Canine interleukin-31 antibodies and uses thereof

Abstract

The present disclosure relates to the field of antibodies, in particular to antibodies binding to animal IL-31 and uses thereof. The present disclosure provides an antibody or antigen-binding fragment thereof specifically binding to animal IL-31, encoding nucleic acid, expression vector, cell, composition comprising the antibody or antigen-binding fragment thereof, and methods and uses for preparing the antibody or antigen-binding fragment thereof. The antibody specifically binding to animal IL-31 of the present disclosure is capable of effectively relieving IL-31-mediated conditions such as pruritus, allergy or asthma.

Description

Technical Field

[0001] This invention relates to the field of antibodies, and more particularly to IL-31 antibodies and their applications. Background Technology

[0002] Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by infiltration of the skin by T cells, eosinophils, and mast cells. Many potential factors are involved in atopic dermatitis, including food components and environmental allergens (such as fleas and dust mites). The prevalence of atopic dermatitis is estimated to be 10% of the total canine population. Itching is a fundamental characteristic and primary symptom of canine atopic dermatitis, severely impacting the quality of life for both the dog and its owner. Therefore, suppressing or reducing itching has become the most important treatment goal for canine atopic dermatitis.

[0003] Cytokines are the most important mediators of pruritus stimulation and inflammation. Interleukin-31 (IL-31) has been identified as a key cytokine inducing pruritus in dogs. Intravenous injection of IL-31 induces pruritus in dogs within 4 hours. IL-31 is a four-helix bundle cytokine belonging to the IL-6 cytokine family, preferentially produced by Th2 cells, but also produced in mast cells and macrophages. IL-31 signals via a heterodimer receptor composed of the oncogene M receptor (OSMR) and the IL-31 receptor α (IL-31RA) subunit. Co-receptor expression has been shown in macrophages, keratinocytes, and dorsal root ganglia. The pro-pruritus and pro-inflammatory cytokine IL-31 binds to its receptor, activating the intracellular JAK-STAT and PI3K-AKT signaling pathways, producing more cellularly active substances that mediate pruritus and inflammation, leading to a vicious cycle of "pruritus-scratching-inflammation."

[0004] Based on existing research on the relationship between IL-31 signaling and atopic dermatitis (AD) pruritus, IL-31 signaling blockade is considered an effective target for treating atopic dermatitis. Therefore, there is a need to provide IL-31 antibodies or their functional fragments to block, inhibit, reduce, limit, or neutralize IL-31 activity and inhibit its cellular transmission, thereby treating IL-31-mediated conditions. Summary of the Invention

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

[0006] According to a first aspect of this disclosure, an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 is provided, said antibody or antigen-binding fragment comprising:

[0007] a1) The amino acid sequences CDR-H1, CDR-H2, and CDR-H3 in the heavy chain variable region as shown in SEQ ID NO:1; and the amino acid sequences CDR-L1, CDR-L2, and CDR-L3 in the light chain variable region as shown in SEQ ID NO:3; or

[0008] a2) The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 in the heavy chain variable region as shown in SEQ ID NO:2; and the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 in the light chain variable region as shown in SEQ ID NO:3; or

[0009] a3) Compared with any of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 described in a1) to a2), at least one CDR contains the substitution, deletion or addition of one or more amino acids;

[0010] The CDR is defined by IMGT, Kabat, Chothia, or the Contact numbering system.

[0011] In some implementations, the animal may be selected from canines, felines, or equines.

[0012] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0013] b1) A heavy chain variable region comprising amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; and a light chain variable region comprising amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively; wherein the CDRs are defined by the IMGT numbering system, or

[0014] The heavy chain variable region comprises amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively; and the light chain variable region comprises amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:20, respectively; wherein the CDRs are defined by the Kabat numbering system, or

[0015] The heavy chain variable region comprises amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:9, respectively; and the light chain variable region comprises amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:20, respectively; wherein the CDRs are defined by the Chothia numbering system, or

[0016] The heavy chain variable region comprises amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:6, respectively; and the light chain variable region comprises amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25, respectively; wherein the CDRs are defined by the Contact numbering system, or

[0017] b2) A heavy chain variable region comprising amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO:4, SEQ ID NO:14, and SEQ ID NO:6, respectively; and a light chain variable region comprising amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively; wherein the CDRs are defined by the IMGT numbering system, or

[0018] The heavy chain variable region comprises amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO:7, SEQ ID NO:15, and SEQ ID NO:9, respectively; and the light chain variable region comprises amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:20, respectively; wherein the CDRs are defined by the Kabat numbering system, or

[0019] The heavy chain variable region comprises amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO:10, SEQ ID NO:16, and SEQ ID NO:9, respectively; and the light chain variable region comprises amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:20, respectively; wherein the CDRs are defined by the Chothia numbering system, or

[0020] The heavy chain variable region comprises amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO:12, SEQ ID NO:17, and SEQ ID NO:6, respectively; and the light chain variable region comprises amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25, respectively; wherein the CDRs are defined by the Contact numbering system, or

[0021] b3) Heavy chain variable region; and, light chain variable region, wherein, compared with any one of the heavy chain variable regions and / or light chain variable regions described in b1)-b2), at least one CDR contains a substitution, deletion or addition of one or more amino acids.

[0022] In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region, which may contain an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2, or an amino acid sequence having at least 75% sequence identity with it.

[0023] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region, which may contain an amino acid sequence as shown in SEQ ID NO:3, or an amino acid sequence having at least 75% sequence identity with it.

[0024] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0025] c1) a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:1, or an amino acid sequence having at least 75% sequence identity with it; and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:3, or an amino acid sequence having at least 75% sequence identity with it.

[0026] c2) a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:2, or an amino acid sequence having at least 75% sequence identity with it; and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:3, or an amino acid sequence having at least 75% sequence identity with it.

[0027] In some embodiments, the antibody may be a monoclonal antibody.

[0028] In some embodiments, the antibody may be a canine, canine-derived, cat, feline-derived, horse, equine-derived, or chimeric antibody.

[0029] In some embodiments, the antibody may be of type IgA, IgD, IgE, IgG, or IgM. In some specific embodiments, the antibody may be of type IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

[0030] In some embodiments, the antibody may include a heavy chain constant region and / or a light chain constant region of an animal-derived antibody. In some embodiments, the antibody may include a heavy chain constant region and / or a light chain constant region of a canine-derived antibody.

[0031] In some embodiments, the heavy chain constant region of the canine-derived antibody may include an amino acid sequence as shown in SEQ ID NO:60, or an amino acid sequence having at least 75% sequence identity with it.

[0032] In some embodiments, the light chain constant region of the canine-derived antibody may include an amino acid sequence as shown in SEQ ID NO:61, or an amino acid sequence having at least 75% sequence identity with it.

[0033] In some embodiments, the heavy chain variable region of the antibody may include a framework region of the heavy chain variable region and / or a framework region of the light chain variable region of an antibody derived from a dog.

[0034] In some embodiments, the antigen-binding fragment may be one known in the art. In specific embodiments, the antigen-binding fragment may include scFv, Fab, Fab', (Fab')2, Fv fragment, Fd, dsFv.

[0035] In some embodiments, the antibody may be a bispecific antibody or a multispecific antibody.

[0036] According to a second aspect of this disclosure, an antibody or antigen-binding fragment thereof specifically binds to animal IL-31 is provided, the antibody or antigen-binding fragment comprising: a heavy chain variable region comprising amino acid sequences CDR-H1 as shown in SEQ ID NO:4, CDR-H2 as shown in INLGXGET, and CDR-H3 as shown in SEQ ID NO:6; and a light chain variable region comprising amino acid sequences CDR-L1 as shown in SEQ ID NO:18, CDR-L2 as shown in SEQ ID NO:19, and CDR-L3 as shown in SEQ ID NO:20, wherein amino acid X is selected from acidic amino acids or polar uncharged amino acids.

[0037] In some embodiments, the amino acid X may be selected from G, S, T, C, Y, N, Q, D, or E. In a preferred embodiment, the amino acid X may be selected from D or N.

[0038] In a preferred embodiment, the antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising the amino acid sequences CDR-H1 as shown in SEQ ID NO:4, CDR-H2 as shown in SEQ ID NO:5, and CDR-H3 as shown in SEQ ID NO:6; and a light chain variable region comprising the amino acid sequences CDR-L1 as shown in SEQ ID NO:18, CDR-L2 as shown in SEQ ID NO:19, and CDR-L3 as shown in SEQ ID NO:20.

[0039] In a preferred embodiment, the antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising the amino acid sequences CDR-H1 as shown in SEQ ID NO:4, CDR-H2 as shown in SEQ ID NO:14, and CDR-H3 as shown in SEQ ID NO:6; and a light chain variable region comprising the amino acid sequences CDR-L1 as shown in SEQ ID NO:18, CDR-L2 as shown in SEQ ID NO:19, and CDR-L3 as shown in SEQ ID NO:20.

[0040] In some embodiments, the heavy chain variable region of the antibody or its antigen-binding fragment includes the framework regions FR-H1, FR-H2, FR-H3 and / or FR-H4 of the antibody heavy chain variable region derived from dogs.

[0041] In some embodiments, the light chain variable region of the antibody or its antigen-binding fragment includes the framework regions FR-L1, FR-L2, FR-L3 and / or FR-L4 of a canine-derived light chain variable region.

[0042] In some embodiments, the antibody may be a monoclonal antibody.

[0043] In some embodiments, the antibody may be a canine, canine-derived, cat, feline-derived, horse, equine-derived, or chimeric antibody.

[0044] In some embodiments, the antibody may be of type IgA, IgD, IgE, IgG, or IgM. In some specific embodiments, the antibody may be of type IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

[0045] In some embodiments, the antibody may include a heavy chain constant region and / or a light chain constant region of an animal-derived antibody. In some embodiments, the antibody may include a heavy chain constant region and / or a light chain constant region of a canine-derived antibody.

[0046] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0047] d1) a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:1, or an amino acid sequence having at least 75% sequence identity with it; and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:3, or an amino acid sequence having at least 75% sequence identity with it, or

[0048] d2) Heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:2, or an amino acid sequence having at least 75% sequence identity with it; and light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:3, or an amino acid sequence having at least 75% sequence identity with it.

[0049] In some embodiments, the antigen-binding fragment may be one known in the art. In specific embodiments, the antigen-binding fragment may include scFv, Fab, Fab', (Fab')2, Fv fragment, Fd, dsFv.

[0050] In some embodiments, the heavy chain constant region of the canine-derived antibody may include an amino acid sequence as shown in SEQ ID NO:60, or an amino acid sequence having at least 75% sequence identity with it.

[0051] In some embodiments, the light chain constant region of the canine-derived antibody may include an amino acid sequence as shown in SEQ ID NO:61, or an amino acid sequence having at least 75% sequence identity with it.

[0052] In some embodiments, the heavy chain variable region of the antibody includes canine-derived framework regions FR-H1, FR-H2, FR-H3 and / or FR-H4, or variants thereof.

[0053] In some embodiments, the heavy chain variable region of the antibody includes: FR-H1 as shown in SEQ ID NO:70; FR-H2 as shown in SEQ ID NO:71; FR-H3 as shown in SEQ ID NO:72; and / or FR-H4 as shown in SEQ ID NO:73.

[0054] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:68, or an amino acid sequence having at least 75% sequence identity with it;

[0055] In some embodiments, the variable region of the antibody's light chain includes canine-derived framework regions FR-L1, FR-L2, FR-L3, and / or FR-L4, or variants thereof.

[0056] In some embodiments, the light chain variable region of the antibody includes: FR-L1 as shown in SEQ ID NO:74; FR-L2 as shown in SEQ ID NO:75; FR-L3 as shown in SEQ ID NO:76; and / or FR-L4 as shown in SEQ ID NO:77;

[0057] In some embodiments, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:69, or an amino acid sequence having at least 75% sequence identity with it.

[0058] According to a third aspect of this disclosure, a nucleic acid molecule is provided that encodes an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to the first aspect of this disclosure.

[0059] According to a fourth aspect of this disclosure, a carrier is provided that includes the nucleic acid molecule of the third aspect of this disclosure.

[0060] In some embodiments, the vector includes prokaryotic expression vectors and eukaryotic expression vectors.

[0061] According to a fifth aspect of this disclosure, a cell is provided comprising an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to a first aspect of this disclosure, an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to a second aspect of this disclosure, a nucleic acid molecule according to a third aspect of this disclosure, or a vector according to a fourth aspect of this disclosure.

[0062] In some embodiments, the cells do not contain reproductive material.

[0063] According to a sixth aspect of this disclosure, a conjugate is provided comprising: an antibody or antigen-binding fragment thereof that specifically binds to canine IL-31 according to a first aspect of this disclosure, or an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to a second aspect of this disclosure; and a conjugation portion, wherein the conjugation portion is selected from detectable substances, small molecule drugs, and / or proteins.

[0064] In some embodiments, the protein may include, but is not limited to, cytokines, antibodies, antibody Fc fragments, antibody scFv fragments, enzymes, and viral capsid proteins.

[0065] In some embodiments, the detectable substance can be any substance conventionally used in the art, such as, but not limited to, fluorescent substances, luminescent markers, or radioactive substances. In a preferred embodiment, the detectable substance can be selected from any one of acridine esters, acridine sulfonamides, luminol, isoluminol, horseradish peroxidase, alkaline phosphatase, and isotopes.

[0066] According to the seventh aspect of this disclosure, a method is provided for preparing an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to the first aspect of this disclosure, or an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to the second aspect of this disclosure.

[0067] In some embodiments, the method includes culturing cells of the fourth aspect of this disclosure and isolating the antibody under conditions that allow expression of the antibody or its antigen-binding fragment.

[0068] According to the eighth aspect of this disclosure, a pharmaceutical composition is provided, comprising: an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to the first aspect of this disclosure, an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to the second aspect of this disclosure, a nucleic acid molecule according to the third aspect of this disclosure, a carrier according to the fourth aspect of this disclosure, a cell according to the fifth aspect of this disclosure, or a conjugate according to the sixth aspect of this disclosure.

[0069] In some embodiments, the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.

[0070] In some embodiments, the pharmaceutically acceptable carrier may be a carrier conventionally used in the art.

[0071] In some embodiments, the route of administration of the pharmaceutical composition may be parenteral, injection, oral, or topical. The pharmaceutical composition may be formulated into a form suitable for administration, such as a solid, semi-solid, or liquid form, and may be an aqueous solution, non-aqueous solution, or suspension, or in the form of powder, tablet, capsule, granules, injection, or infusion.

[0072] According to the ninth aspect of this disclosure, the use of an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to the first aspect of this disclosure, an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to the second aspect of this disclosure, a nucleic acid molecule according to the third aspect of this disclosure, a vector according to the fourth aspect of this disclosure, a cell according to the fifth aspect of this disclosure, a conjugate according to the sixth aspect of this disclosure, or a pharmaceutical composition according to the eighth aspect of this disclosure is provided, said use including one or more of the following:

[0073] d1) Detect the presence or content of IL-31 in the sample;

[0074] d2) Diagnosis or prognostic assessment of IL-31-related diseases;

[0075] d3) Prepare reagents for detecting the presence or content of IL-31 in samples;

[0076] d4) Prepare reagents for the diagnosis or prognostic assessment of IL-31-related diseases.

[0077] In some embodiments, the sample includes whole blood, red blood cell concentrate, platelet concentrate, white blood cell concentrate, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, feces, urine, cultured cells, saliva, oral secretions and / or nasal secretions from the subject.

[0078] In some embodiments, a method for detecting IL-31 in a sample is provided, the method comprising: contacting the sample with an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to a first aspect of the present disclosure, or an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to a second aspect of the present disclosure.

[0079] In some embodiments, a method is provided for diagnosing or prognostically assessing IL-31-related diseases using an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to the first aspect of this disclosure, or an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to the second aspect of this disclosure. In some embodiments, the method includes the step of contacting a sample of the subject with an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to the first aspect of this disclosure, or an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to the second aspect of this disclosure.

[0080] In some implementations, the subject may include a dog, cat, or horse.

[0081] In some embodiments, the IL-31-related diseases include pruritus, allergies, or asthma. In preferred embodiments, the IL-31-related diseases include atopic dermatitis, eczema, psoriasis, scleroderma, allergic dermatitis, urticaria, psoriasis, and asthma.

[0082] According to the tenth aspect of this disclosure, an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to the first aspect of this disclosure, an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to the second aspect of this disclosure, a nucleic acid molecule according to the third aspect of this disclosure, a vector according to the fourth aspect of this disclosure, a cell according to the fifth aspect of this disclosure, a conjugate according to the sixth aspect of this disclosure, or a pharmaceutical composition according to the eighth aspect of this disclosure are provided for the treatment and / or prevention of IL-31-related diseases.

[0083] In some embodiments, this disclosure provides the use of an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to the first aspect of this disclosure, an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to the second aspect of this disclosure, a nucleic acid molecule according to the third aspect of this disclosure, a vector according to the fourth aspect of this disclosure, a cell according to the fifth aspect of this disclosure, a conjugate according to the sixth aspect of this disclosure, or a pharmaceutical composition according to the eighth aspect of this disclosure in the preparation of a medicament for treating and / or preventing IL-31-related diseases.

[0084] In some embodiments, this disclosure provides a method for treating and / or preventing IL-31-related diseases, the method comprising: administering to a subject in need an effective amount of an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to the first aspect of this disclosure, an antibody or antigen-binding fragment thereof that specifically binds to animal IL-31 according to the second aspect of this disclosure, a nucleic acid molecule according to the third aspect of this disclosure, a carrier according to the fourth aspect of this disclosure, a cell according to the fifth aspect of this disclosure, a conjugate according to the sixth aspect of this disclosure, or a pharmaceutical composition according to the eighth aspect of this disclosure.

[0085] In some embodiments, the IL-31-related diseases include pruritus, allergies, or asthma. In preferred embodiments, the IL-31-related diseases include atopic dermatitis, eczema, psoriasis, scleroderma, allergic dermatitis, urticaria, psoriasis, and asthma. Attached Figure Description

[0086] Figure 1 SDS-PAGE results for canine IL31-his and canine IL31-mFc proteins.

[0087] Figure 2 Results of ELISA detection of IL31-his protein in the culture supernatant of hybridoma cells 8D2 and 9D6.

[0088] Figure 3 This is the result of monoclonal antibody subtype identification.

[0089] Figure 4 The results are from SDS-PAGE identification of the chimeric antibody.

[0090] Figure 5 The results are from the in vitro binding experiment of the chimeric antibody.

[0091] Figure 6 The in vivo therapeutic effect of intravenous injection of chimeric antibody CA-9D6.

[0092] Figure 7 The results of SDS-PAGE identification of canine-derived antibody (Can-9D6), chimeric antibody, and cytokinase are presented.

[0093] Figure 8 The results are from in vitro binding experiments of canine-derived antibodies, chimeric antibodies, and cytokines.

[0094] Figure 9 The results show the in vitro competition between canine-derived antibodies and cyproterenol. The assays used biotin-labeled cyproterenol (top image) and biotin-labeled Can-9D6 (bottom image) were performed, respectively.

[0095] Figure 10 The in vivo therapeutic effect of subcutaneous injection of canine-derived antibody Can-9D6. Detailed Implementation

[0096] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0097] Definition

[0098] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0099] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0100] The term "antibody" in this article encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific or trispecific antibodies), single-chain molecules, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0101] The terms "antibody or antigen-binding fragment thereof" and "antibody" are used interchangeably herein, referring to an immunoglobulin capable of specifically binding to a target antigen via at least one of its antigen-binding domains. Antibodies can be classified into different types based on the amino acid sequence of their heavy chain constant regions. There are five major types of immunoglobulins: IgG, IgA, IgM, IgD, and IgE, with heavy chains of γ, α, μ, δ, and ε chains, respectively. Antibodies can also be further classified into subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The light chains of antibodies, based on the amino acid sequence of their constant domains, can be classified into two types: κ and λ.

[0102] The term "variable region" or "variable domain" used in this article refers to the domain of the antibody heavy or light chain in an antigen-binding molecule that participates in antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain is sufficient to confer antigen-binding specificity. The HVRs in each chain are tightly held together by the FR regions and, together with the HVRs of other chains, contribute to the formation of the antibody's antigen-binding site. Constant regions do not directly participate in antibody-antigen binding but have other effector functions, such as participating in antibody-dependent cytotoxicity.

[0103] The term "hypervariant region" or "HVR" in this article refers to a region in the variable domain region of an antibody that is highly variable in sequence and / or forms a structurally defined loop ("hypervariant loop"). Typically, a natural tetrachain antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs typically contain amino acid residues from the hypervariant loop and / or from the "complementarity-determining region (CDR)," the amino acid residues from the "CDR" having the highest sequence variability and / or being involved in antigen recognition.

[0104] The term "framework" or "FR" in this article refers to the variable domain residues other than the hypervariable region (HVR) residues. A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences typically appear in the VH (or VL) in the following sequence: FR-H1(L1)-FR-H2(L2)-FR-H3(L3)-FR4.

[0105] The term "substitution" or "replacement" used in this article to describe amino acids can refer to the substitution of conserved amino acids, in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine (L), arginine (R), histidine (H)), acidic side chains (e.g., aspartic acid (D), glutamic acid (E)), uncharged polar side chains (e.g., glycine (G), asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C)), nonpolar side chains (e.g., alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W)), β-branched side chains (e.g., threonine (T), valine (V), isoleucine (I)), and aromatic side chains (e.g., tyrosine (Y), phenylalanine (F), tryptophan (W), histidine (H)). Therefore, if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, such substitution is considered conserved. In another respect, an amino acid chain can be conservatively replaced by a structurally similar amino acid chain that differs in the order and / or composition of its side chain family members.

[0106] The term "nucleic acid molecule" used in this article refers to one of the most important biological macromolecules in organisms. It is the material basis for carrying and transmitting genetic information and includes two major categories: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0107] The term "antibody fragment" or "antigen-binding fragment" used in this article refers to a portion of a complete antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, bisomatic antibodies, trisomatic antibodies, tetrasomatic antibodies, cross-Fab fragments, linear antibodies, single-chain antibody molecules (e.g., scFv), single-domain antibodies (single-domain antibodies), etc.

[0108] After obtaining the DNA fragments encoding the VH and / or VL of the antibody, these DNA fragments can be further manipulated using recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these operations, the DNA fragment encoding the VH and / or VL is operatively ligated to another DNA fragment encoding a different protein, such as the antibody constant region or a flexible linker. As used herein, the term "operatively ligated" means that two DNA fragments are joined together in such a way that the amino acid sequences encoded by both DNA fragments remain within the reading frame.

[0109] By operatively linking the DNA encoding VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3), isolated DNA encoding the VH region can be converted into a full-length heavy chain gene. Similarly, by operatively linking the DNA encoding VL to another DNA molecule encoding the light chain constant region CL, isolated DNA encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene).

[0110] The terms "antigen-binding domain" or "antigen-binding site" used herein refer to the portion of an antibody or its antigen-binding fragment that specifically binds to an antigenic determinant. More specifically, the term "antigen-binding domain" refers to a region of an antibody that specifically binds to and is complementary to a portion or all of an antigen. In cases where the antigen molecule is large, the antibody may bind only to a specific portion of the antigen, called an epitope. The antigen-binding domain may be provided by, for example, one or more variable domains (also called variable regions). Preferably, the antigen-binding domain comprises a variable region (VL) of the antibody light chain and a variable region (VH) of the antibody heavy chain. In some embodiments, the antigen-binding domain is capable of binding its antigen and blocking or partially blocking the function of said antigen.

[0111] The term "antigenic determinant" used herein is interchangeable with "antigen" and "epitope" and refers to a site on a polypeptide molecule (e.g., a continuous amino acid sequence or a conformation composed of discontinuous amino acids) to which the antigen-binding moiety binds, thereby forming an antigen-binding moiety-antigen complex. Unless otherwise stated, the protein used as an antigen in this disclosure can be any naturally occurring protein of any vertebrate origin, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), dogs, cats, etc.

[0112] The terms "vector" or "expression vector" and "expression construct" used herein are used interchangeably to describe a DNA molecule to which a specific gene, operatively linked, is introduced into a target cell and directed for expression. The vector includes a vector as a self-replicating nucleic acid structure and a vector incorporated into the genome of the host cell into which it has been introduced. The expression vectors disclosed herein contain expression cassettes. Expression vectors can perform transcription of large amounts of stable mRNA. Once the expression vector is within the target cell, the cellular transcription and / or translation mechanisms generate a ribonucleic acid molecule or protein encoded by the gene. The term "expression cassette" herein refers to a recombinant or synthetically produced polynucleotide having a set of nucleic acid elements that allow transcription of a specific nucleic acid in the target cell.

[0113] The term "pharmaceutical composition" herein refers to a mixture containing an antibody or antibody- or antigen-binding fragment thereof, biological material, or conjugate of the present disclosure, and other chemical components, such as physiological / pharmaceutical-grade carriers or excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0114] The term "treatment" herein refers to the administration of an oral or topical therapeutic agent, such as a composition comprising any antibody of the present disclosure or an antibody thereof or an antigen-binding fragment thereof, or a nucleic acid molecule encoding an antibody thereof or an antibody thereof or an antigen-binding fragment thereof, to a subject who has one or more diseases or symptoms, and the therapeutic agent has a therapeutic effect on these diseases or symptoms. Typically, the therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more diseases or symptoms, to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable extent.

[0115] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are included, but those from the reference sequence are excluded.

[0116] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0117] Those skilled in the art will understand that the reference herein to having “at least 75% sequence identity” compared to a sequence is intended to include all sequences having more than 75% sequence identity with that sequence, such as sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity.

[0118] “Affinity” or “binding affinity” refers to the strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Binding affinity is typically expressed as a dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constants (Koff and Kon, respectively). Therefore, equivalent affinity can include different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured using conventional methods known in the art, such as surface plasmon resonance (SPR). When used herein, the antibody molecule “binds” to the antigen, a binding understood by those skilled in the art. In one embodiment, the antibody binds to the antigen with a dissociation constant (KD) of approximately 1 × 10⁻⁶. -5 M or lower, 1×10 -6 M or lower, or 1×10 -7 M or lower, 1×10 -8 M or lower, 1×10 -9M or lower, 1×10 -10 M or lower, 1×10 -11 M or lower.

[0119] The term "chimeric antibody" or "chimerism" herein refers to an antibody in which a portion of the heavy or light chain is derived from a specific source or species, while at least a portion of the remaining portion of the heavy or light chain is derived from a different source or species. In some embodiments, the chimeric antibodies provided herein consist of splicing a variable region of the mouse heavy chain and / or a variable region of the light chain with a constant region of the heavy chain and / or a constant region of the light chain of an antibody from another species (e.g., a dog, cat, or horse, preferably a dog) to form a chimeric structure. In some embodiments, the chimeric antibodies provided herein comprise a constant region of the heavy chain of an antibody derived from a dog.

[0120] In a specific embodiment, the heavy chain constant region of the antibody derived from dogs may include an amino acid sequence as shown in SEQ ID NO:60, or an amino acid sequence having at least 75% sequence identity with it.

[0121] In a specific embodiment, the light chain constant region of the canine-derived antibody may include an amino acid sequence as shown in SEQ ID NO:61, or an amino acid sequence having at least 75% sequence identity with it.

[0122] In other embodiments, the chimeric antibody provided herein comprises splicing CDR1-3 of a mouse heavy chain variable region and / or light chain variable region with frame regions (FR)1-4 of heavy chain variable regions and / or light chain variable regions of other species (e.g., dogs, cats, or horses, preferably dogs) to form a chimeric structure.

[0123] In a specific embodiment, the frame region FR-H1 of the variable region of the antibody heavy chain derived from dogs may include an amino acid sequence as shown in EVQLVQSGAEVKKPGASVKVSCKTS (SEQ ID NO:70), or an amino acid sequence having at least 75% sequence identity with it.

[0124] In a specific embodiment, the frame region FR-H2 of the variable region of the antibody heavy chain derived from dogs may include an amino acid sequence as shown in VNWVRQAPGAGLDWMGR (SEQ ID NO:71), or an amino acid sequence having at least 75% sequence identity with it.

[0125] In a specific embodiment, the frame region FR-H3 of the variable region of the antibody heavy chain derived from dogs may include an amino acid sequence as shown in KYAQKFQGRVTLTADTSTSTAYMELSSLRAGDIAVYFC (SEQ ID NO:72), or an amino acid sequence having at least 75% sequence identity with it.

[0126] In a specific implementation, the frame region FR-H4 of the variable region of the antibody heavy chain derived from dogs may include an amino acid sequence as shown in WGQGTLVTVSS (SEQ ID NO:73), or an amino acid sequence having at least 75% sequence identity with it.

[0127] In a specific embodiment, the frame region FR-L1 of the variable region of the antibody light chain derived from dogs may include an amino acid sequence as shown in EIVMTQSPGSLAGSAGESVSINCKSS (SEQ ID NO:74), or an amino acid sequence having at least 75% sequence identity with it.

[0128] In a specific embodiment, the frame region FR-L2 of the variable region of the antibody light chain derived from dogs may include an amino acid sequence as shown in LAWYQQKPGERPKLLIY (SEQ ID NO:75), or an amino acid sequence having at least 75% sequence identity with it.

[0129] In a specific embodiment, the frame region FR-L3 of the variable region of the antibody light chain derived from dogs may include an amino acid sequence as shown in TRESGVPARFSSSGSGTDFTLTINNLQAEDVGDYYC (SEQ ID NO:76), or an amino acid sequence having at least 75% sequence identity with it.

[0130] In a specific embodiment, the frame region FR-L4 of the variable region of the antibody light chain derived from dogs may include an amino acid sequence as shown in FGQGTKLEIK (SEQ ID NO:77), or an amino acid sequence having at least 75% sequence identity with it.

[0131] In this article, "canine-derived antibody" refers to an antibody in which at least one amino acid in a portion of the non-canine variable region has been replaced by a corresponding amino acid from the canine variable region.

[0132] The term “CDR” in this document refers to the complementary determination region as defined by at least one identification method by those skilled in the art.

[0133] The term "monoclonal antibody" in this article refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, a single antibody that is identical to the population except for a small number of potentially naturally occurring mutations.

[0134] Table 1. Amino acid sequences of the antibody's VH and VL.

[0135]

[0136] Table 2. Amino acid sequences of the antibodies provided in this paper for CDR-H1, CDR-H2, and CDR-H3.

[0137]

[0138] Table 3. CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of the antibodies provided in this paper.

[0139]

[0140] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0141] Example

[0142] Example 1. Expression and purification of recombinant canine IL-31 protein

[0143] 1. Construction of the IL31-his recombinant plasmid

[0144] Referring to the IL-31 sequence (NP_001159386) of canine (Canis lupus familiaris) on NCBI, a mature IL-31 sequence (underlined) was selected, and a signal peptide sequence (sp sequence, which is removed after protein processing and maturation) was added to the N-terminus. A GS linker and 8 His tags were added to the C-terminus. The gene was synthesized by Beijing Tianyi Huiyuan Biotechnology Co., Ltd. and constructed into the eukaryotic expression vector pCMV-Flag.

[0145] Canine IL-31 nucleotide sequence:

[0146] ATGCTGAGCCACACCGGCCCTTCTAGATTCGCCCTGTTCCTGCTGTGCAGCATGGAGACACTGCTGTCC TCTCACATGGCACCGACCCACCAGCTGCCCCCGTCTGATGTTCGGAAAATTATTCTGGAACTGCAACCTCTGAGC CGTGGTCTGCTGGAAGATTACCAGAAAAA AGAAACCGGCGTGCCCGAGAGCAATCGTACCCTGCTGCTGTGCCTG ACAAGCGACAGCCAGCCTCCAAGACTGAATAGTAGCGCTATTCTGCCTTATTTTAGAGCCATTCGTCCGCTGTCCG ACAAGAACATCATCGACAAAATTATCGAGCAGCTGGATAAGCTGAAGTTCCAGCACGAACCTGAAACCGAGATCAG CGTTCCGGCAGATACCTTTGAATGTAAGAGCTTCATCCTGACCATCCTGCAGCAGTTTAGCGCCTGTCTGGAAAGC GTGTTCAAGAGCCTGAACAGCGGTCCGCAG GGCAGCCACCATCATCATCATCACCATCATtga(SEQ ID NO:26) canine IL-31-his amino acid sequence

[0147] SHMAPTHQLPPSDVRKIILELQPLSRGLLEDYQKKETGVPESNRTLLLCLTSDSQPPRLNSSAILPYF RAIRPLSDKNIIDKIIEQLDKLKFQHEPETEISVPADTFECKSFILTILQQFSACLESVFKSLNSGPQ GSHHHHHHHH(SEQ ID NO:27)

[0148] 2. Construction of IL31-mFc recombinant plasmid

[0149] To obtain the secretory IL31-mFc protein, sp-IL-31 and mouse Fc (mFc) fragments were amplified by PCR and sp-IL31-mFc was assembled by overlap PCR. The sp-IL31-mFc was cloned into the pTT3 vector using EcoRI / NotI double digestion sites, and the recombinant plasmid was verified by sequencing.

[0150] mFc nucleotide sequence:

[0151] (SEQ ID NO:28)

[0152] mFc amino acid sequence:

[0153] EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPI ERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ IDNO:29)

[0154] 3. Expression of IL31-his / IL31-mFc recombinant protein

[0155] 1) Preparation of transfection complex

[0156] Preparation method of PEI transfection reagent solution: Add 100 mg of linear PEI transfection reagent (Mw 40000; LABLEAD, catalog number P4000) to 90 mL of Milli-Q ultrapure water, stir until completely dissolved, adjust the pH to 6.9-7.1, and make up to 100 mL with Milli-Q ultrapure water. Then filter through a 0.22 μm filter membrane and collect the filtrate.

[0157] Preparation of transfection complex (per 1L of cell suspension): Add 1mg of recombinant plasmid to 5mL of Hi-exp medium (Opmai, catalog number AC601501), mix well by pipetting, and this is liquid phase A; add 3mL of PEI transfection reagent solution to 5mL of Hi-exp medium, mix well by pipetting, and this is liquid phase B; add liquid phase B to liquid phase A, mix well by pipetting, and then incubate at room temperature for 5min.

[0158] 2) Preparation of cell suspension

[0159] Collect 293F cells from the culture, count the cells, centrifuge at 800 rpm for 5 minutes, discard the supernatant, and resuspend in Hi-exp medium to achieve a cell concentration of 1×10⁻⁶ cells / mL. 6 Cells / mL.

[0160] 3) Add the transfection complex dropwise to 1L of cell suspension and incubate with shaking at 130rpm for 4-5 days (environmental conditions: 37℃, 8% CO2). Then centrifuge at 4000rpm for 20min, collect the supernatant, and then filter it through a 0.45μm filter membrane and collect the filtrate.

[0161] 4. Purification of recombinant proteins

[0162] 1) For IL31-his protein, column chromatography was used (column volume 10 mL; packing material: Ni Sepharose 6FF, Cytiva (GE Life), product catalog number 17531801). First, the column was equilibrated with equilibration buffer (1xPBS buffer; 300 mM NaCl; pH 7.4). Then, the supernatant obtained from expression in step 3 was loaded onto the column (loading volume 1 L). The column was loaded with 5-10 column volumes of washing buffer (1xPBS buffer; 300 mM NaCl; 50 mM imidazole; pH 7.4). The IL31-his protein was obtained by elution with elution buffer (1xPBS buffer; 300 mM NaCl; 500 mM imidazole; pH 7.4).

[0163] 2) For the IL31-mFc protein, an affinity chromatography column (10 mL column volume; packing material: proteinAtBeadsLX, Changzhou Tiandi Renhe Biotechnology Co., Ltd., product catalog number SA08501L) was used. The column was first equilibrated with PBS buffer, then the supernatant obtained from step 3 (1 L loading volume) was loaded. The column was then washed with PBS buffer for 10-15 column volumes, followed by elution with glycine buffer (pH 2.5-3.0, 0.1 M) and the post-column solution was collected. Ten volumes of the post-column solution were mixed with one volume of neutralization buffer (pH 9.0, 1 M Tris-HCl buffer) to obtain a mixture. Subsequently, a 30K ultrafiltration tube (Millipore, UFC903096) was used for concentration and buffer replacement, replacing the buffer system with PBS buffer to obtain the IL31-mFc protein.

[0164] 5. Identification of recombinant proteins

[0165] The concentration of IL31-his / IL31-mFc protein was measured using a nanodropper, and the recombinant protein was identified by SDS-PAGE. Under denaturing conditions, 2 μg of the recombinant protein sample was loaded onto a 12% SDS-PAGE gel. After electrophoresis, Coomassie Brilliant Blue staining was performed, and clear protein bands were revealed after destaining. The results are as follows: Figure 1 As shown, high-purity IL31-his / IL31-mFc proteins were successfully obtained, with IL31-his having a molecular weight of approximately 20 kDa and IL31-mFc having a molecular weight of approximately 50 kDa.

[0166] Example 2. Hybridoma Monoclonal Antibody Screening

[0167] 1. Immunizing mice:

[0168] SPF-grade 6-8 week old Balb / c mice were immunized with canine IL31-mFc protein obtained in Example 1, following the procedure below:

[0169] Day 1: First immunization, multiple subcutaneous injections of immunizing agent (composed of 10μg immunogen, CpG + γ-manganese adjuvant);

[0170] Day 29: Second immunization, multiple subcutaneous injections of immunizing agent (composed of 10 μg immunogen, CpG + γ-manganese adjuvant);

[0171] Day 57: Third immunization, multiple subcutaneous injections of immunizing agent (composed of 10 μg immunogen, CpG + γ-manganese adjuvant);

[0172] Day 64: Orbital venous blood was collected to separate serum, which was used to detect the titer of the test antibody. Mice with high titers were selected for the fourth immunization, and the immunogen was injected into the tail vein.

[0173] Methods for detecting antibody titers in immune serum (ELISA method):

[0174] The canine IL31-his protein obtained in Example 1 was diluted with PBS buffer to a protein concentration of 1 μg / ml to obtain the coating solution. PBST solution: PBS buffer containing 0.05% (v / v) Tween-20. Blocking solution: PBST solution containing 0.2 g / 100 mL BSA. Preparation of antibody dilution buffer: Immune mouse serum was used as the test antibody, and serum from unimmunized normal mice was used as the negative control. The serum was diluted 100-fold, 1000-fold, 10000-fold, and 100000-fold with blocking solution, respectively. The detection was performed according to the following steps:

[0175] 1) Take a 96-well microplate, add coating buffer (100 μL / well), incubate at 4°C for 16 hours (overnight), discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0176] 2) Take the 96-well plate after completing step 1), add blocking solution (200 μL / well), incubate at room temperature for 1 hour, discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0177] 3) Take the 96-well plate from step 2), add antibody dilution buffer (100 μL / well), incubate at room temperature for 1 hour, discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0178] 4) Take the 96-well plate from step 3), add HRP-labeled goat anti-mouse IgG antibody (Zhongshan Jinqiao ZB-2305) (10,000-fold dilution, 100 μL / well), incubate at room temperature for 1 hour, discard the supernatant, wash 5 times with PBST solution, and pat dry.

[0179] 5) Take the 96-well plate after completing step 4), add TMB colorimetric solution (100 μL / well), and react in the dark for 5-10 minutes.

[0180] 6) Take the 96-well plate after completing step 5), add 2M sulfuric acid solution (50μL / well), and then measure the absorbance at 450nm (OD450).

[0181] 2. Hybridoma cell fusion and screening

[0182] 1) Preparation of feeder cells

[0183] The day before fusion, well-developed Balb / c mice were euthanized by cervical dislocation and sterilized in 75% ethanol aqueous solution. Using a pre-chilled syringe, 8-10 ml of pre-chilled 0.34M sucrose aqueous solution was injected through the lower right peritoneum of the mouse, ensuring the needle did not exit the peritoneum. The peritoneal cavity was massaged with a finger for approximately 1 minute. The fluid from the peritoneal cavity was then aspirated and added to a pre-chilled 50 ml centrifuge tube, followed by pre-chilled 1640 complete culture medium. The tube was centrifuged at 1500 rpm for 5 minutes, the supernatant was discarded, and the cell pellet was resuspended in HAT medium to a cell concentration of 1×10⁻⁶. 5 Cells / ml were collected, and the cell suspension was then added to a 96-well cell culture plate (100 μl / well) and cultured.

[0184] 2) Preparation of spleen cells

[0185] Four days after the fourth immunization in step 1, mice were euthanized by enucleation, and blood (polyclonal antibody serum) and spleen were collected. Serum was isolated from the blood as a positive control. A 10cm diameter culture dish was prepared, with 10ml of 1640 medium and DNase added, followed by the addition of mouse spleen. The mixture was ground and pipetted to form a single-cell suspension. The suspension was then filtered through a 70μm filter and collected into a 50ml centrifuge tube. The suspension was centrifuged at 1500rpm for 5min, the supernatant was discarded, and the cell pellet was resuspended in ACK erythrocyte lysis buffer. The cells were incubated at room temperature for 2min, washed with 1640 medium, and then resuspended in 20ml of 1640 medium. After mixing, the cells were counted.

[0186] 3) Cell fusion

[0187] Collect healthy Sp2 / 0 mouse myeloma cells in the logarithmic growth phase and wash them with 1640 medium. Mix Sp2 / 0 mouse myeloma cells and spleen cells at a ratio of 1:1 to 1:3, centrifuge at 1500 rpm for 10 min, discard the supernatant, and agitate the cells to form a paste. Incubate at 37°C, add 1 ml of preheated 50% PEG solution dropwise, then slowly add 40 ml of preheated 1640 medium. Centrifuge at 1500 rpm for 10 min, discard the supernatant, add 10 ml of HAT medium and agitate several times, then add HAT medium to approximately 90 ml. Mix well and drop the mixture into feeder cell culture plates, 2 drops per well, and incubate.

[0188] 4) Screening of hybridoma cell positive wells

[0189] Four days after cell fusion, half of the HAT medium was replaced. After approximately 7-10 days, the hybridoma cell clusters reached a certain size. One day before testing, about 200 μl of medium was aspirated and replaced with 200 μl of fresh HT medium. On the day of testing, the culture medium from the hybridoma cell clusters was used as the test antibody. Positive serum was used as a positive control, and serum from unimmunized mice was used as a negative control. Positive clones were screened by detecting the binding of canine IL-31 using flow cytometry.

[0190] 5) Subcloning of positive hybridoma cells

[0191] The first subcloning was performed using HT medium. Positive cloning wells were selected for the first subcloning, and cell state and cell cluster size were observed under a microscope. Under aseptic conditions, the cells to be subcloned were gently pipetted to mix, avoiding air bubbles. 10 μl of the cell count was taken, and 10 μl of 0.04% trypan blue solution was added. After mixing, the cells were counted in 8 large squares, and the cell concentration was calculated. Based on the cell count results, 100-150 cells were added to 9.5 ml of medium using the limiting dilution method. After mixing, the mixture was added dropwise to a feeder cell culture plate cultured for 1 day.

[0192] After about 5 days of subcloning, the hybridomas in each well are counted under an inverted microscope. Once the hybridoma cells have grown to a suitable size, they are tested again and positive monoclonal cells are selected for a second subcloning. The culture medium is then replaced with 1640 complete medium. Subcloning is repeated 2-3 times until the obtained monoclonal hybridoma cell line can stably secrete the required antibody.

[0193] 6) Expansion culture and cryopreservation of hybridoma cells

[0194] ① The positive hybridoma cells obtained after identification were expanded and transferred to 24-well cell culture plates and cultured until the confluence reached about 80%.

[0195] ② After completing step ①, transfer the cells to a T25 cell culture flask and culture until the cell confluence reaches approximately 80%.

[0196] ③ After completing step ②, repeatedly blow the culture flask with culture medium, then transfer it to a sterile centrifuge tube, centrifuge at 1500 rpm for 5 min, discard the supernatant, resuspend the cells with 3 ml of cell cryopreservation solution, mix thoroughly, transfer the cell suspension to a cryopreservation tube, transfer it to a programmed cooling box, incubate at -80℃ for 24 h, and then transfer it to liquid nitrogen for long-term storage.

[0197] Based on the above steps, hybridoma cells 8D2 and 9D6 that secrete the target monoclonal antibody (i.e., a monoclonal antibody that binds to canine IL-31 protein) were screened and obtained. Figure 2 The results of ELISA detection of IL31-his protein using the steps in the above-described "Antibody Titer Detection Method of Immune Serum" are shown. Positive control PC (fusion mouse serum) and negative control NC (normal Balb / c mouse serum) were diluted 1000-fold and used as controls.

[0198] Example 3. Obtaining the variable region sequence of a monoclonal antibody

[0199] 1) Identification of monoclonal antibody subtypes

[0200] Hybridoma cells of 8D2 and 9D6 were cultured in 1640 complete medium for 2-3 days, and the supernatant was collected. Antibody subtypes were identified using a monoclonal antibody subtype identification kit, following the manufacturer's instructions. The monoclonal antibody subtype identification kit (Isotyping Kit for Mouse Monoclonal Antibody) was manufactured by Beijing Yiqiao Shenzhou Technology Co., Ltd., product catalog number SEK003.

[0201] See results Figure 3 8D2 is the IgG2a subtype, and 9D6 is the IgG1 subtype.

[0202] 2) Monoclonal antibody variable region sequence amplification and sequencing

[0203] Take 1×10 6 ~1×10 7 Hybridoma cells containing anti-canine IL-31 (8D2 and 9D6) were centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were washed with PBS and centrifuged again to remove the supernatant. 1 mL of pre-chilled Trizol (Invitrogen, 15596018CN) was added, and RNA was extracted according to the manufacturer's instructions. cDNA was reverse transcribed using oligo dT primers and M-MLV reverse transcriptase (Progema, M1705).

[0204] Using cDNA as a template, primers targeting the antibody variable region were used. The upstream primer of the mouse VH sequence (Table 4) was paired with the downstream heavy chain primer corresponding to the subtype (Table 5), and the upstream primer of the mouse Vκ sequence (Table 6) was paired with the downstream light chain primer (Table 7). PCR amplification was performed using PrimeSTARMax DNA polymerase (TaKaRa, R045). The amplification system and PCR reaction procedure are shown in Tables 8 and 9 below.

[0205] Table 4. Upstream primers for mouse VH sequence

[0206] Primer Name Nucleotide Sequence of Primer (5'-3') MHVP1 ACTAGTCGACATGAAATGCAGCTGGGTCATSTTCTTC(SEQ ID NO:30) MHVP2 ACTAGTCGACATGGGATGGAGCTRTATCATSYTCTT(SEQ ID NO:31) MHVP3 ACTAGTCGACATGAAGWTGTGGTTAAACTGGGTTTTT(SEQ ID NO:32) MHVP4 ACTAGTCGACATGRACTTTGGGYTCAGCTTGRTTT(SEQ ID NO:33) MHVP5 ACTAGTCGACATGGACTCCAGGCTCAATTTAGTTTTCCTT(SEQ ID NO:34) MHVP6 ACTAGTCGACATGGCTGTCYTRGSGCTRCTCTTCTGC(SEQ ID NO:35) MHVP7 ACTAGTCGACATGGRATGGAGCKGGRTCTTTMTCTT(SEQ ID NO:36) MHVP8 ACTAGTCGACATGAGAGTGCTGATTCTTTTGTG (SEQ ID NO:37) MHVP9 ACTAGTCGACATGGMTTGGGTGGGAMCTTGCTATTCCTG(SEQ ID NO:38) MHVP10 ACTAGTCGACATGGGCAGACTTACATTCTCATTCCTG(SEQ ID NO:39) MHVP11 ACTAGTCGACATGGATTTTGGGCTGATTTTTTTTATTG(SEQ ID NO:40) MHVP12 ACTAGTCGACATGATGGTGTTAAGTCTTCTGTACCTG(SEQ ID NO:41)

[0207] R represents A or G, Y represents C or T, K represents G or T, M represents A or C, S represents G or C, and W represents A or T.

[0208] Table 5. Downstream primers for mouse VH sequence

[0209] Primer name nucleotide sequence of primer IgG1 ATAGACAGATGGGGGTGTCGTTTTGGC(SEQ ID NO:42) IgG2a CTTGACCAGGCATCCTAGAGTCA(SEQ ID NO:43)

[0210] Table 6. Upstream primers for mouse Vκ sequence

[0211]

[0212]

[0213] R represents A or G, Y represents C or T, K represents G or T, M represents A or C, S represents G or C, and W represents A or T.

[0214] Table 7. Downstream primers for mouse Vκ sequence

[0215] Primer name Primer nucleotide sequence (5'-3') Igκ GGATACAGTTGGTGCAGCATC(SEQ ID NO:55)

[0216] Table 8. PCR amplification system

[0217] PrimerStarMaxPremix(2x): 25uL upstream primer 1μL Downstream primer 1μL cDNA template 1μL Sterilized water 22μL Total volume 50μL

[0218] Table 9. PCR Reaction Procedure

[0219]

[0220] PCR amplification products were analyzed by 1% agarose gel electrophoresis. The results showed that the heavy chains 8D2 and 9D6 both had specific bands of the correct size in the MHVP1 amplification products, and the light chains both had specific bands of the correct size in the MκVP7 amplification products. No specific bands were amplified by other primers. The specific bands were excised, the gel products were recovered, and sent to a sequencing company for sequencing to obtain the gene sequences of the variable regions of the antibody's heavy and light chains. Further, the amino acid sequences of the variable regions of the antibody's heavy and light chains were obtained. The amino acid sequence of the variable region of the heavy chain of antibody 8D2 is shown in SEQ ID NO:1, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:3. The amino acid sequence of the variable region of the heavy chain of antibody 9D6 is shown in SEQ ID NO:2, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:3.

[0221] The nucleotide sequence of the variable region of the antibody heavy chain is as follows:

[0222] 8D2:

[0223] CAGGTCCAGCTGCAGCAGTCTGGACCTGAGTTGGTGAAGGCTGGGGCCTCAGTGAAGATTTCCTGCAAAACTTCTGACTACGCATTCAGTGACTCTTGGGTGAATTGGGTGAAGAAGAGGCCTGGACAGGGTCTTGACTGGATTGGACGGATTAATCTTGGGAATGGAGAAACTAAATACAAT GGGAAGTTCAAGGTCAAGGCCACACTGACTGCAGACAAATCCTCCGGCACAGCCTACATGCACCTCAGCAGCCTGACCTCTGTGGACTCTGCGGTCTATTTCTGTGCAAGATCGGGGTACGAAAGTCCCTACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA(SEQ IDNO:56)

[0224] 9D6:

[0225] CAGGTCCAGCTGCAGCAGTCTGGACCTGAGTTGGTGAAGGCTGGGGCCTCAGTGAAGATTTCCTGCAAAACTTCTGACTACGCATTCAGTGACTCTTGGGTGAATTGGGTGAAGAAGAGGCCTGGACAGGGTCTTGACTGGATTGGACGGATTAATCTTGGGGATGGAGAAA CTAAATACAATGGGAAGTTCAAGGTCAAGGCCACACTGACTGCAGACAAATCCTCCGGCACAGCCTACATGTACCTCAGCAGCCTGACCTCTGTGGACTCTGCGGTCTATTTCTGTGCAAGATCGGGGTACGAAAGTCCCTACTGGTACTTCGATGTCTGGGGCGCAGGGTCCACGGTCACCGTCTCCTCA(SEQ IDNO:57)

[0226] The nucleotide sequence of the variable region of the antibody light chain is as follows:

[0227] 8D2:

[0228] GACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTTTGAGCTGCAGGTCCAGTCAGAGTCTTTTATATATTACCACTCAAAAGAACCAATTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACCAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAATTTATTACTGTCAGCAATATTATGGCTATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA(SEQ ID NO:58)

[0229] 9D6:

[0230] GACATTTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTTTGAGCTGCAGGTCCAGTCAGAGTCTTTTATATATTACCACTCAAAAGAACCAGTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAATTGCTGATTTACTGGGCA TCCACCAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAATTTATTACTGTCAGCAATATTATGGCTATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA(SEQ ID NO:59)

[0231] Example 4. Preparation of 8D2 and 9D6 chimeric antibodies

[0232] 1. Construction of a mouse-dog chimeric recombination expression vector

[0233] Antibodies consist of two heterodimers, one heavy chain and one light chain, each with a variable region and a constant region. To construct expression vectors that generate chimeric mouse-dog antibodies or canine-derived antibodies in mammalian expression systems, a universal plasmid vector was first constructed by inserting a signal peptide sequence and either the canine heavy chain or light chain constant region sequence into the pTT3 vector. The amino acid sequences of the canine heavy chain constant region and the canine light chain constant region used are shown below. The Kozak common sequence, signal peptide sequence, and unique restriction endonuclease site (BsmBI) were incorporated into the heavy chain constant region nucleotide sequence and light chain nucleotide sequence, respectively, to promote the expression and secretion of recombinant antibodies from mammalian cell lines. The synthesized nucleotide sequences were Sp-BsmBI-HC (SEQ ID NO:62) and Sp-BsmBI-LC (SEQ ID NO:63), respectively. Then, using HindIII and NotI endonucleases, Sp-BsmBI-HC and Sp-BsmBI-LC were cloned into the pTT3 vector to form pTT3-sp-BsmBI-HC and pTT3-sp-BsmBI-LC vectors, respectively.

[0234] Canine heavy chain constant region amino acid sequence:

[0235] ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSM VTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPE VTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIER TISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYS KLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGK (SEQ ID NO:60)

[0236] Canine light chain constant region amino acid sequence:

[0237] RNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLS STLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD (SEQ ID NO:61)

[0238] Sp-BsmBI-HC nucleotide sequence:

[0239] GCCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCAACTCCGAGACGGGCGGCGGATCTCGTCTCGCCTCCACAACCGCACCTAGCGTGTTTCCACTGGCACCTTCTTGTGGCTCCACAAGCGGTAGCACAGTGGCTCTTGCCTGCTTGGTGAGCGGATACTTCCCTGAACCTGTTACAGTCTCTTGGAACTCTGGCTCTCTGACAAGTGGAGTGCATACCTTCCCTTCTGTGTTGCAGTCTTCTGGTCTGTACAGTCTGAGCAGCATGGTGACAGTGCCTAGCAGTAGGTGGCCTAGCGAAACCTTCACCTGCAACGTGGCTCATCCAGCCTCTAAGACCAAGGTCGATAAGCCTGTGCCTAAGAGGGAGAACGGTAGAGTGCCTAGACCACCTGACTGTCCAAAGTGCCCTGCACCTGAGATGCTGGGTGGACCTTCTGTCTTCATCTTTCCACCTAAGCCTAAAGACACACTGCTGATCGCTCGGACACCTGAGGTCACCTGTGTGGTAGTGGACCTTGATCCAGAAGACCCTGAAGTCCAGATCAGCTGGTTCGTAGACGGCAAGCAGATGCAGACCGCTAAGACACAGCCTAGAGAAGAGCAGTTCAACGGCACCTACAGAGTGGTCAGTGTGTTGCCTATTGGCCATCAAGACTGGCTGAAGGGCAAGCAGTTCACCTGTAAGGTCAACAACAAGGCTTTGCCTAGCCCTATTGAGCGGACAATCAGCAAGGCCAGAGGTCAAGCTCACCAACCAAGCGTGTACGTGTTGCCACCTAGCAGAGAAGAACTGAGCAAGAACACCGTCAGCTTGACATGCCTGATCAAAGACTTCTTTCCACCAGACATCGACGTAGAGTGGCAGAGCAACGGTCAGCAAGAGCCTGAGAGCAAGTACCGGACAACACCACCTCAGCTGGATGAAGATGGCAGCTACTTTCTGTACAGCAAGCTGAGCGTGGACAAGAGCAGATGGCAGAGAGGCGATACCTTCATCTGCGCTGTGATGCACGAAGCCTTGCACAACCACTACACACAAGAGTCTCTGAGCCACTCACCTGGCAAGTGA(SEQ IDNO:62)

[0240] Sp-BsmBI-LC nucleotide sequence:

[0241] GCCACCATGGAGTCACAGACTCAGGTCTTGTATACATGTTGCTGTGGTTGTCTGGTGTTGATGGAGAGACGGGCGGCGGATCTCGTCTCCGGAATGACGCTCAACCAGCTGTCTATCTGTTCCAGCCATCTCCAGACCAGCTGCATACCGGATCAGCCAGCGTCGTGTGCCTTCTGAACTCTTTCTACCCTAAAGACATCAACGTGAAGTGG AAGGTGGACGGAGTGATCCAAGACACCGGCATCCAAGAGAGCGTCACAGAGCAAGACAAAGACAGCACCTATAGCCTGTCCAGCACACTGACAATGTCTAGCACCGAGTATCTGAGTCATGAGCTGTACAGCTGCGAGATCACACATAAATCTCTGCCAAGCACACTCATCAAGAGCTTTCAGAGGTCTGAGTGTCAGAGGGTGGATTAA(SEQ ID NO:63)

[0242] The heavy and light chain variable regions of mouse monoclonal antibodies 8D2 and 9D6 were ligated to the heavy or light chain constant regions of canine IgG molecules, respectively, to form chimeric antibodies. Based on the heavy and light chain variable region sequences of 8D2 and 9D6 obtained in Example 3, forward and reverse amplification primers were designed, and BsmBI restriction enzyme sites were introduced into both primers. Using the cDNA from Example 3 as a template, PCR was performed to amplify each heavy and light chain. The PCR products were cloned into the pTT3-sp-BsmBI-HC and pTT3-sp-BsmBI-LC vectors using the BsmBI enzyme, respectively, to obtain expression plasmids for the heavy chains (pTT3-CA-8D2-H, pTT3-CA-9D6-H) and light chains (pTT3-CA-8D2-L, pTT3-CA-9D6-L) of the chimeric antibodies CA-8D2 and CA-9D6. The recombinant plasmids were sequenced for verification.

[0243] Table 10. Primer sequences for the variable region light and heavy chains:

[0244]

[0245] 2. Preparation of transfection complex

[0246] Preparation of transfection complex (per 1L of cell suspension): Add 500μg of heavy chain expression plasmid and 500μg of light chain expression plasmid to 5mL of Hi-exp medium and mix well by pipetting to obtain phase A; add 3mL of PEI transfection reagent solution to 5mL of Hi-exp medium and mix well by pipetting to obtain phase B; add phase B to phase A, mix well by pipetting, and then incubate at room temperature for 5min.

[0247] 3. Preparation of cell suspension

[0248] Collect 293F cells from the culture, count the cells, centrifuge at 800 rpm for 5 minutes, discard the supernatant, and resuspend in Hi-exp medium to achieve a cell concentration of 1×10⁻⁶ cells / mL. 6 Cells / mL.

[0249] 4. Add the transfection complex dropwise to 1L of cell suspension and incubate with shaking at 130rpm for 5 days (environmental conditions: 37℃, 8% CO2). Then centrifuge at 4000rpm for 20min, collect the supernatant, and filter it through a 0.45μm filter membrane and collect the filtrate.

[0250] 5. Using an affinity chromatography column (column volume 10 mL; packing material proteinAtBeads LX), first equilibrate the column with PBS buffer, then load the filtrate obtained in step 4 (loading volume 1 L), then wash with PBS buffer for 10-15 column volumes, then elute with glycine buffer (pH 2.5-3.0, 0.1 M) and collect the post-column solution. Mix 10 volumes of the post-column solution with 1 volume of neutralization buffer (i.e., pH 9.0, 1 M Tris-HCl buffer) to obtain a mixture.

[0251] 6. Take the mixture obtained in step 5 and concentrate it using a 30K ultrafiltration tube (Millipore, UFC903096). Replace the buffer system with PBS buffer to obtain the genetically engineered antibody solution.

[0252] The expression of chimeric canine IgG antibodies CA-8D2 and CA-9D6 was evaluated using SDS-PAGE. (See electrophoresis images below.) Figure 4 Under both antibody denaturation conditions, only two clear main bands were observed, indicating high purity. The heavy chain band was slightly below 55 kDa, while the light chain band was above 25 kDa. The staining intensity of the heavy chain was approximately twice that of the light chain, indicating successful expression and purification of the chimeric antibody.

[0253] Example 5. In vitro binding assay of chimeric antibodies

[0254] Take canine IL-31 protein and dilute it with PBS buffer to a protein concentration of 1 μg / ml to obtain the coating solution. Preparation of antibody dilution buffer: Take the chimeric antibody solutions CA-8D2 and CA-9D6 prepared in Example 4, first dilute them with PBS buffer to an antibody concentration of 2 μg / ml, and then perform a 2-fold serial dilution with PBS buffer. Detect antibody binding activity according to the following steps:

[0255] 1. Take a 96-well microplate, add coating buffer (100 μL / well), incubate at 4°C for 16 hours (overnight), discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0256] 2. Take the 96-well plate after completing step 1, add blocking solution (200 μL / well), incubate at room temperature for 1 hour, discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0257] 3. Take the 96-well plate from step 2, add antibody dilution buffer (100 μL / well), incubate at room temperature for 1 hour, discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0258] 4. Take the 96-well plate from step 3, add HRP-labeled goat anti-canine IgG (H+L) antibody (SouthernBiotech, catalog number 6070-05) (10000-fold dilution, 100 μL / well), incubate at room temperature for 1 hour, discard the supernatant, wash 5 times with PBST solution, and pat dry.

[0259] 5. Take the 96-well plate after completing step 4, add TMB colorimetric solution (100 μL / well), and react in the dark for 5-10 minutes.

[0260] 6. Take the 96-well plate after completing step 5, add 2M sulfuric acid solution (50μL / well), and then measure the absorbance at 450nm (OD450).

[0261] See results Figure 5 The binding curves of serially diluted CA-8D2 and CA-9D6 to IL-31 proteins largely overlapped, showing no significant difference. A reaction was observed starting at 0.6 ng / mL, and the reaction value increased with increasing antibody concentration, reaching a plateau at approximately 625 ng / mL.

[0262] Example 6. In vivo therapeutic effect of chimeric antibodies

[0263] 1) Select six healthy, 4-6 month old mixed-breed dogs without skin diseases or itching symptoms. Place the experimental animals in individual enclosures and use both manual and video monitoring to identify and score itching behaviors.

[0264] 2) After the experimental animals have been in a single enclosure for ≥1 hour to adapt, each dog was simultaneously assessed and scored for 60 minutes using both manual and video monitoring. The score was used as the “reference baseline”.

[0265] 3) Each experimental dog was intravenously injected with IL-31 at a dose of 4 μg / kg. IL-31 protein can induce a strong itching response in dogs from 20 minutes to 6 hours after injection. Twenty minutes after IL-31 protein injection, the experimental dogs were monitored manually and via video for 2 hours to identify and score itching behavior or to count the number of times they itched.

[0266] Judging itching behavior: Licking / biting the paws, sides of the abdomen and / or anal area, scratching the sides of the abdomen, neck and / or floor, shaking the head, rubbing the buttocks on the cage floor, any of these behaviors occurring within a specified time interval can be rated as "yes".

[0267] Itching behavior assessment and scoring criteria:

[0268] At 1-minute intervals, each dog is judged "yes / no" for whether it is scratching. At the end of the specified time, the number of dogs that answered "yes" is added together to form the "cumulative itching score index".

[0269] All instances of itching during the observation period are counted as "cumulative instances of itching".

[0270] 4) After each observation period, return the dogs to their normal enclosure.

[0271] 5) Subsequently, on day 7, two dogs were intravenously injected with CA-8D2 monoclonal antibody at a dose of 2 mg / kg, two dogs were intravenously injected with CA-9D6 monoclonal antibody, one dog was subcutaneously injected with cytokines (Zotocin, an immunotherapy agent for canine atopic dermatitis) as a positive control, and one dog was injected with saline as a negative control. On days 8 and 15, IL-31 was intravenously injected at a dose of 4 μg / kg to elicit a pruritus response. Simultaneously, 20 minutes after each IL-31 injection, the experimental dogs were monitored manually and via video for 2 hours to assess and score their pruritus behavior (0-120 points).

[0272] Table 11 Results of canine itching behavior scores before medication, 1 day after medication, and 8 days after medication.

[0273]

[0274] Compared with the negative control dogs, injections of IL-31 on days 1 and 8 after intravenous administration of CA-8D2, CA-9D6, and cyproheptadine significantly suppressed pruritus in mixed-breed dogs. Results are shown in Table 11 and... Figure 6As can be seen, both CA-8D2 and CA-9D6 antibodies can significantly improve pruritus response and can be used to treat pruritus and / or allergic diseases in dogs.

[0275] Example 7: Canine-based strategy

[0276] The development of drug-resistant antibodies (ADAs) can lead to a reduction or loss of efficacy for any biological therapeutic protein, including monoclonal antibodies. To minimize the immunogenicity of chimeric antibodies and improve their stability in dogs, further canine-derived modifications were performed on the chimeric antibodies. This primarily involved replacing the frame region (FR) of the variable region (excluding the CDR) of the mouse-derived antibody with a canine sequence. The aim was to retain high-affinity active antibodies using a complete canine frame to minimize the possibility of immunogenicity in vivo.

[0277] Based on the homology with 9D6, the heavy and light chains were screened for canine reproductive sequences in the IGMT database. The FR sequences of the heavy chain variable region and the light chain variable region of 9D6 were replaced with the corresponding canine FR sequences, and canine-derived heavy chain variable region Can-9D6-HV (SEQ ID NO:68) and canine-derived light chain variable region Can-9D6-LV (SEQ ID NO:69) were prepared.

[0278] Canine-derived heavy chain variable region Can-9D6-HV:

[0279] EVQLVQSGAEVKKPGASVKVSCKTSDYAFSDSWVNWVRQAPGAGLDWMGRINLGDGETKYAQKFQGRVTLTADTSSTAYMELSSLRAGDIAVYFCARSGYESPYWYFDVWGQGTLVTVSS(SEQ ID NO:68)

[0280] Canine-derived light chain variable region Can-9D6-LV:

[0281] EIVMTQSPGSLAGSAGESVSINCKSSQSLLYITTQKNQLAWYQQKPGERPKLLIYWASTRESGVPARFSSSGSGTDFTLTINNLQAEDVGDYYCQQYYGYPFTFGQGTKLEIK(SEQ IDNO:69)

[0282] SDS-PAGE was used to identify the quality and purity of the canine-derived 9D6 antibody Can-9D6. Figure 7 It showed no significant difference in band size or staining degree compared to the already marketed product, Cytokine.

[0283] Following the method in Example 5, the in vitro binding curves of Can-9D6, CA-9D6, and cyproheptadine were detected. The three antibodies were serially diluted to the same amount, and their binding curves with IL-31 protein were essentially identical, indicating that the three antibodies have essentially the same affinity for IL-31 protein in vitro. Figure 8 ).

[0284] Example 8: In vitro competition experiment between canine-derived Can-9D6 antibody and cyproheptadine.

[0285] 1. Labeling with cytokines and Can-9D6 antibodies

[0286] 1) Take 2 mg each of the canine-derived antibody Can-9D6 and cytokine prepared in Example 7, with an antibody concentration of 2 mg / mL.

[0287] 2) Take out 2 mg of Sulfo-NHS-LC-Biotin (Thermo 21355) from -20℃, add 360 μL of ultrapure water, and the solution will be biotin solution after complete dissolution.

[0288] 3) Add 27 μL of Biotin solution to each of Can-9D6 and Cytokinin in 1), mix well, and place on ice for 2 hours.

[0289] 4) The above reaction solution was added to a 30K ultrafiltration tube for concentration and buffer replacement. The buffer system was replaced with PBS buffer to obtain biotin-Cetoxamine and biotin-Can-9D6 antibody.

[0290] 2. Can-9D6 antibody and cyproheptadine compete in vitro for ELISA.

[0291] Take canine IL-31 protein and dilute it with PBS buffer to a protein concentration of 1 μg / mL to obtain the coating solution. Antibody dilution preparation method: First, dilute Can-9D6 and Cetirizine separately with PBS buffer to an antibody concentration of 16 μg / mL, then serially dilute each antibody 2-fold with PBS buffer to 0.25 μg / mL. Perform ELISA detection according to the following steps:

[0292] 1) Take a 96-well microplate, add coating buffer (100 μL / well), incubate at 4°C for 16 hours (overnight), discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0293] 2) Take the 96-well plate after completing step 1), add blocking solution (200 μL / well), incubate at room temperature for 1 hour, discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0294] 3) Take the 96-well plate from step 2) and add antibody dilution buffer (100 μL / well) to each well, and incubate at room temperature for 1 hour.

[0295] 4) Take the 96-well plate from step 3) and add 5 μg / mL of biotin-Cetirizine or biotin-Can-9D6 (20 μL / well). Incubate at room temperature with low-speed shaking for 1 hour. Discard the supernatant, wash 5 times with PBST solution, and pat dry.

[0296] 5) Take the 96-well plate from step 4), add HRP-streptavidin (5000-fold dilution, 100 μL / well), incubate at room temperature for 1 hour, discard the supernatant, wash 5 times with PBST solution, and pat dry.

[0297] 6) Take the 96-well plate after completing step 5), add TMB colorimetric solution (100 μL / well), and react in the dark for 5-10 minutes.

[0298] 7) Take the 96-well plate after completing step 6), add 2M sulfuric acid solution (50μL / well), and then measure the absorbance at 450nm (OD450).

[0299] The results are as follows Figure 9 As shown, when using biotin-Cetoxamine for detection, the detection signal value was not affected even when the concentration of Can-9D6 antibody was as high as 16 μg / mL, while the detection signal value of Cetoxamine decreased with increasing concentration and vice versa. This indicates that there is no competition between Can-9D6 and Cetoxamine when binding to IL-31 protein, and it can be inferred that their binding sites are different.

[0300] Example 9: Evaluation of canine 9D6-mu antibody in a canine pruritus model

[0301] Following the method described in Example 6, the antipruritic activity of canine 9D6 (Can-9D6) was evaluated using a canine model of IL-31-induced pruritus. Thirteen healthy, 4-6 month old beagles without skin diseases or pruritus symptoms were selected and divided into three groups: a Can-9D6 group, a cytokine group (n=5 each), and a placebo control group (n=3). Before and after a single subcutaneous injection of 2 mg / kg of Can-9D6 and cytokine (positive control), the animals were repeatedly intravenously injected with 1.75 μg / kg of canine IL-31 protein at one-week intervals to induce pruritus behavior. During each IL-31 challenge period, pruritus behavior was identified and scored simultaneously using both manual and video monitoring, starting 20 minutes after IL-31 protein injection, and observed for 3 hours. All instances of pruritus during the observation period were counted as the "cumulative pruritus count." Another group of dogs received saline injection as a negative control, and the procedure was performed concurrently.

[0302] Figure 10The study showed the cumulative number of itching episodes per dog after each IL-31 attack. Compared to the control group, both the Can-9D6 and cyproheptadine treatments significantly suppressed itching behavior. Therefore, in an IL-31-induced itching model, a single subcutaneous injection of Can-9D6 can significantly suppress itching behavior in dogs and can be used to provide anti-itch protection for dogs with atopic dermatitis and other conditions.

[0303] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An antibody or its antigen-binding fragment that specifically binds to animal IL-31, characterized in that, The antibody or its antigen-binding fragment comprises: a1) The amino acid sequences CDR-H1, CDR-H2, and CDR-H3 in the heavy chain variable region as shown in SEQ ID NO: 1; and the amino acid sequences CDR-L1, CDR-L2, and CDR-L3 in the light chain variable region as shown in SEQ ID NO: 3; or a2) CDR-H1, CDR-H2 and CDR-H3 in the heavy chain variable region as shown in SEQ ID NO: 2; and CDR-L1, CDR-L2 and CDR-L3 in the light chain variable region as shown in SEQ ID NO: 3; The CDR is defined by IMGT, Kabat, Chothia, or the Contact numbering system.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment comprises: b1) A heavy chain variable region comprising amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; and a light chain variable region comprising amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively; wherein the CDRs are defined by the IMGT numbering system, or The heavy chain variable region comprises amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively; and the light chain variable region comprises amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 20, respectively; wherein the CDRs are defined by the Kabat numbering system, or The heavy chain variable region comprises amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 9, respectively; and the light chain variable region comprises amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 20, respectively; wherein the CDRs are defined by the Chothia numbering system, or The heavy chain variable region comprises amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 6, respectively; and the light chain variable region comprises amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, respectively; wherein the CDRs are defined by the Contact numbering system, or b2) Heavy chain variable region, comprising amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 4, SEQ ID NO: 14, and SEQ ID NO: 6; and light chain variable region, comprising amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20; wherein the CDRs are defined by the IMGT numbering system, or The heavy chain variable region comprises amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 7, SEQ ID NO: 15, and SEQ ID NO: 9, respectively; and the light chain variable region comprises amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 20, respectively; wherein the CDRs are defined by the Kabat numbering system, or The heavy chain variable region comprises amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 10, SEQ ID NO: 16, and SEQ ID NO: 9, respectively; and the light chain variable region comprises amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 20, respectively; wherein the CDRs are defined by the Chothia numbering system, or The heavy chain variable region comprises amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 12, SEQ ID NO: 17, and SEQ ID NO: 6, respectively; and the light chain variable region comprises amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, respectively; wherein the CDRs are defined by the Contact numbering system.

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

4. The antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The antibody or its antigen-binding fragment includes: c1) a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having at least 75% sequence identity with it; and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 3, or an amino acid sequence having at least 75% sequence identity with it. c2) a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence having at least 75% sequence identity with it; and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 3, or an amino acid sequence having at least 75% sequence identity with it.

5. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The animal is selected from dogs, cats, or horses.

6. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is a monoclonal antibody.

7. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is a canine, canine-derived, cat, feline-derived, horse, equine-derived, or chimeric antibody.

8. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is of type IgA, IgD, IgE, IgG, or IgM.

9. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibodies are of the IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 types.

10. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody includes the heavy chain constant region and / or light chain constant region derived from animals.

11. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody includes the heavy chain constant region and / or light chain constant region of an antibody derived from dogs.

12. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antigen-binding fragments include scFv, Fab, Fab', (Fab')2, Fv fragment, Fd, and dsFv.

13. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody.

14. The antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The heavy chain variable region of the antibody includes canine-derived framework regions FR-H1, FR-H2, FR-H3 and / or FR-H4, or variants thereof.

15. The antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The heavy chain variable region of the antibody includes: FR-H1 as shown in SEQ ID NO: 70; FR-H2 as shown in SEQ ID NO: 71; FR-H3 as shown in SEQ ID NO: 72; and / or FR-H4 as shown in SEQ ID NO:

73.

16. The antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The heavy chain variable region includes an amino acid sequence as shown in SEQ ID NO: 68, or an amino acid sequence having at least 75% sequence identity with it.

17. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that, The variable regions of the light chain of the antibody include canine-derived framework regions FR-L1, FR-L2, FR-L3 and / or FR-L4, or variants thereof.

18. The antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The light chain variable region of the antibody includes: FR-L1 as shown in SEQ ID NO: 74; FR-L2 as shown in SEQ ID NO: 75; FR-L3 as shown in SEQ ID NO: 76; and / or FR-L4 as shown in SEQ ID NO:

77.

19. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that, The light chain variable region includes an amino acid sequence as shown in SEQ ID NO: 69, or an amino acid sequence having at least 75% sequence identity with it.

20. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof as described in any one of claims 1-19.

21. A carrier, characterized in that, The carrier comprises the nucleic acid molecule of claim 20.

22. A cell, characterized in that, The cell comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1-19, the nucleic acid molecule as described in claim 20, or the vector as described in claim 21.

23. The cell according to claim 22, characterized in that, The cells do not contain reproductive material.

24. A coupling, characterized in that, The conjugate comprises: an antibody or an antigen-binding fragment thereof as described in any one of claims 1-19; and a conjugation portion, wherein the conjugation portion is selected from detectable substances, small molecule drugs, cytokines, antibodies, antibody Fc fragments, antibody scFv fragments, and / or viral capsid proteins.

25. The coupling according to claim 24, characterized in that, The detectable substance is a fluorescent substance, a luminescent marker, or a radioactive substance.

26. The coupling according to claim 25, characterized in that, The detectable substance is selected from any one of acridine ester, acridine sulfonamide, luminol, isoluminol, horseradish peroxidase, alkaline phosphatase, and isotopes.

27. A method for preparing antibodies or antigen-binding fragments thereof, characterized in that, The method includes culturing the cells of claim 22 or 23 and isolating the antibody under conditions that allow expression of the antibody or its antigen-binding fragment.

28. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: an antibody or antigen-binding fragment thereof as described in any one of claims 1-19, a nucleic acid molecule as described in claim 20, or a carrier as described in claim 21, a cell as described in claim 22 or 23, or a conjugate as described in any one of claims 24-26; and a pharmaceutically acceptable carrier.

29. The pharmaceutical composition according to claim 28, characterized in that, The drug composition can be administered via parenteral, injection, or oral routes.

30. The pharmaceutical composition according to claim 28, characterized in that, The pharmaceutical composition is in solid, semi-solid, or liquid form.

31. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-19, the nucleic acid molecule according to claim 20, or the vector according to claim 21, the cell according to claim 22 or 23, the conjugate according to any one of claims 24-26, or the pharmaceutical composition according to any one of claims 28-30, characterized in that, The application includes one or more of the following: d1) Preparation of reagents for detecting the presence or content of IL-31 in a sample; d2) Prepare reagents for the diagnosis or prognostic assessment of IL-31-related diseases in subjects; d3) Prepare a medicine for the prevention or treatment of IL-31-related diseases in subjects, including pruritus, allergy, asthma, atopic dermatitis, eczema, psoriasis, scleroderma, allergic dermatitis, urticaria and psoriasis.

32. The application according to claim 31, characterized in that, The subjects included dogs, cats, or horses.

33. The application according to claim 31, characterized in that, The sample included whole blood from the subject.

34. The application according to claim 31, characterized in that, The sample includes tissue from the subject.

35. The application according to claim 31, characterized in that, The sample included oral secretions from the subject.

36. The application according to claim 31, characterized in that, The samples included bone marrow aspirate, cerebrospinal fluid, feces, urine, cultured cells, and / or nasal secretions from the subject.

37. The application according to claim 31, characterized in that, The samples include red blood cell concentrate, platelet concentrate, white blood cell concentrate, plasma, serum and / or saliva from the subject.

Images