Antibodies or antigen-binding fragments capable of binding to interleukin-31, as well as methods for producing and using the same.

Abstract

The present invention provides an antibody or its antigen-binding fragment capable of binding to interleukin-31, as well as a method for producing the same and its use. The antibody or its antigen-binding fragment has high affinity and activity, and suppresses canine pruritic diseases, particularly canine atopic dermatitis, by blocking the binding of IL-31, especially canine IL-31, to its receptor.

Description

Cross-reference of related applications

[0001] This application claims the benefits of Chinese Patent Application No. 202310894799.2, filed on 20 July 2023, and Chinese Patent Application No. 202310941457.1, filed on 28 July 2023, the contents of which are incorporated herein by reference. [Technical Field]

[0002] The present invention relates to the field of antibodies, and more specifically to antibodies or antigen-binding fragments thereof that can bind to interleukin-31, as well as methods for producing and using the same. [Background technology]

[0003] Skin diseases in dogs and cats are among the most frequently treated conditions in veterinary clinics, and among them, canine atopic dermatitis (CAD) is a common pruritic skin disease in dogs. Canine atopic dermatitis can develop at any age, but clinical symptoms usually appear in the summer around one year of age, and symptoms include itching, redness, swelling, and cracking of the skin, and if the inflammation persists, the affected area of ​​skin may thicken. The causes of canine atopic dermatitis may be related to genetic disorders, immune system dysfunction, environmental exposure, or impaired skin permeability.

[0004] Studies have shown that transgenic mice overexpressing interleukin-31 (IL-31) exhibit symptoms such as dermatitis and pruritus, and that injection of IL-31 can induce acute itching in mice. These studies suggest that IL-31 is closely related to dermatitis and pruritus. IL-31 is primarily produced by activated helper Th2 cells, and its receptor is mainly expressed in macrophages, keratinocytes, and spinal dorsal root ganglia. The IL-31 receptor forms heterodimers in IL-31RA and OSMR. When IL-31 binds to its receptor, it can activate the phosphorylation of downstream JAK kinase and STAT. In atopic dermatitis mouse models, IL-31 mRNA is highly expressed in affected skin areas, and itching can be suppressed by inhibiting the IL-31 signaling pathway. Therefore, IL-31 antagonistic antibodies may suppress itching and alleviate atopic dermatitis. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] The object of the present invention is to provide an antibody or its antigen-binding fragment that can bind to interleukin 31, as well as a method for producing and using the same, in order to overcome the problems present in the prior art. definition

[0006] The ranges and endpoints of values ​​disclosed herein are not limited to these precise ranges or values, and should be understood to include values ​​close to these ranges or values. For numerical ranges, one or more new numerical ranges can be obtained by combining the endpoint values ​​of each range, the endpoint values ​​of each range with individual point values, and the individual point values, and all such numerical ranges should be considered as specifically disclosed herein.

[0007] In this invention, the term "antibody" refers to an immunoglobulin molecule and any form of antibody that expresses a desired biological activity. This includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments. Typically, a full-length antibody structure preferably consists of four polypeptide chains, two heavy chains (H chains) and two light chains (L chains), usually linked by disulfide bonds. Each heavy chain includes a heavy chain variable region and a heavy chain constant region. Each light chain includes a light chain variable region and a light chain constant region. In addition to this typical full-length antibody structure, other derived forms of its structure may also be included.

[0008] The term "antigen-binding fragment" refers to a portion of a complete antibody molecule that retains at least some of the binding specificity of the parent antibody, and typically includes at least a portion of the parent antibody's antigen-binding region or variable region (e.g., one or more CDRs). Examples of antigen-binding fragments include, but are not limited to, Fv fragments, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fd' fragments, single-chain antibody molecules (e.g., scFv, di-scFv, tri-scFv, bispecific antibodies, or scFab), and single-domain antibodies.

[0009] The "Fv fragment" contains variable regions derived from both the heavy and light chains, but does not contain a constant region.

[0010] A "Fab fragment" consists of one light chain, one heavy chain CH1 domain, and a variable region. The heavy chain of a Fab molecule cannot form disulfide bonds with other heavy chain molecules.

[0011] A "Fab' fragment" contains one light chain and one heavy chain portion that includes a region between the VH domain, the CH1 domain, and the CHI domain and the CH2 domain. This allows for the formation of an interchain disulfide bond between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[0012] The "F(ab')2 fragment" consists of two light chains and two heavy chains that include a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Therefore, the F(ab')2 fragment is composed of two Fab' fragments linked by a disulfide bond between the two heavy chains.

[0013] A "single-chain Fv antibody" (or "scFv antibody") is an antibody fragment containing the VH and VL domains of an antibody, where these structures reside within a single polypeptide chain. Generally, Fv polypeptides further contain a polypeptide linker between the VH and VL domains, which allows the scFv to form the structure necessary for antigen binding. For a review of scFv, see Pluckthun (1994), THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269–315. Also see International Patent Application Publication No. WO88 / 01649, and U.S. Patents 4,946,778 and 5,260,203. The contents of the above references are incorporated herein by reference.

[0014] A "bispecific antibody" (also called a "bispecific antibody") is a small antibody fragment with two antigen-binding sites. The fragment contains a heavy chain variable domain (VH) (VH-VL or VL-VH) linked to a light chain variable domain (VL) within the same polypeptide chain. By using a linker too short to allow the two domains on the same chain to pair, the domains pair with a complementary domain on another chain, forming two antigen-binding sites.

[0015] The term "variable region" refers to the domains in the heavy or light chain of an antibody that are involved in antibody binding to an antigen. The variable regions of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures and are further subdivided into hypervariable regions (called complementarity-determining regions (CDRs)) scattered within more conserved regions (called framework regions (FRs)). The term "complementarity-determining regions" (CDRs, e.g., CDR1, CDR2, and CDR3) refers to specific amino acid residues within the antibody variable region that are essential for antigen binding. Each variable region typically has three CDR regions, identified as CDR1, CDR2, and CDR3.

[0016] The term "constant region" refers to specific amino acid sequences on the light and heavy chains of an antibody that do not directly participate in antibody-antigen binding but exert various effector functions, such as antibody-dependent cell-mediated cytotoxicity.

[0017] The term "chimeric antibody" refers to an antibody having at least a portion of the heavy chain variable region and at least a portion of the light chain variable region derived from one species, and at least a portion of the constant region derived from another species. For example, in one embodiment, a chimeric antibody may include a mouse variable region and a canine constant region.

[0018] The term "canine antibody" includes a CDR derived from a non-canine antibody and a framework region and constant region derived from a canine antibody. For example, the anti-IL31 antibody provided herein may include a CDR derived from one or more mouse antibodies, as well as a canine framework region and constant region. The present invention provides exemplary canine antibodies. Another anti-IL31 antibody or variant thereof, comprising heavy-chain and light-chain CDRs according to the present invention, can be prepared using any canine framework sequence and is also included in the present invention. In one embodiment, framework sequences suitable for use in the present invention include those structurally similar to the framework sequences herein. Further modifications can be made to the framework region to improve the properties of the antibody herein. Such other framework modifications include chemical modifications, point mutations to reduce immunogenicity or remove T-cell epitopes, or reverting mutations to residues in the original species lineage sequence. In some examples, such modifications include modifications corresponding to mutations exemplified herein, e.g., revert mutations to the species lineage sequence. For example, in one embodiment, one or more amino acids within the canine framework region of the VH and / or VL of the canine antibody according to this specification are backmutated to the corresponding amino acids in the parent mouse antibody. For example, in the VH and VL of the canine antibody, several sites of the framework amino acids of the template canine antibody described above were backmutated to the corresponding amino acid sequences of the mouse IL31 antibody. In one embodiment, in some examples, one or more amino acid backmutations selected from 2I, 4L, 36Y, 46L, and 68R are preferred, as are one or more amino acid backmutations selected from 68G, 73F, and 78L, and further, one or more amino acid backmutations selected from 2I, 12S, 37Q, 46V, 49K, 59P, 60S, 69S, and 77S. Additional or alternative backmutations in the framework region of the canine antibody according to this specification can improve the properties of the antibody. The present invention also encompasses constant region mutations to improve the properties of the antibody.In some embodiments, the canine IgG B heavy chain constant region is mutated from M to A at position 234, from L to A at position 235, and from G to A at position 237, which reduces the affinity of canine IgG B for canine FcγRI and C1q and avoids the effector function of canine IgG B.

[0019] In this invention, the term "antibody-drug conjugate" refers to a monoclonal antibody targeting a tumor-specific antigen or tumor-associated antigen, conjugated via a linker to a number of low-molecular-weight toxins. This is a complex formed by linking a cytotoxic drug to a monoclonal antibody that targets a tumor. When the antibody-drug conjugate molecule enters the body, it binds to the antigen on the surface of the target cell through the induced action of the monoclonal antibody. Upon entering the target cell, it releases effector molecules through chemical and / or enzymatic action, thereby achieving the objective of eliminating the target cell.

[0020] In this invention, the term “binding affinity” refers to the total strength of non-covalent interactions between a single binding site of a molecule and its binding partner. Unless otherwise specified, “binding affinity” as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., antibody and antigen). “Affinity KD,” “binding rate constant Kon,” and “dissociation rate constant Koff” are typically used to describe the affinity between a molecule (e.g., antibody) and its binding partner (e.g., antigen), i.e., how strongly a ligand binds to a particular protein. Binding affinity is influenced by non-covalent intermolecular interactions such as hydrogen bonds, electrostatic interactions, hydrophobicity between two molecules, and van der Waals forces. Furthermore, the binding affinity between a ligand and its target molecule may be influenced by the presence of other molecules.

[0021] In this invention, the CDR numbering system in antibodies refers to the precise definition of the complementarity-determining region (CDR), framework (FR), and light and heavy chain residues that affect the binding affinity and specificity of the antibody-antigen interaction. Commonly used CDR numbering systems include Kabat, Chothia, IMGT, as well as AbM, Contact, and North, and may vary depending on the numbering system used.

[0022] In this invention, "sequence identity" between two polypeptide or nucleic acid sequences refers to the percentage of identical residues between the sequences relative to the total number of residues. When calculating the identity percentage, the sequences to be compared are aligned to maximize the degree of sequence agreement, and any gaps in the alignment (if any) are resolved using a specific algorithm. Preferred computer programming methods for determining the identity between two sequences include, but are not limited to, GCG packages including GAP, BLASTP, BLASTN, and FASTA (Altschul et al., 1990, J.Mol.Biol.215:403-410). These programs are publicly available from the National Center for Biotechnology Information (NCBI) and other sources. The well-known Smith-Waterman algorithm can also be used to determine identity.

[0023] In the present invention, cell line models are cell-based and are used to study patient diseases, drug development, toxicity testing, and the like by utilizing intracellular changes. In some embodiments of the present invention, HEK293 cells expressing canine IL-31 receptors (canine IL-31RA and canine OSMR) are constructed, and the ability of the IL31 antibody to block the binding of recombinant canine IL-31 protein to its receptors is evaluated. In other embodiments, the IL31 antibody is studied using DH82 cells (canine macrophages / canine malignant histiocytosis cells) to block the binding of IL-31 to the cell surface coreceptors IL-31RA and OSMR, thereby suppressing the downstream JAK / STAT signaling pathway and reducing the degree of STAT3 phosphorylation.

[0024] In the present invention, an animal model refers to using various animals as controllable experimental subjects, and observing various changes in the animals to study fields such as diseases of patients, drug development, and toxicity tests. Commonly used animals are mammals, birds, and reptiles. In some embodiments of the present invention, a recombinant canine IL31-induced canine pruritus model is constructed to evaluate the suppression of the IL31 antibody against canine pruritus. In some other embodiments, a flea-induced canine pruritus model is constructed to evaluate the suppression of the IL31 antibody against canine pruritus.

[0025] In the present invention, the term "recombinant cell" refers to separating cell organelles and their components from living cells, and then recombining cell organelles and their components from different origins under specific conditions in vitro to reconstruct into biologically active cells or cell organelles.

[0026] In the present invention, the term "cutaneous pruritus" refers to a disease mediated by IL-31, that is, a general inflammation, and in particular, a series of diseases accompanied by symptoms of dermatitis and pruritus, including allergic diseases such as atopic dermatitis, contact dermatitis, nodular prurigo, and eczema.

[0027] The term "pharmaceutical composition" refers to a mixture containing one or more of the compounds described in the present invention, or its physiologically / pharmacologically acceptable salts or prodrugs, and other chemical components such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to a living body, thereby promoting the absorption of the active ingredient and enabling it to exert its biological activity. Therapeutic compositions generally must be sterile and stable under manufacturing and storage conditions. The composition can be formulated as a solution, microemulsion, dispersant, liposome, or other regular structure suitable for a high antibody concentration. A sterile injectable solution can be prepared by mixing the required amount of the active compound (i.e., the antibody or its antigen-binding fragment) with the above components or combinations of components in a suitable solvent and filtering and sterilizing as necessary.

[0028] In this invention, the term "nucleotide molecule" refers to a biological macromolecular compound synthesized from a large number of nucleotides, and is one of the most fundamental substances of life. Nucleic acids differ in their chemical composition and the order of their nucleotide sequences. Based on their chemical composition, nucleic acids can be divided into ribonucleic acid (abbreviated as RNA) and deoxyribonucleic acid (abbreviated as DNA). DNA is the primary material basis for the storage, replication, and transmission of genetic information. RNA plays a crucial role in protein synthesis, where transfer RNA (abbreviated as tRNA) carries and transports activated amino acids, messenger RNA (abbreviated as mRNA) functions as a template for protein synthesis, and ribosomal RNA (abbreviated as rRNA) is the primary site for protein synthesis within cells. [Means for solving the problem]

[0029] A first aspect of the present invention provides an antibody or antigen-binding fragment that can bind to interleukin 31, comprising one or more CDR region (antibody complementarity-determining region) sequences or mutant sequences selected from SEQ ID NO: 1 to 6 and / or SEQ ID NO: 7 to 12 and / or SEQ ID NO: 13 to 18.

[0030] In some forms, the IL31 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region CDR or its mutant sequence, which is optionally selected from SEQ ID NO:1-3 and / or SEQ ID NO:7-9 and / or SEQ ID NO:13-15.

[0031] In some forms, the IL31 antibody or its antigen-binding fragment of the present invention comprises a light chain variable region CDR or its mutant sequence, which is optionally selected from SEQ ID NO: 4-6 and / or SEQ ID NO: 10-12 and / or SEQ ID NO: 16-18.

[0032] In some forms, the IL31 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region CDR1, the amino acid sequence of the heavy chain variable region CDR1 having approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the amino acid sequence indicated by SEQ ID NO: 1, 7, or 13.

[0033] In some forms, the IL31 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region CDR2, the amino acid sequence of the heavy chain variable region CDR2 having approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% or more identity with the amino acid sequences indicated by SEQ ID NO: 2, 8, and 14.

[0034] In some forms, the IL31 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region CDR3, the amino acid sequence of the heavy chain variable region CDR3 having approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequences indicated by SEQ ID NO: 3, 9, and 15.

[0035] In some forms, the IL31 antibody or antigen-binding fragment of the present invention comprises a light chain variable region CDR1, the amino acid sequence of the light chain variable region CDR1 having approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequences indicated by SEQ ID NO: 4, 10, and 16.

[0036] In some forms, the IL31 antibody or antigen-binding fragment of the present invention comprises a light chain variable region CDR2, the amino acid sequence of the light chain variable region CDR2 having approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequences indicated by SEQ ID NO: 5, 11, and 17.

[0037] In some forms, the IL31 antibody or its antigen-binding fragment of the present invention comprises a light chain variable region CDR3, the amino acid sequence of the light chain variable region CDR3 having approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequences indicated by SEQ ID NO: 6, 12, and 18.

[0038] In some embodiments, in the IL31 antibody or antigen-binding fragment of the present invention, the amino acid sequence of the heavy chain variable region CDR1 has 80% or more identity with the amino acid sequence indicated by SEQ ID NO: 1, 7, or 13; the amino acid sequence of the heavy chain variable region CDR2 has 80% or more identity with the amino acid sequence indicated by SEQ ID NO: 2, 8, and 14; the amino acid sequence of the heavy chain variable region CDR3 has 80% or more identity with the amino acid sequence indicated by SEQ ID NO: 3, 9, and 15; the amino acid sequence of the light chain variable region CDR1 has 80% or more identity with the amino acid sequence indicated by SEQ ID NO: 4, 10, and 16; the amino acid sequence of the light chain variable region CDR2 has 80% or more identity with the amino acid sequence indicated by SEQ ID NO: 5, 11, and 17; and the amino acid sequence of the light chain variable region CDR3 has 80% or more identity with the amino acid sequence indicated by SEQ ID NO: 6, 12, and 18.

[0039] In the present invention, by the antibody numbering system, SEQ ID NO:1 may be SEQ ID NO:19 or 25, SEQ ID NO:7 may be SEQ ID NO:21 or 30, SEQ ID NO:13 may be SEQ ID NO:23 or 35, SEQ ID NO:2 may be SEQ ID NO:20 or 26, SEQ ID NO:8 may be SEQ ID NO:22 or 31, SEQ ID NO:14 may be SEQ ID NO:24 or 36, SEQ ID NO:3 may be SEQ ID NO:27, SEQ ID NO:9 may be SEQ ID NO:32, SEQ ID NO:15 may be SEQ ID NO:37, SEQ ID NO:4 may be SEQ ID NO:28, SEQ ID NO:10 may be SEQ ID NO:33, SEQ ID NO:16 may be SEQ ID SEQ ID NO:38 may be SEQ ID NO:5, SEQ ID NO:29 (amino acid sequence is RA), SEQ ID NO:11 may be SEQ ID NO:34, and SEQ ID NO:17 may be SEQ ID NO:39. Here, SEQ ID NO:1~18 are CDRs obtained using the Kabat numbering system, SEQ ID NO:3~6, 9~12, and 15~24 are CDRs obtained using the Chothia numbering system, and SEQ ID NO:6, 12, 18, and 25~39 are CDRs obtained using the IMGT numbering system. The amino acid sequence of SEQ ID NO:34 is GA, and the amino acid sequence of SEQ ID NO:39 is YV.In other words, SEQ ID NO:1 is replaced by SEQ ID NO:19 or 25, SEQ ID NO:7 is replaced by SEQ ID NO:21 or 30, SEQ ID NO:13 is replaced by SEQ ID NO:23 or 35, SEQ ID NO:2 is replaced by SEQ ID NO:20 or 26, SEQ ID NO:8 is replaced by SEQ ID NO:22 or 31, SEQ ID NO:14 is replaced by SEQ ID NO:24 or 36, SEQ ID NO:3 is replaced by SEQ ID NO:27, SEQ ID NO:9 is replaced by SEQ ID NO:32, SEQ ID NO:15 is replaced by SEQ ID NO:37, SEQ ID NO:4 is replaced by SEQ ID NO:28, SEQ ID NO:10 is replaced by SEQ ID NO:33, and SEQ ID NO:16 is replaced by SEQ ID NO:38 is substituted, SEQ ID NO:5 is substituted with SEQ ID NO:29 (amino acid sequence: RA), SEQ ID NO:11 is substituted with SEQ ID NO:34 (amino acid sequence: GA), and SEQ ID NO:17 is substituted with SEQ ID NO:39 (amino acid sequence: YV).

[0040] In the present invention, the number of amino acid residues in the heavy chain variable region CDR1 is 10 or less, and / or the number of amino acid residues in the heavy chain variable region CDR2 is 22 or less, and / or the number of amino acid residues in the heavy chain variable region CDR3 is 17 or less, and / or the number of amino acid residues in the light chain variable region CDR1 is 18 or less, and / or the number of amino acid residues in the light chain variable region CDR2 is 10 or less, and / or the number of amino acid residues in the light chain variable region CDR3 is 12 or less.

[0041] In the present invention, the amino acid sequence of the heavy chain variable region CDR1 is shown as SEQ ID NO:1, the amino acid sequence of the heavy chain variable region CDR2 is shown as SEQ ID NO:2, the amino acid sequence of the heavy chain variable region CDR3 is shown as SEQ ID NO:3, the amino acid sequence of the light chain variable region CDR1 is shown as SEQ ID NO:4, the amino acid sequence of the light chain variable region CDR2 is shown as SEQ ID NO:5, and the amino acid sequence of the light chain variable region CDR3 is shown as SEQ ID NO:6.

[0042] In another embodiment of the present invention, the amino acid sequence of the heavy chain variable region CDR1 is shown by SEQ ID NO:7, the amino acid sequence of the heavy chain variable region CDR2 is shown by SEQ ID NO:8, the amino acid sequence of the heavy chain variable region CDR3 is shown by SEQ ID NO:9, the amino acid sequence of the light chain variable region CDR1 is shown by SEQ ID NO:10, the amino acid sequence of the light chain variable region CDR2 is shown by SEQ ID NO:11, and the amino acid sequence of the light chain variable region CDR3 is shown by SEQ ID NO:12.

[0043] In another embodiment of the present invention, the amino acid sequence of the heavy chain variable region CDR1 is shown by SEQ ID NO:13, the amino acid sequence of the heavy chain variable region CDR2 is shown by SEQ ID NO:14, the amino acid sequence of the heavy chain variable region CDR3 is shown by SEQ ID NO:15, the amino acid sequence of the light chain variable region CDR1 is shown by SEQ ID NO:16, the amino acid sequence of the light chain variable region CDR2 is shown by SEQ ID NO:17, and the amino acid sequence of the light chain variable region CDR3 is shown by SEQ ID NO:18.

[0044] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 40-43, 48-51, and 56-59 and having at least about 80%, 85%, 90%, and 95% identity thereto, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 44-47, 52-55, and 60-63 and having at least about 80%, 85%, 90%, and 95% identity thereto.

[0045] In some forms, the antibody according to the present invention is a chimeric antibody, and further, a chimeric anti-IL31 antibody comprising a heavy chain variable region and / or a light chain variable region is provided.

[0046] Preferably, the antibody heavy chain variable region is selected from SEQ ID NO: 40, 48, and 56, and the antibody light chain variable region is selected from SEQ ID NO: 44, 52, and 60.

[0047] Preferably, in the chimeric anti-IL31 antibody, the amino acid sequence of the heavy chain variable region is SEQ ID NO:40 and an amino acid sequence having at least about 80%, 85%, 90%, and 95% identity thereto, and the amino acid sequence of the light chain variable region is SEQ ID NO:44 and an amino acid sequence having at least about 80%, 85%, 90%, and 95% identity thereto.

[0048] Preferably, in the chimeric anti-IL31 antibody, the amino acid sequence of the heavy chain variable region is SEQ ID NO:48 and an amino acid sequence having at least about 80%, 85%, 90%, and 95% identity thereto, and the amino acid sequence of the light chain variable region is SEQ ID NO:52 and an amino acid sequence having at least about 80%, 85%, 90%, and 95% identity thereto.

[0049] Preferably, in the chimeric anti-IL31 antibody, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 56 and an amino acid sequence having at least about 80%, 85%, 90%, and 95% identity thereto, and the amino acid sequence of the light chain variable region is SEQ ID NO: 60 and an amino acid sequence having at least about 80%, 85%, 90%, and 95% identity thereto.

[0050] Preferably, the heavy chain amino acid sequence is selected from SEQ ID NO: 66, 74, or 82, and the light chain amino acid sequence is selected from SEQ ID NO: 70, 78, or 86.

[0051] More preferably, in the antibody, the heavy chain may be indicated by SEQ ID NO:66 and the light chain by SEQ ID NO:70, or the heavy chain by SEQ ID NO:74 and the light chain by SEQ ID NO:78, or the heavy chain by SEQ ID NO:82 and the light chain by SEQ ID NO:86.

[0052] In this invention, the present invention provides a canine antibody or its antigen-binding fragment so that the obtained antibody or antigen-binding fragment can better inhibit IL-31 and have high affinity and high activity, thereby suppressing pruritic skin diseases in canines.

[0053] In some forms, the amino acid sequence of the heavy chain variable region framework FR is selected from canine strain heavy chain sequences and preferably has 0 to 5 reverse mutation residues, which may be 0, 1, 2, 3, 4, or 5 reverse mutations. In some examples, one or more of the amino acid reverse mutations 49G, 71V, 73K, and 78A are preferred, as are the amino acid reverse mutations 54A, 55E, 82D, and 87V.

[0054] In some forms, the amino acid sequence of the light chain variable region framework FR is selected from canine strain light chain sequences and preferably has 0 to 5 reverse mutation residues, and may be 0, 1, 2, 3, 4, or 5 reverse mutation residues. In some examples, one or more amino acid reverse mutations selected from 2I, 4L, 36Y, 46L, and 68R are preferred, as are one or more amino acid reverse mutations selected from 68G, 73F, and 78L, and further, one or more amino acid reverse mutations selected from 2I, 12S, 37Q, 46V, 49K, 59P, 60S, 69S, and 77S. "Reverse mutation residue" refers to the genotype and phenotype that have fully or partially reverted after a second mutation of an amino acid residue.

[0055] In the present invention, the amino acid sequence of the heavy chain variable region is one of those indicated by SEQ ID NO: 41-43, 49-51, and 57-59, and the amino acid sequence of the light chain variable region is one of those indicated by SEQ ID NO: 45-47, 53-55, and 61-63.

[0056] In some embodiments, the present invention provides canine anti-IL31 antibodies comprising amino acid sequences having at least 80%, 85%, 90%, and 95% identity with the heavy chain variable region of the amino acid sequences of SEQ ID NO: 41-43, and amino acid sequences having at least 80%, 85%, 90%, and 95% identity with the light chain variable region of the amino acid sequences of SEQ ID NO: 45-47. Preferably, the amino acid sequence of the heavy chain variable region is one of those indicated by SEQ ID NO: 41-43, and the amino acid sequence of the light chain variable region is one of those indicated by SEQ ID NO: 45-47. More preferably, the amino acid sequence of the heavy chain variable region is one of those indicated by SEQ ID NO: 42 and 43, and the amino acid sequence of the light chain variable region is one of those indicated by SEQ ID NO: 45 and 47.

[0057] In some embodiments, the present invention provides canine anti-IL31 antibodies containing amino acid sequences having at least 80%, 85%, 90%, and 95% identity with the heavy chain variable region of amino acid sequences SEQ ID NO: 49-51, and amino acid sequences having at least 80%, 85%, 90%, and 95% identity with the light chain variable region of amino acid sequences SEQ ID NO: 53-55. Preferably, the amino acid sequence of the heavy chain variable region is one of those indicated by SEQ ID NO: 49-51, and the amino acid sequence of the light chain variable region is one of those indicated by SEQ ID NO: 53-55. More preferably, the amino acid sequence of the heavy chain variable region is one of those indicated by SEQ ID NO: 49 and 50, and the amino acid sequence of the light chain variable region is one of those indicated by SEQ ID NO: 54 and 55.

[0058] In some embodiments, the present invention provides canine anti-IL31 antibodies containing amino acid sequences having at least 80%, 85%, 90%, and 95% identity with the heavy chain variable region of amino acid sequences SEQ ID NO: 57-59, and amino acid sequences having at least 80%, 85%, 90%, and 95% identity with the light chain variable region of amino acid sequences SEQ ID NO: 61-63. Preferably, the amino acid sequence of the heavy chain variable region is one of those indicated by SEQ ID NO: 57-59, and the amino acid sequence of the light chain variable region is one of those indicated by SEQ ID NO: 61-63. More preferably, the amino acid sequence of the heavy chain variable region is one of those indicated by SEQ ID NO: 58 and 59, and the amino acid sequence of the light chain variable region is one of those indicated by SEQ ID NO: 61 and 62.

[0059] In some forms, the present invention provides a chimeric and / or canine antibody or antigen-binding fragment containing the amino acid sequence of the heavy chain constant region or a variant thereof of IgG A, IgG B, IgG C, or IgG D, preferably the amino acid sequence of the heavy chain constant region of an IgG B mutant consisting of SEQ ID NO:64, and the light chain constant region or a variant thereof of a canine κ or λ chain, preferably the amino acid sequence of the κ light chain constant region consisting of SEQ ID NO:65.

[0060] In some embodiments, the present invention provides an anti-IL31 chimeric antibody, the antibody comprising a complete heavy chain, the heavy chain being selected from an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acids of SEQ ID NO: 66, 74, or 82, and the light chain amino acid sequence being selected from an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acids of SEQ ID NO: 70, 78, or 86.

[0061] The chimeric antibody may consist of a heavy chain indicated by SEQ ID NO:66 and a light chain indicated by SEQ ID NO:70, or a heavy chain indicated by SEQ ID NO:74 and a light chain indicated by SEQ ID NO:78, or a heavy chain indicated by SEQ ID NO:82 and a light chain indicated by SEQ ID NO:86.

[0062] In some embodiments, the present invention provides an anti-IL31 canine antibody, the antibody comprising a complete heavy chain and a complete light chain, wherein the amino acid sequence of the heavy chain is one of SEQ ID NO: 67-69, 75-77, and 83-85 and has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity thereto, and the amino acid sequence of the light chain is one of SEQ ID NO: 71-73, 79-81, and 87-89 and has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity thereto.

[0063] The variable region of the aforementioned canine antibody is a combination of the canine variable region heavy chain and canine variable region light chain of the same antibody. For example, combinations of heavy chain 6465-H1 (SEQ ID NO: 41) and light chains 6465-K1, 6465-K2, or 6465-K3 can be described as 6465-H1K1, 6465-H1K2, and 6465-H1K3, respectively, and so on. For example, 6465-H1K1-canIgGB has a canine variable region heavy chain H1, a canine variable region light chain K1, and a canine IgGB constant region, and so on, but specific detailed combinations are shown in Table 1.

[0064] In some embodiments, the antibody consists of a heavy chain indicated by SEQ ID NO:67 and a light chain indicated by SEQ ID NO:71, or a heavy chain indicated by SEQ ID NO:68 and a light chain indicated by SEQ ID NO:71, or a heavy chain indicated by SEQ ID NO:69 and a light chain indicated by SEQ ID NO:71, or a heavy chain indicated by SEQ ID NO:67 and a light chain indicated by SEQ ID NO:72, or a heavy chain indicated by SEQ ID NO:68 and a light chain indicated by SEQ ID NO:72, or a heavy chain indicated by SEQ ID NO:69 and a light chain indicated by SEQ ID NO:73, or a heavy chain indicated by SEQ ID NO:68 and a light chain indicated by SEQ ID It consists of a light chain indicated by NO:73, or a heavy chain indicated by SEQ ID NO:75 and a light chain indicated by SEQ ID NO:79, or a heavy chain indicated by SEQ ID NO:76 and a light chain indicated by SEQ ID NO:79, or a heavy chain indicated by SEQ ID NO:77 and a light chain indicated by SEQ ID NO:79, or a heavy chain indicated by SEQ ID NO:75 and a light chain indicated by SEQ ID NO:80, or a heavy chain indicated by SEQ ID NO:76 and a light chain indicated by SEQ ID NO:80, or a heavy chain indicated by SEQ ID NO:77 and a light chain indicated by SEQ ID NO:80, or a heavy chain indicated by SEQ ID NO:75 and a light chain indicated by SEQ ID NO:81, or a heavy chain indicated by SEQ ID NO:76 and a light chain indicated by SEQ ID NO:81, or SEQ ID It consists of a heavy chain indicated by NO:77 and a light chain indicated by SEQ ID NO:81, or a heavy chain indicated by SEQ ID NO:83 and a light chain indicated by SEQ ID NO:87, or a heavy chain indicated by SEQ ID NO:84 and a light chain indicated by SEQ ID NO:87, or a heavy chain indicated by SEQ ID NO:85 and SEQIt may consist of a light chain indicated by ID NO:87, or a heavy chain indicated by SEQ ID NO:83 and a light chain indicated by SEQ ID NO:88, or a heavy chain indicated by SEQ ID NO:84 and a light chain indicated by SEQ ID NO:88, or a heavy chain indicated by SEQ ID NO:85 and a light chain indicated by SEQ ID NO:88, or a heavy chain indicated by SEQ ID NO:83 and a light chain indicated by SEQ ID NO:89, or a heavy chain indicated by SEQ ID NO:84 and a light chain indicated by SEQ ID NO:89, or a heavy chain indicated by SEQ ID NO:85 and a light chain indicated by SEQ ID NO:89.

[0065] In the present invention, preferably, the antibody or its antigen-binding fragment comprises at least one of a monoclonal antibody, a polyclonal antibody, a multimeric antibody, and a CDR-transplanted antibody, and preferably, the antibody or its antigen-binding fragment comprises one of a single-chain antibody, a Fab antibody, an Fv antibody, a single-domain antibody, a (Fab)2 fragment, an scFv-Fc fusion protein, an scFv-Fv fusion protein, an Fv fragment, and a minimal antigen recognition unit.

[0066] In a second aspect of the present invention, the present invention provides a nucleotide molecule encoding the above-mentioned IL31 antibody or its antigen-binding fragment.

[0067] The present invention is not limited to specific nucleotide sequences, and any sequence that conforms to codon coding rules and is translatable to the above protein sequences is within the scope of protection. If it is necessary to be particularly limited, preferably the nucleotide sequence of the DNA molecule encoding the heavy chain is one of SEQ ID NO: 90-93, 98-101, and 106-109 and a substantially similar sequence having at least 80%, 85%, 90%, 95%, 98%, and 99% sequence identity thereto, and also preferably the nucleotide sequence of the DNA molecule encoding the light chain is one of SEQ ID NO: 94-97, 102-105, and 110-113 and a substantially similar sequence having at least 80%, 85%, 90%, 95%, 98%, and 99% sequence identity thereto.

[0068] More preferably, in DNA encoding an antibody, the heavy chain is indicated by SEQ ID NO:90 and the light chain by SEQ ID NO:94; the heavy chain is indicated by SEQ ID NO:91 and the light chain by SEQ ID NO:95; the heavy chain is indicated by SEQ ID NO:92 and the light chain by SEQ ID NO:95; the heavy chain is indicated by SEQ ID NO:93 and the light chain by SEQ ID NO:95; the heavy chain is indicated by SEQ ID NO:91 and the light chain by SEQ ID NO:96; the heavy chain is indicated by SEQ ID NO:92 and the light chain by SEQ ID NO:96; the heavy chain is indicated by SEQ ID NO:93 and the light chain by SEQ ID NO:96; the heavy chain is indicated by SEQ ID NO:91 and the light chain by SEQ ID NO:97; the heavy chain is indicated by SEQ ID NO:92 and the light chain by SEQ ID The heavy chain is indicated by NO:97, the light chain by SEQ ID NO:93, the heavy chain by SEQ ID NO:97, the heavy chain by SEQ ID NO:98, the light chain by SEQ ID NO:102, the heavy chain by SEQ ID NO:99, the light chain by SEQ ID NO:103, the heavy chain by SEQ ID NO:100, the light chain by SEQ ID NO:103, the heavy chain by SEQ ID NO:101, the light chain by SEQ ID NO:103, the heavy chain by SEQ ID NO:99, the light chain by SEQ ID NO:104, the heavy chain by SEQ ID NO:100, the light chain by SEQ ID NO:104, the heavy chain by SEQ ID NO:101, the light chain by SEQ ID NO:104, the heavy chain by SEQ ID NO:99, the light chain is indicated by SEQ ID NO:105, the heavy chain is indicated by SEQ ID NO:100, the light chain is indicated by SEQ ID NO:105, the heavy chain is indicated by SEQ ID NO:101, the light chain is indicated by SEQ ID NO:105, the heavy chain is indicated by SEQ ID NO:106, the light chain is indicated by SEQ ID NO:110, the heavy chain is indicated by SEQ ID NO:107, the light chain is indicated by SEQ ID NO:111, the heavy chain is indicated by SEQ ID NO:108, the light chain is indicated by SEQ ID NO:111, the heavy chain is indicated by SEQ ID NO:109, the light chain is indicated by SEQ ID NO:111, the heavy chain is indicated by SEQ ID NO:107, the light chain is indicated by SEQ IDThe heavy chain may be indicated by NO:112, the heavy chain by SEQ ID NO:108 and the light chain by SEQ ID NO:112; the heavy chain by SEQ ID NO:109 and the light chain by SEQ ID NO:112; the heavy chain by SEQ ID NO:107 and the light chain by SEQ ID NO:113; the heavy chain by SEQ ID NO:108 and the light chain by SEQ ID NO:113; or the heavy chain by SEQ ID NO:109 and the light chain by SEQ ID NO:113.

[0069] A third aspect of the present invention provides an expression vector that expresses the above-mentioned nucleotide molecule.

[0070] In the present invention, the expression vector refers to a vector to which expression elements (e.g., promoter, RBS, terminator) are added to the basic skeleton of a cloning vector, thereby enabling the expression of a target gene. In the present invention, expression elements are added to the nucleotides of a DNA molecule of the present invention, such as a plasmid containing the nucleotides of the present invention, so that the expression vector can appropriately express it. Preferably, the expression vector is a eukaryotic expression vector such as pCDNA3.1 or pcDNA3.4.

[0071] A fourth aspect of the present invention provides a method for producing an antibody or an antigen-binding fragment thereof, the method comprising transforming host cells with the expression vector described above.

[0072] In the present invention, the transformation method is not particularly limited and may be a general method in the field of introducing an expression vector into host cells for expression.

[0073] In the present invention, the host cell is not limited, but any cell capable of smoothly expressing the nucleotide molecule of the present invention is acceptable. Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell, and includes Chinese hamster ovary cells (CHO), NSO, NP2 / 0, HeLa cells, 293T cells, HEK293 cells, and other cell lines.

[0074] A fifth aspect of the present invention provides a pharmaceutical composition comprising an auxiliary material and the above-mentioned antibody or antigen-binding fragment, or the above-mentioned nucleotide molecule or expression vector. Preferably, the auxiliary material is selected from pharmaceutically acceptable carriers or excipients.

[0075] In the present invention, the drug carrier and excipient include, but are not limited to, physiological saline, buffer solution, glucose, water, glycerol, ethanol, or combinations thereof, as long as they are pharmaceutically acceptable.

[0076] A sixth aspect of the present invention provides a method for treating pruritic diseases of mammals, the method comprising administering the above-mentioned antibody or its antigen-binding fragment, or nucleic acid molecule, or expression vector, or pharmaceutical composition to a mammal.

[0077] The dose of the antibody or its antigen-binding fragment is 1 to 5 mg / kg, and may be within a range of 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, or any two of these values, or within that range.

[0078] The aforementioned administration methods include, but are not limited to, intravenous injection, intracutaneous injection, subcutaneous injection, sublingual administration, rectal perfusion, inhalation administration, and aerosol administration, and are more preferably subcutaneous injection.

[0079] The aforementioned pruritic skin diseases include, but are not limited to, atopic dermatitis, contact dermatitis, pruritus nodularis, and eczema, and are preferably atopic dermatitis.

[0080] The mammal is at least one species from the Felidae, Canidae, Suidae, Bovidae, Goatidae, Cercopithecidae, Muridae, and Equidae families. Preferably, the mammal is at least one species from the Dog, Wolf, Dhole, and Fox families, more preferably a dog.

[0081] A seventh aspect of the present invention provides the use of the above-mentioned antibody or its antigen-binding fragment, or nucleic acid molecule, or vector in the manufacture of a drug for treating pruritic diseases of mammals.

[0082] The present invention provides for the use of the above-mentioned antibody or its antigen-binding fragment, or nucleic acid molecule, or vector in the treatment of pruritic diseases of mammals.

[0083] Regarding pruritic skin diseases, the dosage and method of drug administration, as well as their effects on mammals, have been explained above, but will not be discussed in detail here. [Effects of the Invention]

[0084] According to the above technical solution, the antibody or antigen-binding fragment capable of binding to interleukin 31 according to the present invention has high affinity and high activity, and suppresses mammalian pruritic diseases by blocking the binding of IL-31, particularly canine IL-31, to its receptor. [Brief explanation of the drawing]

[0085] [Figure 1] This is the percentage of APC-positive cells in the presence of antibodies 6465, 7475, or B2. [Figure 2] This study involved the inhibitory effect of three chimeric antibodies and lokivetmab on canine IL-31 pruritic attacks. [Figure 3] This study describes the inhibitory effect of four canine-like antibodies, constructed based on the 6465 antibody, on canine IL-31 pruritic attacks. [Figure 4] This study describes the inhibitory effect of four canine antibodies, constructed based on the 7475 antibody, on canine IL-31 pruritic attacks. [Figure 5] This study describes the inhibitory effect of four canine-like antibodies, constructed based on the B2 antibody, on canine IL-31 pruritic attacks. [Modes for carrying out the invention]

[0086] Example 1: Production and identification of mouse anti-canine interleukin 31 (IL-31) antibody (1) Preparation of antigen HEK293 cells were transiently transfected with His-labeled canine IL-31 (interleukin 31) recombinant protein and expressed. The culture supernatant of the transiently transfected cells was collected and subjected to affinity chromatography using a Ni column. The eluate was purified canine IL-31 recombinant protein.

[0087] (2) Mouse immunity Balb / c mice and C57 mice were immunized with canine IL-31 recombinant protein as the antigen. In the first immunization, Freund's complete adjuvant emulsion antigen was used, with 100 μg of antigen administered per mouse. In subsequent immunizations, Freund's incomplete adjuvant emulsion antigen was used, with 50 μg of antigen administered per mouse. The immunization interval was 2 weeks, and the injection method was multiple injections intraperitoneally and subcutaneously. After the fourth immunization, serum was collected and antibody titers were analyzed using enzyme-linked immunosorbent assay (ELISA). Mice with serum immunosorbent titers exceeding 360,000 were selected to construct an antibody library.

[0088] (3) Spleen RNA extraction and reverse transcription RNA was extracted from mouse spleen tissue using the TRIzol method. RNA integrity was evaluated by agarose gel electrophoresis, and RNA concentration and OD were measured using an ultramicrospectrophotometer. 260 / OD 280 The following measurements were taken. Qualified RNA was selected and reverse transcribed according to the instructions for Superscript II reverse transcriptase (Thermo Scientific), and cDNA was obtained.

[0089] (4) Construction of antibody libraries and phage display The heavy and light chains of antibodies were amplified from cDNA, and scFv antibody libraries and Fab antibody libraries were constructed, each with a library volume of 1E8 cfu. The antibody libraries were electroporated into TG1 E. coli, and the TG1 E. coli was infected with helper phage M13KO7. After incubation overnight, the phage was precipitated using PEG / NaCl to produce a phage display antibody library.

[0090] (5) Screening of phage display antibody libraries Solid-phase screening and solid-phase / liquid-phase cross-screening were performed on phage display antibody libraries using immunotubes and magnetic bead sorting devices, respectively. For solid-phase screening, immunotubes were coated with canine IL-31 recombinant protein, and for liquid-phase screening, Dynabeads magnetic beads were coated with biotinylated canine IL-31 recombinant protein. After three screenings, enriched phage aggregates and single clones isolated therefrom were identified using phage ELISA.

[0091] (6) Construction, expression, and purification of antibodies DNA sequencing was performed on phagemides from phage ELISA-positive single clones. From the sequencing results, the variable region of a mouse-derived antibody with the correct reading frame was selected and subjected to gene synthesis. Subsequently, the variable region of the mouse-derived antibody was fused with the constant region of human IgG1 and inserted into a pcDNA3.4 vector to construct a full-length antibody expression plasmid. This full-length antibody was transiently transfected into HEK293 cells, and the culture supernatant was purified by protein A affinity chromatography to obtain purified full-length antibody.

[0092] (7) Screening for antibodies that block the binding of canine IL-31 to its receptor. Biotin-labeled canine IL-31 recombinant protein was incubated with different antibody concentrations for 5 minutes, then added to HEK293 cells expressing the canine IL-31 receptor (canine IL-31RA and canine OSMR) on the membrane. The cells were incubated on ice for 30 minutes, rinsed three times with PBS, and then streptavidin-APC fluorescent secondary antibody was added. The percentage of APC-labeled cells was analyzed by flow cytometry. The APC-positive cell rate reflects the binding ability of biotin-labeled canine IL-31 recombinant protein to cells expressing the canine IL-31 receptor on the membrane. A lower APC-positive cell rate indicates a stronger ability of the antibody to block the binding of biotin-labeled canine IL-31 recombinant protein to its receptor.

[0093] Results: With test antibodies 6465, 7475, and B2, a decrease in APC-positive cells was observed with increasing antibody concentration, indicating that all antibodies possessed a blocking effect. Specific results are shown in Figure 1. The amino acid sequences of complementarity-determining regions (CDRs) 1, 2, and 3 of the heavy chain variable region of antibody 6465, numbered by Kabat, are SEQ ID NO: 1 to 3, respectively, and the amino acid sequences of the light chain variable region CDRs 1, 2, and 3 are SEQ ID NO: 4 to 6, respectively. The amino acid sequences of the heavy chain variable region CDRs 1, 2, and 3 of antibody 7475 are SEQ ID NO: 7 to 9, respectively, and the amino acid sequences of the light chain variable region CDRs 1, 2, and 3 are SEQ ID NO: 10 to 12, respectively. The amino acid sequences of the heavy chain variable region CDRs 1, 2, and 3 of antibody B2 are SEQ ID NO: 13 to 15, respectively, and the amino acid sequences of the light chain variable region CDRs 1, 2, and 3 are SEQ ID NO: 16 to 18, respectively. The amino acid sequences of the heavy chain variable regions CDR1, 2, and 3 of antibody 6465, numbered by Chothia, are SEQ ID NO: 19, 20, and 3, respectively, and the amino acid sequences of the light chain variable regions CDR1, 2, and 3 are SEQ ID NO: 4 to 6, respectively. The amino acid sequences of the heavy chain variable regions CDR1, 2, and 3 of antibody 7475 are SEQ ID NO: 21, 22, and 9, respectively, and the amino acid sequences of the light chain variable regions CDR1, 2, and 3 are SEQ ID NO: 10 to 12, respectively. The amino acid sequences of the heavy chain variable regions CDR1, 2, and 3 of antibody B2 are SEQ ID NO: 23, 24, and 15, respectively, and the amino acid sequences of the light chain variable regions CDR1, 2, and 3 are SEQ ID NO: 16 to 18, respectively.The amino acid sequences of the heavy chain variable regions CDR1, 2, and 3 of antibody 6465, numbered by IMGT, are SEQ ID NO: 25-27, respectively, and the amino acid sequences of the light chain variable regions CDR1, 2, and 3 are SEQ ID NO: 28, 29, and 6, respectively. The amino acid sequences of the heavy chain variable regions CDR1, 2, and 3 of antibody 7475 are SEQ ID NO: 30-32, respectively, and the amino acid sequences of the light chain variable regions CDR1, 2, and 3 are SEQ ID NO: 33, 34, and 12, respectively. The amino acid sequences of the heavy chain variable regions CDR1, 2, and 3 of antibody B2 are SEQ ID NO: 35-37, respectively, and the amino acid sequences of the light chain variable regions CDR1, 2, and 3 are SEQ ID NO: 38, 39, and 18, respectively. Example 2: Construction of chimeric and canine antibodies of mouse anti-canine IL-31 antibody

[0094] (1) Chimeric antibody For 6465, 7475, and B2, canine chimeric antibody clones containing mouse-derived heavy and light chain variable regions were constructed. Based on the 6465 chimeric antibody (6465-mHvKv-canIgG B), heavy chain variable region variants (SEQ ID NO: 40) and light chain variable region variants (SEQ ID NO: 44) were constructed. Based on the 7475 chimeric antibody (7475-mHvKv-canIgG B), heavy chain variable region variants (SEQ ID NO: 48) and light chain variable region variants (SEQ ID NO: 52) were constructed. Based on the B2 chimeric antibody (B2-mHvKv-canIgG B), heavy chain variable region variants (SEQ ID NO: 56) and light chain variable region variants (SEQ ID NO: 60) were constructed. The constant region (including CL, CH1, CH2, and CH3) derived from the canine IgG B antibody was also included. The amino acid sequence of the canine IgG B heavy chain constant domain is shown in SEQ ID NO:64. The amino acid sequence of the canine IgG B light chain constant domain is shown in SEQ ID NO:65. Specific detailed combinations are shown in Table 1.

[0095] (2) Canine antibody 1) Three canine heavy chain variable region variants (SEQ ID NO: 41-43) and three canine light chain variable region variants (SEQ ID NO: 45-47) of 6465 were constructed, and these contained different modifications or substitutions. The canine strategy for 6465-H1 was CDR transplantation and 49G, 71V, and 73K reverse mutations; for 6465-H2, it was CDR transplantation and 49G, 71V, 73K, and 78A reverse mutations; and for 6465-H3, it was CDR transplantation only. The canine transformation strategy for 6465-K1 was CDR transplantation and reverse mutations of 2I, 4L, 46L, and 68R; the canine transformation strategy for 6465-K2 was CDR transplantation and reverse mutations of 4L, 36Y, 46L, and 68R; and the canine transformation strategy for 6465-K3 was CDR transplantation and reverse mutation of 36Y.

[0096] 2) Three canine heavy chain variable region variants (SEQ ID NO: 49-51) and three canine light chain variable region variants (SEQ ID NO: 53-55) of 7475 were constructed, and these contained different modifications or substitutions. The canine strategy for 7475-H1, 7475-H2, and 7475-H3 was CDR transplantation only. The canine strategy for 7475-K1 and 7475-K3 was CDR transplantation only, while the canine strategy for 7475-K2 was CDR transplantation and 68G, 73F, and 78L reverse mutations.

[0097] 3) Three canine heavy chain variable region variants (SEQ ID NO: 57-59) and three canine light chain variable region variants (SEQ ID NO: 61-63) of B2 were constructed, and these contained different modifications or substitutions. The canine strategy for B2-H1 was CDR transplantation and reverse mutations of 54A, 82D, and 87V. The canine strategy for B2-H2 was CDR transplantation and reverse mutations of 55E, 82D, and 87V. The canine strategy for B2-H3 was CDR transplantation and reverse mutations of 55E, 82D, and 87V. The canine transformation strategy for B2-K1 was CDR transplantation and reverse mutations of 2I, 46V, 49K, and 69S; the canine transformation strategy for B2-K2 was CDR transplantation and reverse mutations of 46V, 49K, and 69S; and the canine transformation strategy for B2-K3 was CDR transplantation and reverse mutations of 2I, 12S, 37Q, 46V, 49K, 59P, 60S, 69S, and 77S.

[0098] The variable region of a canine antibody is a combination of the canine variable region heavy chain and canine variable region light chain of the same antibody. For example, combinations of heavy chain 6465-H1 (SEQ ID NO: 41) and light chains 6465-K1, 6465-K2, or 6465-K3 are denoted as 6465-H1K1, 6465-H1K2, and 6465-H1K3, respectively, and so on. The constant region of a canine antibody consists of the constant domain of the canine IgG B heavy chain and the constant domain of the canine Kappa light chain (the amino acid sequence of the constant domain of the canine IgG B heavy chain is shown in SEQ ID NO: 64, and the amino acid sequence of the constant domain of the canine Kappa light chain is shown in SEQ ID NO: 65). For example, 6465-H1K1-canIgG B has a canine IgG variable region heavy chain H1, a canine IgG variable region light chain K1, and a canine IgG B constant region, and so on. Specific detailed combinations are shown in Table 1.

[0099] The constant region of the canine IgG B heavy chain contains three amino acid mutations, M234A, L235A, and G237A, with the aim of reducing the affinity of canine IgG B for canine FcγRI and C1q, thereby evading the effector function of canine IgG B.

[0100] [Table 1-1] [Table 1-2] Example 3: In vitro evaluation of chimeric antibodies and canine antibodies

[0101] (1) Evaluation of the in vitro activity of novel antibodies constructed based on 6465, 7475, and B2 antibodies.

[0102] Measurement of the suppression of a novel antibody against canine IL-31-induced STAT (signaling and transcriptional activation factor) phosphorylation.

[0103] IL-31 binds to its cell surface co-receptors, IL-31RA and OSMR, and activates the downstream JAK / STAT signaling pathway, thereby increasing the degree of STAT3 phosphorylation. When an IL-31 neutralizing antibody blocks IL-31 from binding to its receptor, it also suppresses STAT3 phosphorylation in the downstream signaling pathway. The IC50 values ​​of antibody suppression against STAT phosphorylation were evaluated in a DH82 cell assay using anti-canine IL-31 chimeric and canine antibodies at different concentration gradients. The specific procedure was as follows: 50,000 DH82 cells were seeded per well, 10 ng / mL of IFN-γ was added, and the cells were stimulated at 37°C for 24 hours, followed by 2 hours of serum starvation. 2.86 μg / mL of recombinant canine IL-31 protein was incubated with different concentrations of canine IL-31 antibody for 1 hour, after which the above DH82 cells were added and stimulated at 37°C for 5 minutes. Subsequently, HTRF (Heatstroke-Resistant Fibre-Fibre) was performed. (R)According to the instructions of the STAT3 p-Y705 kit, the STAT3 phosphorylation level in DH82 cells was detected, and the IC50 value was calculated based on the antibody concentration. As shown in Table 2, all three chimeric antibodies and 27 canineized antibodies could inhibit the activity of pSTAT, and the IC50 was 6.7 - 9.4 μg / mL.

[0104]

Table 2

[0105] (2) Measurement of the affinity of the new antibodies constructed based on 6465, 7475, and B2 antibodies The binding affinity of the antibodies was measured using a Gator device based on the biomembrane interference (BLI) technique. When the immobilized substance on the biosensor interacts with the analyte in the solution, the thickness of the biomembrane layer on the sensor increases, the curve of the interference spectrum shifts to the long-wavelength side, and this phase shift is detected and analyzed by the workstation, and the change in the number of molecules on the sensor surface, and the dissociation rate K off , the association rate K on , the affinity KD (KD = K off / K on ) and other related concentration and kinetic data can be quantitatively measured.

[0106] The binding affinities of three chimeric antibodies, 27 canineized antibodies, and the positive control rociletumab were detected respectively. The specific process is as follows. First, the antibodies were labeled with biotin, and then, using the biotin-labeled antibody as the immobilized substance, the biosensor was used to bind to the biotin-labeled antibody immobilized with streptavidin probes (Gator TM Streptavidin Probes). Using recombinant canine IL-31 protein at different concentration gradients as the analyte, the kinetic data of the binding to the biotin-labeled antibody were measured, and the affinity was calculated. As shown in Table 3, the binding affinities (KD) of the three chimeric antibodies and 27 canineized antibodies reached 10 -9 M, which was about 10 times that of the positive control rociletumab.

[0107] [Table 3-1] [Table 3-2] Example 4: Evaluation of in vivo efficacy of chimeric and canine antibodies based on 6465, 7475, and B2.

[0108] The in vivo efficacy of canine IL-31 chimeric antibodies and canine antibodies was evaluated using a canine IL-31 pruritic attack model.

[0109] First, a model (weighing approximately 10 kg) was created using Beagle dogs, and appropriate test dogs were selected. Beagle dogs were intravenously injected with 2.5 μg / kg of recombinant canine IL-31 protein. Itchy behavior (biting, scratching, licking, rolling, rubbing, etc.) of the Beagle dogs was observed using video monitoring 30 minutes before administration and 4 hours after administration. One point was scored for each itchy behavior observed within one minute, and zero points were given for all others. Scoring was performed every minute. The cumulative itchy score 30 minutes before administration was used as the baseline, and the cumulative itchy score 4 hours after administration was used as the measured value. The final itchy score was obtained by subtracting the baseline from the measured value. Due to individual differences, the itchy scores of each Beagle dog after modeling varied considerably. To ensure uniformity of the model, Beagle dogs with itchy scores between 100 and 150 were selected and grouped.

[0110] Twenty-five Beagle dogs were selected for successful modeling and randomly divided into five groups of five: three treatment groups (6465, 7475, and B2 chimeric antibodies), a PBS control group, and a positive control group (lokivetomab). After seven days of adaptation, the treatment groups received subcutaneous injections of 2 mg / kg of 6465, 7475, and B2 chimeric antibodies, respectively. The positive control group received subcutaneous injections of 2 mg / kg of lokivetomab. The negative control group received subcutaneous injections of an equal volume of PBS. The following day, all Beagle dogs received intravenous administration of 2.5 μg / kg of canine IL-31 recombinant protein. Based on the above scoring criteria, the pruritus scores of the Beagle dogs in each group were recorded, and it was evaluated whether the administered antibodies suppressed IL-31-mediated pruritus. As shown in Figure 2, when PBS was administered to beagle dogs, the pruritus symptoms caused by IL-31 were not reduced, and the pruritus score remained at 100-150. However, administration of any of the three chimeric antibodies or lokivetomab significantly reduced pruritus caused by IL-31 in dogs, demonstrating a significant inhibitory effect.

[0111] The efficacy of 6465, 7475, and B2 canine antibodies (6465-H2K1-canIgGB, 6465-H3K1-canIgGB, 6465-H2K3-canIgGB, 6465-H3K3-canIgGB, 7475-H1K2-canIgGB, 7475-H2K2-canIgGB, 7475-H1K3-canIgGB, 7475-H2K3-canIgGB, B2-H2K1-canIgGB, B2-H3K1-canIgGB, B2-H2K2-canIgGB, B2-H3K2-canIgGB) was evaluated using the same experimental method. The results are shown in Figures 3, 4, and 5, respectively. Similar to the chimeric antibodies, the 12 canine antibodies showed a significant inhibitory effect, with the pruritus score decreasing by more than 75% during the 4-hour observation period after modeling. The other canine antibodies shown in Table 1 also showed similar effects to these 12 types of canine antibodies.

[0112] Furthermore, the effectiveness of canine IL-31 canine antibodies was evaluated using a flea-induced atopic dermatitis model. Preliminary results showed that canine antibodies significantly suppressed flea-induced pruritic behavior. Efficacy results in dogs with atopic dermatitis also showed that canine antibodies reduced pruritic symptoms in dogs suffering from atopic dermatitis.

[0113] No adverse reactions were observed in the test dogs during the above-mentioned experiment or for one month thereafter.

[0114] The results above demonstrate that the antibody constructed according to the present invention has high affinity and high activity, and can suppress pruritic skin diseases in dogs caused by IL-31 and fleas by blocking the binding of canine IL-31 to its receptor, thereby reducing the itching symptoms in dogs suffering from atopic dermatitis.

[0115] While preferred embodiments of the present invention have been described in detail above, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications are possible to the technical solutions of the present invention, such as combining each technical feature in any other suitable way. These simple modifications and combinations are also considered to be within the scope of disclosure of the present invention and are all covered by the protection of the present invention.

[0116] In this invention, the sequences of SEQ ID NO:1 to 39 are as follows. Of these, SEQ ID NO:29, 34, and 39 are too short to be recognized by the WIPO sequence and are therefore "intentionally skipped" by the software, but this does not affect the search. The original sequences of SEQ ID NO:1 to 39 are adopted as the original disclosure. SEQ ID NO:1:NYYLY SEQ ID NO:2:ELNPRNGGTNLNAKFKT SEQ ID NO:3:GGTTVVVRDVMDF SEQ ID NO:4:RASESVDSYGNSFMH SEQ ID NO:5:RASNLES SEQ ID NO:6:QQSNEDPYT SEQ ID NO:7:SYAMS SEQ ID NO:8:YISNGGDYIFYADTVKG SEQ ID NO:9:RGHYNTSSYWYFDV SEQ ID NO:10:KASENVGTYVS SEQ ID NO:11:GASNRYT SEQ ID NO:12:GQSYSYPPT SEQ ID NO:13:DAWMD SEQ ID NO:14:EIRSRAESHATYYAESVKG SEQ ID NO:15:ASTMITTGWFAY SEQ ID NO:16:RASQNISVYLH SEQ ID NO:17:YVSQSIS SEQ ID NO:18:QNGHSFPYT SEQ ID NO:19:GYSFTNY SEQ ID NO:20:NPRNGG SEQ ID NO:21:GFTFSSY SEQ ID NO:22:SNGGDY SEQ ID NO:23:GFTFSDA SEQ ID NO:24:RSRAESHA SEQ ID NO:25:GYSFTNYY SEQ ID NO:26:LNPRNGGT SEQ ID NO:27:TRGGTTVVVRDVMDF SEQ ID NO:28:ESVDSYGNSF SEQ ID NO:29:RA SEQ ID NO:30:GFTFSSYA SEQ ID NO:31:ISNGGDYI SEQ ID NO:32:TRRGHYNTSSYWYFDV SEQ ID NO:33:ENVGTY SEQ ID NO:34:GA SEQ ID NO:35:GFTFSDAW SEQ ID NO:36:IRSRAESHAT SEQ ID NO:37:TPASTMITTGWFAY SEQ ID NO:38:QNISVY SEQ ID NO:39:YV

Claims

1. An antibody or antigen-binding fragment capable of binding to interleukin 31, The amino acid sequence of the heavy chain variable region CDR1 of the antibody or its antigen-binding fragment has 80% or more identity with the amino acid sequence indicated by SEQ ID NO: 1, 7, or 13; the amino acid sequence of the heavy chain variable region CDR2 has 80% or more identity with the amino acid sequence indicated by SEQ ID NO: 2, 8, and 14; the amino acid sequence of the heavy chain variable region CDR3 has 80% or more identity with the amino acid sequence indicated by SEQ ID NO: 3, 9, and 15; the amino acid sequence of the light chain variable region CDR1 has 80% or more identity with the amino acid sequence indicated by SEQ ID NO: 4, 10, and 16; the amino acid sequence of the light chain variable region CDR2 has 80% or more identity with the amino acid sequence indicated by SEQ ID NO: 5, 11, and 17; and the amino acid sequence of the light chain variable region CDR3 is SEQ ID An antibody or antigen-binding fragment capable of binding to interleukin 31, characterized by having 80% or more identity with the amino acid sequences indicated by NO: 6, 12, and 18.

2. The number of amino acid residues in the heavy chain variable region CDR1 is 10 or less. and / or, the number of amino acid residues in the heavy chain variable region CDR2 is 22 or less. and / or, the number of amino acid residues in the heavy chain variable region CDR3 is 17 or less. and / or, the number of amino acid residues in the light chain variable region CDR1 is 18 or less. and / or, the number of amino acid residues in the light chain variable region CDR2 is 10 or less. and / or, the antibody or antigen-binding fragment according to claim 1, wherein the number of amino acid residues in the light chain variable region CDR3 is 12 or less.

3. The amino acid sequence of the heavy chain variable region CDR1 is shown by SEQ ID NO: 1, the amino acid sequence of the heavy chain variable region CDR2 is shown by SEQ ID NO: 2, the amino acid sequence of the heavy chain variable region CDR3 is shown by SEQ ID NO: 3, the amino acid sequence of the light chain variable region CDR1 is shown by SEQ ID NO: 4, the amino acid sequence of the light chain variable region CDR2 is shown by SEQ ID NO: 5, and the amino acid sequence of the light chain variable region CDR3 is shown by SEQ ID NO:

6. Alternatively, the amino acid sequence of the heavy chain variable region CDR1 is shown by SEQ ID NO: 7, the amino acid sequence of the heavy chain variable region CDR2 is shown by SEQ ID NO: 8, the amino acid sequence of the heavy chain variable region CDR3 is shown by SEQ ID NO: 9, the amino acid sequence of the light chain variable region CDR1 is shown by SEQ ID NO: 10, the amino acid sequence of the light chain variable region CDR2 is shown by SEQ ID NO: 11, and the amino acid sequence of the light chain variable region CDR3 is shown by SEQ ID NO:

12. Alternatively, the amino acid sequence of the heavy chain variable region CDR1 is shown by SEQ ID NO: 13, the amino acid sequence of the heavy chain variable region CDR2 is shown by SEQ ID NO: 14, the amino acid sequence of the heavy chain variable region CDR3 is shown by SEQ ID NO: 15, the amino acid sequence of the light chain variable region CDR1 is shown by SEQ ID NO: 16, the amino acid sequence of the light chain variable region CDR2 is shown by SEQ ID NO: 17, and the amino acid sequence of the light chain variable region CDR3 is shown by SEQ ID NO:

18. Alternatively, SEQ ID NO: 1 is replaced by SEQ ID NO: 19 or 25, SEQ ID NO: 7 is replaced by SEQ ID NO: 21 or 30, SEQ ID NO: 13 is replaced by SEQ ID NO: 23 or 35, SEQ ID NO: 2 is replaced by SEQ ID NO: 20 or 26, SEQ ID NO: 8 is replaced by SEQ ID NO: 22 or 31, SEQ ID NO: 14 is replaced by SEQ ID NO: 24 or 36, SEQ ID NO: 3 is replaced by SEQ ID NO: 27, SEQ ID NO: 9 is replaced by SEQ ID NO: 32, and SEQ ID NO: 15 is replaced by SEQ ID The antibody or antigen-binding fragment according to claim 1, wherein NO: 37 is substituted, SEQ ID NO: 4 is substituted with SEQ ID NO: 28, SEQ ID NO: 10 is substituted with SEQ ID NO: 33, SEQ ID NO: 16 is substituted with SEQ ID NO: 38, SEQ ID NO: 5 is substituted with SEQ ID NO: 29 (amino acid sequence: RA), SEQ ID NO: 11 is substituted with SEQ ID NO: 34 (amino acid sequence: GA), and SEQ ID NO: 17 is substituted with SEQ ID NO: 39 (amino acid sequence: YV).

4. The aforementioned antibody is a chimeric antibody, Preferably, the antibody heavy chain variable region is selected from SEQ ID NO: 40, 48, 56, and the antibody light chain variable region is selected from SEQ ID NO: 44, 52, 60. Preferably, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 40, and the amino acid sequence of the light chain variable region is SEQ ID NO:

44. Alternatively, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 48, and the amino acid sequence of the light chain variable region is SEQ ID NO:

52. Alternatively, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 56, and the amino acid sequence of the light chain variable region is SEQ ID NO:

60. Preferably, the heavy chain amino acid sequence is selected from SEQ ID NO: 66, 74, or 82, and the light chain amino acid sequence is selected from SEQ ID NO: 70, 78, or 86. More preferably, the antibody or its antigen-binding fragment according to any one of claims 1 to 3, wherein the heavy chain is indicated by SEQ ID NO: 66 and the light chain by SEQ ID NO: 70, or the heavy chain is indicated by SEQ ID NO: 74 and the light chain by SEQ ID NO: 78, or the heavy chain is indicated by SEQ ID NO: 82 and the light chain by SEQ ID NO:

86.

5. The aforementioned antibody is a canine antibody, Preferably, the amino acid sequence of the heavy chain variable region framework FR is selected from canine strain heavy chain sequences and preferably has 0 to 5 reverse mutation residues. Preferably, the amino acid sequence of the light chain variable region framework FR is selected from canine strain light chain sequences and preferably has 0 to 5 reverse mutation residues. Preferably, the amino acid sequence of the heavy chain variable region is one of those indicated by SEQ ID NO: 41-43, 49-51, and 57-59, and the amino acid sequence of the light chain variable region is one of those indicated by SEQ ID NO: 45-47, 53-55, and 61-63. Preferably, the amino acid sequence of the heavy chain variable region is one of those indicated by SEQ ID NO: 41 to 43, and the amino acid sequence of the light chain variable region is one of those indicated by SEQ ID NO: 45 to 47, more preferably, the amino acid sequence of the heavy chain variable region is one of those indicated by SEQ ID NO: 42 and 43, and the amino acid sequence of the light chain variable region is one of those indicated by SEQ ID NO: 45 and 47. Preferably, the amino acid sequence of the heavy chain variable region is one of those indicated by SEQ ID NO: 49 to 51, and the amino acid sequence of the light chain variable region is one of those indicated by SEQ ID NO: 53 to 55, more preferably, the amino acid sequence of the heavy chain variable region is one of those indicated by SEQ ID NO: 49 and 50, and the amino acid sequence of the light chain variable region is one of those indicated by SEQ ID NO: 54 and 55. Preferably, the amino acid sequence of the heavy chain variable region is one of those indicated by SEQ ID NO: 57 to 59, and the amino acid sequence of the light chain variable region is one of those indicated by SEQ ID NO: 61 to 63, more preferably, the amino acid sequence of the heavy chain variable region is one of those indicated by SEQ ID NO: 58 and 59, and the amino acid sequence of the light chain variable region is one of those indicated by SEQ ID NO: 61 and 62. Preferably, the amino acid sequence of the heavy chain is one of SEQ ID NO: 67-69, 75-77, and 83-85. Preferably, the amino acid sequence of the light chain is one of SEQ ID NO: 71-73, 79-81, and 87-89. More preferably, the antibody consists of a heavy chain indicated by SEQ ID NO: 67 and a light chain indicated by SEQ ID NO: 71, or a heavy chain indicated by SEQ ID NO: 68 and a light chain indicated by SEQ ID NO: 71, or a heavy chain indicated by SEQ ID NO: 69 and a light chain indicated by SEQ ID NO: 71, or a heavy chain indicated by SEQ ID NO: 67 and a light chain indicated by SEQ ID NO: 72, or a heavy chain indicated by SEQ ID NO: 68 and a light chain indicated by SEQ ID NO: 72, or a heavy chain indicated by SEQ ID NO: 69 and a light chain indicated by SEQ ID NO: 72, or a heavy chain indicated by SEQ ID NO: 67 and a light chain indicated by SEQ ID Consists of a light chain indicated by NO: 73, or a heavy chain indicated by SEQ ID NO: 68 and a light chain indicated by SEQ ID NO: 73, or a heavy chain indicated by SEQ ID NO: 69 and a light chain indicated by SEQ ID NO: 73, or a heavy chain indicated by SEQ ID NO: 75 and a light chain indicated by SEQ ID NO: 79, or a heavy chain indicated by SEQ ID NO: 76 and a light chain indicated by SEQ ID NO: 79, or a heavy chain indicated by SEQ ID NO: 77 and a light chain indicated by SEQ ID NO: 79, or a heavy chain indicated by SEQ ID NO: 75 and a light chain indicated by SEQ ID NO: 80, or a heavy chain indicated by SEQ ID NO: 76 and a light chain indicated by SEQ ID Consists of a light chain indicated by NO: 80, or a heavy chain indicated by SEQ ID NO: 77 and a light chain indicated by SEQ ID NO: 80, or a heavy chain indicated by SEQ ID NO: 75 and a light chain indicated by SEQ ID NO: 81, or a heavy chain indicated by SEQ ID NO: 76 and a light chain indicated by SEQ ID NO: 81, or a heavy chain indicated by SEQ ID NO: 77 and a light chain indicated by SEQ ID NO: 81, or a heavy chain indicated by SEQ ID NO: 83 and a light chain indicated by SEQ ID NO: 87, or a heavy chain indicated by SEQ ID NO: 84 and a light chain indicated by SEQ ID NO: 87, or a heavy chain indicated by SEQ ID NO: 85 and a light chainConsists of a light chain indicated by NO: 87, or a heavy chain indicated by SEQ ID NO: 83 and a light chain indicated by SEQ ID NO: 88, or a heavy chain indicated by SEQ ID NO: 84 and a light chain indicated by SEQ ID NO: 88, or a heavy chain indicated by SEQ ID NO: 85 and a light chain indicated by SEQ ID NO: 88, or a heavy chain indicated by SEQ ID NO: 83 and a light chain indicated by SEQ ID NO: 89, or a heavy chain indicated by SEQ ID NO: 84 and a light chain indicated by SEQ ID NO: 89, or a heavy chain indicated by SEQ ID NO: 85 and a light chain indicated by SEQ ID NO: 89, Preferably, the antibody or its antigen-binding fragment is at least one of a monoclonal antibody, a polyclonal antibody, a multimeric antibody, and a CDR-transplanted antibody. Preferably, the antibody or its antigen-binding fragment is one of a single-chain antibody, a Fab antibody, an Fv antibody, a single-domain antibody, a (Fab)2 fragment, an scFv-Fc fusion protein, an scFv-Fv fusion protein, an Fv fragment, and a minimal antigen recognition unit, according to any one of claims 1 to 3.

6. A nucleotide molecule, wherein the nucleotide molecule encodes an antibody or antigen-binding fragment according to any one of claims 1 to 5, Preferably, the nucleotide sequence of the DNA molecule encoding the heavy chain is one of SEQ ID NO: 90-93, 98-101, and 106-109. Preferably, the nucleotide sequence of the DNA molecule encoding the light chain is one of SEQ ID NO: 94-97, 102-105, and 110-113. More preferably, in the DNA encoding the antibody, the heavy chain is indicated by SEQ ID NO: 90 and the light chain by SEQ ID NO: 94; the heavy chain is indicated by SEQ ID NO: 91 and the light chain by SEQ ID NO: 95; the heavy chain is indicated by SEQ ID NO: 92 and the light chain by SEQ ID NO: 95; the heavy chain is indicated by SEQ ID NO: 93 and the light chain by SEQ ID NO: 95; the heavy chain is indicated by SEQ ID NO: 91 and the light chain by SEQ ID NO: 96; the heavy chain is indicated by SEQ ID NO: 92 and the light chain by SEQ ID NO: 96; the heavy chain is indicated by SEQ ID NO: 93 and the light chain by SEQ ID NO: 96; and the heavy chain is SEQ ID NO: 91, light chain is indicated by SEQ ID NO: 97, heavy chain is indicated by SEQ ID NO: 92, light chain is indicated by SEQ ID NO: 97, heavy chain is indicated by SEQ ID NO: 93, light chain is indicated by SEQ ID NO: 97, heavy chain is indicated by SEQ ID NO: 98, light chain is indicated by SEQ ID NO: 102, heavy chain is indicated by SEQ ID NO: 99, light chain is indicated by SEQ ID NO: 103, heavy chain is indicated by SEQ ID NO: 100, light chain is indicated by SEQ ID NO: 103, heavy chain is indicated by SEQ ID NO: 101, light chain is indicated by SEQ ID NO: 103, heavy chain is indicated by SEQ ID NO: 99, light chain is indicated by SEQ ID NO: 104, heavy chain is indicated by SEQ ID NO: 100, light chain is indicated by SEQ ID NO: 104, heavy chain is indicated by SEQ ID NO: 101, light chain is indicated by SEQ ID NO: 104, heavy chain is indicated by SEQ ID NO: 99, light chain is indicated by SEQ ID NO: 105, heavy chain is indicated by SEQ ID NO: 100, light chain is indicated by SEQ ID NO: 105, heavy chain is indicated by SEQ ID NO: 101, light chain is indicated by SEQ ID NO: 105, heavy chain is indicated by SEQ ID NO: 106, light chain is indicated by SEQ ID NO: 110, heavy chain is indicated by SEQ ID NO: 107, light chain is indicated by SEQ ID NO: 111, heavy chain is indicated by SEQ ID NO: 108, light chain is indicated by SEQ ID NO: 111, heavy chain is indicated by SEQ ID NO: 109, light chain is indicated by SEQ ID NO: 111, heavy chain is indicated by SEQ ID NO: 107, light chain is indicated by SEQ IDA nucleotide molecule characterized by being represented by NO: 112, with the heavy chain being SEQ ID NO: 108, the light chain being represented by SEQ ID NO: 112, with the heavy chain being SEQ ID NO: 109, the light chain being represented by SEQ ID NO: 112, with the heavy chain being SEQ ID NO: 107, the light chain being represented by SEQ ID NO: 113, with the heavy chain being SEQ ID NO: 108, the light chain being SEQ ID NO: 113, and the heavy chain being SEQ ID NO: 109, the light chain being SEQ ID NO:

113.

7. An expression vector characterized in that the expression vector expresses the nucleotide molecule described in claim 6.

8. A method for producing an antibody or an antigen-binding fragment thereof, the method comprising transforming a host cell with the expression vector described in claim 7, Preferably, the host cell is a mammalian cell, more preferably a CHOK1 cell or a HEK293 cell.

9. A pharmaceutical composition comprising an auxiliary material, an antibody or antigen-binding fragment according to any one of claims 1 to 5, a nucleotide molecule according to claim 6, or an expression vector according to claim 7, Preferably, the auxiliary material is at least one selected from a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, and an adjuvant, characterized in that the pharmaceutical composition.

10. A method for treating a mammalian pruritic disease, comprising administering to a mammal an antibody or antigen-binding fragment according to any one of claims 1 to 5, or a nucleic acid molecule according to claim 6, or an expression vector according to claim 7, or a pharmaceutical composition according to claim 9. Preferably, the dose of the antibody or its antigen-binding fragment is 1 to 5 mg / kg. More preferably, the administration method is at least one of intravenous injection, intramuscular injection, subcutaneous injection, sublingual administration, rectal perfusion, inhalation administration, or aerosol administration, more preferably subcutaneous injection. More preferably, the pruritic skin disorder is a disorder related to abnormal expression of IL-31, More preferably, the pruritic skin disease is at least one of atopic dermatitis, contact dermatitis, pruritus nodularis, and eczema. More preferably, the mammal is at least one of the animals belonging to the Felidae, Canidae, Suidae, Bovidae, Goatidae, Cercopithecidae, Muridae, and Equidae. Preferably, the mammal is at least one of the following: dog, wolf, dhole, and fox, more preferably a dog.

11. The use of an antibody or antigen-binding fragment according to any one of claims 1 to 5, or a nucleic acid molecule according to claim 6, or an expression vector according to claim 7, in the manufacture of a drug for treating mammalian pruritic diseases, Preferably, the pruritic skin disorder is a lesion associated with abnormal expression of IL-31, More preferably, the pruritic skin disease is at least one of atopic dermatitis, contact dermatitis, pruritus nodularis, and eczema. More preferably, the mammal is at least one of the animals belonging to the Felidae, Canidae, Suidae, Bovidae, Goatidae, Cercopithecidae, Muridae, and Equidae. Preferably, the mammal used is at least one of dogs, wolves, dholes, and foxes, more preferably a dog.

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