence having at least 75% sequence identity thereto.

[0019] In some embodiments, the heavy chain variable region of the antibody can comprise a framework region of a heavy chain variable region of a caninized antibody and / or a framework region of a light chain variable region.

[0020] In some embodiments, the antigen binding fragment can be known in the art. In particular embodiments, the antigen binding fragment can comprise a scFv, a Fab, a Fab’, a (Fab’)2, a Fv fragment, a Fd, a dsFv.

[0021] In some embodiments, the heavy chain variable region of the antibody comprises a caninized framework region FR-H1, FR-H2, FR-H3 and / or FR-H4, or a variant thereof.

[0022] In some embodiments, the heavy chain variable region of the antibody comprises: a FR-H1 comprising SEQ ID NO: 62; a FR-H2 comprising SEQ ID NO: 63; a FR-H3 comprising SEQ ID NO: 64; and / or, a FR-H4 comprising SEQ ID NO: 65.

[0023] In some embodiments, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 60, or an amino acid sequence having at least 75% sequence identity thereto; In some embodiments, the light chain variable region of the antibody comprises a caninized framework region FR-L1, FR-L2, FR-L3, and / or FR-L4, or a variant thereof, In some embodiments, the light chain variable region of the antibody comprises: a FR-L1 comprising an amino acid sequence as set forth in SEQ ID NO: 66; a FR-L2 comprising an amino acid sequence as set forth in SEQ ID NO: 67; a FR-L3 comprising an amino acid sequence as set forth in SEQ ID NO: 68; and / or, a FR-L4 comprising an amino acid sequence as set forth in SEQ ID NO: 69; In some embodiments, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 61, or an amino acid sequence having at least 75% sequence identity thereto.

[0024] According to a third aspect of the present disclosure, there is provided a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to the first aspect or the second aspect of the present disclosure.

[0025] According to a fourth aspect of the present disclosure, there is provided a vector comprising the nucleic acid molecule according to the third aspect of the present disclosure.

[0026] In some embodiments, the vector comprises a prokaryotic expression vector and a eukaryotic expression vector.

[0027] According to a fifth aspect of the present disclosure, there is provided a cell comprising the antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to the first aspect or the second aspect of the present disclosure, the nucleic acid molecule according to the third aspect of the present disclosure, or the vector according to the fourth aspect of the present disclosure.

[0028] In some embodiments, the cell does not comprise reproductive material.

[0029] According to a sixth aspect of the present disclosure, there is provided a conjugate comprising: the antibody or antigen-binding fragment thereof that specifically binds to canine IL-31 according to the first aspect or the second aspect of the present disclosure; and, a conjugating moiety, wherein the conjugating moiety is selected from the group consisting of a detectable substance, a small molecule drug, and / or a protein.

[0030] In some embodiments, the protein can include, but is not limited to, a cytokine, an antibody, an antibody Fc fragment, an antibody scFv fragment, an enzyme, and a viral coat protein.

[0031] In some embodiments, the detectable substance can be any substance conventionally used in the art, such as, but not limited to, a fluorescent substance, a luminescent label, or a radioactive substance. In preferred embodiments, the detectable substance can be selected from the group consisting of acridinium ester, acridinium sulfonamide, luminol, isoluminol, horseradish peroxidase, alkaline phosphatase, and any of the isotopes.

[0032] According to a seventh aspect of the present disclosure, there is provided a method of preparing an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to the first or second aspect of the present disclosure.

[0033] In some embodiments, the method comprises culturing the cell according to the present disclosure under conditions that allow expression of the antibody or antigen-binding fragment thereof and isolating the antibody.

[0034] According to an eighth aspect of the present disclosure, there is provided a pharmaceutical composition comprising: an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to the first or second aspect of the present disclosure, a nucleic acid molecule according to the third aspect of the present disclosure, a vector according to the fourth aspect of the present disclosure, a cell according to the fifth aspect of the present disclosure, or a conjugate according to the sixth aspect of the present disclosure.

[0035] In some embodiments, the pharmaceutical composition can further comprise a pharmaceutically acceptable carrier.

[0036] In some embodiments, the pharmaceutically acceptable carrier can be any carrier conventionally used in the art.

[0037] In some embodiments, the pharmaceutical composition can be administered parenterally, by injection, orally, or topically. The pharmaceutical composition can be prepared in a form suitable for administration, such as a solid, semi-solid, or liquid form, and can be in the form of an aqueous solution, non-aqueous solution, or suspension, a powder, tablet, capsule, granule, injection, or infusion.

[0038] According to a ninth aspect of the present disclosure, there is provided use of an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to the first or second aspect of the present disclosure, a nucleic acid molecule according to the third aspect of the present disclosure, a vector according to the fourth aspect of the present disclosure, a cell according to the fifth aspect of the present disclosure, a conjugate according to the sixth aspect of the present disclosure, or a pharmaceutical composition according to the eighth aspect of the present disclosure, the use comprising one or more of: d1) detecting the presence or amount of IL-31 in a sample; d2) diagnosing or prognosticating an IL-31 -related disease; d3) preparing a reagent for detecting the presence or amount of IL-31 in a sample; d4) preparing a reagent for diagnosing or prognosticating an IL-31 -related disease.

[0039] In some embodiments, the sample comprises whole blood, red blood cell concentrate, platelet concentrate, white blood cell concentrate, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, fecal matter, urine, cultured cells, saliva, oral secretion, and / or nasal secretion from the subject.

[0040] In some embodiments, there is provided a method of detecting IL-31 in a sample, the method comprising the step of contacting an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 as described in the first aspect or the second aspect of the present disclosure with the sample.

[0041] In some embodiments, there is provided a method of diagnosing or prognostic evaluation of IL-31 -related diseases using an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 as described in the first aspect or the second aspect of the present disclosure. In some embodiments, the method comprises the step of contacting an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 as described in the first aspect or the second aspect of the present disclosure with a sample from the subject.

[0042] In some embodiments, the subject can comprise a dog, a cat, or a horse.

[0043] In some embodiments, the IL-31 -related diseases comprise pruritus, allergy, or asthma. In preferred embodiments, the IL-31 -related diseases comprise atopic dermatitis, eczema, psoriasis, scleroderma, allergic dermatitis, urticaria, psoriasiform dermatitis, and asthma.

[0044] According to a tenth aspect of the present disclosure, there is provided use of an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 as described in the first aspect or the second aspect of the present disclosure, the nucleic acid molecule as described in the third aspect, the vector as described in the fourth aspect, the cell as described in the fifth aspect, the conjugate as described in the sixth aspect, or the pharmaceutical composition as described in the eighth aspect for the treatment and / or prevention of IL-31 -related diseases.

[0045] In some embodiments, the present disclosure provides use of an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 as described in the first aspect or the second aspect of the present disclosure, the nucleic acid molecule as described in the third aspect, the vector as described in the fourth aspect, the cell as described in the fifth aspect, the conjugate as described in the sixth aspect, or the pharmaceutical composition as described in the eighth aspect for the manufacture of a medicament for the treatment and / or prevention of IL-31 -related diseases.

[0046] In some embodiments, the present disclosure provides a method for treating and / or preventing an IL-31 related disease, comprising: administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof specifically binding to animal IL-31 according to the first aspect or the second aspect, the nucleic acid molecule according to the third aspect, the vector according to the fourth aspect, the cell according to the fifth aspect, the conjugate according to the sixth aspect or the pharmaceutical composition according to the eighth aspect of the present disclosure.

[0047] In some embodiments, the IL-31 related disease comprises pruritus, allergy or asthma. In preferred embodiments, the IL-31 related disease comprises atopic dermatitis, eczema, psoriasis, scleroderma, allergic dermatitis, urticaria, psoriasiform and asthma. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 SDS-PAGE results of canine IL31-his protein and canine IL31-mFc protein.

[0049] Figure 2 ELISA results of hybridoma cell 12G10 culture supernatant and IL31-his protein.

[0050] Figure 3 Subtype identification results of 12G10 monoclonal antibody.

[0051] Figure 4 SDS-PAGE identification results of chimeric antibody CA-12G10.

[0052] Figure 5 In vitro binding experiment results of chimeric antibody CA-12G10.

[0053] Figure 6 SDS-PAGE identification results of chimeric antibody CA-12G10, caninized antibody Can-12G10 and cetuximab.

[0054] Figure 7 In vitro binding experiment results of chimeric antibody CA-12G10, caninized antibody Can-12G10 and cetuximab.

[0055] Figure 8 In vitro competition experiment results of caninized antibody and cetuximab. Biotin-labeled Can-12G10 (upper panel) and biotin-labeled cetuximab (lower panel) were used for detection, respectively.

[0056] Figure 9 In vivo treatment effect of subcutaneous injection of caninized antibody Can-12G10. DETAILED DESCRIPTION

[0057] To make the objectives, technical solutions, and advantages of the present application clearer, the present application will be described in further detail below with reference to embodiments. The specific embodiments described herein are intended for illustration only and are not intended to be limiting in any way. Moreover, in the following description, descriptions of well-known structures and techniques are omitted to avoid obscuring the concept of the present disclosure. Such structures and techniques are described in many publications.

[0058] Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The following definitions are applied to the descriptions of the present application and throughout this document, and apply equally whether the terms are in the singular or plural.

[0059] The term "about" as used herein means a range of ±20% of the value that follows. In some embodiments, the term "about" means a range of ±10% of the value that follows. In some embodiments, the term "about" means a range of ±5% of the value that follows.

[0060] The term "antibody" herein encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific or trispecific antibodies), single chain molecules, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0061] The terms "antibody or antigen-binding fragment thereof" and "antibody" are used interchangeably herein to refer to an immunoglobulin that is capable of specifically binding to a target antigen via at least one antigen-binding domain therein. Antibodies can be classified into different types based on the amino acid sequences of their heavy chain constant regions. There are five major classes of immunoglobulins, namely IgG, IgA, IgM, IgD, IgE, whose heavy chains are γ, α, μ, δ, ε, respectively. Antibodies can be further divided into subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The light chains of antibodies can be classified into two types, κ and λ, based on the amino acid sequences of their constant domains.

[0062] The term "variable region" or "variable domain" herein refers to the domain of an antibody heavy or light chain in an antigen binding molecule that is involved in binding to an antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain can be sufficient to confer antigen binding specificity. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen binding site of antibodies. The constant regions do not participate directly in binding of antibody to antigen, have other effector functions, for example, involvement in antibody-dependent cellular cytotoxicity.

[0063] The term "hypervariable region" or "HVR" herein refers to the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops ("hypervariable loops") that are believed to be involved in antigen binding. In general, native four-chain antibodies comprise six HVRs: three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). The HVRs generally comprise amino acid residues from the hypervariable loops and / or from the "complementarity determining regions" (CDRs), which have the highest sequence variability and / or are involved in antigen recognition.

[0064] The term "framework" or "FR" herein refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of a variable domain generally consist of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the following sequence in VHand (or VL): FR-H1 (L1)-FR-H2 (L2)-FR-H3 (L3)-FR-H4.

[0065] The expression "replacement" or "substitution" of an amino acid as used herein can refer to a substitution of an amino acid residue with an amino acid residue having a similar side chain, where the amino acid substitution is a substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine (L), arginine (R), histidine (H)), acidic side chains (e.g., aspartic acid (D), glutamic acid (E)), uncharged polar side chains (e.g., glycine (G), asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), cysteine (C)), nonpolar side chains (e.g., alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W)), beta-branched side chains (e.g., threonine (T), valine (V), isoleucine (I)), and aromatic side chains (e.g., tyrosine (Y), phenylalanine (F), tryptophan (W), histidine (H)). Thus, if one amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered to be conservative. In another aspect, an amino acid chain can be conservatively replaced with a structurally similar amino acid chain that differs in the order and / or composition of side chain family members.

[0066] The term "nucleic acid molecule" herein is one of the most important biological macromolecules in organisms, which is the material basis for carrying and transmitting genetic information, including two major categories of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0067] The term "antibody fragment" or "antigen binding fragment" herein encompasses a portion of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, diabodies, triabodies, tetrabodies, cross-Fab fragments, linear antibodies, single-chain antibody molecules (e.g., scFv), single-domain antibodies (dAbs), and the like.

[0068] After obtaining the DNA fragments encoding the VHand / or VLof an antibody, these DNA fragments can be further manipulated by recombinant DNA technology, for example, to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes, or to scFv genes. In these manipulations, the DNA fragments encoding the VHand / or VLare operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" as used herein means that two DNA fragments are linked in such a way that the amino acid sequences encoded by these two DNA fragments remain in reading frame.

[0069] By operatively linking the DNA encoding VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3), isolated DNA encoding the VH region can be converted into a full-length heavy chain gene. Similarly, by operatively linking the DNA encoding VL to another DNA molecule encoding the light chain constant region CL, isolated DNA encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene).

[0070] The terms "antigen-binding domain" or "antigen-binding site" used herein refer to the portion of an antibody or its antigen-binding fragment that specifically binds to an antigenic determinant. More specifically, the term "antigen-binding domain" refers to a region of an antibody that specifically binds to and is complementary to a portion or all of an antigen. In cases where the antigen molecule is large, the antibody may bind only to a specific portion of the antigen, called an epitope. The antigen-binding domain may be provided by, for example, one or more variable domains (also called variable regions). Preferably, the antigen-binding domain comprises a variable region (VL) of the antibody light chain and a variable region (VH) of the antibody heavy chain. In some embodiments, the antigen-binding domain is capable of binding its antigen and blocking or partially blocking the function of said antigen.

[0071] The term "antigenic determinant" used herein is interchangeable with "antigen" and "epitope" and refers to a site on a polypeptide molecule (e.g., a continuous amino acid sequence or a conformation composed of discontinuous amino acids) to which an antigen-binding moiety binds, thereby forming an antigen-binding moiety-antigen complex. Unless otherwise stated, the protein used as an antigen in this disclosure can be any naturally occurring protein of any vertebrate origin, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), dogs, cats, etc.

[0072] The terms "vector" or "expression vector" and "expression construct" used herein are used interchangeably to describe a DNA molecule to which a specific gene, operatively linked, is introduced into a target cell and directed for expression. The vector includes a vector as a self-replicating nucleic acid structure and a vector incorporated into the genome of the host cell into which it has been introduced. The expression vectors disclosed herein contain expression cassettes. Expression vectors can perform transcription of large amounts of stable mRNA. Once the expression vector is within the target cell, the cellular transcription and / or translation mechanisms generate a ribonucleic acid molecule or protein encoded by the gene. The term "expression cassette" herein refers to a recombinant or synthetically produced polynucleotide having a set of nucleic acid elements that allow transcription of a specific nucleic acid in the target cell.

[0073] The term "pharmaceutical composition" herein refers to a mixture containing an antibody or antibody- or antigen-binding fragment thereof, biological material, or conjugate of the present disclosure, and other chemical components, such as physiological / pharmaceutical-grade carriers or excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0074] The term "treatment" herein refers to the administration of an oral or topical therapeutic agent, such as a composition comprising any antibody of the present disclosure or an antibody thereof or an antigen-binding fragment thereof, or a nucleic acid molecule encoding an antibody thereof or an antibody thereof or an antigen-binding fragment thereof, to a subject who has one or more diseases or symptoms, and the therapeutic agent has a therapeutic effect on these diseases or symptoms. Typically, the therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more diseases or symptoms, to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable extent.

[0075] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are counted, but those from the reference sequence are not.

[0076] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0077] Those skilled in the art will understand that the reference herein to having “at least 75% sequence identity” compared to a sequence is intended to include all sequences having more than 75% sequence identity with that sequence, such as sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity.

[0078] The term "chimeric antibody" or "chimerism" herein refers to an antibody in which a portion of the heavy or light chain is derived from a specific source or species, while at least a portion of the remaining portion of the heavy or light chain is derived from a different source or species. In some embodiments, the chimeric antibodies provided herein consist of splicing a murine heavy chain variable region and / or light chain variable region with a heavy chain constant region and / or light chain constant region of an antibody from another species (e.g., a dog, cat, or horse, preferably a dog) to form a chimeric structure. In some embodiments, the chimeric antibodies provided herein comprise a heavy chain constant region derived from a dog.

[0079] In a specific embodiment, the heavy chain constant region of the antibody derived from dogs may include an amino acid sequence as shown in SEQ ID NO:52, or an amino acid sequence having at least 75% sequence identity with it.

[0080] In a specific implementation, the light chain constant region of the canine-derived antibody may include an amino acid sequence as shown in SEQ ID NO:53, or an amino acid sequence having at least 75% sequence identity with it.

[0081] In other embodiments, the chimeric antibody provided herein comprises splicing CDR1-3 of a mouse heavy chain variable region and / or light chain variable region with frame regions (FR)1-4 of heavy chain variable regions and / or light chain variable regions of other species (e.g., dogs, cats, or horses, preferably dogs) to form a chimeric structure.

[0082] In a specific implementation, the frame region FR-H1 of the variable region of the antibody heavy chain derived from dogs may include an amino acid sequence as shown in EVQLVESGGDLVKPGGSLRLSCVAS (SEQ ID NO: 62), or an amino acid sequence having at least 75% sequence identity with it.

[0083] In a specific implementation, the frame region FR-H2 of the variable region of the antibody heavy chain derived from dogs may include an amino acid sequence as shown in MSWVRQAPGKGLQWVAT (SEQ ID NO: 63), or an amino acid sequence having at least 75% sequence identity with it.

[0084] In a specific embodiment, the frame region FR-H3 of the variable region of the antibody heavy chain derived from dogs may include an amino acid sequence as shown in HYADAVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 64), or an amino acid sequence having at least 75% sequence identity with it.

[0085] In a specific implementation, the frame region FR-H4 of the variable region of the antibody heavy chain derived from dogs may include an amino acid sequence as shown in WGQGTLVTVS (SEQ ID NO: 65), or an amino acid sequence having at least 75% sequence identity with it.

[0086] In a specific embodiment, the frame region FR-L1 of the variable region of the antibody light chain derived from dogs may include an amino acid sequence as shown in EIVMTQSPASLSLSQEEKVTITCRAS (SEQ ID NO: 66), or an amino acid sequence having at least 75% sequence identity with it.

[0087] In a specific implementation, the frame region FR-L2 of the variable region of the antibody light chain derived from dogs may include an amino acid sequence as shown in LNWYQQKPGQAPKLLIY (SEQ ID NO: 67), or an amino acid sequence having at least 75% sequence identity with it.

[0088] In a specific embodiment, the frame region FR-L3 of the variable region of the antibody light chain derived from dogs may include an amino acid sequence as shown in RRATGVPSRFSGSGSGTDFSFTISSLEPEDVAVYFC (SEQ ID NO: 68), or an amino acid sequence having at least 75% sequence identity with it.

[0089] In a specific implementation, the frame region FR-L4 of the variable region of the antibody light chain derived from dogs may include an amino acid sequence as shown in FGQGTKLEIK (SEQ ID NO: 69), or an amino acid sequence having at least 75% sequence identity with it.

[0090] The term "canine-derived antibody" in this article refers to an antibody in which at least one amino acid in a portion of the non-canine variable region has been replaced by a corresponding amino acid from the canine variable region.

[0091] The term “CDR” in this document refers to the complementary determination region as defined by at least one identification method by those skilled in the art.

[0092] The term "monoclonal antibody" in this article refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, a single antibody that is identical to the population except for a small number of potentially naturally occurring mutations.

[0093] Table 1. Amino acid sequences of VH and VL of the 12G10 antibody

[0094] Table 2.12 shows the amino acid sequences of the G10 antibody's CDR-H1, CDR-H2, and CDR-H3.

[0095] Table 3.12 shows the amino acid sequences of the CDR-L1, CDR-L2, and CDR-L3 of the G10 antibody.

[0096] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0097] Example Example 1. Expression and purification of recombinant canine IL-31 protein 1. Construction of the IL31-his recombinant plasmid Referring to the IL-31 sequence (NP_001159386) of canine (Canis lupus familiaris) on NCBI, a mature IL-31 sequence (underlined) was selected, and a signal peptide sequence (sp sequence, which is removed after protein processing and maturation) was added to the N-terminus. A GS linker and 8 His tags were added to the C-terminus. The gene was synthesized by Beijing Tianyi Huiyuan Biotechnology Co., Ltd. and constructed into the eukaryotic expression vector pCMV-Flag.

[0098] Canine IL-31-his nucleotide sequence: ATGCTGAGCCACACCGGCCCTTCTAGATTCGCCCTGTTCCTGCTGTGCAGCATGGAGACACTGCTGTCC TCTCACATGGCACCGACCCACCAGCTGCCCCCGTCTGATGTTCGGAAAATTATTCTGGAACTGCAACCTCTGAGC CGTGGTCTGCTGGAAGATTACCAGAAAAAAGAAACCGGCGTGCCCGAGAGCAATCGTACCCTGCTGCTGTGCCTGA CAAGCGACAGCCAGCCTCCAAGACTGAATAGTAGCGCTATTCTGCCTTATTTTAGAGCCATTCGTCCGCTGTCCGA CAAGAACATCATCGACAAAATTATCGAGCAGCTGGATAAGCTGAAGTTCCAGCACGAACCTGAAACCGAGATCAGC GTTCCGGCAGATACCTTTGAATGTAAGAGCTTCATCCTGACCATCCTGCAGCAGTTTAGCGCCTGTCTGGAAAGCG TGTTCAAGAGCCTGAACAGCGGTCCGCAG GGCAGCCACCATCATCATCATCACCATCATtga(SEQ ID NO: 21) Canine IL-31-his amino acid sequence: SHMAPTHQLPPSDVRKIILELQPLSRGLLEDYQKKETGVPESNRTLLLCLTSDSQPPRLNSSAILPYF RAIRPLSDKNIIDKIIEQLDKLKFQHEPETEISVPADTFECKSFILTILQQFSACLESVFKSLNSGPQ GSHHHHHHHH (SEQ ID NO: 22) 2. Construction of IL31-mFc recombinant plasmid To obtain the secretory IL31-mFc protein, sp-IL-31 and mouse Fc (mFc) fragments were amplified by PCR and sp-IL31-mFc was assembled by overlap PCR. The sp-IL31-mFc was cloned into the pTT3 vector using EcoRI / NotI double digestion sites, and the recombinant plasmid was verified by sequencing.

[0099] mFc nucleotide sequence: GAGCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATGCAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTAAA(SEQ ID NO: 23) Amino acid sequence of mFc: EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO: 24) 3. Expression of IL31-his / IL31-mFc recombinant protein 1) Preparation of transfection complex Preparation method of PEI transfection reagent solution: Add 100 mg of linear PEI transfection reagent (Mw 40000; LABLEAD, catalog number P4000) to 90 mL of Milli-Q ultrapure water, stir until completely dissolved, adjust the pH to 6.9-7.1, and make up to 100 mL with Milli-Q ultrapure water. Then filter through a 0.22 μm filter membrane and collect the filtrate.

[0100] Preparation of transfection complex (per 1L of cell suspension): Add 1mg of recombinant plasmid to 5mL of Hi-exp medium (Opmai, catalog number AC601501), mix well by pipetting, and this is liquid phase A; add 3mL of PEI transfection reagent solution to 5mL of Hi-exp medium, mix well by pipetting, and this is liquid phase B; add liquid phase B to liquid phase A, mix well by pipetting, and then incubate at room temperature for 5min.

[0101] 2) Preparation of cell suspension Collect 293F cells from the culture, count the cells, centrifuge at 800 rpm for 5 minutes, discard the supernatant, and resuspend the cell pellet in Hi-exp medium to achieve a cell concentration of 1×10⁻⁶ cells / mL. 6 Cells / mL.

[0102] 3) Add the transfection complex dropwise to 1L of cell suspension and incubate with shaking at 130rpm for 4-5 days (environmental conditions: 37℃, 8% CO2). Then centrifuge at 4000rpm for 20min, collect the supernatant, filter it through a 0.45μm filter membrane, and collect the filtrate.

[0103] 4. Purification of recombinant proteins 1) For IL31-his protein, column chromatography was used (column volume 10 mL; packing material: Ni Sepharose 6FF, Cytiva (GE Life), product catalog number 17531801). First, the column was equilibrated with equilibration buffer (1xPBS buffer; 300 mM NaCl; pH 7.4). Then, the supernatant obtained from expression in step 3 was loaded onto the column (loading volume 1 L). The column was loaded with 5-10 column volumes of washing buffer (1xPBS buffer; 300 mM NaCl; 50 mM imidazole; pH 7.4). The IL31-his protein was obtained by elution with elution buffer (1xPBS buffer; 300 mM NaCl; 500 mM imidazole; pH 7.4).

[0104] 2) For the IL31-mFc protein, an affinity chromatography column (10 mL column volume; packing material: Protein At Beads LX, Changzhou Tiandi Renhe Biotechnology Co., Ltd., product catalog number SA08501L) was used. The column was first equilibrated with PBS buffer, then the supernatant obtained from step 3 (1 L loading volume) was loaded. The column was then washed with PBS buffer for 10-15 column volumes, followed by elution with glycine buffer (pH 2.5-3.0, 0.1 M) and collection of the post-column solution. Ten volumes of the post-column solution were mixed with one volume of neutralization buffer (pH 9.0, 1 M Tris-HCl buffer) to obtain a mixture. Subsequently, a 30K ultrafiltration tube (Millipore, UFC903096) was used for concentration and buffer replacement, replacing the buffer system with PBS buffer to obtain the IL31-mFc protein.

[0105] 5. Identification of recombinant proteins The concentration of IL31-his / IL31-mFc protein was measured using a nanodropper, and the recombinant protein was identified by SDS-PAGE. Under denaturing conditions, 2 μg of the recombinant protein sample was loaded onto a 12% SDS-PAGE gel. After electrophoresis, Coomassie Brilliant Blue staining was performed, and clear protein bands were revealed after destaining. The results are as follows: Figure 1 As shown, high-purity IL31-his / IL31-mFc proteins were successfully obtained, with IL31-his having a molecular weight of approximately 20 kDa and IL31-mFc having a molecular weight of approximately 50 kDa.

[0106] Example 2. Hybridoma Monoclonal Antibody Screening 1. Immunizing mice: SPF-grade 6-8 week old Balb / c mice were immunized with canine IL-31-mFc protein obtained in Example 1, following the procedure below: Day 1: First immunization, multiple subcutaneous injections of immunizing agent (composed of 10 μg immunogen, CpG + γ-manganese adjuvant). Day 29: Second immunization, multiple subcutaneous injections of immunizing agent (composed of 10 μg immunogen, CpG + γ-manganese adjuvant). Day 57: Third immunization, multiple subcutaneous injections of immunizing agent (composed of 10 μg immunogen, CpG + γ-manganese adjuvant). Day 64: Orbital venous blood was collected to separate serum, which was used to detect the titer of the test antibody. Mice with high titers were selected for the fourth immunization, and the immunogen was injected into the tail vein.

[0107] Methods for detecting antibody titers in immune serum (ELISA method): Take canine IL31-his protein and dilute it with PBS buffer to a protein concentration of 1 μg / ml; this is the coating solution. PBST solution: PBS buffer containing 0.05% (v / v) Tween-20. Blocking solution: PBST solution containing 0.2 g / 100 mL BSA. Antibody dilution preparation: Immune mouse serum was used as the test antibody, and serum from unimmunized normal mice was used as the negative control. The serum was diluted 100-fold, 1000-fold, 10000-fold, and 100000-fold with blocking solution, respectively. Perform the detection according to the following steps: 1) Take a 96-well microplate, add coating buffer (100 μL / well), incubate at 4°C for 16 hours (overnight), discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0108] 2) Take the 96-well plate after completing step 1), add blocking solution (200 μL / well), incubate at room temperature for 1 hour, discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0109] 3) Take the 96-well plate from step 2), add antibody dilution buffer (100 μL / well), incubate at room temperature for 1 hour, discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0110] 4) Take the 96-well plate from step 3), add HRP-labeled goat anti-mouse IgG antibody (Zhongshan Jinqiao ZB-2305) (10000-fold dilution, 100 μL / well), incubate at room temperature for 1 hour, discard the supernatant, wash 5 times with PBST solution, and pat dry.

[0111] 5) Take the 96-well plate after completing step 4), add TMB colorimetric solution (100 μL / well), and react in the dark for 5-10 minutes.

[0112] 6) Take the 96-well plate after completing step 5), add 2M sulfuric acid solution (50μL / well), and then measure the absorbance at 450nm (OD450).

[0113] 2. Hybridoma cell fusion and screening 1) Preparation of feeder cells The day before fusion, well-developed Balb / c mice were euthanized by cervical dislocation and sterilized in 75% ethanol aqueous solution. Using a pre-chilled syringe, 8-10 ml of pre-chilled 0.34M sucrose aqueous solution was injected through the lower right peritoneum of the mouse, ensuring the needle did not exit the peritoneum. The peritoneal cavity was massaged with a finger for approximately 1 minute. The fluid from the peritoneal cavity was then aspirated and added to a pre-chilled 50 ml centrifuge tube. Pre-chilled 1640 complete culture medium was added, and the tube was centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded, and the cell pellet was resuspended in 1640 complete culture medium containing HAT (Sigma, catalog number H0262) (HAT medium) to a cell concentration of 1×10⁻⁶.5 Cells / ml were then added to a 96-well cell culture plate (100 μl / well) and cultured.

[0114] 2) Preparation of spleen cells Four days after the fourth immunization in step 1, mice were euthanized by enucleation, and blood and spleen were collected separately. Serum was isolated from the blood and used as a positive control (i.e., fusion mouse serum). A 10 cm diameter culture dish was prepared, and 10 ml of 1640 medium and DNase were added. Mouse spleens were then added, and the mixture was ground and pipetted to form a single-cell suspension. The suspension was filtered through a 70 μm filter, and the filtrate was collected into a 50 ml centrifuge tube. The tube was then centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. The cell pellet was resuspended in 5 ml of ACK erythrocyte lysis buffer and incubated at room temperature for 2 min. Cells were washed with 1640 medium, centrifuged, and the supernatant was discarded. The cells were then resuspended in 20 ml of 1640 medium, mixed thoroughly, and counted.

[0115] 3) Cell fusion Collect healthy Sp2 / 0 mouse myeloma cells in the logarithmic growth phase and wash with 1640 medium. Mix Sp2 / 0 mouse myeloma cells and spleen cells at a ratio of 1:1 to 1:3, centrifuge at 1500 rpm for 10 min, and discard the supernatant. Pat the cells to form a paste, then place in a 37°C water bath. Add 1 ml of preheated 50% PEG solution dropwise, followed by 40 ml of preheated 1640 medium. Centrifuge at 1500 rpm for 10 min, and discard the supernatant. Break up the cell pellet, add 10 ml of HAT medium and agitate several times, then add more HAT medium to approximately 90 ml. Mix well and drop the mixture 2 drops per well into feeder cell culture plates for incubation.

[0116] 4) Screening of hybridoma cell positive wells Four days after cell fusion, half of the HAT medium was replaced. After approximately 7-10 days, the hybridoma cell clusters reached a certain size. One day before testing, about 200 μl of medium was aspirated and replaced with 200 μl of fresh 1640 complete medium containing HT (Sigma, catalog number H0137) (HT medium). On the day of testing, the culture medium from the hybridoma cell clusters was used as the test antibody. Serum from fused mice was used as a positive control, and serum from unimmunized mice as a negative control. Binding to canine IL-31 was detected using ELISA to screen for positive clones.

[0117] 5) Subcloning of positive hybridoma cells The first subcloning was performed using HT medium. Positive cloning wells were selected for the first subcloning, and cell state and cell cluster size were observed under a microscope. Under aseptic conditions, the cells to be subcloned were gently pipetted to mix, avoiding air bubbles. 10 μl of the cell count was aspirated, and 10 μl of 0.04% trypan blue solution was added. After mixing, the cells were counted in 8 large squares, and the cell concentration was calculated. Based on the cell count results, 100-150 cells were added to 9.5 ml of medium using the limiting dilution method. After mixing, the mixture was dropped into feeder cell culture plates cultured for 1 day.

[0118] After about 5 days of subcloning, the number of hybridoma clones in each well is counted under an inverted microscope. Once the hybridoma cells have grown to a suitable size, they are tested again and positive single clones are screened for a second subcloning. The culture medium is then replaced with 1640 complete medium. Subcloning is repeated 2-3 times until the obtained single-clone hybridoma cell line can stably secrete the required antibody.

[0119] 6) Expansion culture and cryopreservation of hybridoma cells ① The positive hybridoma cells obtained after identification were expanded and transferred to 24-well cell culture plates and cultured until the confluence reached about 80%.

[0120] ②After completing step ①, transfer the cells to a T25 cell culture flask and culture them until the cell confluence reaches about 80%.

[0121] ③ After completing step ②, repeatedly blow the culture flask with culture medium, then transfer the blown cells to a sterile centrifuge tube, centrifuge at 1500 rpm for 5 min, discard the supernatant, resuspend the cells with 3 ml of cell cryopreservation solution, mix thoroughly, transfer the cell suspension to a cryopreservation tube, place it in a programmed cooling box, incubate at -80℃ for 24 h, and then transfer it to liquid nitrogen for long-term storage.

[0122] Based on the above steps, hybridoma cells 12G10 that secrete the target monoclonal antibody (i.e., a monoclonal antibody that binds to canine IL-31 protein) were screened and obtained. Figure 2 The results of ELISA detection of IL31-his protein using the steps in the above-mentioned "Antibody Titer Detection Method of Immune Serum" are shown. Positive control PC (fusion mouse serum) and negative control NC (normal Balb / c mouse serum) were diluted 1000-fold and used as controls.

[0123] Example 3. Obtaining the variable region sequence of a monoclonal antibody 1) Identification of monoclonal antibody subtypes After culturing 12G10 hybridoma cells in 1640 complete medium for 2-3 days, the supernatant was collected. Antibody isotypes were identified using a monoclonal antibody isotype identification kit, following the manufacturer's instructions. The monoclonal antibody isotype identification kit (Isotyping Kit for Mouse Monoclonal Antibody) was manufactured by Beijing Yiqiao Shenzhou Technology Co., Ltd., product catalog number SEK003.

[0124] See results Figure 3 12G10 is the IgG2a subtype.

[0125] 2) Monoclonal antibody variable region sequence amplification and sequencing Take 1×10 6 ~1×10 7 Hybridoma cells containing 12G10 anti-canine IL-31 were centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were washed with PBS and centrifuged again to remove the supernatant. 1 mL of pre-chilled Trizol (Invitrogen, 15596018CN) was added, and RNA was extracted according to the manufacturer's instructions. cDNA was reverse transcribed using oligo dT primers and M-MLV reverse transcriptase (Progema, M1705).

[0126] Using cDNA as a template, primers targeting the antibody variable region were used. The upstream primer of the mouse VH sequence (Table 4) was paired with the downstream heavy chain primer corresponding to the subtype (Table 5), and the upstream primer of the mouse Vκ sequence (Table 6) was paired with the downstream light chain primer (Table 7). PCR amplification was performed using PrimeSTAR Max DNA polymerase (TaKaRa, R045). The amplification system and PCR reaction procedure are shown in Tables 8 and 9 below.

[0127] Table 4. Upstream primers for mouse VH sequence

[0128] R represents A or G, Y represents C or T, K represents G or T, M represents A or C, S represents G or C, and W represents A or T.

[0129] Table 5. Downstream primers for mouse VH sequence

[0130] Table 6. Upstream primers for mouse Vκ sequence

[0131] R represents A or G, Y represents C or T, K represents G or T, M represents A or C, S represents G or C, and W represents A or T.

[0132] Table 7. Downstream primers for mouse Vκ sequence

[0133] Table 8. PCR amplification system

[0134] Table 9. PCR reaction procedure

[0135] PCR amplification products were analyzed by 1% agarose gel electrophoresis. The results showed that the 12G10 heavy chain had a specific band of the correct size in the amplification product of MHVP3, and the light chain had a specific band of the correct size in the amplification product of MκVP9. Other primers did not amplify any specific bands. The specific bands were excised, the gel products were recovered, and sent to a sequencing company for sequencing to obtain the gene sequences of the variable regions of the antibody heavy chain and light chain. The amino acid sequences of the variable regions of the antibody heavy chain (SEQ ID NO: 1) and light chain (SEQ ID NO: 2) were further obtained.

[0136] The nucleotide sequence of the variable region of the 12G10 antibody heavy chain is as follows: GAAGTGATGCTGGTGGAGTCTGGGGGAGACTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGTTGCCTCTGGCTTCACTTTCAGTACCTATGCCATGTCTTGGGTTCGCCAGATTCCGGAGAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGTGCTTACACCCA CTATTCAGACAGTGTGAAGGGACGATTCACCGTCTCCAGAGACAATGCCAAGAACACCCTGTCCCTGCAAATGAGCAGTCTGAGGACTGCGGACTCGGCCATATATTACTGTGCAAGACTTGATTACGACTGGTTTCCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCT(SEQ ID NO: 50) The nucleotide sequence of the variable region of the light chain of the 12G10 antibody is as follows: GATATCCAGATGACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAATCACCATCAGTTGCAGGACAAGTCAGGACATTAGCAATTCTTTGAACTGGTATCAGCAGAAACCAGGTGGATCTATTAAACTCCTTATCTACTACACATCAAGATTA CCCTCAGGAGTCCCATCAAGATTCAGTGGCAGTGGGTCTGGAACAGATTACTCTCTCACCATTAGCAACCTGGAGGAAGAAGATATTGCCACGTACTTTTGCCAACAAGGTAAAACGCTTCCGTTCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAG(SEQ ID NO: 51) Example 4. Preparation of 12G10 mouse-canine chimeric antibody 1. Construction of a mouse-dog chimeric antibody recombinant expression vector Antibodies consist of heterodimers of two heavy and light chains, each with a variable and a constant region. To construct expression vectors that generate chimeric mouse-dog antibodies or canine-derived antibodies in mammalian expression systems, a universal plasmid vector was first constructed by inserting a signal peptide sequence and a canine heavy or light chain constant region sequence into the pTT3 vector. The amino acid sequences of the canine heavy and light chain constant regions are shown below. The Kozak common sequence, signal peptide sequence, and unique restriction endonuclease site (BsmBI) were incorporated into the heavy and light chain constant region nucleotide sequences, respectively, to promote the expression and secretion of recombinant antibodies from mammalian cell lines. The resulting nucleotide sequences were sp-BsmBI-HC and sp-BsmBI-LC, respectively. Then, sp-BsmBI-HC and sp-BsmBI-LC were cloned into the pTT3 vector using HindIII and NotI endonucleases, forming the pTT3-sp-BsmBI-HC and pTT3-sp-BsmBI-LC vectors, respectively.

[0137] Canine heavy chain constant region amino acid sequence: ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGK(SEQ ID NO: 52) Amino acid sequence of canine light chain constant region: RNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD(SEQ ID NO: 53) sp-BsmBI-HC nucleotide sequence: sp-BsmBI-LC nucleotide sequence: GCCACCATGGAGTCACAGACTCAGGTCTTGTATACATGTTGCTGTGGTTGTCTGGTGTTGATGGAGAGACGGGCGGCGGATCTCGTCTCCGGAATGACGCTCAACCAGCTGTCTATCTGTTCCAGCCATCTCCAGACCAGCTGCATACCGGATCAGCCAGCGTCGTGTGCCTTCTGAACTCTTTCTACCCTAAAGACATCAACGTGAAGTGG AAGGTGGACGGAGTGATCCAAGACACCGGCATCCAAGAGAGCGTCACAGAGCAAGACAAAGACAGCACCTATAGCCTGTCCAGCACACTGACAATGTCTAGCACCGAGTATCTGAGTCATGAGCTGTACAGCTGCGAGATCACACATAAATCTCTGCCAAGCACACTCATCAAGAGCTTTCAGAGGTCTGAGTGTCAGAGGGTGGATTAA(SEQ ID NO: 55) The heavy and light chain variable regions of the mouse monoclonal antibody 12G10 were ligated to the heavy or light chain constant regions of canine IgG molecules, respectively, to form chimeric antibodies. Based on the sequences of the 12G10 heavy and light chain variable regions, forward and reverse amplification primers were designed, and BsmBI restriction enzyme sites were introduced into both primers. Using cDNA from 12G10 hybridoma cells as templates, PCR amplification of the heavy and light chains was performed. The PCR products of the heavy and light chain variable regions were cloned into the vectors pTT3-sp-BsmBI-HC and pTT3-sp-BsmBI-LC using the BsmBI enzyme, respectively, to obtain the expression plasmids of the chimeric antibody CA-12G10 heavy chain (pTT3-CA-12G10-H) and light chain (pTT3-CA-12G10-L). The recombinant plasmids have been sequenced and verified.

[0138] Table 10. Primer sequences for the variable region light and heavy chains:

[0139] 2. Preparation of transfection complex Preparation of transfection complex (per 1L of cell suspension): Add 500μg of heavy chain expression plasmid and 500μg of light chain expression plasmid to 5mL of Hi-exp medium and mix well by pipetting to obtain phase A; add 3mL of PEI transfection reagent solution to 5mL of Hi-exp medium and mix well by pipetting to obtain phase B; add phase B to phase A, mix well by pipetting, and then incubate at room temperature for 5min.

[0140] 3. Preparation of cell suspension Collect 293F cells from the culture, count the cells, centrifuge at 800 rpm for 5 minutes, discard the supernatant, and resuspend the cell pellet in Hi-exp medium to achieve a cell concentration of 1×10⁻⁶ cells / mL. 6 Cells / mL.

[0141] 4. Add the transfection complex dropwise to 1L of cell suspension and incubate at 130rpm for 5 days (environmental conditions: 37℃, 8% CO2). Then centrifuge at 4000rpm for 20min, collect the supernatant, filter it through a 0.45μm filter membrane, and collect the filtrate.

[0142] 5. Using an affinity chromatography column (column volume 10 mL; packing material: protein At Beads LX), first equilibrate the column with PBS buffer, then load the filtrate obtained in step 4 (loading volume 1 L), wash with PBS buffer for 10-15 column volumes, then elute with glycine buffer (pH 2.5-3.0, 0.1 M) and collect the post-column solution. Mix 10 volumes of the post-column solution with 1 volume of neutralization buffer (i.e., pH 9.0, 1 M Tris-HCl buffer) to obtain a mixture.

[0143] 6. Take the mixture obtained in step 5 and concentrate it using a 30K ultrafiltration tube (Millipore, UFC903096). Replace the buffer system with PBS buffer to obtain the genetically engineered antibody solution.

[0144] The expression of chimeric canine IgG antibody CA-12G10 was evaluated using SDS-PAGE. (See electrophoresis image below.) Figure 4 Under denaturing conditions, only two clear main bands were observed, indicating high purity. The heavy chain band was slightly below 55 kDa, while the light chain band was above 25 kDa. The staining intensity of the heavy chain was approximately twice that of the light chain, indicating successful expression and purification of the chimeric antibody.

[0145] Example 5. In vitro binding assay of chimeric antibodies Take canine IL-31 protein and dilute it with PBS buffer to a protein concentration of 1 μg / ml; this is the coating solution. Prepare the antibody dilution buffer: Take the prepared chimeric antibody CA-12G10 solution, first dilute it with PBS buffer to an antibody concentration of 10 μg / ml, then perform a 2-fold serial dilution with PBS buffer. Detect antibody binding activity according to the following steps: 1. Take a 96-well microplate, add coating buffer (100 μL / well), incubate at 4°C for 16 hours (overnight), discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0146] 2. Take the 96-well plate from step 1, add blocking buffer (200 μL / well), incubate at room temperature for 1 hour, discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0147] 3. Take the 96-well plate from step 2, add antibody dilution buffer (100 μL / well), incubate at room temperature for 1 hour, discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0148] 4. Take the 96-well plate from step 3, add HRP-labeled goat anti-canine IgG (H+L) antibody (SouthernBiotech, catalog number 6070-05) (10000-fold dilution, 100 μL / well), incubate at room temperature for 1 hour, discard the supernatant, wash 5 times with PBST solution, and pat dry.

[0149] 5. Take the 96-well plate after completing step 4, add TMB colorimetric solution (100μL / well), and react in the dark for 5-10 minutes.

[0150] 6. Take the 96-well plate after completing step 5, add 2M sulfuric acid solution (50μL / well), and then measure the absorbance at 450nm (OD450).

[0151] See results Figure 5 The binding curves of serially diluted CA-12G10 and IL-31 proteins showed a reaction starting from 0.6 ng / mL, with the reaction value increasing as the antibody concentration increased, reaching a plateau at around 1250 ng / mL.

[0152] Example 6. Canine-based strategy The development of drug-resistant antibodies (ADAs) can lead to a reduction or loss of efficacy for any biological therapeutic protein, including monoclonal antibodies. To minimize the immunogenicity of chimeric antibodies and improve their stability in dogs, further canine-derived modifications were performed on the chimeric antibodies. This primarily involved replacing the frame region (FR) of the variable region (excluding the CDR) of the mouse-derived antibody with a canine sequence. The aim was to retain high-affinity active antibodies using a complete canine frame to minimize the potential for immunogenicity in vivo.

[0153] Based on the homology with 12G10, the heavy and light chains were screened for canine reproductive sequences in the IMGT database. The FR sequences of the heavy and light chain variable regions of 12G10 were replaced with the corresponding canine FR sequences. Individual amino acids were then replaced according to antibody expression and biological characteristics, resulting in the canine-derived heavy chain variable region Can-12G10-HV (SEQ ID NO: 60) and the canine-derived light chain variable region Can-12G10-LV (SEQ ID NO: 61).

[0154] Canine-derived heavy chain variable region Can-12G10-HV: EVQLVESGGDLVKPGGSLRLSCVASGFTFSTYAMSWVRQAPGKGLQWVATISSGGAYTHYADAVKGRFTISSRDNAKNTLYLQMNSLRAEDTAVYYCARLDYDWFPYWGQGTLVTVS(SEQ ID NO: 60) Canine-derived light chain variable region Can-12G10-LV: EIVMTQSPASLSLSQEEKVTITCRASQDISNSLNWYQQKPGQAPKLLIYYTSRRATGVPSRFSGSGSGTDFSFTISSLEPEDVAVYFCQQGKTLPFTFGQGTKLEIK(SEQ ID NO: 61) SDS-PAGE was used to identify the quality and purity of the canine-derived 12G10 antibody Can-12G10. Figure 6 There was no significant difference in band size or staining degree between CA-12G10 and the already marketed product Cytokinin.

[0155] Following the method in Example 5, the in vitro binding curves of Can-12G10, CA-12G10, and cyproheptadine were detected. The three antibodies were serially diluted to the same amount, and their binding curves with IL-31 protein were essentially identical, indicating that the three antibodies have essentially the same affinity for IL-31 protein in vitro. Figure 7 ).

[0156] Example 7. In vitro competition experiment between canine-derived Can-12G10 antibody and cyproheptadine. 1. Labeling with Can-12G10 and cytokinase antibodies 1) Take 2 mg each of the canine-derived antibody Can-12G10 and cytokine prepared above, with an antibody concentration of 2 mg / mL.

[0157] 2) Take out the 2mg Sulfo-NHS-LC-Biotin (Thermo 21355) dispensed from -20℃, add 360μL of ultrapure water, and after complete dissolution, you will have a biotin solution.

[0158] 3) Add 27 μL of Biotin solution to each of Can-12G10 and Cytokinin in 1), mix well, and place on ice for 2 hours.

[0159] 4) The above reaction solution was added to a 30K ultrafiltration tube for concentration and buffer replacement. The buffer system was replaced with PBS buffer to obtain biotin-Can-12G10 and biotin-Cetylamine antibody.

[0160] 2. In vitro competitive ELISA assay of Can-12G10 and Cetirizine Take canine IL-31 protein and dilute it with PBS buffer to a protein concentration of 1 μg / mL to obtain the coating solution. Antibody dilution preparation method: First, dilute Can-12G10 and Cetirizine separately with PBS buffer to an antibody concentration of 16 μg / mL, then serially dilute each with PBS buffer to 0.25 μg / mL. Perform ELISA detection according to the following steps: 1) Take a 96-well microplate, add coating buffer (100 μL / well), incubate at 4°C for 16 hours (overnight), discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0161] 2) Take the 96-well plate after completing step 1), add blocking solution (200 μL / well), incubate at room temperature for 1 hour, discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0162] 3) Take the 96-well plate from step 2) and add antibody dilution buffer (100 μL / well) to each well, and incubate at room temperature for 1 hour.

[0163] 4) Take the 96-well plate from step 3) and add 5 μg / mL of biotin-Can-12G10 or biotin-Cetylphenidate (20 μL / well). Incubate at room temperature with low-speed shaking for 1 hour. Discard the supernatant, wash 5 times with PBST solution, and pat dry.

[0164] 5) Take the 96-well plate from step 4), add HRP-streptavidin working solution (5000 times dilution, 100 μL / well), incubate at room temperature for 1 hour, discard the supernatant, wash 5 times with PBST solution, and pat dry.

[0165] 6) Take the 96-well plate after completing step 5), add TMB colorimetric solution (100 μL / well), and react in the dark for 5-10 minutes.

[0166] 7) Take the 96-well plate after completing step 6), add 2M sulfuric acid solution (50μL / well), and then measure the absorbance at 450nm (OD450).

[0167] The results are as follows Figure 8 As shown, when using biotin-Can12G10 for detection, a concentration of cytokinin up to 16 μg / mL did not affect the detected signal value, while the detected signal value of Can12G10 decreased with increasing concentration, and vice versa. This indicates that Can-12G10 and cytokinin do not compete with each other when binding to IL-31 protein, suggesting that their binding sites are different.

[0168] Example 8. Evaluation of canine-derived Can-12G10 antibody in a canine pruritus model 1) Eight healthy beagle dogs aged 4-6 months without skin diseases or itching symptoms were selected. The experimental animals were placed in single-enclosed enclosures, and itching behavior was identified and scored using both manual and video monitoring.

[0169] 2) After the experimental animals have been in a single enclosure for ≥1 hour for acclimatization, each dog was simultaneously assessed and scored for 60 minutes using both manual and video monitoring. The score was used as the “reference baseline”.

[0170] 3) One week prior to medication, verify whether IL-31 protein can induce basal itching in experimental dogs: Each experimental dog was intravenously injected with IL-31 protein at a dose of 1.75 μg / kg. IL-31 protein injection induced a strong basal itching response in dogs from 20 minutes to 6 hours post-injection. Twenty minutes after IL-31 protein injection, the experimental dogs were monitored manually and via video for 2 hours to identify and score basal itching behavior or to count the number of basal itching episodes. Judging itching behavior: Licking / biting the paws, sides of the abdomen and / or anal area, scratching the sides of the abdomen, neck and / or floor, shaking the head, rubbing the buttocks on the cage floor, any of these behaviors occurring within a specified time interval can be rated as "yes".

[0171] Itching behavior assessment and scoring criteria: At 1-minute intervals, each dog is judged "yes / no" for whether it is scratching. At the end of the specified time, the number of dogs that answered "yes" is added together to form the "cumulative itching score index".

[0172] All instances of itching during the observation period are counted as "cumulative instances of itching".

[0173] 4) After each observation period, return the dogs to their normal enclosure.

[0174] 5) Five dogs received a single subcutaneous injection of Can-12G10 monoclonal antibody at a dose of 2 mg / kg, while three dogs served as a placebo control group and received a subcutaneous injection of saline. Pruritus was then induced by intravenous injection of IL-31 at a dose of 1.75 μg / kg on days 1, 8, 15, 22, and 29. A reference baseline was obtained before each IL-31 protein injection, as per step 2). During each IL-31 challenge period, pruritus behavior was identified and scored using both manual and video monitoring, starting 20 minutes after IL-31 protein injection. This observation continued for 2 hours, and all instances of pruritus during the observation period were counted as the "cumulative pruritus count."

[0175] Table 11 Results of the pruritus behavior scores in experimental dogs

[0176] The statistical results of the itch behavior scores of the experimental dogs (Table 11) and the statistical results of the cumulative number of itch occurrences after each IL-31 attack ( Figure 9 Definitions TCTCACATGGCACCGACCCACCAGCTGCCCCCGTCTGATGTTCGGAAAATTATTCTGGAACTGCAACCTCTGAGC CGTGGTCTGCTGGAAGATTACCAGAAAAAAGAAACCGGCGTGCCCGAGAGCAATCGTACCCTGCTGCTGTGCCTGA CAAGCGACAGCCAGCCTCCAAGACTGAATAGTAGCGCTATTCTGCCTTATTTTAGAGCCATTCGTCCGCTGTCCGA CAAGAACATCATCGACAAAATTATCGAGCAGCTGGATAAGCTGAAGTTCCAGCACGAACCTGAAACCGAGATCAGC GTTCCGGCAGATACCTTTGAATGTAAGAGCTTCATCCTGACCATCCTGCAGCAGTTTAGCGCCTGTCTGGAAAGCG TGTTCAAGAGCCTGAACAGCGGTCCGCAG SHMAPTHQLPPSDVRKIILELQPLSRGLLEDYQKKETGVPESNRTLLLCLTSDSQPPRLNSSAILPYF RAIRPLSDKNIIDKIIEQLDKLKFQHEPETEISVPADTFECKSFILTILQQFSACLESVFKSLNSGPQ Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Definitions Compared to the control group, Can-12G10 treatment significantly suppressed itching behavior. Therefore, in an IL-31-induced pruritus model, a single subcutaneous injection of Can-12G10 can significantly suppress canine pruritus and can be used to provide anti-pruritus protection for dogs with atopic dermatitis, etc.

[0177] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An antibody or its antigen-binding fragment that specifically binds to animal IL-31, characterized in that, The antibody or its antigen-binding fragment comprises: a1) The amino acid sequences CDR-H1, CDR-H2, and CDR-H3 in the heavy chain variable region as shown in SEQ ID NO: 1; and the amino acid sequences CDR-L1, CDR-L2, and CDR-L3 in the light chain variable region as shown in SEQ ID NO: 2; or a2) Compared with CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 described in a1), at least one CDR contains one or more amino acid substitutions, deletions or additions; The CDR is defined by IMGT, Kabat, Chothia, or the Contact numbering system.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment comprises: b1) A heavy chain variable region comprising the amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; and a light chain variable region comprising the amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 14, YTS, and SEQ ID NO: 15, respectively; wherein the CDRs are defined by the IMGT numbering system, or The heavy chain variable region comprises amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; and the light chain variable region comprises amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 15, respectively; wherein the CDRs are defined by the Kabat numbering system, or The heavy chain variable region comprises amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 8, respectively; and the light chain variable region comprises amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 15, respectively; wherein the CDRs are defined by the Chothia numbering system, or The heavy chain variable region comprises amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; and the light chain variable region comprises amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively; wherein the CDRs are defined by the Contact numbering system, or b2) Heavy chain variable region; and, light chain variable region, wherein, compared with the heavy chain variable region and / or light chain variable region of b1), at least one CDR contains a substitution, deletion or addition of one or more amino acids.

3. The antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The antibody or its antigen-binding fragment includes a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having at least 75% sequence identity with it; and / or The antibody or its antigen-binding fragment includes a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence having at least 75% sequence identity with it.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, characterized in that, The animal is selected from dogs, cats, or horses. Preferably, the antibody is a monoclonal antibody; Preferably, the antibody is a canine, canine-derived, cat, feline-derived, equine, equine-derived, or chimeric antibody. Preferably, the antibody is of type IgA, IgD, IgE, IgG, or IgM; more preferably, the antibody is of type IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. Preferably, the antibody comprises a heavy chain constant region and / or a light chain constant region derived from an animal; more preferably, the antibody comprises a heavy chain constant region and / or a light chain constant region derived from a canine antibody. Preferably, the antigen-binding fragment includes scFv, Fab, Fab', (Fab')2, Fv fragment, Fd, and dsFv. Preferably, the antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, characterized in that, The heavy chain variable region of the antibody includes canine-derived framework regions FR-H1, FR-H2, FR-H3 and / or FR-H4, or variants thereof, and / or The variable regions of the light chain of the antibody include canine-derived framework regions FR-L1, FR-L2, FR-L3 and / or FR-L4, or variants thereof.

6. An antibody or its antigen-binding fragment that specifically binds to animal IL-31, characterized in that, The antibody or its antigen-binding fragment is canine-derived, and the antibody or its antigen-binding fragment comprises: c1) A heavy chain variable region comprising amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; and a light chain variable region comprising amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 14, YTS, and SEQ ID NO: 15; wherein the CDRs are defined by the IMGT numbering system, or c2) a heavy chain variable region; and a light chain variable region, wherein, compared with the heavy chain variable region and / or the light chain variable region described in b1), at least one CDR contains a substitution, deletion, or addition of one or more amino acids. Preferably, the heavy chain variable region of the antibody comprises: FR-H1, which contains the amino acid sequence shown in SEQ ID NO: 62, or an amino acid sequence having at least 75% sequence identity with it; FR-H2, which contains the amino acid sequence shown in SEQ ID NO: 63, or an amino acid sequence having at least 75% sequence identity with it; FR-H3, which contains the amino acid sequence shown in SEQ ID NO: 64, or an amino acid sequence having at least 75% sequence identity with it; and / or, FR-H4, which contains the amino acid sequence shown in SEQ ID NO: 65, or an amino acid sequence having at least 75% sequence identity with it; Preferably, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 60, or an amino acid sequence having at least 75% sequence identity with it. Preferably, the light chain variable region of the antibody comprises: FR-L1, which contains the amino acid sequence shown in SEQ ID NO: 66, or an amino acid sequence having at least 75% sequence identity with it; FR-L2, which contains the amino acid sequence shown in SEQ ID NO: 67, or an amino acid sequence having at least 75% sequence identity with it; FR-L3, which contains the amino acid sequence shown in SEQ ID NO: 68, or an amino acid sequence having at least 75% sequence identity with it; and / or, FR-L4, which contains the amino acid sequence shown in SEQ ID NO: 69, or an amino acid sequence having at least 75% sequence identity with it; Preferably, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 61, or an amino acid sequence having at least 75% sequence identity with it.

7. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 as described in any one of claims 1-6.

8. A carrier, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 7.

9. A cell, characterized in that, The cell comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1-6, the nucleic acid molecule as described in claim 7, or the vector as described in claim 8. Preferably, the cells do not contain reproductive material.

10. A coupling, characterized in that, The conjugate comprises: an antibody or an antigen-binding fragment thereof as described in any one of claims 1-6; and a conjugation portion, wherein the conjugation portion is selected from detectable substances, small molecule drugs, and / or proteins. Preferably, the protein includes cytokines, antibodies, antibody Fc fragments, antibody scFv fragments, enzymes, and viral capsid proteins. Preferably, the detectable substance includes fluorescent substances, luminescent markers, or radioactive substances; more preferably, the detectable substance is selected from any one of acridine esters, acridine sulfonamides, luminol, isoluminol, horseradish peroxidase, alkaline phosphatase, and isotopes.

11. A method for preparing an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31, characterized in that, The method includes culturing the cells of claim 9 and isolating the antibody under conditions that allow expression of the antibody or its antigen-binding fragment.

12. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: an antibody or antigen-binding fragment thereof as described in any one of claims 1-6; a nucleic acid molecule as described in claim 7; a carrier as described in claim 8; a cell as described in claim 9; or a conjugate as described in claim 10; and a pharmaceutically acceptable carrier. Preferably, the route of administration of the pharmaceutical composition is parenteral, injection, oral, or topical. Preferably, the pharmaceutical composition is in solid, semi-solid, or liquid form.

13. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-6, the nucleic acid molecule according to claim 7, the vector according to claim 8, the cell according to claim 9, the conjugate according to claim 10, or the pharmaceutical composition according to claim 12, characterized in that, The application includes one or more of the following: d1) Detect the presence or content of IL-31 in the sample; d2) Diagnosis or prognostic assessment of IL-31-related diseases; d3) To prevent or treat IL-31-related diseases in the subject; d4) Prepare reagents for detecting the presence or content of IL-31 in a sample; d5) Prepare reagents for the diagnosis or prognostic assessment of IL-31-related diseases in subjects; d6) Prepare drugs for the prevention or treatment of IL-31-related diseases in subjects.

14. The application according to claim 13, characterized in that, The samples include whole blood, red blood cell concentrate, platelet concentrate, white blood cell concentrate, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, feces, urine, cultured cells, saliva, oral secretions and / or nasal secretions from the subject.

15. The application according to claim 13 or 14, characterized in that, The IL-31-related diseases include pruritus, allergies, or asthma. In a preferred embodiment, the IL-31-related diseases include atopic dermatitis, eczema, psoriasis, scleroderma, allergic dermatitis, urticaria, psoriasis, and asthma.

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