Antibody capable of binding to interleukin-31 or antigen binding fragment thereof, and preparation method therefor and use thereof

Abstract

Provided are an antibody capable of binding to interleukin-31 or an antigen binding fragment thereof, and a preparation method therefor and a use thereof. The antibody or the antigen binding fragment thereof has high affinity and high activity, and by blocking the binding of IL-31 and a receptor thereof, especially canine IL-31, the antibody or the antigen binding fragment thereof inhibits canine pruritic skin diseases, especially canine atopic dermatitis.

Description

Antibodies capable of binding to interleukin-31 or antigen-binding fragments thereof, and preparation methods and applications thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of Chinese patent application 202310894799.2 filed on July 20, 2023 and Chinese patent application 202310941457.1 filed on July 28, 2023, the contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of antibodies, and in particular to an antibody capable of binding to interleukin-31 or an antigen-binding fragment thereof, and a preparation method and application thereof. Background Art

[0004] Skin diseases in dogs and cats are common in veterinary hospitals. Canine atopic dermatitis (CAD) is a common itchy skin disease in dogs. It can occur at any age, but clinical symptoms typically appear in the summer around one year old. These symptoms include itching, redness, swelling, and cracking. As inflammation persists, the lesions thicken. The causes of canine CAD may be related to genetic disorders, immune system dysfunction, environmental exposure, or impaired skin permeability.

[0005] Studies have found that transgenic mice overexpressing interleukin-31 (IL-31) develop symptoms such as skin inflammation and pruritus. Furthermore, injection of IL-31 can induce acute pruritus in mice. These studies suggest that IL-31 is closely associated with skin inflammation and pruritus. IL-31 is primarily produced by activated helper Th2 cells, and the IL-31 receptor is primarily expressed in macrophages, keratinocytes, and dorsal root ganglia. The IL-31 receptor forms a heterodimer consisting of IL-31RA and OSMR. Binding of IL-31 to its receptor activates downstream JAK kinases and STAT phosphorylation. In mouse models of atopic dermatitis, IL-31 mRNA is highly expressed in lesional skin, and inhibiting the IL-31 signaling pathway can suppress pruritus. Therefore, IL-31 antagonistic antibodies have the potential to inhibit pruritus and alleviate atopic dermatitis.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to overcome the problems existing in the prior art and to provide an antibody or antigen-binding fragment thereof that can bind to interleukin-31, and a preparation method and application thereof.

[0008] definition

[0009] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0010] In the present invention, the term "antibody" means an immunoglobulin molecule and refers to any form of an antibody that exhibits the desired biological activity. This includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), and even antibody fragments. Typically, a full-length antibody structure preferably comprises four polypeptide chains, usually two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region and a heavy chain constant region. Each light chain comprises a light chain variable region and a light chain constant region. In addition to this typical full-length antibody structure, its structure may also include other derivative forms.

[0011] The term "antigen-binding fragment" comprises a portion of an intact antibody molecule that retains at least some of the binding specificity of the parent antibody, typically including at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. 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 or tri-scFv, diabodies or scFab), and single-domain antibodies.

[0012] An "Fv fragment" contains the variable regions from both the heavy and light chains, but lacks the constant regions.

[0013] A "Fab fragment" consists of a light chain and the CH1 domain and variable region of a heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0014] A "Fab' fragment" contains one light chain and a portion of one heavy chain including the VH domain and the CHI domain, and the region between the CHI and CH2 domains, such that an interchain disulfide bond can form between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule.

[0015] A "F(ab')2 fragment" contains two light chains and two heavy chains comprising a portion of the constant region between the CH1 and CH2 domains, with an interchain disulfide bond formed between the two heavy chains. Thus, a F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0016] "Single-chain Fv antibody" (or "scFv antibody") refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally speaking, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315. See also International Patent Application Publication No. WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203. The contents of the above-mentioned documents are incorporated herein by reference.

[0017] "Diabodies," also known as "bispecific antibodies," are small antibody fragments with two antigen-binding sites. These fragments contain a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and form two antigen-binding sites.

[0018] The term "variable region" refers to the domain of an antibody heavy or light chain that is involved in the antibody's antigen binding. The variable regions of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures and can be further subdivided into hypervariable regions (called complementary determining regions (CDRs)) interspersed with more conserved regions (called framework regions (FRs)). The term "complementarity determining regions" (CDRs, such as CDR1, CDR2, and CDR3) refers to the amino acid residues in an antibody variable region whose presence is essential for antigen binding. Each variable region typically has three CDR regions identified as CDR1, CDR2, and CDR3.

[0019] The term "constant region" refers to amino acid sequences on the light and heavy chains of antibodies that are not directly involved in binding the antibody to the antigen, but exhibit various effector functions, such as antibody-dependent cellular toxicity.

[0020] The term "chimeric antibody" has at least a portion of a heavy chain variable region and at least a portion of a light chain variable region derived from one species; and at least a portion of a constant region derived from another species. For example, in one embodiment, a chimeric antibody can include a murine variable region and a canine constant region.

[0021] The term "caninized antibody" comprises CDRs derived from a non-canine antibody and framework regions and constant regions derived from a canine antibody. For example, the anti-IL31 antibodies provided herein may comprise CDRs derived from one or more murine antibodies and canine framework regions and constant regions. The present invention provides exemplary caninized antibodies. Additional anti-IL31 antibodies or variants thereof comprising the heavy chain CDRs and light chain CDRs provided herein can be generated using any canine framework sequence and are also encompassed by the present invention. In one embodiment, framework sequences suitable for use in the present invention include those structurally similar to the framework sequences provided herein. Additional modifications may be made in the framework regions to improve the properties of the antibodies provided herein. Such additional framework modifications may include chemical modifications; point mutations to reduce immunogenicity or remove T cell epitopes; or reverting mutations to residues in the original germline sequence. In some embodiments, such modifications include those corresponding to the mutations exemplified herein, including backmutations to the germline sequence. For example, in one embodiment, one or more amino acids in the canine framework regions of the VH and / or VL of the caninized antibodies provided herein are backmutated to the corresponding amino acids in the parent murine antibody. For example, in the VH and VL regions of the caninized antibodies, several amino acid positions in the framework of the template canine antibody are backmutated to the corresponding amino acid sequences in the mouse IL31 antibody. In one embodiment, in some examples, one or more amino acid backmutations are preferably selected from 2I, 4L, 36Y, 46L, and 68R; another preferred amino acid backmutation is 68G, 73F, and 78L; and another preferred amino acid backmutation is 2I, 12S, 37Q, 46V, 49K, 59P, 60S, 69S, and 77S. Additional or alternative backmutations can be made in the framework regions of the caninized antibodies provided herein to improve the antibody's properties. The present invention also includes constant region mutations to improve the properties of antibodies. In some embodiments, the constant region of the canine IgG B heavy chain is mutated from M to A at position 234, from L to A at position 235, and from G to A at position 237 to reduce the affinity of canine IgG B to canine FcγRI and C1q, thereby avoiding the effector function of canine IgG B.

[0022] In the present invention, the term "Antibody Drug Conjugate" refers to a complex composed of a monoclonal antibody targeting a tumor-specific antigen or a tumor-associated antigen coupled to a varying number of small molecule toxins via a linker. It is a complex formed by linking a cytotoxic drug to a tumor-targeting monoclonal antibody. After entering the body, the Antibody Drug Conjugate molecule can bind to the antigen on the surface of the target cell through the guidance effect of the monoclonal antibody and enter the target cell. The Antibody Drug Conjugate molecule inside the cell can release effector molecules through chemical and / or enzymatic action to achieve the purpose of eliminating the target cell.

[0023] In the present invention, the term "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule and its binding partner. Unless otherwise indicated, "binding affinity" as used herein refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). "Affinity KD," "association rate constant Kon," and "dissociation rate constant Koff" are commonly used to describe the affinity between a molecule (e.g., an antibody) and its binding partner (e.g., an antigen), i.e., how tightly a ligand binds to a specific protein. Binding affinity is affected by non-covalent intermolecular interactions, such as hydrogen bonds, electrostatic interactions, hydrophobic and van der Waals forces between two molecules. In addition, the binding affinity between a ligand and its target molecule may be affected by the presence of other molecules.

[0024] As used herein, the CDR numbering system in antibodies precisely defines the complementarity determining regions (CDRs), framework (FRs), and residues in the light and heavy chains that influence the binding affinity and specificity of antibody-antigen interactions. Commonly used CDR numbering systems include Kabat, Chothia, IMGT, AbM, Contact, and North. Discrepancies may occur when using different numbering systems.

[0025] As used herein, "sequence identity" between two polypeptide or nucleic acid sequences refers to the percentage of identical residues between the sequences as a percentage of the total number of residues. In calculating percent identity, the sequences being compared are aligned to produce the largest match between the sequences, and gaps in the alignment, if any, are resolved by a specific algorithm. Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG program package, including GAP, BLASTP, BLASTN, and FASTA (Altschul et al., 1990, J. Mol. Biol. 215: 403-410). The aforementioned 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.

[0026] In the present invention, the cell line model is based on cells and is used to study patient diseases, drug development, and toxicity testing through changes within cells. In some embodiments of the present invention, HEK293 cells expressing canine IL-31 receptors (canine IL-31RA and canine OSMR) are constructed to evaluate the ability of the IL31 antibody to block the binding of canine IL-31 recombinant protein to its receptor. In other embodiments, DH82 cells (canine macrophages / canine renal malignant histiocytosis cells) are used to study the IL31 antibody, which blocks the binding of IL-31 to the cell surface co-receptors IL-31RA and OSMR, thereby inhibiting the downstream JAK / STAT signaling pathway and reducing the degree of STAT3 phosphorylation.

[0027] In the present invention, animal models refer to the use of different animals as controllable experimental subjects for studying patient diseases, drug development, and toxicity testing through the use of different animal changes. Commonly used animals include mammals, birds, and reptiles. In some embodiments, a canine pruritus model induced by recombinant canine IL31 is constructed to evaluate the inhibitory effect of the IL31 antibody on canine pruritus. In other embodiments, a canine pruritus model induced by fleas is constructed to evaluate the inhibitory effect of the IL31 antibody on canine pruritus.

[0028] In the present invention, the term "recombinant cell" refers to separating organelles and their components from living cells, and then recombining organelles and their components from different sources under certain in vitro conditions to reassemble them into cells or organelles with biological activity.

[0029] In the present invention, the term "pruritus" refers to diseases mediated by IL-31, i.e., general inflammation; in particular, a series of diseases with dermatitis and pruritus symptoms, including atopic dermatitis, contact dermatitis, nodular pruritus, eczema and other allergic diseases.

[0030] The term "pharmaceutical composition" refers to a mixture containing one or more compounds of the present invention or their physiologically / pharmaceutically acceptable salts or prodrugs with other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity. The therapeutic composition should generally be sterile and stable under manufacturing and storage conditions. The composition can be formulated as a solution, microemulsion, dispersant, liposome, or other ordered structure suitable for high antibody concentration. The active compound (i.e., antibody or antigen-binding fragment thereof) can be incorporated in the required amount together with one of the ingredients or combinations of ingredients listed above in a suitable solvent, and then filtered and sterilized as needed to prepare a sterile injectable solution.

[0031] In the present invention, the term "nucleotide molecule" refers to a biological macromolecular compound composed of many nucleotides, which is one of the most basic substances of life. Different nucleic acids have different chemical compositions, nucleotide arrangement orders, etc. According to the different chemical compositions, nucleic acids can be divided into ribonucleic acid (abbreviated as RNA) and deoxyribonucleic acid (abbreviated as DNA). DNA is the main material basis for storing, replicating and transmitting genetic information. RNA plays an important role in the process of protein synthesis - among which transfer RNA, abbreviated as tRNA, plays the role of carrying and transferring activated amino acids; messenger RNA, abbreviated as mRNA, is the template for protein synthesis; ribosomal RNA, abbreviated as rRNA, is the main site for cell protein synthesis.

[0032] In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof that can bind to interleukin-31, which comprises one or more CDR region (antibody complementarity determining region) sequences or mutant sequences thereof selected from SEQ ID NOs: 1-6 and / or SEQ ID NOs: 7-12 and / or SEQ ID NOs: 13-18.

[0033] In some embodiments, the IL31 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region CDR or a mutant sequence thereof selected from SEQ ID NOs: 1-3 and / or SEQ ID NOs: 7-9 and / or SEQ ID NOs: 13-15.

[0034] In some embodiments, the IL31 antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable region CDR or a mutant sequence thereof selected from SEQ ID NOs: 4-6 and / or SEQ ID NOs: 10-12 and / or SEQ ID NOs: 16-18.

[0035] In some embodiments, the IL31 antibody or antigen-binding fragment thereof described herein comprises a heavy chain variable region CDR1, wherein the amino acid sequence of the heavy chain variable region CDR1 is not less than about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, 7, or 13.

[0036] In some embodiments, the IL31 antibody or antigen-binding fragment thereof described herein comprises a heavy chain variable region CDR2, wherein the amino acid sequence of the heavy chain variable region CDR2 is not less than about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NOs: 2, 8, and 14.

[0037] In some embodiments, the IL31 antibody or antigen-binding fragment thereof described herein comprises a heavy chain variable region CDR3, and the amino acid sequence of the heavy chain variable region CDR3 is approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NOs: 3, 9, and 15.

[0038] In some embodiments, the IL31 antibody or antigen-binding fragment thereof described herein comprises a light chain variable region CDR1, and the amino acid sequence of the light chain variable region CDR1 is approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NOs: 4, 10, and 16.

[0039] In some embodiments, the IL31 antibody or antigen-binding fragment thereof described herein comprises a light chain variable region CDR2, and the amino acid sequence of the light chain variable region CDR2 is approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NOs: 5, 11, and 17.

[0040] In some embodiments, the IL31 antibody or antigen-binding fragment thereof described herein comprises a light chain variable region CDR3, and the amino acid sequence of the light chain variable region CDR3 is approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NOs: 6, 12, and 18.

[0041] In some embodiments, the IL31 antibody or antigen-binding fragment thereof of the present invention, the amino acid sequence of the heavy chain variable region CDR1 is at least 80% identical to the amino acid sequence of SEQ ID NO: 1, 7 or 13, the amino acid sequence of the heavy chain variable region CDR2 is at least 80% identical to the amino acid sequence of SEQ ID NO: 2, 8 and 14, and the amino acid sequence of the heavy chain variable region CDR3 is at least 80% identical to the amino acid sequence of SEQ ID NO: 3, 9 and 15; and the amino acid sequence of the light chain variable region CDR1 is at least 80% identical to the amino acid sequence of SEQ ID NO: 4, 10 and 16, the amino acid sequence of the light chain variable region CDR2 is at least 80% identical to the amino acid sequence of SEQ ID NO: 5, 11 and 17, and the amino acid sequence of the light chain variable region CDR3 is at least 80% identical to the amino acid sequence of SEQ ID NO: 6, 12 and 18.

[0042] In the present invention, according to different antibody numbering systems, SEQ ID NO: 1 may also be SEQ ID NO: 19 or 25; SEQ ID NO: 7 may also be SEQ ID NO: 21 or 30, SEQ ID NO: 13 may also be SEQ ID NO: 23 or 35, SEQ ID NO: 2 may also be SEQ ID NO: 20 or 26, SEQ ID NO: 8 may also be SEQ ID NO: 22 or 31, SEQ ID NO: 14 may also be SEQ ID NO: 24 or 36, SEQ ID NO: 3 may also be SEQ ID NO: 27, SEQ ID NO: 9 may also be SEQ ID NO: 32, SEQ ID NO: 15 may also be SEQ ID NO: 37, SEQ ID NO: 4 may also be SEQ ID NO: 28, SEQ ID NO: 10 may also be SEQ ID NO: 33, SEQ ID NO: 16 may also be SEQ ID NO: 38, SEQ ID NO: 5 may also be SEQ ID NO: 29 (amino acid sequence is RA), SEQ ID NO: 11 may also be SEQ ID NO: 34, SEQ ID NO: 17 may also be SEQ ID NO: NO: 39. SEQ ID NOs: 1-18 are CDRs using the Kabat numbering system, SEQ ID NOs: 3-6, 9-12, and 15-24 are CDRs using the Chothia numbering system, and SEQ ID NOs: 6, 12, 18, and 25-39 are CDRs 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.That is, SEQ ID NO: 1 is substituted with SEQ ID NO: 19 or 25; SEQ ID NO: 7 is substituted with SEQ ID NO: 21 or 30, SEQ ID NO: 13 is substituted with SEQ ID NO: 23 or 35, SEQ ID NO: 2 is substituted with SEQ ID NO: 20 or 26, SEQ ID NO: 8 is substituted with SEQ ID NO: 22 or 31, SEQ ID NO: 14 is substituted with SEQ ID NO: 24 or 36, SEQ ID NO: 3 is substituted with SEQ ID NO: 27, SEQ ID NO: 9 is substituted with SEQ ID NO: 32, SEQ ID NO: 15 is substituted with SEQ ID NO: 37, 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 is RA), SEQ ID NO: 11 is substituted with SEQ ID NO: 34 (amino acid sequence is GA), SEQ ID NO: 17 is substituted with SEQ ID NO: 29 (amino acid sequence is RA), NO: 39 (amino acid sequence is YV) is substituted.

[0043] In the present invention, the number of amino acid residues in the heavy chain variable region CDR1 does not exceed 10; and / or, the number of amino acid residues in the heavy chain variable region CDR2 does not exceed 22; and / or, the number of amino acid residues in the heavy chain variable region CDR3 does not exceed 17; and / or, the number of amino acid residues in the light chain variable region CDR1 does not exceed 18; and / or, the number of amino acid residues in the light chain variable region CDR2 does not exceed 10; and / or, the number of amino acid residues in the light chain variable region CDR3 does not exceed 12.

[0044] In the present invention, in order to enable the obtained antibody or antigen-binding fragment thereof to better inhibit IL-31 and have high affinity and high activity, thereby inhibiting canine pruritus, the amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO: 2, the amino acid sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO: 3, the amino acid sequence of the light chain variable region CDR1 is shown in SEQ ID NO: 4, the amino acid sequence of the light chain variable region CDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region CDR3 is shown in SEQ ID NO: 6.

[0045] In another embodiment of the present invention, the amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO: 7, the amino acid sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO: 8, the amino acid sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO: 9, the amino acid sequence of the light chain variable region CDR1 is shown in SEQ ID NO: 10, the amino acid sequence of the light chain variable region CDR2 is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region CDR3 is shown in SEQ ID NO: 12.

[0046] In another embodiment of the present invention, the amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO: 13, the amino acid sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO: 14, the amino acid sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO: 15, the amino acid sequence of the light chain variable region CDR1 is shown in SEQ ID NO: 16, the amino acid sequence of the light chain variable region CDR2 is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region CDR3 is shown in SEQ ID NO: 18.

[0047] In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 40-43, 48-51, 56-59 and at least about 80%, 85%, 90%, 95% identical; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 44-47, 52-55, 60-63 and at least about 80%, 85%, 90%, 95% identical.

[0048] In some embodiments, the antibody provided by the present invention is a chimeric antibody, and further provides a chimeric anti-IL31 antibody comprising a heavy chain variable region and / or a light chain variable region.

[0049] 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.

[0050] Preferably, the amino acid sequence of the heavy chain variable region of the chimeric anti-IL31 antibody is SEQ ID NO: 40 and an amino acid sequence at least about 80%, 85%, 90%, or 95% identical thereto, and the amino acid sequence of the light chain variable region is SEQ ID NO: 44 and an amino acid sequence at least about 80%, 85%, 90%, or 95% identical thereto.

[0051] Preferably, the amino acid sequence of the heavy chain variable region of the chimeric anti-IL31 antibody is SEQ ID NO: 48 and an amino acid sequence with at least about 80%, 85%, 90%, or 95% identity, and the amino acid sequence of the light chain variable region is SEQ ID NO: 52 and an amino acid sequence with at least about 80%, 85%, 90%, or 95% identity.

[0052] Preferably, the amino acid sequence of the heavy chain variable region of the chimeric anti-IL31 antibody is SEQ ID NO: 56 and an amino acid sequence at least about 80%, 85%, 90%, or 95% identical thereto, and the amino acid sequence of the light chain variable region is SEQ ID NO: 60 and an amino acid sequence at least about 80%, 85%, 90%, or 95% identical thereto.

[0053] Preferably, the heavy chain amino acid sequence is selected from SEQ ID NO: 66, 74 or 82; the light chain amino acid sequence is selected from SEQ ID NO: 70, 78 or 86.

[0054] More preferably, the antibody may have a heavy chain as shown in SEQ ID NO: 66 and a light chain as shown in SEQ ID NO: 70, or a heavy chain as shown in SEQ ID NO: 74 and a light chain as shown in SEQ ID NO: 78, or a heavy chain as shown in SEQ ID NO: 82 and a light chain as shown in SEQ ID NO: 86.

[0055] In the present invention, in order to enable the obtained antibodies or antigen-binding fragments thereof to better inhibit IL-31 and have high affinity and high activity, thereby inhibiting canine pruritus, the present invention provides caninized antibodies or antigen-binding fragments thereof.

[0056] In some embodiments, the heavy chain variable region framework FR amino acid sequence is selected from a canine germline heavy chain sequence, preferably having 0-5 back mutation residues, which can be 0, 1, 2, 3, 4 or 5 back mutations; in some embodiments, preferably one or more amino acid back mutations are selected from 49G, 71V, 73K, 78A, and another preferably amino acid back mutation is 54A, 55E, 82D, 87V.

[0057] In some embodiments, the light chain variable region framework FR amino acid sequence is selected from canine germline light chain sequences, preferably having 0-5 reverse mutation residues, and may be 0, 1, 2, 3, 4, or 5 reverse mutation residues. In some embodiments, preferably, one or more amino acid reverse mutations are selected from 2I, 4L, 36Y, 46L, and 68R; another preferably, one or more amino acid reverse mutations are selected from 68G, 73F, and 78L; and another preferably, one or more amino acid reverse mutations are selected from 2I, 12S, 37Q, 46V, 49K, 59P, 60S, 69S, and 77S. "Reverse mutation residues" refer to residues that fully or partially restore the original genotype and phenotype after a second amino acid mutation.

[0058] In the present invention, the amino acid sequence of the heavy chain variable region is one of SEQ ID NOs: 41-43, 49-51 and 57-59, and the amino acid sequence of the light chain variable region is one of SEQ ID NOs: 45-47, 53-55 and 61-63.

[0059] In some embodiments, the present invention provides a caninized anti-IL31 antibody comprising a heavy chain variable region having an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NOs: 41-43, and a light chain variable region having an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NOs: 45-47. Preferably, the amino acid sequence of the heavy chain variable region is one of SEQ ID NOs: 41-43, and the amino acid sequence of the light chain variable region is one of SEQ ID NOs: 45-47. More preferably, the amino acid sequence of the heavy chain variable region is one of SEQ ID NOs: 42 and 43, and the amino acid sequence of the light chain variable region is one of SEQ ID NOs: 45 and 47.

[0060] In some embodiments, the present invention provides a caninized anti-IL31 antibody comprising a heavy chain variable region having an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NOs: 49-51, and a light chain variable region having an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NOs: 53-55. Preferably, the amino acid sequence of the heavy chain variable region is one of SEQ ID NOs: 49-51, and the amino acid sequence of the light chain variable region is one of SEQ ID NOs: 53-55; more preferably, the amino acid sequence of the heavy chain variable region is one of SEQ ID NOs: 49 and 50, and the amino acid sequence of the light chain variable region is one of SEQ ID NOs: 54 and 55.

[0061] In some embodiments, the present invention provides a caninized anti-IL31 antibody comprising a heavy chain variable region having an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NOs: 57-59, and a light chain variable region having an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NOs: 61-63. Preferably, the amino acid sequence of the heavy chain variable region is one of SEQ ID NOs: 57-59, and the amino acid sequence of the light chain variable region is one of SEQ ID NOs: 61-63; more preferably, the amino acid sequence of the heavy chain variable region is one of SEQ ID NOs: 58 and 59, and the amino acid sequence of the light chain variable region is one of SEQ ID NOs: 61 and 62.

[0062] In some embodiments, the present invention provides chimeric and / or caninized antibodies or antigen-binding fragments comprising an IgG A, IgG B, IgG C, IgG D heavy chain constant region or a variant thereof, preferably an IgG B mutant heavy chain constant region amino acid sequence consisting of SEQ ID NO: 64; and a canine κ or λ chain light chain constant region or a variant thereof, preferably a κ light chain constant region amino acid sequence consisting of SEQ ID NO: 65.

[0063] In some embodiments, the present invention provides an anti-IL31 chimeric antibody, wherein the antibody comprises a complete heavy chain, wherein the heavy chain is selected from an amino acid sequence of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acids consisting of SEQ ID NO: 66, 74, or 82; and the light chain amino acid sequence is selected from an amino acid sequence of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acids consisting of SEQ ID NO: 70, 78, or 86.

[0064] The chimeric antibody may be composed of the heavy chain shown in SEQ ID NO: 66 and the light chain shown in SEQ ID NO: 70; or the heavy chain shown in SEQ ID NO: 74 and the light chain shown in SEQ ID NO: 78; or the heavy chain shown in SEQ ID NO: 82 and the light chain shown in SEQ ID NO: 86.

[0065] In some embodiments, the present invention provides an anti-IL31 caninized antibody, wherein the antibody comprises a complete heavy chain having an amino acid sequence of one of SEQ ID NOs: 67-69, 75-77, and 83-85, and an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical thereto; and a complete light chain having an amino acid sequence of one of SEQ ID NOs: 71-73, 79-81, and 87-89, and an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical thereto.

[0066] The variable regions of the caninized antibodies are freely combined with caninized heavy and light chains from the same antibody. For example, heavy chain 6465-H1 (SEQ ID NO: 41) can be combined with light chains 6465-K1, 6465-K2, or 6465-K3, labeled 6465-H1K1, 6465-H1K2, and 6465-H1K3, respectively, and so on. For example, 6465-H1K1-canIgGB comprises a caninized heavy chain H1 variable region, a caninized light chain K1 variable region, and a canine IgGB constant region, and so on. Specific combinations are detailed in Table 1.

[0067] In some embodiments, the antibody may be composed of a heavy chain of SEQ ID NO: 67 and a light chain of SEQ ID NO: 71, or a heavy chain of SEQ ID NO: 68 and a light chain of SEQ ID NO: 71, or a heavy chain of SEQ ID NO: 69 and a light chain of SEQ ID NO: 71, or a heavy chain of SEQ ID NO: 67 and a light chain of SEQ ID NO: 72, or a heavy chain of SEQ ID NO: 68 and a light chain of SEQ ID NO: 72, or a heavy chain of SEQ ID NO: 69 and a light chain of SEQ ID NO: 72, or a heavy chain of SEQ ID NO: 67 and a light chain of SEQ ID NO: 73, or a heavy chain of SEQ ID NO: 68 and a light chain of SEQ ID NO: 73, or a heavy chain of SEQ ID NO: 69 and a light chain of SEQ ID NO: 73, or a heavy chain of SEQ ID NO: 75 and a light chain of SEQ ID NO: 79, or a heavy chain of SEQ ID NO: 76 and a light chain of SEQ ID NO: 77. NO:79, or the heavy chain of SEQ ID NO:77 and the light chain of SEQ ID NO:79, or the heavy chain of SEQ ID NO:75 and the light chain of SEQ ID NO:80, or the heavy chain of SEQ ID NO:76 and the light chain of SEQ ID NO:80, or the heavy chain of SEQ ID NO:77 and the light chain of SEQ ID NO:80, or the heavy chain of SEQ ID NO:75 and the light chain of SEQ ID NO:81, or the heavy chain of SEQ ID NO:76 and the light chain of SEQ ID NO:81, or the heavy chain of SEQ ID NO:77 and the light chain of SEQ ID NO:81, or the heavy chain of SEQ ID NO:83 and the light chain of SEQ ID NO:87, or the heavy chain of SEQ ID NO:84 and the light chain of SEQ ID NO:87, or the heavy chain of SEQ ID NO:85 and the light chain of SEQ ID NO:87, or the heavy chain of SEQ ID NO:83 and the light chain of SEQ ID NO:87. The heavy chain of SEQ ID NO: 84 and the light chain of SEQ ID NO: 88, or the heavy chain of SEQ ID NO: 85 and the light chain of SEQ ID NO: 88, or the heavy chain of SEQ ID NO: 83 and the light chain of SEQ ID NO: 89,or the heavy chain shown in SEQ ID NO: 84 and the light chain shown in SEQ ID NO: 89, or the heavy chain shown in SEQ ID NO: 85 and the light chain shown in SEQ ID NO: 89.

[0068] In the present invention, preferably, the antibody or its antigen-binding fragment includes at least one of a monoclonal antibody, a polyclonal antibody, a multimeric antibody and a CDR-grafted antibody; preferably, the antibody or its antigen-binding fragment includes one of a single-chain antibody, a Fab antibody, an Fv antibody, a single-domain antibody, a (Fab)2 fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein, an Fv fragment and an antigen minimum recognition unit.

[0069] In a second aspect, the present invention provides a nucleotide molecule encoding the above-mentioned IL31 antibody or an antigen-binding fragment thereof.

[0070] The present invention is not limited to a specific nucleotide sequence. As long as the codon encoding rules are met and the above-mentioned protein sequence can be translated, it is within the scope of protection of the present invention. If specific limitations are required, preferably, the nucleotide sequence of the DNA molecule encoding the heavy chain is one of SEQ ID NOs: 90-93, 98-101 and 106-109, and substantially similar sequences thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity therewith; and preferably, the nucleotide sequence of the DNA molecule encoding the light chain is one of SEQ ID NOs: 94-97, 102-105 and 110-113, and substantially similar sequences thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity therewith.

[0071] More preferably, the DNA encoding the antibody may be, the heavy chain is SEQ ID NO: 90, the light chain is SEQ ID NO: 94; the heavy chain is SEQ ID NO: 91, the light chain is SEQ ID NO: 95; the heavy chain is SEQ ID NO: 92, the light chain is SEQ ID NO: 95; the heavy chain is SEQ ID NO: 93, the light chain is SEQ ID NO: 95; the heavy chain is SEQ ID NO: 91, the light chain is SEQ ID NO: 96; the heavy chain is SEQ ID NO: 92, the light chain is SEQ ID NO: 96; the heavy chain is SEQ ID NO: 93, the light chain is SEQ ID NO: 96; the heavy chain is SEQ ID NO: 91, the light chain is SEQ ID NO: 97; the heavy chain is SEQ ID NO: 92, the light chain is SEQ ID NO: 97; the heavy chain is SEQ ID NO: 93, the light chain is SEQ ID NO: 97; the heavy chain is SEQ ID NO: 98, the light chain is SEQ ID NO: 102; the heavy chain is SEQ ID NO: : SEQ ID NO: 99, the light chain is set forth in SEQ ID NO: 103; the heavy chain is set forth in SEQ ID NO: 100, the light chain is set forth in SEQ ID NO: 103; the heavy chain is set forth in SEQ ID NO: 101, the light chain is set forth in SEQ ID NO: 103; the heavy chain is set forth in SEQ ID NO: 99, the light chain is set forth in SEQ ID NO: 104; the heavy chain is set forth in SEQ ID NO: 100, the light chain is set forth in SEQ ID NO: 104; the heavy chain is set forth in SEQ ID NO: 101, the light chain is set forth in SEQ ID NO: 104; the heavy chain is set forth in SEQ ID NO: 99, the light chain is set forth in SEQ ID NO: 105; the heavy chain is set forth in SEQ ID NO: 100, the light chain is set forth in SEQ ID NO: 105; the heavy chain is set forth in SEQ ID NO: 101, the light chain is set forth in SEQ ID NO: 105; the heavy chain is set forth in SEQ ID NO: 106, the light chain is set forth in SEQ ID NO: 110; the heavy chain is set forth in SEQ ID NO: 107, the light chain is set forth in SEQ ID NO: NO: 111; the heavy chain is SEQ ID NO: 108, the light chain is SEQ ID NO: 111; the heavy chain is SEQ ID NO: 109, the light chain is SEQ ID NO: 111; the heavy chain is SEQ ID NO: 107, the light chain is SEQ ID NO: 112; the heavy chain is SEQ ID NO: 108, the light chain is SEQ ID NO: 112; the heavy chain is SEQ ID NO: 109, the light chain is SEQ ID NO: 112; the heavy chain is SEQ ID NO: 107, the light chain is SEQ ID NO: 113;The heavy chain is SEQ ID NO: 108, and the light chain is SEQ ID NO: 113; the heavy chain is SEQ ID NO: 109, and the light chain is SEQ ID NO: 113.

[0072] The third aspect of the present invention provides an expression vector, which expresses the above-mentioned nucleotide molecule.

[0073] In the present invention, the expression vector refers to a vector that adds expression elements (such as a promoter, RBS, terminator, etc.) to the basic cloning vector skeleton to enable the expression of the target gene. In the present invention, the expression elements are added to the nucleotide sequence so that the expression vector can successfully express the DNA molecule of the present invention, such as a plasmid containing the nucleotide sequence of the present invention. Preferably, the expression vector is a eukaryotic expression vector, such as pCDNA3.1 or pcDNA3.4.

[0074] A fourth aspect of the present invention provides a method for producing an antibody or an antigen-binding fragment thereof, the method comprising: transforming a host cell with the above-mentioned expression vector.

[0075] In the present invention, the transformation method is not limited and is a common method in the art of introducing an expression vector into a host cell for expression.

[0076] In the present invention, the host cell is not limited and can successfully express the nucleotide molecules of the present invention. Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell, including Chinese hamster ovary cells (CHO), NS0, NP2 / 0, Hela cells, 293T cells, HEK293 and other cell lines.

[0077] The fifth aspect of the present invention provides a pharmaceutical composition comprising an adjuvant and the above-mentioned antibody or antigen-binding fragment thereof or the above-mentioned nucleotide molecule or expression vector. Preferably, the adjuvant is selected from a pharmaceutically acceptable carrier or excipient.

[0078] In the present invention, the drug carrier and excipient are not limited as long as they are pharmaceutically acceptable, including but not limited to saline, buffer, glucose, water, glycerol, ethanol or a combination thereof.

[0079] A sixth aspect of the present invention provides a method for treating pruritus in mammalian skin, comprising administering the above-mentioned antibody or antigen-binding fragment thereof or nucleic acid molecule or expression vector or pharmaceutical composition to the mammal.

[0080] The dosage of the antibody or antigen-binding fragment thereof is 1-5 mg / kg, which can be 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg or a range formed by any two of the above values ​​and a value within the range.

[0081] The administration method includes but is not limited to intravenous injection, intramuscular injection, subcutaneous injection, sublingual administration, rectal perfusion, inhalation administration, spray administration, etc., and subcutaneous injection is more preferred.

[0082] The pruritic skin diseases include but are not limited to atopic dermatitis, contact dermatitis, nodular pruritus and eczema, preferably atopic dermatitis.

[0083] The mammal is at least one of feline, canine, porcine, bovine, ovine, monkey, murine and equine. Preferably, the mammal is at least one of dog, wolf, jackal and fox, more preferably dog.

[0084] A seventh aspect of the present invention provides a use of the above-mentioned antibody or antigen-binding fragment thereof or nucleic acid molecule or vector in the preparation of a medicament for treating pruritic skin diseases in mammals.

[0085] The present invention provides a use of the above-mentioned antibody or antigen-binding fragment thereof or nucleic acid molecule or vector in treating pruritic skin diseases in mammals.

[0086] The itchy skin disease, the dosage and method of administration of the drug, and mammals have been explained above and will not be repeated here.

[0087] Through the above technical solution, the antibody or antigen-binding fragment thereof that can bind to interleukin-31 provided by the present invention has high affinity and high activity, and inhibits pruritic skin diseases in mammals by blocking the binding of IL-31 and its receptor, especially canine IL-31. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 shows the APC-positive cell rate in the presence of antibodies 6465, 7475, or B2;

[0089] FIG2 shows the inhibitory effects of three chimeric antibodies and Lokivetmab on canine IL-31 pruritus challenge;

[0090] FIG3 shows the inhibitory effects of four caninized antibodies constructed based on the 6465 antibody on canine IL-31 itch challenge;

[0091] FIG4 shows the inhibitory effects of four caninized antibodies constructed based on the 7475 antibody on canine IL-31 itch challenge;

[0092] FIG5 shows the inhibitory effects of four caninized antibodies constructed based on antibody B2 on canine IL-31 itch challenge. DETAILED DESCRIPTION

[0093] Example 1. Production and identification of mouse anti-canine interleukin 31 (IL-31) antibodies

[0094] (1) Antigen preparation

[0095] HEK293 cells were transiently transfected with His-tagged canine IL-31 (interleukin 31) recombinant protein, and the culture supernatant of the transiently transfected cells was collected and affinity chromatographed using a Ni column. The eluate was the purified canine IL-31 recombinant protein.

[0096] (2) Mouse immunization

[0097] Balb / c mice and C57 mice were immunized with canine IL-31 recombinant protein as the antigen. For the first immunization, 100 μg of antigen was emulsified with Freund's complete adjuvant, and 50 μg of antigen was administered to each mouse. The interval between immunizations was two weeks, and the injection method was intraperitoneal and subcutaneous injection at multiple sites. After the fourth immunization, serum was collected and analyzed for antibody titer using an enzyme-linked immunosorbent assay (ELISA). Mice with serum immune titers exceeding 360,000 were selected for antibody library construction.

[0098] (3) Spleen RNA extraction and reverse transcription

[0099] RNA was extracted from mouse spleen tissue using the TRIzol method, and the integrity of RNA was determined by agarose gel electrophoresis. The concentration and OD of RNA were determined using an ultra-micro spectrophotometer. 260 / OD 280 RNA of qualified quality was selected and reverse transcribed according to the instructions of Superscript II reverse transcriptase (Thermo Scientific) to obtain cDNA.

[0100] (4) Antibody library construction and phage display

[0101] Antibody heavy and light chains were amplified from cDNA to construct scFv and Fab libraries, respectively, with a library capacity of 1E8 cfu. The antibody libraries were electroporated into TG1 E. coli and then infected with helper phage M13KO7. After overnight incubation, phage were precipitated using PEG / NaCl to prepare phage-displayed antibody libraries.

[0102] (5) Phage display antibody library screening

[0103] Phage-displayed antibody libraries were screened using immunotubes and magnetic bead screeners for solid-phase screening and solid-phase and liquid-phase cross-screening, respectively. For solid-phase screening, immunotubes were coated with canine IL-31 recombinant protein, while for liquid-phase screening, Dynabeads magnetic beads were coated with biotinylated canine IL-31 recombinant protein. After three rounds of screening, the enriched phage pool and the isolated single clones were identified using Phage ELISA.

[0104] (6) Antibody construction, expression and purification

[0105] Phage ELISA-positive monoclonal phagemids were sequenced. Mouse antibody variable regions with the correct reading frame were selected from the sequencing results for gene synthesis. The mouse antibody variable regions were then fused to the human IgG1 constant region and inserted into the pcDNA3.4 vector to construct a full-length antibody expression plasmid. The full-length antibody was transiently expressed in HEK293 cells, and the culture supernatant was purified by Protein A affinity chromatography.

[0106] (7) Screening of antibodies that block the binding of canine IL-31 to its receptor

[0107] Biotinylated canine IL-31 recombinant protein was incubated with various concentrations of antibody for 5 minutes. The cells were then added to HEK293 cells expressing membrane-expressed canine IL-31 receptors (canine IL-31RA and canine OSMR). The cells were incubated on ice for 30 minutes. After three rinses with PBS, streptavidin-APC fluorescent secondary antibody was added, and the proportion of cells labeled with APC was analyzed by flow cytometry. The APC-positive cell rate reflects the ability of biotinylated canine IL-31 recombinant protein to bind to cells expressing membrane-expressing canine IL-31 receptors. A lower APC-positive cell rate indicates a greater ability of the antibody to block the binding of biotinylated canine IL-31 recombinant protein to its receptor.

[0108] Results: As the concentration of the tested antibodies 6465, 7475, and B2 increased, all of them led to a decrease in APC-positive cells, indicating that the antibodies all had a blocking effect. Specific results are shown in Figure 1. Using Kabat encoding, the amino acid sequences of the heavy chain variable region complementarity determining regions (CDRs) 1, 2, and 3 of antibody 6465 were SEQ ID NOs: 1-3, and the amino acid sequences of the light chain variable region CDRs 1, 2, and 3 were SEQ ID NOs: 4-6, respectively; the amino acid sequences of the heavy chain variable region CDRs 1, 2, and 3 of antibody 7475 were SEQ ID NOs: 7-9, and the amino acid sequences of the light chain variable region CDRs 1, 2, and 3 were SEQ ID NOs: 10-12, respectively; the amino acid sequences of the heavy chain variable region CDRs 1, 2, and 3 of antibody B2 were SEQ ID NOs: 13-15, and the amino acid sequences of the light chain variable region CDRs 1, 2, and 3 were SEQ ID NOs: 16-18, respectively. Using Chothia encoding, the amino acid sequences of the heavy chain variable region CDR1, 2, and 3 of antibody 6465 were SEQ ID NOs: 19, 20, and 3, respectively, and the amino acid sequences of the light chain variable region CDR1, 2, and 3 were SEQ ID NOs: 4-6, respectively; the amino acid sequences of the heavy chain variable region CDR1, 2, and 3 of antibody 7475 were SEQ ID NOs: 21, 22, and 9, respectively, and the amino acid sequences of the light chain variable region CDR1, 2, and 3 were SEQ ID NOs: 10-12, respectively; the amino acid sequences of the heavy chain variable region CDR1, 2, and 3 of antibody B2 were SEQ ID NOs: 23, 24, and 15, respectively, and the amino acid sequences of the light chain variable region CDR1, 2, and 3 were SEQ ID NOs: 16-18, respectively. Using IMGT encoding, the amino acid sequences of the heavy chain variable region CDR1, 2, and 3 of antibody 6465 were SEQ ID NOs: 25-27, and the amino acid sequences of the light chain variable region CDR1, 2, and 3 were SEQ ID NOs: 28, 29, and 6, respectively; the amino acid sequences of the heavy chain variable region CDR1, 2, and 3 of antibody 7475 were SEQ ID NOs: 30-32, and the amino acid sequences of the light chain variable region CDR1, 2, and 3 were SEQ ID NOs: 33, 34, and 12, respectively; the amino acid sequences of the heavy chain variable region CDR1, 2, and 3 of antibody B2 were SEQ ID NOs: 35-37, and the amino acid sequences of the light chain variable region CDR1, 2, and 3 were SEQ ID NOs: 38, 39, and 18, respectively.

[0109] Example 2: Construction of chimeric and caninized murine anti-canine IL-31 antibodies

[0110] (1)Chimeric antibodies

[0111] Canine chimeric antibody clones were constructed for 6465, 7475, and B2. These chimeric antibodies possess mouse heavy chain variable regions and mouse light chain variable regions. Based on the chimeric antibody 6465 (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 chimeric antibody 7475 (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; and based on the chimeric antibody B2 (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. These antibodies also contain constant regions (including CL, CH1, CH2, and CH3) from a canine IgG B antibody. The amino acid sequence of the constant region domain of the canine IgG B heavy chain is shown in SEQ ID NO: 64. The amino acid sequence of the constant region domain of canine IgG B light chain is shown in SEQ ID NO: 65. Specific combinations are shown in Table 1.

[0112] (2) Caninized antibodies

[0113] 1) Three caninized heavy chain variable region variants (SEQ ID NOs: 41-43) and three caninized light chain variable region variants (SEQ ID NOs: 45-47) of 6465 were constructed, containing different modifications or substitutions. The caninization strategy for 6465-H1 involved CDR grafting and backmutations to 49G, 71V, and 73K, 6465-H2 involved CDR grafting and backmutations to 49G, 71V, 73K, and 78A, and 6465-H3 involved only CDR grafting. The caninization strategy for 6465-K1 involved CDR grafting and backmutations to 2I, 4L, 46L, and 68R, 6465-K2 involved CDR grafting and backmutations to 4L, 36Y, 46L, and 68R, and 6465-K3 involved CDR grafting and backmutation to 36Y.

[0114] 2) Three caninized heavy chain variable region variants (SEQ ID NOs: 49-51) and three caninized light chain variable region variants (SEQ ID NOs: 53-55) of 7475 were constructed, containing different modifications or substitutions. The caninization strategy for 7475-H1, 7475-H2, and 7475-H3 all involved CDR grafting alone. The caninization strategy for 7475-K1 and 7475-K3 involved CDR grafting alone, while the caninization strategy for 7475-K2 involved CDR grafting and backmutations 68G, 73F, and 78L.

[0115] 3) Constructing three caninized heavy chain variable region variants (SEQ ID NOs: 57-59) and three caninized light chain variable region variants (SEQ ID NOs: 61-63) of B2, containing different modifications or substitutions. The caninization strategy for B2-H1 involved CDR grafting and backmutations 54A, 82D, and 87V; the caninization strategy for B2-H2 involved CDR grafting and backmutations 55E, 82D, and 87V; and the caninization strategy for B2-H3 involved CDR grafting and backmutations 55E, 82D, and 87V. The caninization strategy for B2-K1 was CDR grafting and back mutations to 2I, 46V, 49K and 69S; the caninization strategy for B2-K2 was CDR grafting and back mutations to 46V, 49K and 69S; and the caninization strategy for B2-K3 was CDR grafting and back mutations to 2I, 12S, 37Q, 46V, 49K, 59P, 60S, 69S and 77S.

[0116] The variable region of a caninized antibody is composed of a caninized heavy chain and a caninized light chain from the same antibody. For example, heavy chain 6465-H1 (SEQ ID NO: 41) can be combined with light chains 6465-K1, 6465-K2, or 6465-K3, labeled 6465-H1K1, 6465-H1K2, and 6465-H1K3, respectively, and so on. The constant region of a caninized antibody is composed of the constant domain of a canine IgG B heavy chain and the constant domain of a 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. 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 comprises a caninized heavy chain H1, a caninized light chain K1, and a canine IgG B constant domain, and so on. Specific combinations are detailed in Table 1.

[0117] The constant region of the canine IgG B heavy chain contains three amino acid mutations, namely M234A, L235A and G237A, the purpose of which is to reduce the affinity of canine IgG B to canine FcγRI and C1q, thereby avoiding the effector function of canine IgG B.

[0118] Table 1 Sequence list of chimeric antibodies and caninized antibodies

[0119] Example 3. In vitro evaluation of chimeric antibodies and caninized antibodies

[0120] (1) In vitro activity evaluation of new antibodies constructed based on 6465, 7475, and B2 antibodies

[0121] Inhibition assay of canine IL-31-induced STAT (signal transducer and activator of transcription) phosphorylation by the new antibody:

[0122] IL-31 activates the downstream JAK / STAT signaling pathway by binding to its cell surface co-receptors IL-31RA and OSMR, leading to an increase in STAT3 phosphorylation. When IL-31 neutralizing antibodies block the binding of IL-31 to its receptor, STAT3 phosphorylation in its downstream signaling pathway is also inhibited. Using anti-canine IL-31 chimeric antibodies and caninized antibodies with different concentration gradients, the IC50 value of antibody inhibition of STAT phosphorylation was evaluated in a DH82 cell assay. The specific process is to plate 50,000 DH82 cells per well, add 10 ng / mL IFN-γ, stimulate at 37°C for 24 hours, and then serum starve for 2 hours. After incubating 2.86 μg / mL canine IL-31 recombinant protein with different concentrations of canine IL-31 antibodies for 1 hour, the above-mentioned DH82 cells were added and stimulated at 37°C for 5 minutes. Then refer to The p-Y705 kit was used to detect STAT3 phosphorylation in DH82 cells, and IC50 values ​​were calculated based on the antibody concentration. As shown in Table 2, all three chimeric antibodies and 27 caninized antibodies were able to inhibit pSTAT activity, with IC50 values ​​ranging from 6.7 to 9.4 μg / mL.

[0123] Table 2 pSTAT activity induced by caninized antibodies

[0124] (2) Affinity determination of new antibodies constructed based on 6465, 7475 and B2 antibodies

[0125] The binding affinity of antibodies is determined using a Gator instrument based on biofilm interferometry (BLI) technology. When the solidified substance on the biosensor interacts with the analyte in the solution, the thickness of the biofilm layer on the sensor increases, and the interference spectrum curve shifts toward increasing wavelengths. The phase shift is detected and analyzed by the workstation, and the change in the number of molecules on the sensor surface and related concentration and kinetic data, such as the dissociation rate K, can be quantitatively obtained. off , binding rate K on and affinity KD, where KD = K off / K on .

[0126] The binding affinity of 3 chimeric antibodies, 27 caninized antibodies and the positive control Lokivetmab were tested. The specific process is to first label the antibody with biotin, and then use the biotin-labeled antibody as a solid to bind to the biosensor using a streptavidin probe (Gator TMStreptavidin probes) were used to immobilize biotin-labeled antibodies. Different concentration gradients of canine IL-31 recombinant protein were then used as analytes to measure the binding kinetics of the biotin-labeled antibodies and calculate the affinity. The results are shown in Table 3. The binding affinity KD of the three chimeric antibodies and 27 caninized antibodies reached 10 -9 M, which is about 10 times the affinity of the positive control Lokivetmab.

[0127] Table 3 Affinity data of IL31 antibody

[0128] Note: E is scientific notation, which is ×10. The value after E is the power of 10.

[0129] Example 4. In vivo efficacy evaluation of chimeric antibodies and caninized antibodies based on 6465, 7475, and B2

[0130] The in vivo efficacy of anti-canine IL-31 chimeric antibodies and caninized antibodies was evaluated using the canine IL-31 pruritus challenge model.

[0131] First, beagle dogs (weighing about 10 kg) were modeled and suitable test dogs were screened. 2.5 μg / kg canine IL-31 recombinant protein was injected intravenously, and the beagle dogs' itch behavior (such as chewing, scratching, biting, licking, rolling and rubbing, etc.) was monitored by video half an hour before and four hours after administration. If itch behavior was observed within 1 minute, 1 point was given, and if not, 0 point was given. Scoring was performed every 1 minute. The cumulative itch score half an hour before administration was used as the baseline value, and the cumulative itch score four hours after administration was used as the measured value. The measured value was deducted from its baseline value to obtain the final itch score. Due to individual differences, the itch score of each beagle dog after modeling fluctuated greatly. For the uniformity of the model, beagles with itch scores between 100-150 points were screened and grouped.

[0132] After successfully establishing the model, 25 beagle dogs were randomly divided into five groups: three treatment groups (6465, 7475, and B2 chimeric antibodies), a PBS control group, and a positive control group (Lokivetmab), with five animals in each group. After seven days of acclimation, the beagle dogs in the treatment groups received a subcutaneous injection of 2 mg / kg of 6465, 7475, and B2 chimeric antibodies, the positive control group received a subcutaneous injection of 2 mg / kg of Lokivetmab, and the negative control group received an equal volume of PBS. The following day, all beagle dogs received an intravenous injection of 2.5 μg / kg of canine IL-31 recombinant protein. The pruritus score of each beagle group was recorded according to the aforementioned scoring criteria to evaluate whether the administered antibodies inhibited IL-31-mediated pruritus. The results are shown in Figure 2. Administration of PBS to beagles could not reduce the pruritus symptoms caused by IL31, and the pruritus score remained between 100 and 150. However, administration of three chimeric antibodies and Lokivetmab significantly reduced the pruritus caused by IL31 in beagles, showing a significant inhibitory effect.

[0133] Using the same experimental method, the pharmacological efficacy of caninized antibodies 6465, 7475, and B2 (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, and B2-H3K2-canIgGB) was evaluated. The results are shown in Figures 3, 4, and 5, respectively. The results showed that, similar to chimeric antibodies, the 12 caninized antibodies had significant inhibitory effects, with the itch scores decreasing by more than 75% during the four-hour observation period after modeling. The performance of the other caninized antibodies shown in Table 1 was similar to that of these 12 caninized antibodies.

[0134] Furthermore, preliminary results from evaluating the efficacy of a caninized antibody against canine IL-31 in a flea-induced allergic dermatitis model showed that the caninized antibody also significantly inhibited flea-induced pruritus. Efficacy results in dogs with atopic dermatitis also showed that the caninized antibody reduced pruritus in these dogs.

[0135] During the above-mentioned trial period and the subsequent one month, no adverse reactions were found in the experimental dogs.

[0136] The above results show that the antibody constructed by the present invention has high affinity and high activity, and can inhibit canine pruritic skin diseases caused by canine IL-31 and fleas by blocking the binding of canine IL-31 and its receptor, and can reduce the itching symptoms of dogs with atopic dermatitis.

[0137] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

[0138] In the present invention, the sequences of SEQ ID NOs: 1-39 are as follows. SEQ ID NOs: 29, 34, and 39 are too short to be recognized by WIPO Sequence, and therefore are intentionally skipped in the software. However, this does not affect the search, and the original sequences of SEQ ID NOs: 1-39 are hereby deemed as the original disclosure.

[0139] SEQ ID NO: 1 is: NYYLY

[0140] SEQ ID NO: 2 is: ELNPRNNGGTNLNAKFKT

[0141] SEQ ID NO: 3 is: GGTTVVVRDVMDF

[0142] SEQ ID NO: 4 is: RASESVDSYGNSFMH

[0143] SEQ ID NO: 5 is: RASNLES

[0144] SEQ ID NO: 6 is: QQSNEDPYT

[0145] SEQ ID NO: 7 is: SYAMS

[0146] SEQ ID NO: 8 is: YISNGGDYIFYADTVKG

[0147] SEQ ID NO: 9 is: RGHYNTSSYWYFDV

[0148] SEQ ID NO: 10 is: KASENVGTYVS

[0149] SEQ ID NO: 11 is: GASNRYT

[0150] SEQ ID NO: 12 is: GQSYSYPPT

[0151] SEQ ID NO: 13 is: DAWMD

[0152] SEQ ID NO: 14 is: EIRSRAESHATYYAESVKG

[0153] SEQ ID NO: 15 is: ASTMITTGWFAY

[0154] SEQ ID NO: 16 is: RASQNISVYLH

[0155] SEQ ID NO: 17 is: YVSQSIS

[0156] SEQ ID NO: 18 is: QNGHSFPYT

[0157] SEQ ID NO: 19 is: GYSFTNY

[0158] SEQ ID NO: 20 is: NPRNGG

[0159] SEQ ID NO: 21 is: GFTFSSY

[0160] SEQ ID NO: 22 is: SNGGDY

[0161] SEQ ID NO: 23 is: GFTFSDA

[0162] SEQ ID NO: 24 is: RSRAESHA

[0163] SEQ ID NO: 25 is: GYSFTNYY

[0164] SEQ ID NO: 26 is: LNPRNGGT

[0165] SEQ ID NO: 27 is: TRGGTTVVVRDVMDF

[0166] SEQ ID NO: 28 is: ESVDSYGNSF

[0167] SEQ ID NO: 29 is: RA

[0168] SEQ ID NO: 30 is: GFTFSSYA

[0169] SEQ ID NO: 31 is: ISNGGDYI

[0170] SEQ ID NO: 32 is: TRRGHYNTSSYWYFDV

[0171] SEQ ID NO: 33 is: ENVGTY

[0172] SEQ ID NO: 34 is: GA

[0173] SEQ ID NO: 35 is: GFTFSDAW

[0174] SEQ ID NO: 36 is: IRSRAESHAT

[0175] SEQ ID NO: 37 is: TPASTMITTGWFAY

[0176] SEQ ID NO: 38 is: QNISVY

[0177] SEQ ID NO: 39 is: YV

Claims

1. An antibody or antigen-binding fragment thereof that can bind to interleukin-31, characterized in that: The amino acid sequence of the heavy chain variable region CDR1 of the antibody or antigen-binding fragment thereof is not less than 80% identical to the amino acid sequence shown in SEQ ID NO: 1, 7 or 13, the amino acid sequence of the heavy chain variable region CDR2 is not less than 80% identical to the amino acid sequence shown in SEQ ID NO: 2, 8 and 14, the amino acid sequence of the heavy chain variable region CDR3 is not less than 80% identical to the amino acid sequence shown in SEQ ID NO: 3, 9 and 15, the amino acid sequence of the light chain variable region CDR1 is not less than 80% identical to the amino acid sequence shown in SEQ ID NO: 4, 10 and 16, the amino acid sequence of the light chain variable region CDR2 is not less than 80% identical to the amino acid sequence shown in SEQ ID NO: 5, 11 and 17, and the amino acid sequence of the light chain variable region CDR3 is not less than 80% identical to the amino acid sequence shown in SEQ ID NO: 6, 12 and 18.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The number of amino acid residues in the heavy chain variable region CDR1 does not exceed 10; and / or, the number of amino acid residues in the heavy chain variable region CDR2 does not exceed 22; and / or, the number of amino acid residues in the heavy chain variable region CDR3 does not exceed 17; and / or, the number of amino acid residues in the light chain variable region CDR1 is no more than 18; and / or, the number of amino acid residues in the light chain variable region CDR2 is no more than 10; And / or, the number of amino acid residues in the light chain variable region CDR3 does not exceed 12.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO: 2, the amino acid sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO: 3, the amino acid sequence of the light chain variable region CDR1 is shown in SEQ ID NO: 4, the amino acid sequence of the light chain variable region CDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region CDR3 is shown in SEQ ID NO: 6; or, the amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO: 7, the amino acid sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO: 8, the amino acid sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO: 9, the amino acid sequence of the light chain variable region CDR1 is shown in SEQ ID NO: 10, the amino acid sequence of the light chain variable region CDR2 is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region CDR3 is shown in SEQ ID NO: 12; Or, the amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO: 13, and the heavy chain variable region CDR2 The amino acid sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO: 15, the amino acid sequence of the light chain variable region CDR1 is shown in SEQ ID NO: 16, the amino acid sequence of the light chain variable region CDR2 is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region CDR3 is shown in SEQ ID NO: 18; or, SEQ ID NO: 1 is substituted with SEQ ID NO: 19 or 25; SEQ ID NO: 7 is substituted with SEQ ID NO: 21 or 30, SEQ ID NO: 13 is substituted with SEQ ID NO: 23 or 35, SEQ ID NO: 2 is substituted with SEQ ID NO: 20 or 26, SEQ ID NO: 8 is substituted with SEQ ID NO: 22 or 31, SEQ ID NO: 14 is substituted with SEQ ID NO: 24 or 36, SEQ ID NO: 3 is substituted with SEQ ID NO: 27, SEQ ID NO: 9 is substituted with SEQ ID NO: 32, SEQ ID NO: 15 is substituted with SEQ ID NO: 37, 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 is RA), SEQ ID NO: 11 is substituted with SEQ ID NO: 34 (amino acid sequence is GA), SEQ ID NO: 17 is substituted with SEQ ID NO: NO: 39 (amino acid sequence is YV) is substituted.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein The antibody is a chimeric antibody; Preferably, the antibody heavy chain variable region is selected from SEQ ID NOs: 40, 48, 56, and the antibody light chain variable region is selected from SEQ ID NOs: 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; or, 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; or, 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; the light chain amino acid sequence is selected from SEQ ID NO: 70, 78 or 86; More preferably, the antibody may have a heavy chain as shown in SEQ ID NO: 66 and a light chain as shown in SEQ ID NO: 70, or a heavy chain as shown in SEQ ID NO: 74 and a light chain as shown in SEQ ID NO: 78, or a heavy chain as shown in SEQ ID NO: 82 and a light chain as shown in SEQ ID NO:

86.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein The antibody is a caninized antibody; Preferably, the heavy chain variable region framework FR amino acid sequence is selected from canine germline heavy chain sequences, preferably having 0-5 back mutation residues; Preferably, the light chain variable region framework FR amino acid sequence is selected from canine germline light chain sequences, preferably having 0-5 back mutation residues; Preferably, the amino acid sequence of the heavy chain variable region is one of SEQ ID NOs: 41-43, 49-51 and 57-59, and the amino acid sequence of the light chain variable region is one of SEQ ID NOs: 45-47, 53-55 and 61-63; Preferably, the amino acid sequence of the heavy chain variable region is one of SEQ ID NOs: 41-43, and the amino acid sequence of the light chain variable region is one of SEQ ID NOs: 45-47. More preferably, the amino acid sequence of the heavy chain variable region is one of SEQ ID NOs: 42 and 43, and the amino acid sequence of the light chain variable region is one of SEQ ID NOs: 45 and 47. Preferably, the amino acid sequence of the heavy chain variable region is one of SEQ ID NOs: 49-51, and the amino acid sequence of the light chain variable region is one of SEQ ID NOs: 53-55; more preferably, the amino acid sequence of the heavy chain variable region is one of SEQ ID NOs: 49 and 50, and the amino acid sequence of the light chain variable region is one of SEQ ID NOs: 54 and 55; Preferably, the amino acid sequence of the heavy chain variable region is one of SEQ ID NOs: 57-59, and the amino acid sequence of the light chain variable region is one of SEQ ID NOs: 61-63; more preferably, the amino acid sequence of the heavy chain variable region is one of SEQ ID NOs: 58 and 59, and the amino acid sequence of the light chain variable region is one of SEQ ID NOs: 61 and 62; Preferably, the amino acid sequence of the heavy chain is one of SEQ ID NOs: 67-69, 75-77 and 83-85; Preferably, the amino acid sequence of the light chain is one of SEQ ID NOs: 71-73, 79-81 and 87-89; More preferably, the antibody may be composed of the heavy chain of SEQ ID NO: 67 and the light chain of SEQ ID NO: 71, or the heavy chain of SEQ ID NO: 68 and the light chain of SEQ ID NO: 71, or the heavy chain of SEQ ID NO: 69 and the light chain of SEQ ID NO: 71, or the heavy chain of SEQ ID NO: 67 and the light chain of SEQ ID NO: 72, or the heavy chain of SEQ ID NO: 68 and the light chain of SEQ ID NO: 72, or the heavy chain of SEQ ID NO: 69 and the light chain of SEQ ID NO: 72, or the heavy chain of SEQ ID NO: 67 and the light chain of SEQ ID NO: 73, or the heavy chain of SEQ ID NO: 68 and the light chain of SEQ ID NO: 73, or the heavy chain of SEQ ID NO: 69 and the light chain of SEQ ID NO: 73, or the heavy chain of SEQ ID NO: 75 and the light chain of SEQ ID NO: 79, or the heavy chain of SEQ ID NO: 76 and the light chain of SEQ ID NO:

77. The heavy chain of SEQ ID NO:77 and the light chain of SEQ ID NO:79, or the heavy chain of SEQ ID NO:75 and the light chain of SEQ ID NO:80, or the ...

80. The heavy chain of SEQ ID NO:76 and the light chain of SEQ ID NO:80, or the heavy chain of SEQ ID NO:77 and the light chain of SEQ ID NO:80, or the heavy chain of SEQ ID NO:75 and the light chain of SEQ ID NO:81, or the heavy chain of SEQ ID NO:76 and the light chain of SEQ ID NO:81, or the heavy chain of SEQ ID NO:77 and the light chain of SEQ ID NO:81, or the heavy chain of SEQ ID NO:83 and the light chain of SEQ ID NO:87, or the heavy chain of SEQ ID NO:84 and the light chain of SEQ ID NO:87, or the heavy chain of SEQ ID NO:85 and the light chain of SEQ ID NO:87, or the heavy chain of SEQ ID NO:83 and the light chain of SEQ ID NO:88, or the heavy chain of SEQ ID NO:84 and the light chain of SEQ ID NO:88, or the heavy chain of SEQ ID NO:85 and the light chain of SEQ ID NO:88, or the heavy chain of SEQ ID NO:86 and the light chain of SEQ ID NO:

87. The heavy chain shown in SEQ ID NO:83 and the light chain shown in SEQ ID NO:89, or the heavy chain shown in SEQ ID NO:84 and the light chain shown in SEQ ID NO:89, or the heavy chain shown in SEQ ID NO:85 and the light chain shown in SEQ ID NO:89; Preferably, the antibody or antigen-binding fragment thereof is at least one of a monoclonal antibody, a polyclonal antibody, a multimeric antibody and a CDR-grafted antibody; Preferably, the antibody or antigen-binding fragment thereof is one of a single-chain antibody, a Fab antibody, a Fv antibody, a single-domain antibody, a (Fab)2 fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein, a Fv fragment and an antigen minimum recognition unit.

6. A nucleotide molecule, characterized in that The nucleotide molecule encodes the antibody or antigen-binding fragment thereof 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 NOs: 90-93, 98-101 and 106-109; Preferably, the nucleotide sequence of the DNA molecule encoding the light chain is one of SEQ ID NOs: 94-97, 102-105 and 110-113; More preferably, the DNA encoding the antibody is: the heavy chain is SEQ ID NO: 90, and the light chain is SEQ ID NO: 94; the heavy chain is SEQ ID NO: 91, and the light chain is SEQ ID NO: 95; the heavy chain is SEQ ID NO: 92, and the light chain is SEQ ID NO: 95; the heavy chain is SEQ ID NO: 93, and the light chain is SEQ ID NO: 95; the heavy chain is SEQ ID NO: 91, and the light chain is SEQ ID NO: 96; the heavy chain is SEQ ID NO: 92, and the light chain is SEQ ID NO: 96; the heavy chain is SEQ ID NO: 93, and the light chain is SEQ ID NO: 96; the heavy chain is SEQ ID NO: 91, and the light chain is SEQ ID NO: 97; the heavy chain is SEQ ID NO: 92, and the light chain is SEQ ID NO: 97; the heavy chain is SEQ ID NO: 93, and the light chain is SEQ ID NO: 97; the heavy chain is SEQ ID NO: 98, and the light chain is SEQ ID NO: 102; the heavy chain is SEQ ID NO: ID NO: 99, the light chain is shown in SEQ ID NO: 103; the heavy chain is SEQ ID NO: 100, the light chain is shown in SEQ ID NO: 103; the heavy chain is SEQ ID NO: 101, the light chain is shown in SEQ ID NO: 103 : the heavy chain is SEQ ID NO: 99, and the light chain is SEQ ID NO: 104; the heavy chain is SEQ ID NO: 100, and the light chain is SEQ ID NO: 104; the heavy chain is SEQ ID NO: 101, and the light chain is SEQ ID NO: 104; the heavy chain is SEQ ID NO: 99, and the light chain is SEQ ID NO: 105; the heavy chain is SEQ ID NO: 100, and the light chain is SEQ ID NO: 105; the heavy chain is SEQ ID NO: 101, and the light chain is SEQ ID NO: 105; the heavy chain is SEQ ID NO: 106, and the light chain is SEQ ID NO: 110; the heavy chain is SEQ ID NO: 107, and the light chain is SEQ ID NO: 111; the heavy chain is SEQ ID NO: 108, and the light chain is SEQ ID NO: 111; the heavy chain is SEQ ID NO: 109, and the light chain is SEQ ID NO: 111; the heavy chain is SEQ ID NO: 107, and the light chain is SEQ ID NO:

112. ID NO: 112; the heavy chain is SEQ ID NO: 108, and the light chain is SEQ ID NO: 112; the heavy chain is SEQ ID NO: 109, and the light chain is SEQ ID NO: 112; the heavy chain is SEQ ID NO: 107, and the light chain is SEQ ID NO: 113; the heavy chain is SEQ ID NO: 108, and the light chain is SEQ ID NO: 113; the heavy chain is SEQ ID NO: 109, and the light chain is SEQ ID NO:

113.

7. An expression vector, characterized in that: The expression vector expresses the nucleotide molecule according to claim 6.

8. A method for producing an antibody or an antigen-binding fragment thereof, characterized in that: The method comprises: transforming a host cell with the expression vector of claim 7; Preferably, the host cell is a mammalian cell, more preferably a CHOK1 cell or a HEK293 cell.

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

10. A method for treating pruritus of mammalian skin, characterized in that: The method comprises: administering to a mammal the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the expression vector according to claim 7, or the pharmaceutical composition according to claim 9; Preferably, the antibody or antigen-binding fragment thereof is administered in an amount of 1-5 mg / kg; More preferably, the administration method is at least one of intravenous injection, intramuscular injection, subcutaneous injection, sublingual administration, rectal perfusion, inhalation administration, and spray administration, and more preferably subcutaneous injection; Further preferably, the pruritic skin disease is a disease associated with abnormal expression of IL-31; Further preferably, the pruritic skin disease is at least one of atopic dermatitis, contact dermatitis, nodular pruritus and eczema; More preferably, the mammal is at least one of feline, canine, porcine, bovine, ovine, monkey, murine and equine; Preferably, the mammal is at least one of a dog, a wolf, a jackal and a fox, more preferably a dog.

11. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, or the expression vector according to claim 7 in the preparation of a medicament for treating pruritus of the skin of a mammal; Preferably, the pruritic skin disease is a lesion associated with abnormal expression of IL-31; Further preferably, the pruritic skin disease is at least one of atopic dermatitis, contact dermatitis, nodular pruritus, and eczema; More preferably, the mammal is at least one of feline, canine, porcine, bovine, ovine, monkey, murine and equine; Preferably, the mammal is at least one of a dog, a wolf, a jackal and a fox, more preferably a dog.

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