Antibodies against IL-31 and uses thereof

Abstract

The present disclosure relates to an anti-IL-31 antibody and uses thereof. Specifically, provided is an antibody specifically binding to canine IL-31 or an antigen binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises an amino acid sequence shown in any one of SEQ ID NO: 1-10 or an HCDR1 of any variant thereof, an amino acid sequence shown in any one of SEQ ID NO: 11-20 or an HCDR2 of any variant thereof, and an amino acid sequence shown in any one of SEQ ID NO: 21-30 or an HCDR3 of any variant thereof; the light chain variable region comprises an amino acid sequence shown in any one of SEQ ID NO: 31-40 or an LCDR1 of any variant thereof, an amino acid sequence shown in any one of SEQ ID NO: 41-50 or an LCDR2 of any variant thereof, and an amino acid sequence shown in any one of SEQ ID NO: 51-60 or an LCDR3 of any variant thereof. The anti-canine IL-31 antibody disclosed by the invention has better affinity compared with cetumazine, and can effectively relieve clinical symptoms related to specific dermatitis of model animals.

Description

Technical Field

[0001] This disclosure relates to the fields of genetic engineering and immunology, and specifically to an antibody against IL-31 and its uses. Background Technology

[0002] Canine atopic dermatitis is a genetically predisposed inflammatory and itchy allergic skin disease in dogs. It is estimated that approximately 450 million dogs worldwide suffer from atopic dermatitis, with an incidence rate as high as 10%–15% (Kinga Gortel et al., Can Vet J, 2018 Sep; 59(9):1013-1016). In the UK, due to genetic factors, Labradors and Golden Retrievers have an almost 50% risk of developing atopic dermatitis. Certain breeds, such as Golden Retrievers, Labradors, Pit Bulls, Pugs, Boxers, and German Shepherds, have a significantly higher risk than other breeds, and breed susceptibility varies geographically (Natalie Katharina YvonneGedon et al., Clin Transl Allergy, 2018 Oct; 8:41). Atopic dermatitis typically develops between 6 months and 3 years of age. Food, environmental allergens, and genetic factors are the main causes of atopic dermatitis, which differs from parasitic allergies. Affected dogs often present with erythema and itching, with secondary symptoms including pain and inflammatory infections. Itching and inflammatory infections can lead to spontaneous hair loss, abrasions, and secondary infections, manifesting as papules, pustules, and crusts. Common sites of onset include the armpits, abdomen, distal extremities, inner ear, and the perioral, perioral, and perianal areas.

[0003] Dogs with atopic dermatitis have a defective skin barrier. Allergens can penetrate the skin through these defects and interact with immune cells, which then release various itch-inducing chemicals, resulting in itching. During an allergic reaction, multiple other chemicals are released simultaneously, leading to inflammation on the skin. This inflammation further degrades the barrier function, increasing allergen permeability and causing secondary infections and bacterial infections. IL-31 is the most important cytokine associated with atopic inflammation. Others, such as IL-4, IL-13, and TNF-α, can also affect lipid synthesis, further weakening the epidermal barrier function (Jackeline Franco et al., Metabolit, 2021 Sep 30; 11(10):670). Serum L-31 concentration is closely related to the severity of canine skin lesions. When dogs are exposed to allergens, helper T cells (Th2) in the skin produce the cytokine IL-31. IL-31 binds to a co-receptor composed of IL-31 receptor A (IL-31RA) and oncogene M receptor (OSMR), which activates the STAT signaling cascade via Janus kinase phosphorylation, leading to upregulation of target genes and causing itching behavior in dogs (Eniko Sonkoly et al., J Allergy Clin Immunol, 2006 Feb; 117(2):411-417).

[0004] In addition, IL-31 can stimulate the production of inflammatory mediators by promoting epithelial cell responses, as demonstrated in transgenic mice overexpressing IL-31. The expression of IL-31 receptor mRNA is highest in the dorsal root ganglion. IL-31 can coordinate the interaction between immune cells (T cells, mast cells, eosinophils) and epithelial cells (Jan J. Cornelissen et al., Nat Rev Clin Oncol, 2012 Oct; 9(10):579-590). IL-31 concentrations are elevated under pruritic allergic skin conditions and can induce scratching behavior in various species (e.g., mice, monkeys, and dogs). Studies on laboratory beagles have shown that when IL-31 is administered via several routes (intradermal, subcutaneous, and intravenous), it induces intense itching behavior within minutes to hours, suggesting that experimental models of IL-31 administration via injection can be used to evaluate the antipruritic effect of canine therapeutics in dogs (Matthew Krautmann et al., Vet Immunol Immunopathol, 2023 Apr; 258:110574).

[0005] Therefore, canine atopic dermatitis is not a single disease, but a complex syndrome composed of multiple subtypes. Advances in the monitoring and treatment of atopic dermatitis may include the use of the Canine Atopic Dermatitis Severity Index (CADESI) to assess lesion severity, evaluate pruritus, and measure skin biophysical parameters (JR Calesso et al., Pol J Vet Sci, 2023 Jun; 26(2):231-238). Current chemical treatments include corticosteroids, cyclosporine, essential fatty acids, and antihistamines. For long-term treatment, cyclosporine may have a slow onset of action and increase costs for pet owners. While corticosteroids can effectively control allergic pruritus and inflammation in the short term, they can cause secondary adverse reactions with a high incidence rate, and are therefore not suitable for long-term treatment (Clarissa P Souza et al., Vet Dermatol, 2018 Dec; 29(6):489). In addition, desensitization therapy can be used to treat allergic reactions. This involves injecting the allergen 1-2 times per month for 1-2 years. This reduces allergic reactions and increases the body's tolerance to the allergen. It is a non-drug treatment, harmless to the body, and has a low probability of preventing the dog from ever being allergic to that allergen again. However, desensitization therapy should only be performed by a dermatology specialist veterinarian.

[0006] Currently, the latest treatment for canine atopic dermatitis involves the use of selective Janus kinase 1 inhibitors and canine anti-IL-31 monoclonal antibodies. Anti-IL-31 antibodies have been shown to reduce or eliminate the pruritus associated with IL-31 in NC / Nga mice (a mouse model of human atopic dermatitis). Anti-canine IL-31 antibody (Cetirizine) has been proven effective in treating canine atopic dermatitis and allergic dermatitis in 87.8% of cases (Gober M et al., Front Vet Sci, 2022 July; 9:909776). Zoetis' exclusive product, Cetirizine, has achieved retail sales of $800-1 billion in three years since its launch. Currently, the domestic market specification is 20mg (1ml) / vial, with a market price of around 600 yuan. Due to its current monopoly in the domestic market, the treatment cost for pets is relatively high. The dosage of Cetirizine is 2mg / kg. For dogs weighing over 30kg, more than three 20mg (1ml) / vial injections are required subcutaneously, which is inconvenient for clinical use.

[0007] Given the challenges posed by the expensive diagnostic tests, frequent clinical flare-ups, and lifelong treatment required for canine atopic dermatitis, there is an urgent need to develop a new, more effective, safer, and less expensive canine IL-31 antibody to improve the quality of life for pet dogs. Summary of the Invention

[0008] In order to address the problems existing in the prior art, the purpose of this disclosure is to provide an antibody against IL-31 or its antigen-binding fragment, its preparation method and its pharmaceutical use.

[0009] To achieve the above objectives, this disclosure adopts the following specific solutions:

[0010] In one aspect, this disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to canine IL-31, comprising a heavy chain variable region and a light chain variable region, wherein,

[0011] The heavy chain variable region comprises: HCDR1 having an amino acid sequence shown in any one of SEQ ID NO:1-10 or any variant thereof, HCDR2 having an amino acid sequence shown in any one of SEQ ID NO:11-20 or any variant thereof, and HCDR3 having an amino acid sequence shown in any one of SEQ ID NO:21-30 or any variant thereof.

[0012] The light chain variable region comprises: LCDR1 having an amino acid sequence shown in any one of SEQ ID NO:31-40 or any variant thereof, LCDR2 having an amino acid sequence shown in any one of SEQ ID NO:41-50 or any variant thereof, and LCDR3 having an amino acid sequence shown in any one of SEQ ID NO:51-60 or any variant thereof.

[0013] On the other hand, this disclosure provides a biological material selected from any of the following:

[0014] (1) A polynucleotide encoding the aforementioned antibody or its antigen-binding fragment; or

[0015] (2) An expression vector containing the polynucleotide described in (1); or

[0016] (3) A host cell containing the polynucleotide described in (1) or the expression vector described in (2).

[0017] On the other hand, this disclosure provides a method for producing an antibody or antigen-binding fragment thereof that specifically binds to canine IL-31, comprising the steps of: culturing the aforementioned host cells, isolating the antibody from the cultured species, and purifying the antibody.

[0018] On the other hand, this disclosure provides a pharmaceutical composition comprising the aforementioned antibody or its antigen-binding fragment, and a pharmaceutically acceptable excipient, diluent, or carrier.

[0019] On the other hand, this disclosure provides the use of the aforementioned antibody or its antigen-binding fragment, the aforementioned biological material, the aforementioned pharmaceutical composition, and the aforementioned kit in the preparation of a medicament for treating pruritus or allergies in dogs.

[0020] On the other hand, this disclosure provides the use of the aforementioned antibody or its antigen-binding fragment, the aforementioned biological material, the aforementioned pharmaceutical composition, and the aforementioned kit in the preparation of a medicament for reducing, inhibiting, or neutralizing IL-31 activity in dogs.

[0021] Compared with the prior art, this disclosure has at least the following beneficial effects:

[0022] The disclosed anti-canine IL-31 antibody or its antigen-binding fragment specifically binds to canine IL-31, exhibiting comparable or better affinity compared to existing commercial products like Cetirizine. It has been validated to possess good biological activity in both cells and animals, effectively alleviating clinical symptoms associated with atopic dermatitis in canine pruritus models. Furthermore, the modified canine antibody carries a low risk of immunogenicity. Attached Figure Description

[0023] Figure 1 Results of SDS-PAGE electrophoresis for canine IL-31.

[0024] Figure 2 ELISA results comparing B cell culture supernatant with cyproheptadine (positive control).

[0025] Figure 3 This is an SDS-PAGE image of the rabbit-derived antibody. Columns 1-10 represent the following groups: 38E2-1 (non-reduced), 47C1-1 (non-reduced), 35C9-1 (non-reduced), 19D1-1 (non-reduced), 5H4-2 (non-reduced), 38E2-1 (reduced), 47C1-1 (reduced), 35C9-1 (reduced), 19D1-1 (reduced), and 5H4-2 (reduced). M represents the marker group.

[0026] Figure 4 The image shows the Western blot (WB) results of the rabbit antibody candidates. Columns 1-5 represent groups 38E2-1 (non-reducing), 47C1-1 (non-reducing), 35C9-1 (non-reducing), 19D1-1 (non-reducing), and 5H4-2 (non-reducing), respectively.

[0027] Figure 5 The graph shows the BLI affinity assay for rabbit-derived antibodies.

[0028] Figure 6 Scoring of canine pruritus models mediated by different doses of IL-31.

[0029] Figure 7 The pruritus score 8 days after administration of cyproheptadine indirectly confirmed the successful establishment of the canine pruritus model. Simultaneously, cyproheptadine was able to inhibit IL-31-mediated pruritus in the model dogs.

[0030] Figure 8 Itching scores in a canine pruritus model after treatment with rabbit-derived antibodies.

[0031] Figure 9 The strategy for constructing canine-derived antibodies.

[0032] Figure 10 This is an SDS-PAGE result image of the chimeric antibody. Columns 1-6 represent the groups 38E2-1 (non-reduced), 47C1-1 (non-reduced), 35C9-1 (non-reduced), 38E2-1 (reduced), 47C1-1 (reduced), and 35C9-1 (reduced), respectively.

[0033] Figure 11 This is a graph showing the Western blot (WB) results of the chimeric antibody. Columns 1-3 represent the 38E2-1 (non-reducing), 47C1-1 (non-reducing), and 35C9-1 (non-reducing) groups, respectively.

[0034] Figure 12 This is the ELISA result for chimeric antibodies. "Weighting fixed" indicates fixed weighting.

[0035] Figure 13 The results are the assay results of the cell biological activity of the chimeric antibody.

[0036] Figure 14 Itching score for a canine pruritus model after treatment with chimeric antibody.

[0037] Figure 15 This is an SDS-PAGE result of the canine-derived antibody. Figure 15 The first eight columns of the electrophoresis image on the left are groups 38E2-1-H3L3 (non-reduced), 38E2-1-H3L2 (non-reduced), 47C1-1-H2L2 (non-reduced), 47C1-1-H2L3 (non-reduced), 38E2-1-H3L3 (reduced), 38E2-1-H3L2 (reduced), 47C1-1-H2L2 (reduced), and 47C1-1-H2L3 (reduced). Figure 15 In the electrophoresis image on the right, columns 1-4 represent groups 35C9-1-H1L1 (non-reduced), 35C9-1-H1L2 (non-reduced), 35C9-1-H1L1 (reduced), and 35C9-1-H1L2 (reduced), respectively.

[0038] Figure 16 This is a Western blot (WB) image showing the results of canine-derived antibodies. Figure 16 The first four columns of the electrophoresis image on the left are groups 38E2-1-H3L3 (non-reducing), 38E2-1-H3L2 (non-reducing), 47C1-1-H2L2 (non-reducing), and 47C1-1-H2L3 (non-reducing), respectively. Figure 16 The first and second columns of the electrophoresis image on the right are groups 35C9-1-H1L1 (non-reduced) and 35C9-1-H1L2 (non-reduced), respectively.

[0039] Figure 17 This is the result of an ELISA assay for canine-derived antibodies. "Weighting fixed" indicates a fixed weighting.

[0040] Figure 18 The results are the results of the cell biological activity assay for canine-derived antibodies.

[0041] Figure 19 Itching scores for a canine pruritus model after treatment with canine-derived antibodies.

[0042] Figure 20 The results validated the drug efficacy within 28 hours after administration of different concentrations of canine-derived antibody 38E2-1-H3L3.

[0043] Figure 21 The results are from repeated dosing of 9 mg / kg canine antibody 38E2-1-H3L3 to determine blood drug concentration. Detailed Implementation

[0044] To more clearly illustrate the technical solutions in the embodiments of this disclosure or the prior art, the terms and implementation methods used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the implementation methods described below are only one implementation method of this disclosure. For those skilled in the art, other implementation methods can be obtained based on these drawings.

[0045] I. Terminology

[0046] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0047] The articles “a” and “a kind” used in this article refer to one or more (i.e., at least one) grammatical objects. For example, “a component” means one or more components.

[0048] As used herein, the term "about" indicates and covers a specified value and a range greater than and less than that value. In some embodiments, the term "about" may indicate a variation of ±0.1%, ±0.5%, ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%. In some embodiments, where applicable, the term "about" indicates a specified value ± one standard deviation of that value.

[0049] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0050] The term “consistently of” or variations thereof, used throughout the specification and claims, means that all said components or groups of components are included, and optionally include other components that are similar to or different in nature from said components, which do not significantly alter or introduce new properties to the specified dosing regimen, method or composition.

[0051] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

[0052] The term "antibody" refers to any form of antibody that exhibits desired biological or binding activity. Therefore, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, humanized, fully human antibodies, and chimeric antibodies.

[0053] Typically, the basic antibody structural unit comprises a tetramer. Each tetramer consists of two pairs of identical polypeptide chains, each pair having a "light" chain (approximately 25 kDa) and a "heavy" chain (approximately 50–70 kDa). The amino-terminal portion of each chain includes a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxyl-terminal portion of the heavy chain defines a constant region primarily responsible for effector function. Human light chains are typically classified as kappa light chains and lambda light chains. Furthermore, human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. In both the light and heavy chains, the variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also includes a "D" region of approximately 10 or more amino acids. See Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)).

[0054] The variable region of each light / heavy chain pair forms the antibody binding site. Therefore, in general, a complete antibody has two binding sites. Except for bifunctional or bispecific antibodies, these two binding sites are usually the same.

[0055] Typically, both heavy and light chains have variable regions containing three highly variable regions, also known as complementarity-determining regions, located within relatively conservative frame regions. CDRs are usually aligned through frame regions, enabling them to bind specific epitopes. Generally, from the N-terminus to the C-terminus, the variable regions of both light and heavy chains include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The allocation of amino acids to each domain generally conforms to the following definition: Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32: 1-75; Kabat, et al., (1977) J. Biol. Chem. 252: 6609-6616; Chothia, et al., (1987) J Mol. Biol. 196: 901-917 or Chothia, et al., (1989) Nature 342: 878-883.

[0056] As used herein, "antibody fragment" or "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, that is, an antibody fragment that retains the ability to specifically bind to an antigen that binds to the full-length antibody, such as a fragment that retains one or more CDR regions. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments.

[0057] As used in this article, "CDR" or "CDR" refers to the complementarity-determining region in the variable region of immunoglobulins, and is usually defined using the Kabat numbering system.

[0058] As used herein, an antibody that "specifically binds" to a particular target protein is an antibody that exhibits preferential binding to that target compared to other proteins, but this specificity does not require absolute binding specificity. An antibody is considered "specific" to its intended target if its binding determines the presence of the target protein in the sample, for example, without producing undesirable results such as false positives. The antibodies or their binding fragments used in this invention will bind to the target protein with an affinity at least two times, preferably at least ten times, more preferably at least 20 times, and most preferably at least 100 times higher than that with non-target proteins. As used herein, an antibody is said to specifically bind to a polypeptide containing a given amino acid sequence, such as the amino acid sequence of a mature human PD-1 or human PD-L1 molecule, but not to a protein lacking that sequence.

[0059] As used herein, a "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species (e.g., human) or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species (e.g., mouse) or belonging to another antibody class or subclass, and fragments of such antibodies, provided they exhibit the desired biological activity.

[0060] As used herein, a “variant of conserved modification” or “conservative substitution” refers to the substitution of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity), allowing for frequent alterations without changing the protein’s biological activity or other desired properties (e.g., antigen affinity and / or specificity). Those skilled in the art will recognize that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not significantly alter its biological activity (see Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to disrupt biological activity. Exemplary conserved substitutions are listed in Table 1.

[0061] Table 1. Exemplary Conserved Amino Acid Replacements

[0062] Original residues Conservative replacement Ala(A) Gly;Ser Arg(R) Lys;His Asn(N) Gln;His Asp(D) Glu;Asn Cys(C) Ser;Ala Gln(Q) Asn Glu(E) Asp; Gln Gly(G) Ala His(H) Asn;Gln Ile(I) Leu; Val Leu(L) Ile; Val Lys(K) Arg; His Met(M) Leu; Ile; Tyr Phe(F) Tyr; Met; Leu Pro(P) Ala Ser(S) Thr Thr(T) Ser Trp(W) Tyr; Phe Tyr(Y) Trp; Phe Val(V) Ile; Leu

[0063] As used herein, the terms “cell,” “cell line,” and “cell culture” are used interchangeably, and all such names include their progeny. Therefore, the words “transformer” and “transformed cell” include the primary test cell and the cultures derived from it, regardless of the number of transfections. It should also be understood that, due to intentional or unintentional mutations, all progeny cannot be exactly identical in terms of DNA content. This includes mutant progeny with the same function or biological activity as those screened in the original transformed cells. Where different names are intended, this is clearly evident from the context. In the context of expressing a heterologous nucleic acid sequence, “host cell” refers to a prokaryotic or eukaryotic cell (e.g., bacterial cells, yeast cells, mammalian cells, and insect cells) whether in vitro or in vivo. For example, a host cell can be located within a transgenic animal. A host cell can serve as a recipient of a vector and can include any transformable organism capable of replicating the vector and / or expressing the heterologous nucleic acid encoded by the vector.

[0064] As used herein, the term "pharmaceutical composition" refers to a composition administered to a mammalian patient (preferably a canine patient). In a preferred embodiment, the pharmaceutical composition comprises a composition for parenteral injection or infusion. This parenteral injection or infusion can take advantage of a reabsorption process, such as intradermal, subcutaneous, intramuscular, and / or intraperitoneal injection or infusion. Alternatively, the parenteral injection or infusion can bypass the reabsorption process and is in the form of intracardiac, intraarterial, intravenous, intralumbar, and / or intramembranous injection or infusion. In another preferred embodiment, the pharmaceutical composition comprises a composition for transdermal administration. An example of transdermal administration is epidermal administration, wherein the pharmaceutical composition is administered in the form of a solution, suspension, emulsion, foam, ointment, cream, paste, and / or patch applied to the skin. Alternatively, administration of the pharmaceutical composition can be achieved through one or more mucous membranes. For example, administration may be buccal, lingual, or sublingual, i.e., through the mucous membranes of the mouth and / or tongue, and may take the form of tablets, lozenges, sugar-coated tablets (i.e., sugar-coated pills), and / or mouthwash. Alternatively, administration may be enteric, i.e., through the gastric and / or intestinal mucosa, and may take the form of tablets, sugar-coated tablets (i.e., sugar-coated pills), capsules, solutions, suspensions, and / or emulsions. Alternatively, administration may be rectal, and may take the form of suppositories, rectal capsules, and / or ointments or creams. Alternatively, administration may be intranasal, and may take the form of drops, ointments or creams, and / or sprays. Alternatively, administration may be pulmonary, i.e., through the bronchi and / or alveoli, and may take the form of aerosols and / or inhalers. Alternatively, administration may be conjunctival, and may take the form of eye drops, eye ointments, and / or eye washes. Optionally, administration can be achieved through the mucous membranes of the genitourinary tract, such as vaginally or urethra, and can take the form of suppositories, ointments, and / or pens. It should be understood that the above-mentioned alternative forms of administration are not mutually exclusive and can be combined in any number to form an effective treatment regimen.

[0065] The pharmaceutical compositions disclosed herein may further include pharmaceutically acceptable carriers. Examples of suitable pharmaceutical carriers are those already known, including phosphate-buffered saline solutions, water, emulsions (such as oil / water emulsions), various wetting agents, sterile solutions, etc. Compositions containing these carriers can be formulated using known conventional methods. These pharmaceutical compositions can be administered to subjects at appropriate doses. Dosing regimens can be determined by the participating physicians and clinical factors. As is known in the medical field, the dose for any given patient depends on many factors, including the patient's size, body surface area, age, the specific compound being administered, sex, time and route of administration, overall health status, and other drugs being administered concurrently. For example, parenteral formulations include sterile water or non-aqueous solutions, suspensions, emulsions, and liposomes. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable organic lipids (such as ethyl oleate). Carriers include sodium chloride solutions, Ringer's glucose, glucose and sodium chloride, lactated Ringer's, or non-volatile oils. Suitable carriers for intravenous or intra-arterial administration include fluids and nutrient supplements, electrolyte supplements (such as those based on Ringer's glucose), etc. Preservatives and other additives may also be included, such as antimicrobial, antioxidant, chelating agents, inert gases, etc. Additionally, the pharmaceutical compositions of the present invention may contain protein carriers, such as serum albumin or immunoglobulins, preferably human. It is of interest that, in addition to humanized monoclonal antibodies or fragments thereof (as described herein), the pharmaceutical compositions of the present invention may also contain other bioactive agents, depending on the intended use of the pharmaceutical composition. These agents may be drugs acting on the gastrointestinal system, drugs as cell inhibitors, drugs preventing polyuricemia, drugs inhibiting immune responses (such as corticosteroids), drugs modulating inflammatory responses, drugs acting on the circulatory system, and / or existing known agents such as cytokines.

[0066] As used herein, "object" or "patient" refers to an animal in need of treatment that can be affected by the molecules of the present invention. Animals that can be treated according to the present invention include vertebrates, with mammals such as dogs, cats, and equines being particularly preferred examples.

[0067] As used herein, a “therapeutic effective amount” (or “effective amount”) refers to an amount of active ingredient (e.g., the pharmaceutical agent of the present invention) sufficient to produce a beneficial or desired result when applied to a subject or patient. An effective amount may be administered as a single or multiple dose, application, or dosage. The therapeutic effective amount of the compositions of the present invention can be readily determined by those skilled in the art. In the context of the present invention, a “therapeutic effective amount” is an amount that produces an objectively observed change in one or more parameters related to the treatment of pruritus or allergic diseases (including clinical improvement of symptoms). Of course, the therapeutic effective amount will vary depending on the specific subject and disease being treated, the subject’s weight and age, the severity of the disease symptoms, the specific compound chosen, the dosing regimen to be followed, the time of administration, the route of administration, etc., all of which can be readily determined by those skilled in the art.

[0068] As used herein, “therapeutic” encompasses the comprehensive treatment of a disease or condition. The “therapeutic” agents of this invention may act in a preventative or protective manner, including comprising a procedure designed for animals identified as at risk (pharmacologically). They may also act in a modified or substantially therapeutic manner, or may act to slow the rate or extent of progression of at least one symptom of the disease or condition to be treated. “Treatment,” “treating,” etc., refer to therapeutic treatments and preventative or protective measures. Animals requiring treatment include those already suffering from the condition and those for which prevention is desired. The terms “treat” or “cure” a disease or condition include prevention or protection against the disease or condition (i.e., preventing the development of clinical symptoms); suppression of the disease or condition (i.e., halting or inhibiting the development of clinical symptoms); and / or relief of the disease or condition (i.e., causing the clinical symptoms to subside). As to be understood, it is not always possible to distinguish between “prevention” and “suppression” of a disease or condition, as the eventual triggering event may be unknown or potential. Therefore, the term "prevention" should be understood as constituting a class of treatments that encompass both "prevention" and "inhibition." The term "treatment" thus includes "prevention."

[0069] As used herein, the term "allergic disease" is defined as a condition or illness resulting from the interaction between the immune system and external substances. These external substances are called "allergens." Common allergens include airborne allergens such as pollen, dust, mold, dust mite proteins, and saliva injected through insect bites. Examples of allergic diseases include, but are not limited to, the following: allergic dermatitis, summer eczema, urticaria, equine respiratory distress syndrome, inflammatory airway diseases, recurrent airway obstruction, airway hyperresponsiveness, chronic obstructive pulmonary disease, and autoimmune inflammatory processes such as irritable bowel syndrome (IBS).

[0070] As used herein, the terms "pruritus" and "pruritus" are defined as diseases or conditions characterized by an intense itching sensation that produces an urge to rub or scratch the skin for relief. Examples of pruritus include, but are not limited to, atopic dermatitis, eczema, psoriasis, scleroderma, and pruritus.

[0071] II. Detailed Implementation Plan

[0072] In one aspect, this disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to canine IL-31, comprising a heavy chain variable region and a light chain variable region, wherein,

[0073] The heavy chain variable region comprises: HCDR1 having an amino acid sequence shown in any one of SEQ ID NO:1-10 or any variant thereof, HCDR2 having an amino acid sequence shown in any one of SEQ ID NO:11-20 or any variant thereof, and HCDR3 having an amino acid sequence shown in any one of SEQ ID NO:21-30 or any variant thereof.

[0074] The light chain variable region comprises: LCDR1 having an amino acid sequence shown in any one of SEQ ID NO:31-40 or any variant thereof, LCDR2 having an amino acid sequence shown in any one of SEQ ID NO:41-50 or any variant thereof, and LCDR3 having an amino acid sequence shown in any one of SEQ ID NO:51-60 or any variant thereof.

[0075] In some embodiments, the heavy chain variable region of the antibody or its antigen-binding fragment includes:

[0076] (1) HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:11, and HCDR3 shown in SEQ ID NO:21; or,

[0077] (2) HCDR1 shown in SEQ ID NO:2, HCDR2 shown in SEQ ID NO:12, and HCDR3 shown in SEQ ID NO:22; or,

[0078] (3) HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:13, and HCDR3 shown in SEQ ID NO:23; or,

[0079] (4) HCDR1 shown in SEQ ID NO:4, HCDR2 shown in SEQ ID NO:14, and HCDR3 shown in SEQ ID NO:24; or,

[0080] (5) HCDR1 shown in SEQ ID NO:5, HCDR2 shown in SEQ ID NO:15, and HCDR3 shown in SEQ ID NO:25; or,

[0081] (6) HCDR1 shown in SEQ ID NO:6, HCDR2 shown in SEQ ID NO:16, and HCDR3 shown in SEQ ID NO:26; or,

[0082] (7) HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:17, and HCDR3 shown in SEQ ID NO:27; or,

[0083] (8) HCDR1 shown in SEQ ID NO:8, HCDR2 shown in SEQ ID NO:18, and HCDR3 shown in SEQ ID NO:28; or,

[0084] (9) HCDR1 shown in SEQ ID NO:9, HCDR2 shown in SEQ ID NO:19, and HCDR3 shown in SEQ ID NO:29; or,

[0085] (10) HCDR1 shown in SEQ ID NO:10, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:30;

[0086] The light chain variable region includes:

[0087] (1) LCDR1 shown in SEQ ID NO:31, LCDR2 shown in SEQ ID NO:41, and LCDR3 shown in SEQ ID NO:51; or,

[0088] (2) LCDR1 shown in SEQ ID NO:32, LCDR2 shown in SEQ ID NO:42, and LCDR3 shown in SEQ ID NO:52; or,

[0089] (3) LCDR1 shown in SEQ ID NO:33, LCDR2 shown in SEQ ID NO:43, and LCDR3 shown in SEQ ID NO:53; or,

[0090] (4) LCDR1 shown in SEQ ID NO:34, LCDR2 shown in SEQ ID NO:44, and LCDR3 shown in SEQ ID NO:54; or,

[0091] (5) LCDR1 shown in SEQ ID NO:35, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:55; or,

[0092] (6) LCDR1 shown in SEQ ID NO:36, LCDR2 shown in SEQ ID NO:46, and LCDR3 shown in SEQ ID NO:56; or,

[0093] (7) LCDR1 shown in SEQ ID NO:37, LCDR2 shown in SEQ ID NO:47, and LCDR3 shown in SEQ ID NO:57; or,

[0094] (8) LCDR1 shown in SEQ ID NO:38, LCDR2 shown in SEQ ID NO:48, and LCDR3 shown in SEQ ID NO:58; or,

[0095] (9) LCDR1 shown in SEQ ID NO:39, LCDR2 shown in SEQ ID NO:49, and LCDR3 shown in SEQ ID NO:59; or,

[0096] (10) LCDR1 shown in SEQ ID NO:40, LCDR2 shown in SEQ ID NO:50, and LCDR3 shown in SEQ ID NO:60.

[0097] In some embodiments, the antibody or its antigen-binding fragment,

[0098] (1) The heavy chain variable region includes HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:11, and HCDR3 shown in SEQ ID NO:21; the light chain variable region includes LCDR1 shown in SEQ ID NO:31, LCDR2 shown in SEQ ID NO:41, and LCDR3 shown in SEQ ID NO:51; or,

[0099] (2) The heavy chain variable region includes HCDR1 shown in SEQ ID NO:2, HCDR2 shown in SEQ ID NO:12, and HCDR3 shown in SEQ ID NO:22; the light chain variable region includes LCDR1 shown in SEQ ID NO:32, LCDR2 shown in SEQ ID NO:42, and LCDR3 shown in SEQ ID NO:52; or,

[0100] (3) The heavy chain variable region includes HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:13, and HCDR3 shown in SEQ ID NO:23; the light chain variable region includes LCDR1 shown in SEQ ID NO:33, LCDR2 shown in SEQ ID NO:43, and LCDR3 shown in SEQ ID NO:53; or,

[0101] (4) The heavy chain variable region includes HCDR1 shown in EQ ID NO:4, HCDR2 shown in SEQ ID NO:14, and HCDR3 shown in SEQ ID NO:24; the light chain variable region includes LCDR1 shown in SEQ ID NO:34, LCDR2 shown in SEQ ID NO:44, and LCDR3 shown in SEQ ID NO:54; or,

[0102] (5) The heavy chain variable region includes HCDR1 shown in SEQ ID NO:5, HCDR2 shown in SEQ ID NO:15, and HCDR3 shown in SEQ ID NO:25; the light chain variable region includes LCDR1 shown in SEQ ID NO:35, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:55; or,

[0103] (6) The heavy chain variable region includes HCDR1 shown in SEQ ID NO:6, HCDR2 shown in SEQ ID NO:16, and HCDR3 shown in SEQ ID NO:26; the light chain variable region includes LCDR1 shown in SEQ ID NO:36, LCDR2 shown in SEQ ID NO:46, and LCDR3 shown in SEQ ID NO:56; or,

[0104] (7) The heavy chain variable region includes HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:17, and HCDR3 shown in SEQ ID NO:27; the light chain variable region includes LCDR1 shown in SEQ ID NO:37, LCDR2 shown in SEQ ID NO:47, and LCDR3 shown in SEQ ID NO:57; or,

[0105] (8) The heavy chain variable region includes HCDR1 shown in SEQ ID NO:8, HCDR2 shown in SEQ ID NO:18, and HCDR3 shown in SEQ ID NO:28; the light chain variable region includes LCDR1 shown in SEQ ID NO:38, LCDR2 shown in SEQ ID NO:48, and LCDR3 shown in SEQ ID NO:58; or,

[0106] (9) The heavy chain variable region includes HCDR1 shown in SEQ ID NO:9, HCDR2 shown in SEQ ID NO:19, and HCDR3 shown in SEQ ID NO:29; the light chain variable region includes LCDR1 shown in SEQ ID NO:39, LCDR2 shown in SEQ ID NO:49, and LCDR3 shown in SEQ ID NO:59; or,

[0107] (10) The heavy chain variable region includes HCDR1 shown in SEQ ID NO:10, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:30; the light chain variable region includes LCDR1 shown in SEQ ID NO:40, LCDR2 shown in SEQ ID NO:50, and LCDR3 shown in SEQ ID NO:60.

[0108] In some embodiments, the antibody or its antigen-binding fragment, (1) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:11, and HCDR3 shown in SEQ ID NO:21; the light chain variable region comprises LCDR1 shown in SEQ ID NO:31, LCDR2 shown in SEQ ID NO:41, and LCDR3 shown in SEQ ID NO:51; or,

[0109] (2) The heavy chain variable region includes HCDR1 shown in SEQ ID NO:2, HCDR2 shown in SEQ ID NO:12, and HCDR3 shown in SEQ ID NO:22; the light chain variable region includes LCDR1 shown in SEQ ID NO:32, LCDR2 shown in SEQ ID NO:42, and LCDR3 shown in SEQ ID NO:52; or,

[0110] (3) The heavy chain variable region includes HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:13, and HCDR3 shown in SEQ ID NO:23; the light chain variable region includes LCDR1 shown in SEQ ID NO:33, LCDR2 shown in SEQ ID NO:43, and LCDR3 shown in SEQ ID NO:53.

[0111] In some embodiments, the antibody or its antigen-binding fragment is selected from any one of rabbit antibodies or their antigen-binding fragments, chimeric antibodies or their antigen-binding fragments, or canine antibodies or their antigen-binding fragments.

[0112] In some embodiments, the antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment that specifically binds to canine IL-31 comprises a heavy chain selected from the sequences shown below, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with the following sequences: SEQ ID NO: 61-70, 163-165, 175-180; and / or

[0113] It includes light chains selected from the sequences shown below, or light chains that have at least 80%, 85%, 90%, 95% or 99% identity with the sequences shown below: SEQ ID NO:71-80, 166-168, 181-186.

[0114] In some embodiments, the antibody or its antigen-binding fragment, wherein,

[0115] (1) The heavy chain comprises the amino acid sequence described in SEQ ID NO:61 or any variant thereof; the light chain comprises the amino acid sequence described in SEQ ID NO:71 or any variant thereof; or,

[0116] (2) The heavy chain comprises the amino acid sequence described in SEQ ID NO:62 or any variant thereof; the light chain comprises the amino acid sequence described in SEQ ID NO:72 or any variant thereof; or,

[0117] (3) The heavy chain comprises the amino acid sequence described in SEQ ID NO:63 or any variant thereof; the light chain comprises the amino acid sequence described in SEQ ID NO:73 or any variant thereof; or,

[0118] (4) The heavy chain comprises the amino acid sequence described in SEQ ID NO:64 or any variant thereof; the light chain comprises the amino acid sequence described in SEQ ID NO:74 or any variant thereof; or,

[0119] (5) The heavy chain comprises the amino acid sequence described in SEQ ID NO:65 or any variant thereof; the light chain comprises the amino acid sequence described in SEQ ID NO:75 or any variant thereof; or,

[0120] (6) The heavy chain comprises the amino acid sequence described in SEQ ID NO:66 or any variant thereof; the light chain comprises the amino acid sequence described in SEQ ID NO:76 or any variant thereof; or,

[0121] (7) The heavy chain comprises the amino acid sequence described in SEQ ID NO:67 or any variant thereof; the light chain comprises the amino acid sequence described in SEQ ID NO:77 or any variant thereof; or,

[0122] (8) The heavy chain comprises the amino acid sequence described in SEQ ID NO:68 or any variant thereof; the light chain comprises the amino acid sequence described in SEQ ID NO:78 or any variant thereof; or,

[0123] (9) The heavy chain comprises the amino acid sequence described in SEQ ID NO:69 or any variant thereof; the light chain comprises the amino acid sequence described in SEQ ID NO:79 or any variant thereof; or,

[0124] (10) The heavy chain comprises the amino acid sequence described in SEQ ID NO:70 or any variant thereof; the light chain comprises the amino acid sequence described in SEQ ID NO:80 or any variant thereof; or,

[0125] (11) The heavy chain comprises the amino acid sequence described in SEQ ID NO:163 or any variant thereof; the light chain comprises the amino acid sequence described in SEQ ID NO:166 or any variant thereof; or,

[0126] (12) The heavy chain comprises the amino acid sequence described in SEQ ID NO:164 or any variant thereof; the light chain comprises the amino acid sequence described in SEQ ID NO:167 or any variant thereof; or,

[0127] (13) The heavy chain comprises the amino acid sequence described in SEQ ID NO:165 or any variant thereof; the light chain comprises the amino acid sequence described in SEQ ID NO:168 or any variant thereof; or,

[0128] (14) The heavy chain comprises the amino acid sequence described in SEQ ID NO:175 or any variant thereof; the light chain comprises the amino acid sequence described in SEQ ID NO:181 or any variant thereof; or,

[0129] (15) The heavy chain comprises the amino acid sequence described in SEQ ID NO:176 or any variant thereof; the light chain comprises the amino acid sequence described in SEQ ID NO:182 or any variant thereof; or,

[0130] (16) The heavy chain comprises the amino acid sequence described in SEQ ID NO:177 or any variant thereof; the light chain comprises the amino acid sequence described in SEQ ID NO:183 or any variant thereof; or,

[0131] (17) The heavy chain comprises the amino acid sequence described in SEQ ID NO:178 or any variant thereof; the light chain comprises the amino acid sequence described in SEQ ID NO:184 or any variant thereof; or,

[0132] (18) The heavy chain comprises the amino acid sequence described in SEQ ID NO:179 or any variant thereof; the light chain comprises the amino acid sequence described in SEQ ID NO:185 or any variant thereof; or,

[0133] (19) The heavy chain comprises the amino acid sequence described in SEQ ID NO:180 or any variant thereof; the light chain comprises the amino acid sequence described in SEQ ID NO:186 or any variant thereof.

[0134] On the other hand, this disclosure provides a biological material selected from any of the following:

[0135] (1) A polynucleotide encoding the aforementioned antibody or its antigen-binding fragment; or

[0136] (2) An expression vector containing the polynucleotide described in (1); or

[0137] (3) A host cell containing the polynucleotide described in (1) or the expression vector described in (2).

[0138] On the other hand, this disclosure provides a method for producing an antibody or antigen-binding fragment thereof that specifically binds to canine IL-31, comprising the steps of: culturing the aforementioned host cells, isolating the antibody from the cultured species, and purifying the antibody.

[0139] On the other hand, this disclosure provides a pharmaceutical composition comprising the aforementioned antibody or its antigen-binding fragment, and a pharmaceutically acceptable excipient, diluent, or carrier.

[0140] On the other hand, this disclosure provides the use of the aforementioned antibody or its antigen-binding fragment, the aforementioned biological material, the aforementioned pharmaceutical composition, and the aforementioned kit in the preparation of a medicament for treating pruritus or allergies in dogs.

[0141] In some embodiments, the application includes reducing, suppressing, or neutralizing pruritus or allergies in dogs. In some embodiments, the pruritus is atopic dermatitis, eczema, psoriasis, or scleroderma. In some preferred embodiments, the pruritus is atopic dermatitis. In some embodiments, the application includes reducing the clinical manifestations of atopic dermatitis. In some embodiments, the clinical manifestation of atopic dermatitis is pruritus.

[0142] On the other hand, this disclosure provides the use of the aforementioned antibody or its antigen-binding fragment, the aforementioned biological material, the aforementioned pharmaceutical composition, and the aforementioned kit in the preparation of a medicament for reducing, inhibiting, or neutralizing IL-31 activity in dogs.

[0143] On the other hand, this disclosure provides a treatment method for canine pruritus or allergy, characterized by using a therapeutically effective amount of the aforementioned antibody or its antigen-binding fragment or the aforementioned pharmaceutical composition to reduce, inhibit or neutralize IL-31 activity in the dog.

[0144] Example

[0145] A further understanding of this disclosure can be obtained by referring to some specific embodiments given herein, which are for illustrative purposes only and are not intended to limit the scope of this disclosure in any way. It will be apparent that various modifications and variations can be made to this disclosure without departing from its essence, and therefore such modifications and variations are also within the scope of protection claimed in this application.

[0146] Example 1: Preparation of rabbit anti-canine IL-31 monoclonal antibody

[0147] 1. Immunogen preparation and identification

[0148] The nucleotide sequence (GeneID: 100302725, SEQ ID NO: 200) and amino acid sequence (SEQ ID NO: 199) of canine IL-31 were retrieved from the NCBI database. Recombinant canine IL-31 was generated in CHO cells using a transient transfection system to produce canine IL-31 protein. This recombinant canine IL-31 protein was subsequently used as an immunogen. The purity and molecular weight of the recombinant canine IL-31 protein were analyzed by SDS-PAGE, and its activity was identified by ELISA.

[0149] 1.1. SDS-PAGE determination

[0150] The specific experimental procedure is as follows:

[0151] (1) Loading the gel plates: Remove the comb, load the gel plates (in pairs) into the mold, and then place the whole assembly in the electrophoresis tank. The bottom sealing film needs to be removed from the reduced gel plates.

[0152] (2) Sample preparation: Non-reducing sample: Take an appropriate amount of sample and mix it with non-reducing 5× loading buffer (4:1), then boil in a water bath for 5 minutes. Reducing sample: Take an appropriate amount of sample and mix it with reducing 5× loading buffer (4:1), then boil in a water bath for 5 minutes.

[0153] (3) Sample loading: Fill the inner and outer tanks of the electrophoresis tank with electrophoresis buffer, and add the sample and marker. Cover the electrophoresis tank, making sure that the red electrode is for the positive electrode and the black electrode is for the negative electrode.

[0154] (4) Electrophoresis: Turn on the power and start electrophoresis. When using gradient gel, set the initial voltage to 90V, and change it to 120V after 30 minutes until the end of electrophoresis; when using non-gradient gel, set a single voltage (120V) until the end of electrophoresis.

[0155] (5) Staining: Place the gel in a petri dish containing staining solution and stain on a shaker for about 15 minutes.

[0156] (6) Decolorization: Remove the gel from the staining solution, immerse it in the decolorization solution, and place it on a shaker until the base color is colorless.

[0157] (7) Use a Bio-Rad chemiluminescence imaging system to photograph the sample and then label the information.

[0158] The protein electrophoresis results are shown in Figure 1 In this column, column M represents protein biomarkers (Bio-rad, Cat. no. 16103573S), with molecular weights indicated in the left-hand notes. Column R represents the reaction results for reduced samples, and column NR represents the reaction results for non-reduced samples. Figure 1 The results showed that the purity of the prepared recombinant canine IL-31 protein expression met the requirements, with a molecular weight of 16.1965 kDa.

[0159] 1.2. ELISA assay

[0160] Add 1 μg / mL of cyproheptadine (canine monoclonal antibody for IL-31) to a 96-well plate, 100 μL / well, and coat overnight at 4°C. Wash the plate three times with 300 μL / well of 0.1% PBST, then add blocking buffer and incubate at 37°C for 1 h. After washing the plate three times, add serially diluted IL-31 and incubate at 37°C for 1 h. After washing the plate three times, add HRP-labeled 6xHis-IgG (H+L) (Abcam) diluted 1000, 5000, and 10000 times for testing, and incubate at 37°C for 1 h. After washing the plate three times, develop the color with TMB chromogenic solution, add 1M H2SO4 solution to stop the reaction, and read the absorbance of the plate at 450 nm using a microplate reader.

[0161] The results of the ELISA binding activity test are shown in Table 2. The results in Table 2 show that the recombinant canine IL-31 protein has good binding activity with cyproheptadine.

[0162] Table 2. Canine IL-31 ELISA Binding Activity Assay

[0163]

[0164] 2. Animal immunization

[0165] In this embodiment, a rapid immunization protocol (Phoenix MonRab) was used. Four New Zealand rabbits (named R10265, R10266, R10267, and R10268, respectively) were immunized three times with recombinant canine IL-31 protein. Seven days after the third immunization, the serum of the immunized animals was detected by indirect ELISA to determine the level of immune response. The ELISA detection procedure can be referred to the experimental steps in 1.2.

[0166] The test results are shown in Table 3. Finally, two rabbits, R10266 and R10267, were selected for B cell screening.

[0167] Table 3. Indirect ELISA detection of animal serum titers

[0168]

[0169]

[0170] Note: The titer is highest when (signal / blank) >= 2.1, and the blank OD is the highest. 450 The value is the mean of two replicates. A represents canine IL31, and B represents irrelevant negative proteins with a His tag.

[0171] 3. B cell clone screening

[0172] 3.1. PBMC Collection and B Cell Screening

[0173] PBMCs were collected from rabbits with high immune response levels to obtain antigen-positive B cells. These antigen-positive B cells were then seeded into 96-well plates for screening.

[0174] 3.2. Initial screening

[0175] Cell culture supernatant was collected, and the supernatant was screened using an indirect ELISA method to select the supernatant that was positive for canine IL-31 protein.

[0176] 3.3. Confirm Filtering

[0177] All positive B cells obtained in the initial screening stage were screened using indirect ELISA with recombinant canine IL-31 protein. Subsequent screening with His-tag irrelevant proteins yielded clones targeting group A (positive for canine IL-31 protein) and group B (negative proteins with His tags). Table 4 shows the results of screening B cell supernatants using indirect ELISA. Cell clones with high binding affinity to group A and low binding affinity to group B were used for subsequent experiments.

[0178] Table 4. Indirect ELISA screening of B cell supernatant

[0179]

[0180]

[0181] 3.4. Epitope Confirmation

[0182] B cell culture supernatant was co-incubated with cyproheptadine (positive control), and a competitive ELISA was used to confirm whether the selected positive clones and cyproheptadine shared the same epitope. The epitope confirmation results of the B cell culture supernatant are shown in Tables 5-1 and 5-2. R10268#1:100 represents a 100-fold dilution of the selected rabbit serum with the highest titer as a positive clone control.

[0183] The formula for calculating the inhibition rate is: Inhibition rate (%) = 100% - (clone's own OD) 450 / Blank culture medium OD 450 )*100%

[0184] Table 5-1. Identification of epitopes in B cell supernatant

[0185]

[0186]

[0187] Table 5-2. Identification of epitopes in B cell supernatant

[0188]

[0189]

[0190] As shown in Tables 5-1 and 5-2, B cell clones 5H4-2, 18E8-1, 19D1-1, 19C7-1, 30G5-1, 35C9-1, 38E2-1, 44B4-1, 47C1-1, and 30A11-1 have a good binding ability with canine IL-31 protein.

[0191] Depend on Figure 2It is known that 5H4-2 and 30A11-1 can compete with the positive control for binding to canine IL-31. Therefore, it is speculated that the B cell clones of 5H4-2 and 30A11-1 may have the same epitopes as the positive control.

[0192] 3.5. ELISA assay

[0193] Ten B cell clones with good binding ability to canine IL-31, as selected above, were subjected to ELISA assay. The specific procedure was as follows: 100 μL / well of 1 μg / mL canine IL-31 was added to a 96-well plate and coated overnight at 4°C; the plate was washed three times with 300 μL / well of 0.1% PBST washing buffer, and then incubated at 37°C for 1 h with blocking buffer; after washing three times, B cell culture supernatant was added and incubated at 37°C for 1 h; after washing three times, Goat-anti-Canine-igG(H+L)-HRP was added and incubated at 37°C for 1 h; after washing three times, TMB chromogenic solution was used for color development, and the assay was stopped with 1M H2SO4 solution. The absorbance was read at 450 nm using an ELISA plate.

[0194] The binding activities of different cell samples are shown in Table 6. As can be seen from the results in Table 6, the binding activities of all cell types were good. The binding activities of the 10 cell types were ranked, with B cell clone 19D1-1 showing the strongest binding activity.

[0195] Table 6. ELISA Measurement Results

[0196] Experiment number sample <![CDATA[EC 50 Value / (ng / ml)]]> Combining activity ranking 1 5H4-2 0.841 3 2 18E8-1 1.95 6 3 19D1-1 0.196 1 4 19C7-1 3.21 10 5 30G5-1 2.08 7 6 35C9-1 2.3 8 7 38E2-1 0.915 4 8 44B4-1 0.232 2 9 47C1-1 1.79 5 10 30A11-1 2.82 9

[0197] 3.6. Assay for Cell Biological Activity

[0198] As previously mentioned, the pro-inflammatory cytokine interleukin-31 (IL-31) is produced by activated T lymphocytes from various species and can cause itching on the skin of dogs. In canine atopic dermatitis, when affected dogs are exposed to allergens, helper T cells (Th2) in the skin produce the cytokine IL-31. IL-31 binds to a co-receptor composed of IL-31 receptor A (IL-31RA) and oncogene M receptor (OSMR), activating the STAT signaling cascade via Janus kinase phosphorylation and leading to upregulation of target genes, thus causing itching behavior in dogs. Anti-IL-31 monoclonal antibodies can neutralize IL-31, thereby relieving itching.

[0199] To assess whether the 10 screened B cell clones could affect the ability of IL-31 to mediate pSTAT signaling, a method for measuring cell biological activity was established in this embodiment. The method involved pretreating canine DH82 monocytes for 24 h with 10 ng / mL canine interferon-gamma (R&D Systems, 781-CG-050) and serum starvation for 2 h before IL-31 treatment to enhance IL-31 receptor expression and pSTAT signal transduction. After pretreatment, recombinant canine IL-31 was added at 1 μg / mL for 5 min, and the ability to phosphorylate STAT was evaluated using HTRF technology (PerkinElmer, 62AT3PET). The ability of the mAb to inhibit STAT phosphorylation was qualitatively measured after co-incubating B cell supernatant with canine IL-31 for 1 h.

[0200] The specific experimental steps are as follows:

[0201] (1) Cell sensitization: DH82 (canine macrophages / canine kidney malignant histiocytic proliferative cells) (Chinese Academy of Sciences Cell Bank, catalog number TCO 3) were sensitized at 1x10 cells per well. 5 Cells / 100μL were seeded in 96-well cell culture plates and sensitized for 24 hours in MEM medium containing 15% FBS, 2mmol / L GlutaMax, and 1mmol / L sodium pyruvate at 37°C and 5% CO2. Canine interferon-γ was added at 10ng / mL and maintained for 24 hours.

[0202] (2) Washing: Remove the culture medium and wash the cells once with 200 μL of 1xPBS per well;

[0203] (3) Serum starvation: Replace with serum-free medium 100 μl / well and starve for 2 h;

[0204] (4) Sample processing: IL-31 cytokine was diluted to 2 μg / ml with serum-free medium. B cell culture supernatant and IL-31 were mixed and incubated together for one hour. Canine IL-31 containing 1 μg / ml was used as a negative control.

[0205] (5) Add inhibitors and stimulants: After removing the culture medium, add the pre-incubated mAb and IL-31 cytokine mixture directly at 100 μl / well and continue culturing in a 37°C carbon dioxide incubator for 1 h;

[0206] (6) Washing: Remove the culture medium and wash the cells once with 200 μl / well of 1xPBS;

[0207] (7) Cell lysis: Add 30 μL of 1x cell lysis buffer to each well, lyse at 100 rpm for 30 min, and the lysis is complete. Sample preparation is finished.

[0208] (8) Add samples: Add 16 μL of cell lysis supernatant and 4 μL of premixed antibody to the 96-well plate provided with the HTRF, incubate overnight at room temperature, and then take readings. Add 16 μL of phosphorylated control and negative control. Cover the 96-well plate with sealing film.

[0209] (9) Incubation: Incubate overnight at room temperature.

[0210] (10) Reading: Eu*Crypatate was used to set up the reader and read the fluorescence emission at two different wavelengths (665nm and 620nm) in a compatible HTRF reader to calculate the antibody inhibition rate.

[0211] (11) Data processing: Ratio = (665nm signal / 620nm signal) * 10000

[0212] Inhibition rate = 100% - (sample ratio / ratio of canine IL-31 negative control) * 100%

[0213] The criteria for a valid trial: a phosphorylated control ratio / negative control ratio > 2.

[0214] (12) Results Analysis

[0215] The calculated ratio of phosphorylated control to negative control was 3.8, indicating that the experiment was successful.

[0216] The results of cell viability assay are shown in Table 7. As can be seen from Table 7, the B cell clones with an inhibition rate of more than 20% are 38E2-1, 47C1-1 and 35C9-1.

[0217] Table 7. Assay of Cell Biological Activity

[0218]

[0219]

[0220] 4. Antibody sequencing

[0221] Ten ELISA-positive B cell clones were sequenced for region V using Sanger sequencing. The amino acid sequences of the ten antibodies are shown in Table 8, and the nucleotide sequences are shown in [Table 8]. Figure 9 .

[0222] Table 8. Amino acid sequence numbering information of antibodies

[0223]

[0224] Table 9. Nucleotide sequence numbering information of antibodies

[0225]

[0226] 5. BLI Affinity Test

[0227] 5.1. Transient expression of rabbit-derived anti-IL-31 monoclonal antibody

[0228] (1) Gene sequence design

[0229] Five clones, 38E2-1, 47C1-1, 35C9-1, 19D1-1, and 5H4-2, were selected from 10 B cell clones. The heavy chain variable region and light chain variable region of these five molecules were spliced ​​with the heavy chain constant region and light chain constant region of rabbit IgG, respectively, to obtain the complete heavy chain and light chain sequences of these five molecules.

[0230] (2) Sequence synthesis and expression vectors

[0231] The heavy and light chains of five molecules—38E2-1, 47C1-1, 35C9-1, 19D1-1, and 5H4-2—were fused and synthesized separately. First, the genes were amplified by PCR. Then, the pCDNA3.1 vector was linearized using NheI and ApaI restriction endonucleases. Finally, the genes were ligated into the NheI and ApaI restriction sites of the pCDNA3.1 vector using a seamless cloning method. The ligation products were transformed into *E. coli* Top10 competent cells. After plating, single colonies were picked for sequencing verification. Successfully transformed single colonies were expanded and endotoxin-free plasmids were extracted and integrated into the pcDNA3.1 plasmid. Finally, the following plasmids were obtained: 38E2-1-pcDNA3.1, 47C1-1-pcDNA3.1, 35C9-1-pcDNA3.1, 19D1-1-pcDNA3.1, and 5H4-2-pcDNA3.1.

[0232] (3) Transient transfection of CHO cells to express rabbit-derived anti-IL-31 monoclonal antibody

[0233] 1) CHO-S cell culture: Under normal culture conditions in 125mL shake flasks, when the cell density grows to 1*102... 7 When the number of cells / mL is 2×10 5 Inoculate with an initial density of 5 × 10⁶ cells / mL into new 125mL shake flasks and incubate at 37°C with 5% CO₂ for 2 days. When the density reaches 5 × 10⁶ cells / mL, inoculate with the culture medium. 5 When the number of cells / mL is 1, begin transient transfection according to the instructions for using the lipo2000 transfection reagent;

[0234] 2) After 7 days, collect the cell supernatant, centrifuge at 5000 rpm for 30 min to remove cells and debris, filter at 0.45 μm and store at 4℃ for subsequent antibody purification.

[0235] 5.2. Purification of rabbit-derived anti-IL-31 monoclonal antibody

[0236] Cell culture supernatant was purified using Protein A affinity chromatography. The specific procedure was as follows: Equilibrate with affinity chromatography equilibration buffer (20 mM PB, 0.15 M NaCl, pH 7.2); load the clarified filtrate onto the supernatant; equilibrate again with affinity chromatography equilibration buffer (20 mM PB, 0.15 M NaCl, pH 7.2); elute with affinity chromatography eluent (20 mM PB, 1 M NaCl, pH 7.2); and finally elute with elution buffer (0.1 M glycine-HCl, pH 3.0). Protein electrophoresis results are shown below. Figure 3 ,Depend on Figure 3 The eluted sample showed an SDS-PAGE purity >95%, with a target molecular weight of approximately 150 kDa. Results of Western blotting for rabbit antibody candidates are shown below. Figure 4 ,Depend on Figure 4 It can be seen that all rabbit-derived antibody candidates can bind to canine IL-31.

[0237] 5.3. Affinity Measurement

[0238] Five clones (38E2-1, 47C1-1, 35C9-1, 19D1-1, and 5H4-2) were selected. Using cytokine as a positive control, the purified antibody was immobilized on a ProA biosensor, and canine IL-31 protein was used as the mobile phase for BLI affinity assay.

[0239] The affinity assay results are shown in Table 10. As can be seen from Table 10, three clones showed greater affinity than the positive control Canine IgG: 38E2-1, 47C1-1, and 35C9-1. 38E2-1 exhibited the best affinity, with a value of 2.547E-09. The BLI affinity assay results are shown in... Figure 5 The results were consistent with those of the cell biological activity assay.

[0240] Table 10. BLI Affinity Determination

[0241]

[0242] Example 2: Establishment of a canine pruritus model

[0243] 1. Preliminary establishment of a canine pruritus model

[0244] To confirm the association between the inhibition of IL-31-mediated cell signaling observed in DH82 (canine renal malignant histiocytosis cells) assays and the inhibition of IL-31-mediated pruritus in dogs, the applicant, referring to relevant literature and patents, established a canine pruritus model using recombinant canine IL-31 protein to evaluate the in vivo activity of the antibody. In this model, recombinantly expressed canine IL-31 was intravenously administered to experimental dogs at two doses: 3 μg / kg for the low-dose group and 8 μg / kg for the high-dose group. Sterile PBS was used as a negative control. Five dogs were selected from each group. Clinical observation began 30 minutes before inoculation and was recorded continuously for 30 minutes. A second clinical observation began 30 minutes after inoculation and was recorded continuously for 2 hours, recording the frequency of pruritus in both observations. The 30-minute clinical observation before inoculation was used to establish a baseline. During the clinical observation period, behaviors such as licking, scratching, biting, rubbing, frequent head shaking, and digging were all considered pruritus behaviors. Specifically, at consecutive 1-minute intervals, each dog was judged "yes / no" to indicate whether it exhibited itching behavior. Itching behavior, such as licking / chewing paws, flanks, and / or anal area; scratching flanks, neck, and / or floor; head shaking; and rubbing their hindquarters on the cage floor, was sufficient to elicit a "yes" response at the specified time interval. At the end of the period, the number of "yes" responses was summed to obtain the cumulative itching score index (PSI). The PSI results were statistically analyzed at 30 minutes before injection and 2 hours after injection. One-way ANOVA using SPSS software was used to compare the significance of PSI differences between the dose groups and the negative control group. A P>0.05 indicated no significant difference in PSI levels between the dose groups and the control group; a P<0.05 indicated a significant difference in PSI levels between the dose groups and the control group.

[0245] Statistical analysis has been seen before Figure 6 ,Depend on Figure 6 It can be seen that the cumulative PSI results of the high-dose group and the low-dose group after half an hour of injection for 2 hours were significantly different from those of the negative control, and there was no significant difference in PSI results between the high-dose group and the low-dose group. Therefore, a dosage of 3 μg / kg was initially selected to establish a canine pruritus model.

[0246] 2. Validation experiment of the pruritus model

[0247] After determining the intravenous dosage of canine IL-31, we further validated the model. On day 7, canine IL-31 was administered intravenously to the experimental dogs at a dose of 3 μg / kg. Sterile PBS was used as a negative control, and 10 dogs showing significant differences from the negative control were selected. On day 0, the treatment group (5 dogs / group) received a subcutaneous injection of 2 mg / kg cyproheptadine in the neck, while the control group (5 dogs / group) received an equal volume of sterile PBS subcutaneously in the neck. On day 8, canine IL-31 was administered intravenously at a dose of 3 μg / kg. Clinical observation began 30 minutes before inoculation and was recorded continuously for 30 minutes. Clinical observation also began 30 minutes after inoculation and was recorded continuously for 2 hours, recording the frequency of itching twice. The clinical observation 30 minutes before inoculation was used to establish a baseline. The PSI results for the cumulative 30 minutes before injection and the cumulative 2 hours after injection were statistically analyzed. One-way ANOVA using SPSS software was used to compare the significant differences in PSI between the treatment group and the control group. When P>0.05, it indicates that the PSI levels in the treatment group and the control group are not significantly different; when P<0.05, it indicates that there is a significant difference in PSI levels between the treatment group and the control group.

[0248] Statistical results are shown below Figure 7 ,Depend on Figure 7 It can be seen that there was a significant difference between the treatment group and the control group after day 8, indicating that cytokine can inhibit IL-31-mediated itching symptoms in dogs after 8 days of administration.

[0249] Finally, a canine pruritus model was successfully established by administering IL-31 intravenously to dogs, and the intravenous dose of IL-31 in dogs was determined to be 3 μg / kg.

[0250] Example 3: In vivo efficacy study of rabbit-derived anti-IL-31 monoclonal antibody

[0251] After establishing the canine pruritus model, the applicant further investigated the in vivo efficacy of the rabbit-derived anti-L-31 monoclonal antibody. A certain amount of rabbit-derived antibodies 38E2-1, 47C1-1, and 35C9-1 were transiently expressed in samples. Cytokine was used as a positive control, and sterile PBS as a negative control. Five dogs were selected for each group, for a total of 25 experimental dogs. Abnormal dogs were excluded using the canine pruritus model. On day 0, the treatment group (5 dogs / group) received subcutaneous injections of rabbit-derived antibodies 38E2-1, 47C1-1, and 35C9-1 via the neck, respectively, at a dose of 2 mg / kg. The positive control group (5 dogs / group) received a subcutaneous injection of 2 mg / kg cytokine via the neck, and the negative control group (5 dogs / group) received an equal volume of sterile PBS via the neck. Referring to relevant literature, dogs were intravenously injected with 3 μg / kg canine IL-31 2.5 hours later. Clinical observation began 30 minutes before injection and was recorded continuously for 30 minutes. Clinical observation began again 30 minutes after injection and was recorded continuously for 2 hours, recording the frequency of itching in both cases. The clinical observation 30 minutes before injection was used to establish a baseline. The PSI results were statistically analyzed at 30 minutes before injection and 2 hours after injection. One-way ANOVA using SPSS software was used to compare the significance of PSI differences between the treatment group and the control group. P>0.05 indicated no significant difference in PSI levels between the treatment group and the control group; P<0.05 indicated a significant difference in PSI levels between the treatment group and the control group.

[0252] The experimental results are shown in Figure 8 ,Depend on Figure 8 It was found that, after 30 minutes and a cumulative 2 hours following injection, there were significant differences between the treatment group and the negative control group (sterile PBS). Rabbit antibody 38E2-1 showed no significant difference compared to the positive control group (Cetirizine) (P>0.05); rabbit antibody 47C1-1 showed no significant difference compared to the positive control group (P>0.05); and rabbit antibody 35C9-1 showed a significant difference compared to the positive control group (P=0.00014). Among the three rabbit antibodies, 38E2-1 showed the best in vivo activity, followed by 47C1-1 and 35C9-1.

[0253] Example 4: Canine Antibody Modification Strategy

[0254] Drug-resistant antibodies (ADAs) can be associated with the loss of efficacy of any biological therapeutic protein, including monoclonal antibodies. To help mitigate the risks associated with the formation of ADAs for the rabbit anti-IL-31 monoclonal antibody presented herein, a canine derivatization strategy is proposed to construct a chimeric antibody. See [link to relevant documentation] Figure 9This canine derivatization strategy is based on identifying the most suitable canine antibody sequence for CDR transplantation. After extensive analysis of the heavy and light chains of all available canine sequences, candidate lines were selected based on their homology with the rabbit mAbs. Specifically, the steps involved finding the canine derivatization template with the highest FV region match to the rabbit antibody on the IMGT website, replacing the CDR region in the canine template with the rabbit CDR region, analyzing the sequence using bioinformatics software, and performing reverse mutations on several amino acids to obtain several canine antibodies with affinity and activity comparable to the maternal antibody.

[0255] Example 5: Construction and preparation of chimeric antibodies against 38E2-1, 47C1-1, and 35C9-1

[0256] Construction and transient expression of 1.38E2-1, 47C1-1 and 35C9-1 chimeric antibodies

[0257] Following the canine-derived modification strategy of Example 4, the heavy chain variable regions and light chain variable regions of the three molecules 38E2-1, 47C1-1, and 35C9-1 were spliced ​​with the heavy chain constant region and light chain constant region of canine IgGB, respectively, to obtain the complete heavy chain and light chain sequences of these three molecules. The amino acid sequences and DNA sequences of the canine IgGB heavy chain and light chain were obtained from the GenBank database. The accession number for the IgGB heavy chain amino acids is AAL35302.1 (SEQ ID NO: 161), and the accession number for the DNA is AF354265.1 (SEQ ID NO: 162); the accession number for the canine antibody κ light chain amino acids is ABY 57289.1, and the accession number for the DNA is EU305402.1. The amino acid and nucleotide sequence coding information of the chimeric antibody is shown in Table 11.

[0258] Table 11. Sequence coding information of chimeric antibodies

[0259]

[0260] Sequence synthesis and transient transfection expression in CHO cells were performed according to the method in Example 1.5, and the purity of the one-step affinity chimeric antibody was evaluated. The experimental results are shown in [Figure 1]. Figure 10 ,Depend on Figure 10 It can be seen that the SDS-PAGE purity is >95%, the target molecular weight is around 150KD, and the protein immunoblotting results are shown in [reference needed]. Figure 11 ,Depend on Figure 11 It can be seen that all chimeric antibody candidates can bind to canine IL-31.

[0261] 2. Evaluation of chimeric antibody affinity

[0262] 2.1 ELISA assay

[0263] Add 100 μL / well of 1 μg / mL canine IL-31 to a 96-well plate and coat overnight at 4°C. Wash the plate three times with 300 μL / well of 0.1% PBST, then add blocking buffer and incubate at 37°C for 1 h. After washing the plate three times, add serially diluted chimeric antibody and incubate at 37°C for 1 h. After washing the plate three times, add Goat-anti-Canine-igG(H+L)-HRP and incubate at 37°C for 1 h. After washing the plate three times, develop the color with TMB chromogenic solution, stop with 1M H2SO4 solution, and read the absorbance at 450 nm using an ELISA plate.

[0264] The test results are shown in Table 12 and Figure 12 As shown in Table 12, the binding activity of the three chimeric antibodies to canine IL-31 was higher than that of the positive control. 38E2-1 was the best, followed by 47C1-1, and 35C9-1 was the worst.

[0265] Table 12. ELISA assay for chimeric antibodies

[0266] Serial Number Antibody <![CDATA[EC 50 Value / (ng / mL)]]> 1 Chimeric antibody 38E2-1 93.99 2 Chimeric antibody 35C9-1 105.50 3 Chimeric antibody 47C1-1 98.45 4 Cytokine 125.70

[0267] 2.2 BLI Affinity Determination

[0268] The purified antibody was immobilized on the ProA biosensor, and BLI affinity was determined using canine IL-31 protein as the mobile phase. Table 13 shows that the affinity of the chimeric antibody was not significantly different from or slightly lower (within 3-fold) compared to the corresponding rabbit antibody, and both exhibited higher affinity than the cytokinin antibody.

[0269] Table 13. Affinity assay of chimeric antibody BLI

[0270] Antibody Affinity determination KD(M) Rabbit antibody 38E2-1 8.15E-10 Chimeric antibody 38E2-1 2.49E-09 Rabbit antibody 47C1-1 3.75E-09 Chimeric antibody 47C1-1 8.32E-09 Rabbit-derived antibody 35C9-1 1.27E-08 Chimeric antibody 35C91 1.19E-08 Cytokine 3.19E-08

[0271] 3. Evaluation of the cellular biological activity of chimeric antibodies

[0272] 3.1 In vitro activity evaluation

[0273] The purified chimeric antibody sample was initially diluted to 10 μg / mL, and then serially diluted 1.75-fold to obtain 8 concentration points. Each serially diluted sample was mixed with an equal volume of IL-31 protein and incubated for 1 h. In vitro activity was evaluated according to the cell biological activity assay in Example 1.6. The results are shown in Table 14 and... Figure 13 The results above show that all three chimeric antibodies possess in vitro activity. Among them, antibody 38E2-1 exhibits the highest IC50 value. 50 It has a low value, a high inhibition rate, and the best in vitro activity.

[0274] Table 14. Assay of chimeric antibody cell biological activity

[0275] Serial Number Antibody <![CDATA[IC 50 Value / (ng / ml)]]> 1 Rabbit antibody 38E2-1 1250 2 Chimeric antibody 38E2-1 1323 3 Rabbit antibody 47C1-1 1569 4 Chimeric antibody 47C1 1630 5 Rabbit antibody 35C9 1638 6 Chimeric antibody 35C9 1714 7 Cytokine 1548

[0276] Example 6: In vivo efficacy evaluation of chimeric antibodies

[0277] The in vivo efficacy of the chimeric antibodies was evaluated according to the method in Example 3. The applicant used samples with transient transexpression to assess the in vivo efficacy of chimeric antibodies 38E2-1, 47C1-1, and 35C9-1. Cytokine was used as a positive control, and sterile PBS as a negative control. Five dogs were selected for each group, for a total of 25 experimental dogs. Abnormal dogs were excluded using a canine pruritus model. On day 0, the treatment group (5 dogs / group) received subcutaneous injections of chimeric antibodies 38E2-1, 47C1-1, and 35C9-1 via the neck, respectively, at a dose of 2 mg / kg. The positive control group (5 dogs / group) received a subcutaneous injection of 2 mg / kg cytokine via the neck, and the negative control group (5 dogs / group) received an equal volume of sterile PBS via the neck. Referring to relevant literature, dogs were intravenously injected with 3 μg / kg canine IL-31 2.5 hours later. Clinical observation began 30 minutes before injection and was recorded continuously for 30 minutes. Clinical observation began again 30 minutes after injection and was recorded continuously for 2 hours, recording the frequency of itching in both cases. The clinical observation 30 minutes before injection was used to establish a baseline. The PSI results were statistically analyzed at 30 minutes before injection and 2 hours after injection. One-way ANOVA using SPSS software was used to compare the significance of PSI differences between the treatment group and the control group. A P>0.05 indicated no significant difference in PSI levels between the treatment group and the control group; a P<0.05 indicated a significant difference in PSI levels between the treatment group and the control group.

[0278] The experimental results are statistically presented as follows: Figure 14 As shown, by Figure 14 It was found that there were significant differences between the treatment group and the negative control group. Chimeric antibody 38E2 showed a significant difference compared to the positive control group (P = 0.000302). Chimeric antibody 35C9-1 showed no significant difference compared to the positive control group (P > 0.05). Chimeric antibody 47C1-1 showed a significant difference compared to the positive control group (P = 0.000596). Among the three chimeric antibodies, chimeric antibody 38E2-1 showed the best in vivo activity, followed by chimeric antibodies 47C1-1 and 35C9-1.

[0279] Example 7: Construction and preparation of canine-derived antibodies

[0280] Construction and transient expression of canine antibodies derived from 1.38E2-1, 47C1-1, and 35C9-1

[0281] Following the canine-derived modification strategy of Example 4, 38E2-1, 47C1-1, and 35C9-1 were canine-derived, yielding two canine-derived antibodies for each maternal antibody. The sequence coding information of the canine-derived antibodies is shown in Table 15.

[0282] Table 15. Sequence coding information of canine antibodies

[0283]

[0284] Sequence synthesis and transient transfection expression in CHO cells were performed according to the method in Example 1.5, and the purity of the one-step affinity chimeric antibody was evaluated. The experimental results are shown in [Figure 1]. Figure 15 ,Depend on Figure 15 It can be seen that the SDS-PAGE purity is >95%, the target molecular weight is around 150KD, and the results of the Western blotting assay are shown below. Figure 16 ,Depend on Figure 16 It is known that all canine-derived antibody candidates can bind to canine IL-31.

[0285] 2. Evaluation of canine antibody affinity

[0286] (1) ELISA assay

[0287] The canine-derived antibodies were measured according to the ELISA assay method described in Example 4.2. The detection results are shown below. Figure 17 As shown in Table 16, the binding activity of the six canine antibodies to canine IL-31 was lower than that of the corresponding chimeric antibodies. The binding activity of canine antibodies 38E2-1 and 47C1-1 was not significantly different from that of the positive control cytokine. The binding activity of canine antibodies 35C9-1-H1L1 and 35C9-1-H1L2 was not as good as that of the positive control cytokine.

[0288] Table 16. ELISA assay for canine-derived antibodies

[0289] Sample Name <![CDATA[ELISA(EC 50 (ng / ml)]]> Chimeric antibody 38E2-1 92.85 Canine-derived antibody 38E2-1-H3L3 93.41 Canine-derived antibody 38E2-1-H3L2 97.75 Chimeric antibody 47C1-1 102.2 Canine-derived antibody 47C1-1-H2L2 113.4 Canine-derived antibody 47C1-1-H3L3 105.1 Chimeric antibody 35C9-1 102.7 Canine-derived antibody 35C9-1-H1L1 189.7 Canine-derived antibody 35C9-1-H1L2 157.6 Cytokine 123.8

[0290] (2) BLI affinity determination

[0291] The canine-derived antibodies were tested according to the BLI affinity assay method in Example 4.2. As shown in Table 17, the affinity of the canine-derived antibodies was one order of magnitude lower than that of the rabbit-derived antibodies. However, the affinity of the modified 38E2-1 and 47C1-1 was still higher than that of the positive control cytokine, while the affinity of the modified 35C9-1 was lower than that of the positive control cytokine.

[0292] Table 17. Affinity assay of canine-derived antibodies

[0293] name Affinity determination KD(M) Rabbit antibody 38E2-1 8.15E-10 Chimeric antibody 38E2-1 2.49E-09 Canine-derived antibody 38E2-1-H3L3 5.35E-09 Canine-derived antibody 38E2-1-H3L2 8.04E-09 Rabbit antibody 47C1-1 3.75E-09 Chimeric antibody 47C1-1 8.32E-09 Canine-derived antibody 47C1-1-H2L2 1.51E-08 Canine-derived antibody 47C1-1-H3L3 1.55E-08 Rabbit-derived antibody 35C9-1 1.27E-08 Chimeric antibody 35C9-1 1.19E-08 Canine-derived antibody 35C9-1-H1L1 4.95E-07 Canine-derived antibody 35C9-1-H1L2 3.89E-07 Cytokine 3.19E-08

[0294] 3. Evaluation of the cellular biological activity of canine-derived antibodies

[0295] (1) In vitro activity evaluation

[0296] The purified canine-derived sample was evaluated for in vitro activity according to Example 4.3, and the results are shown in Table 18. Figure 18 The results above show that the canine-derived 38E2-1 and 47C1-1 cells are not significantly different from their corresponding chimeric antibodies and cytokine, while the modified 35C9-1 cells show a decrease in activity compared to cytokine.

[0297] Table 18. Results of Cell Biological Activity Assay for Canine-Derived Antibodies

[0298] Sample Name <![CDATA[In vitro activity (IC 50 value ng / ml)]]> Chimeric antibody 38E2 1296 Canine-derived antibody 38E2-1-H3L3 1397 Canine-derived antibody 38E2-1-H3L2 1500 Chimeric antibody 47C1 1558 Canine-derived antibody 47C1-1-H2L2 1448 Canine-derived antibody 47C1-1-H3L3 1612 Chimeric antibody 35C9 1711 Canine-derived antibody 35C9-1-H1L1 2256 Canine-derived antibody 35C9-1-H1L2 2045 Cytokine 1508

[0299] Example 8: In vivo efficacy evaluation of canine-derived antibodies

[0300] 1. In vivo activity assessment of canine-derived antibodies

[0301] The in vivo efficacy of canine antibodies was evaluated according to the method in Example 3. A certain amount of sample transiently transexpressed was used to assess the in vivo efficacy of canine antibodies 38E2-1, 47C1-1, and 35C9-1. Cytokine was used as a positive control, and sterile PBS as a negative control. Five dogs were selected for each group, for a total of 40 experimental dogs. Abnormal dogs were excluded using a canine pruritus model. On day 0, the treatment group (5 dogs / group) received subcutaneous injections of canine antibodies 38E2-1-H3L3, 38E2-1-H3L2, 47C1-1-H2L2, 47C1-1-H3L3, 35C9-1-H1L1, and 35C9-1-H1L2 via the neck, respectively, at a dose of 2 mg / kg. The positive control group (5 dogs / group) received a subcutaneous injection of 2 mg / kg cytokine via the neck, and the negative control group (5 dogs / group) received an equal volume of sterile PBS via the neck. Referring to relevant literature, dogs were intravenously injected with 3 μg / kg canine IL-31 2.5 hours later. Clinical observation began 30 minutes before injection and was recorded continuously for 30 minutes. Clinical observation began again 30 minutes after injection and was recorded continuously for 2 hours, recording the frequency of itching in both cases. The clinical observation 30 minutes before injection was used to establish a baseline. The PSI results were statistically analyzed at 30 minutes before injection and 2 hours after injection. One-way ANOVA using SPSS software was used to compare the significance of PSI differences between the treatment group and the control group. A P>0.05 indicated no significant difference in PSI levels between the treatment group and the control group; a P<0.05 indicated a significant difference in PSI levels between the treatment group and the control group.

[0302] The experimental results are statistically presented as follows: Figure 19 As shown, by Figure 19It can be seen that there are significant differences between the treatment group and the negative control group. The canine antibodies 38E2-1-H3L3, 38E2-1-H3L2, and 47C1-1-H2L2 are comparable in efficacy to cytokine. The canine antibody 47C1-1-H3L3 is significantly different from cytokine, while the canine antibodies 35C9-1-H1L1 and 35C9-1-H1L2 are extremely significantly different and less effective than cytokine.

[0303] 2. Study on the efficacy of canine-derived antibody 38E2-1-H3L3

[0304] To determine the minimum effective dose and duration of action of canine antibody 38E2-1-H3L3, the efficacy of the antibody was studied according to the method described in Example 3. The experimental groups received 0.5 mg / kg, 1 mg / kg, and 2 mg / kg of canine antibody 38E2-1-H3L3, with cytokine as a positive control and sterile PBS as a negative control. Five dogs were selected for each group, for a total of 25 dogs. Abnormal dogs were excluded using a canine pruritus model. On day 0, the treatment groups (5 dogs / group) received subcutaneous injections of 0.5 mg / kg, 1 mg / kg, and 2 mg / kg of canine antibody 38E2-1-H3L3 via the neck, respectively. The positive control group (5 dogs / group) received a subcutaneous injection of 2 mg / kg cytokine via the neck, and the negative control group (5 dogs / group) received an equal volume of sterile PBS via the neck. Referring to relevant literature, dogs were intravenously injected with 3 μg / kg canine IL-31 on days 1, 7, 14, 21, and 28. Clinical observation began 30 minutes before each injection and was recorded continuously for 30 minutes. Clinical observation also began 30 minutes after each injection and was recorded continuously for 2 hours, recording the frequency of itching. The clinical observation 30 minutes before injection on day 1 was used to establish a baseline. PSI results were statistically analyzed for a cumulative 30 minutes before injection on day 1 and for a cumulative 2 hours after injection on days 1, 7, 14, 21, and 28. One-way ANOVA using SPSS software was used to compare the significance of PSI differences between the treatment and control groups. A P > 0.05 indicated no significant difference in PSI levels between the treatment and control groups; a P < 0.05 indicated a significant difference in PSI levels between the treatment and control groups.

[0305] Statistical experimental results are shown in Figure 20 ,Depend on Figure 20 It can be seen that there is a significant difference between the treatment group and the negative control group. The canine antibody 38E2-1-H3L3 at doses of 0.5 mg / kg, 1 mg / kg and 2 mg / kg had the same efficacy as cytokine on day 28 after administration, indicating that the minimum dose of canine antibody 38E2-1-H3L3 is 0.5 mg / kg and the duration of drug efficacy is at least 28 days.

[0306] Example 9: Immunogenicity evaluation of canine antibody 38E2-1-H3L3

[0307] Drug resistance antibody (ADA) production can affect drug activity and half-life. To evaluate the immunogenicity of canine antibody 38E2-1-H3L3, 10 healthy dogs were selected and divided into experimental and control groups, based on relevant patents and literature. Dogs in the experimental group (n=5 / group) received a subcutaneous injection of 9 mg / kg canine antibody 38E2-1-H3L3 via the neck on days 0, 14, and 28. Dogs in the negative control group (n=5 / group) received a subcutaneous injection of an equal volume of sterile PBS via the neck. Blood samples were collected on days 0, 7, 14, 21, and 28 to detect serum drug concentration and ADA levels.

[0308] 1. Blood drug concentration measurement

[0309] Add 100 μL / well of 1 μg / mL canine IL-31 to a 96-well plate and coat overnight at 4°C. Wash the plate three times with 300 μL / well of 0.1% PBST, then add blocking buffer containing 5% skim milk and incubate at 37°C for 1 h. After washing three times, dilute the negative serum 200 times with PBS containing 1% skim milk as a diluent, then add serially diluted canine-derived antibody and incubate at 37°C for 1 h. After washing three times, add Goat-anti-Canine-igG(H+L)-HRP 1:2000 and incubate at 37°C for 1 h. After washing three times, develop the color with TMB chromogenic solution, stop with 1M H2SO4 solution, and read the absorbance at 450 nm using an ELISA plate.

[0310] Depend on Figure 21 It can be seen that the drug metabolism in the experimental groups was basically the same.

[0311] 2. Preparation of ADA-positive serum

[0312] (1) Dilute the canine antibody 38E2-1-H3L3 with physiological saline to 1 mg / mL, mix it with an equal volume of adjuvant (QuickAntibody-Mouse5W), and immunize 5 mice (Balb / c female mice, 6 weeks old) with 100 μL / mouse via intramuscular injection.

[0313] (2) Three weeks later, the canine antibody 38E2-1-H3L3 was diluted with physiological saline to 1 mg / mL, mixed with an equal volume of adjuvant, and then injected intramuscularly into 5 mice again at a dose of 100 μL / mouse.

[0314] (3) About two weeks later, blood was collected from 5 mice, and serum was obtained by centrifugation at 3000 rpm for 5 min. The serum titer of the immunized mice was determined by ELISA.

[0315] 3. ELISA measurement of positive serum titer

[0316] Add 100 μl / well of 1 μg / mL canine-derived antibody 38E2-1-H3L3 to a 96-well plate and coat overnight at 4°C. Wash the plate three times with 300 μl / well of 0.1% PBST, then add blocking buffer and incubate at 37°C for 1 h. After washing the plate three times, dilute the serum of five immunized mice 100-fold with blocking buffer, then serially dilute 3-fold to 10 spots, and add 100 μL / well of each sample to a 96-well plate and incubate at 37°C for 1 h, using the blocking buffer as a blank control. After washing the plate three times, add Rabbit-anti-mouse-IgG(H+L)-HRP and incubate at 37°C for 1 h. After washing the plate three times, develop the color with TMB chromogenic solution, stop with 1M H2SO4 solution, and read the absorbance at 450 nm using an ELISA plate.

[0317] As shown in Table 19, the average value of the blank control group was about 0.45. Using twice the value of 0.45 (i.e., greater than 0.9) as the criterion for positive results, the serum titers of immunized mice 1-5 were 1:24300, 1:72900, 1:72900, 1:218700, and 1:72900, respectively.

[0318] Table 19. Determination of Positive Serum Titer

[0319]

[0320]

[0321] 3. Antidote antibody assay

[0322] Add 100 μL / well of 1 μg / mL canine-derived antibody to a 96-well plate and coat overnight at 4°C. Wash the plate three times with 300 μL / well of 0.1% PBST, then add blocking buffer containing 5% skim milk and incubate at 37°C for 1 h. After washing three times, use PBS containing 1% skim milk as diluent to perform a 10-fold dilution followed by a 2-fold serial dilution for 10 spots of mouse polyclonal positive serum containing anti-drug antibodies. All test sera and canine negative sera were diluted 10-fold using sample diluent and incubated at 37°C for 1 h. After washing three times, add biotin-labeled canine-derived antibody. After washing three times, add Streptavidin-HRP. After washing three times, develop the color with TMB chromogenic solution, stop with 1M H2SO4 solution, and read the absorbance at 450 nm using an ELISA plate.

[0323] The results of the positive serum anti-drug antibody assay are shown in Table 20, and the results of the anti-drug antibody assay in the experimental group and the negative control group are shown in Table 21. As can be seen from the tables above, no anti-drug antibodies were detected in any of the tested samples, indicating that the immunogenicity risk of canine-derived antibody 38E2-1-H3L3 is very low.

[0324] Table 20. Results of positive serum anti-drug antibody assay

[0325] valence 32000 16000 8000 4000 2000 1000 500 250 125 62.5 31.25 <![CDATA[OD 450 ]]> 3.625 3.663 3.688 3.668 3.675 3.640 3.458 3.564 1.309 0.435 0.163

[0326] Table 21. Results of anti-drug antibody assay in the experimental group and negative control group

[0327]

[0328] In summary, the anti-canine IL-31 monoclonal antibodies of the present invention can effectively alleviate the clinical symptoms associated with canine atopic dermatitis, while the immunogenicity risk of the canine-derived antibodies is very low.

[0329] The above description is merely an embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. An antibody that specifically binds to canine IL-31 or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region comprises: HCDR1 having the amino acid sequence shown in SEQ ID NO:2 or any variant thereof, HCDR2 having the amino acid sequence shown in SEQ ID NO:12 or any variant thereof, and HCDR3 having the amino acid sequence shown in SEQ ID NO:22 or any variant thereof. The light chain variable region comprises: LCDR1 having the amino acid sequence shown in SEQ ID NO:32 or any variant thereof, LCDR2 having the amino acid sequence shown in SEQ ID NO:42 or any variant thereof, and LCDR3 having the amino acid sequence shown in SEQ ID NO:52 or any variant thereof.

2. The antigen-binding fragment of the antibody according to claim 1, wherein, The heavy chain variable region includes: HCDR1 shown in SEQ ID NO:2, HCDR2 shown in SEQ ID NO:12, and HCDR3 shown in SEQ ID NO:22; or, The light chain variable region includes: LCDR1 shown in SEQ ID NO:32, LCDR2 shown in SEQ ID NO:42, and LCDR3 shown in SEQ ID NO:

52.

3. The antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The antibody or its antigen-binding fragment is selected from any one of rabbit-derived antibodies or their antigen-binding fragments, chimeric antibodies or their antigen-binding fragments, or canine-derived antibodies or their antigen-binding fragments.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein, The antibody that specifically binds to canine IL-31 or its antigen-binding fragment comprises a heavy chain selected from the sequences shown below, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with the sequences shown below: SEQ ID NO: 62, 164, 177-178; and / or It includes light chains selected from the sequences shown below, or light chains that have at least 80%, 85%, 90%, 95% or 99% identity with the sequences shown below: SEQ ID NO: 72, 167, 183-184.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein, (1) The heavy chain comprises the amino acid sequence described in SEQ ID NO:62 or any variant thereof; the light chain comprises the amino acid sequence described in SEQ ID NO:72 or any variant thereof; or, (2) The heavy chain comprises the amino acid sequence described in SEQ ID NO:164 or any variant thereof; the light chain comprises the amino acid sequence described in SEQ ID NO:167 or any variant thereof; or, (3) The heavy chain comprises the amino acid sequence described in SEQ ID NO:177 or any variant thereof; the light chain comprises the amino acid sequence described in SEQ ID NO:183 or any variant thereof; or, (4) The heavy chain contains the amino acid sequence described in SEQ ID NO:178 or any variant thereof; the light chain contains the amino acid sequence described in SEQ ID NO:184 or any variant thereof.

6. A biological material selected from any one of the following: (1) A polynucleotide encoding the antibody or antigen-binding fragment thereof as described in any one of claims 1-5; or (2) An expression vector containing the polynucleotide described in (1); or (3) A host cell containing the polynucleotide described in (1) or the expression vector described in (2).

7. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-5, and a pharmaceutically acceptable excipient, diluent, or carrier.

8. A kit for detecting or quantifying canine IL-31 protein in clinical or biological samples, comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-5, optionally further comprising one or more reagents for detecting the binding of said antibody or antigen-binding fragment thereof to canine IL-31 or its epitopes.

9. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-5, the biomaterial of claim 6, the pharmaceutical composition of claim 7, and the kit of claim 8 in the preparation of a medicament for treating pruritus or allergies in dogs; Preferably, the pruritus is atopic dermatitis, eczema, psoriasis, or scleroderma; preferably, the pruritus is atopic dermatitis.

10. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-5, the biological material of claim 6, the pharmaceutical composition of claim 7, or the kit of claim 8 in the preparation of a medicament for reducing, inhibiting, or neutralizing IL-31 activity in dogs.

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