Anti-IL-31 antibodies and uses thereof

Abstract

The present disclosure relates to an anti-IL-31 antibody and its use. Specifically, an antibody or antigen-binding fragment thereof that specifically binds to canine IL-31 is provided, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: a HCDR1 having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-10 or any variant thereof, a HCDR2 having an amino acid sequence as set forth in any one of SEQ ID NOs: 11-20 or any variant thereof, and a HCDR3 having an amino acid sequence as set forth in any one of SEQ ID NOs: 21-30 or any variant thereof; and the light chain variable region comprises: a LCDR1 having an amino acid sequence as set forth in any one of SEQ ID NOs: 31-40 or any variant thereof, a LCDR2 having an amino acid sequence as set forth in any one of SEQ ID NOs: 41-50 or any variant thereof, and a LCDR3 having an amino acid sequence as set forth in any one of SEQ ID NOs: 51-60 or any variant thereof. The anti-canine IL-31 antibody disclosed herein has better affinity than ceramide and can effectively alleviate clinical symptoms associated with atopic dermatitis in model animals.

Description

Technical Field

[0001] The present disclosure relates to the fields of genetic engineering and immunology, and particularly to an anti-IL-31 antibody and uses thereof. Background Art

[0002] Canine atopic dermatitis is a genetically susceptible inflammatory and pruritic allergic skin disease in dogs. According to statistics, approximately 450 million dogs worldwide suffer from atopic dermatitis, with an incidence rate as high as 10% to 15% (Kinga Gortel et al., Can Vet J, 2018 Sep;59(9):1013-1016). In the UK, Labradors and Golden Retrievers have an almost 50% risk of developing atopic dermatitis due to their genetic background. Certain breeds, such as Golden Retrievers, Labradors, Pit Bulls, Pugs, Boxers, and German Shepherds, have a significantly higher risk than other dogs, and the degree of susceptibility varies by breed and geographic location (Natalie Katharina Yvonne Gedon et al., Clin Transl Allergy, 2018 Oct;8:41). Atopic dermatitis typically develops between six months and three years of age. Food, environmental allergens, and genetic factors are the primary causes of atopic dermatitis. Distinct from parasitic allergies, affected dogs often experience erythema and pruritus, with secondary symptoms including pain and inflammation. These itching and inflammation can lead to spontaneous hair loss, abrasions, and secondary infections, manifesting as papules, pustules, and crusting. Common sites of disease include the axillae, ventral flanks, distal extremities, the inner ear, and the periocular, oral, and anal areas.

[0003] The skin barrier of dogs with atopic dermatitis is defective. When allergens pass through the skin defects and interact with immune cells, the immune cells release various itchy chemicals, causing itching. During the allergic reaction, a variety of other chemicals are released at the same time, causing inflammation on the skin. Inflammation further degrades the barrier function, and then the permeability of allergens increases, leading to secondary infection and bacteria. Among them, 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 and further weaken the barrier function of the epidermis (Jackeline Franco et al., Metabolit, 2021 Sep 30; 11 (10): 670). The serum concentration of L-31 is closely related to the severity of canine skin lesions. When dogs are exposed to allergens, the helper T cells (Th2) in the skin will produce the cytokine IL-31. IL-31 binds to the co-receptor composed of IL-31 receptor A (IL-31RA) and oncostatin M receptor (OSMR), activates the STAT signaling cascade through Janus kinase phosphorylation, leads to upregulation of target genes, and causes itchy 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, which has been confirmed 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). The concentration of IL-31 increases under pruritic allergic skin conditions and can induce scratching behavior in a variety of species (such as mice, monkeys and dogs). Studies in laboratory beagles have shown that when IL-31 is administered by several routes (intradermal, subcutaneous, and intravenous), it induces intense pruritic behavior within minutes to hours, suggesting that the experimental model of IL-31 administration by injection can be used to evaluate the antipruritic effects 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 consisting of multiple types. Monitoring the treatment progress of atopic dermatitis may include using the Canine Atopic Dermatitis Extent and Severity Index (CADESI) to assess the severity of lesions, evaluate itching, and measure biophysical parameters of the skin (JR Calesso et al., Pol J Vet Sci, 2023 Jun; 26(2): 231-238). Current chemical treatments include glucocorticoids, cyclosporine, essential fatty acids, and antihistamines. For long-term treatment, cyclosporine may be slow to take effect and increase the cost to pet owners. Although glucocorticoids can effectively control allergic pruritus and inflammation in the short term, they may produce secondary adverse reactions and have a high incidence rate, making them unsuitable for long-term treatment (Clarissa P Souza et al., Vet Dermatol, 2018 Dec; 29(6): 489). Alternatively, desensitization therapy can be used. Injecting the allergen once or twice a month for one to two years can reduce allergic reactions and build tolerance to the allergen. This non-drug treatment is harmless and has a low likelihood of permanently eliminating allergies to the allergen. However, desensitization therapy should only be performed by veterinarians specializing in dermatology.

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

[0007] Given the challenges of expensive diagnostic testing, frequent clinical episodes, and the need for lifelong treatment, canine atopic dermatitis is challenging for owners, pets, and veterinarians. Therefore, 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 of pet dogs. Summary of the Invention

[0008] In order to solve the problems existing in the prior art, the present disclosure aims to provide an anti-IL-31 antibody or an antigen-binding fragment thereof, a preparation method thereof, and medical use thereof.

[0009] In order to achieve the above objectives, the present disclosure adopts the following specific solutions:

[0010] In one aspect, the present 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: a HCDR1 having an amino acid sequence as shown in any one of SEQ ID NOs: 1-10 or any variant thereof, a HCDR2 having an amino acid sequence as shown in any one of SEQ ID NOs: 11-20 or any variant thereof, and a HCDR3 having an amino acid sequence as shown in any one of SEQ ID NOs: 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 NOs: 31-40 or any variant thereof, LCDR2 having an amino acid sequence shown in any one of SEQ ID NOs: 41-50 or any variant thereof, and LCDR3 having an amino acid sequence shown in any one of SEQ ID NOs: 51-60 or any variant thereof.

[0013] In another aspect, the present disclosure provides a biomaterial selected from any one of the following:

[0014] (1) a polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6; 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] In another aspect, the present disclosure provides a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to canine IL-31, comprising the steps of culturing the aforementioned host cell, isolating the antibody from the culture medium, and purifying the antibody.

[0018] In another aspect, the present disclosure provides a pharmaceutical composition comprising the aforementioned antibody or antigen-binding fragment thereof, and a pharmaceutically acceptable excipient, diluent, or carrier.

[0019] In another aspect, the present disclosure provides a use of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned biomaterial, the aforementioned pharmaceutical composition, and the aforementioned kit in preparing a medicament for treating pruritus or allergy in dogs.

[0020] On the other hand, the present disclosure provides a use of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned biomaterial, the aforementioned pharmaceutical composition, and the aforementioned kit in the preparation of a drug for reducing, inhibiting, or neutralizing IL-31 activity in dogs.

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

[0022] The anti-canine IL-31 antibodies or antigen-binding fragments disclosed herein can specifically bind to canine IL-31 with comparable or better affinity than the currently available commercial product, ceramide. They have demonstrated excellent biological activity in both cells and animals, effectively alleviating clinical symptoms associated with atopic dermatitis in a canine pruritus model. Furthermore, the engineered caninized antibodies have a low immunogenicity risk. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the result of SDS-PAGE electrophoresis identification of canine IL-31.

[0024] Figure 2 The results are shown in ELISA test of B cell culture supernatant competing with ceramide (positive control).

[0025] Figure 3 This is an SDS-PAGE image of rabbit antibodies. Columns 1-10 represent the 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) groups, respectively. M represents a marker group.

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

[0027] Figure 5 This is the BLI affinity determination graph of the rabbit antibody.

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

[0029] Figure 7 The pruritus score after 8 days of ceramide administration indirectly verifies the successful establishment of the canine pruritus model. Ceramide can also inhibit IL-31-mediated pruritus in the model dogs.

[0030] Figure 8 The itch score of the dog pruritus model after treatment with rabbit-derived antibodies.

[0031] Figure 9 The strategy for constructing caninized antibodies.

[0032] Figure 10 The SDS-PAGE results of the chimeric antibody are shown in Figure 1. Columns 1 to 6 represent the 38E2-1 (non-reduced), 47C1-1 (non-reduced), 35C9-1 (non-reduced), 38E2-1 (reduced), 47C1-1 (reduced), and 35C9-1 (reduced) groups, respectively.

[0033] Figure 11 The figure shows the results of WB detection of chimeric antibodies. Columns 1-3 represent the 38E2-1 (non-reduced), 47C1-1 (non-reduced), and 35C9-1 (non-reduced) groups, respectively.

[0034] Figure 12 The ELISA results of chimeric antibodies are shown in Table 1. “Weighting fixed” indicates a fixed weight.

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

[0036] Figure 14 The pruritus score of the canine pruritus model after chimeric antibody administration was obtained.

[0037] Figure 15 This is the SDS-PAGE result of caninized antibody. Figure 15 Columns 1-8 of A are the 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) groups, respectively. Figure 15 Columns 1-4 in B are the 35C9-1-H1L1 (non-reduced), 35C9-1-H1L2 (non-reduced), 35C9-1-H1L1 (reduced), and 35C9-1-H1L2 (reduced) groups, respectively.

[0038] Figure 16 This is the WB test result of caninized antibody. Figure 16Columns 1-4 of A are the 38E2-1-H3L3 (non-reduced), 38E2-1-H3L2 (non-reduced), 47C1-1-H2L2 (non-reduced), and 47C1-1-H2L3 (non-reduced) groups, respectively. Figure 16 Columns 1 and 2 of B are the 35C9-1-H1L1 (non-reduced) and 35C9-1-H1L2 (non-reduced) groups, respectively.

[0039] Figure 17 The ELISA results for caninized antibodies are shown in Table 1. Weighting fixed refers to fixed weights.

[0040] Figure 18 These are the results of cell biological activity assays of caninized antibodies.

[0041] Figure 19 The pruritus score of the canine pruritus model after treatment with caninized antibodies was obtained.

[0042] Figure 20 The results show the drug efficacy verification results within 28 days after administration of different concentrations of caninized antibody 38E2-1-H3L3.

[0043] Figure 21 These are the results of repeated administration of 9 mg / kg of the caninized antibody 38E2-1-H3L3 to the blood. DETAILED DESCRIPTION

[0044] In order to more clearly illustrate the embodiments of the present disclosure or the technical solutions in the prior art, the terms and implementation methods required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the implementation method described below is only one implementation method of the present disclosure. For ordinary technicians in this field, other implementation methods can also be obtained based on these drawings.

[0045] I. Terminology

[0046] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present disclosure belongs.

[0047] The articles "a" and "an" used herein refer to one or more than one (ie, at least one) of the grammatical object to which the article refers. For example, "a component" means one component or more than one component.

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

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

[0050] As used throughout the specification and claims, the term "consisting essentially of" or variations thereof means that all recited components or groups of components are included, and optionally, other components of similar or different properties from the recited components that do not significantly alter the basic or novel properties of a specified dosage regimen, method, or composition.

[0051] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and where it does not.

[0052] The term "antibody" refers to any form of antibody that exhibits the 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 is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "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 can define a constant region primarily responsible for effector function. Typically, human light chains are divided into kappa light chains and lambda light chains. Furthermore, human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and the antibody isotype is defined as IgM, IgD, IgG, IgA, and IgE, respectively. In both light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, while the heavy chain also includes a "D" region of approximately 10 or more amino acids. See generally Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)).

[0054] The variable regions of each light / heavy chain pair form the antibody binding site. Thus, in general, an intact antibody has two binding sites. Except for bifunctional or bispecific antibodies, these two binding sites are usually identical.

[0055] Typically, the variable regions of both heavy and light chains contain three hypervariable regions, also known as complementarity-determining regions, located within relatively conserved framework regions. The CDRs are typically aligned within the framework regions, enabling them to bind to specific epitopes. Generally speaking, from N-terminus to C-terminus, both light and heavy chain variable domains include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain generally conforms to the following definitions: 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, i.e., an antibody fragment that retains the ability to specifically bind to the antigen bound by the full-length antibody, e.g., 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 herein, "CDR" or "CDR" refers to the complementarity determining region in an immunoglobulin variable region, generally 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 the target compared to other proteins, but this specificity does not require absolute binding specificity. If the binding of the antibody determines the presence of the target protein in the sample, for example without producing undesirable results such as false positives, the antibody is considered to be "specific" for its intended target. The antibodies or binding fragments thereof used in the present invention will bind to the target protein with an affinity that is at least twice as high as that with non-target proteins, preferably at least ten times as high, more preferably at least 20 times as high, and most preferably at least 100 times as high. As used herein, if an antibody binds to a polypeptide comprising a given amino acid sequence, such as the amino acid sequence of a mature human PD-1 or human PD-L1 molecule, but does not bind to a protein lacking the sequence, the antibody is said to specifically bind to a polypeptide comprising the sequence.

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

[0060] As used herein, "conservatively modified variants" or "conservative substitutions" refer to amino acids in a protein that are replaced by other amino acids with similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity, etc.), so that changes can be made frequently without changing the biological activity or other desired properties (e.g., antigen affinity and / or specificity) of the protein. Those skilled in the art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not significantly change biological activity (see Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.)). In addition, substitutions of amino acids with similar structure or function are less likely to destroy biological activity. Exemplary conservative substitutions are listed in Table 1.

[0061] Table 1 Exemplary conservative amino acid substitutions

[0062]

[0063]

[0064] As used herein, "cell," "cell line," and "cell culture" are used interchangeably, and all such names include their offspring. Thus, the words "transformants" and "transformed cells" include primary test cells and cultures derived therefrom, without regard to the number of transfers. It should also be understood that, due to intentional or unintentional mutations, all offspring may not be precisely identical in terms of DNA content. Mutant offspring having the same function or biological activity as that screened in the original transformed cells are included. Where different names are intended, this is clearly evident from the context. In the context of expressing heterologous nucleic acid sequences, "host cell" refers to a prokaryotic or eukaryotic cell (e.g., a bacterial cell, a yeast cell, a mammalian cell, and an insect cell) in vitro or in vivo. For example, a host cell can be located in a transgenic animal. A host cell can be used as a receptor for a vector and can include any transformable organism capable of replicating the vector and / or expressing the heterologous nucleic acid encoded by the vector.

[0065] As used herein, the term "pharmaceutical composition" relates to a composition for administration to a mammalian patient (preferably a canine patient). In a preferred embodiment, the pharmaceutical composition comprises a composition for parenteral injection or infusion. The parenteral injection or infusion can take advantage of the resorption process in the form of intradermal, subcutaneous, intramuscular and / or intraperitoneal injection or infusion. Alternatively, the parenteral injection or infusion can bypass the resorption process and be in the form of intracardiac, intraarterial, intravenous, lumbar and / or intrathecal injection or infusion. In another preferred embodiment, the pharmaceutical composition comprises a composition for administration through the skin. An example of administration through the skin is epidermal administration, wherein the pharmaceutical composition is administered in the form of a solution, suspension, emulsion, foam, ointment, salves, paste and / or a patch applied to the skin. Alternatively, administration of the pharmaceutical composition can be achieved through one or more mucous membranes. For example, administration can be buccal, lingual or sublingual, i.e. by the mucous membrane of the mouth and / or tongue, and application forms can be such as tablets, lozenges, sugar-coated tablets (i.e. dragees) and / or mouthwash solutions. Optionally, administration can be enteric, i.e. by the stomach and / or intestinal mucosa, and application forms can be such as tablets, sugar-coated tablets (i.e. dragees), capsules, solutions, suspensions and / or emulsions. Optionally, administration can be rectal, and application forms can be such as suppositories, rectal capsules and / or ointments or salves. Optionally, administration can be intranasal, and application forms can be such as drops, ointments or salves and / or sprays. Optionally, administration can be pulmonary, i.e. by bronchial and / or alveolar, and application forms can be such as aerosols and / or inhalants. Optionally, administration can be conjunctival, and application forms can be such as eye drops, eye ointments and / or eyewashes. Alternatively, administration can be achieved through the urogenital tract mucosa, such as intravaginal or intraurethral, and the application form can be, for example, a suppository, an ointment and / or a pen. It should be understood that the above optional forms of administration are not mutually exclusive, and any number of combinations thereof can be used to form an effective treatment regimen.

[0066] The pharmaceutical compositions of the present disclosure may further include a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known and include phosphate buffered saline, water, emulsions (such as oil / water emulsions), various wetting agents, sterile solutions, and the like. Compositions containing these carriers can be formulated by conventional methods. These pharmaceutical compositions can be administered to subjects at appropriate doses. The dosage regimen can be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any one patient depends on many factors, including the patient's size, body surface area, age, specific compound being administered, sex, time and route of administration, overall health, and other drugs being administered concurrently. For example, formulations for parenteral administration 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 solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Carriers suitable for intravenous or intra-arterial administration include fluid and nutrient supplements, electrolyte supplements (such as those based on Ringer's dextrose), etc. Preservatives and other additives may also be contained, for example, antimicrobial, antioxidant, chelating agent, inert gas, etc. In addition, the pharmaceutical composition of the present invention may include a protein carrier, such as serum albumin or immunoglobulin, preferably human. It is of concern that, in addition to humanized monoclonal antibodies (such as those described in the present invention), the pharmaceutical composition of the present invention may also include other bioactive agents, depending on the desired use of the pharmaceutical composition. The reagent can be a drug acting on the gastrointestinal system, a drug as a cell inhibitor, a drug to prevent polyuricemia, a drug that suppresses immune response (such as corticosteroids), a drug that regulates inflammatory response, a drug acting on the circulatory system and / or an existing known reagent such as a cytokine.

[0067] As used herein, "subject" 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.

[0068] As used herein, a "therapeutically effective amount" (or "effective amount") refers to an amount of an active ingredient (e.g., an agent of the present invention) that is sufficient to produce a beneficial or desired result when administered to a subject or patient. An effective amount can be administered in one or more administrations, applications, or dosages. A therapeutically effective amount of a composition of the present invention can be readily determined by one of ordinary skill in the art. In the context of the present invention, a "therapeutically effective amount" is an amount that produces an objectively observed change in one or more parameters associated with the treatment of pruritus or an allergic disease, including clinical improvement of symptoms. Of course, the therapeutically effective amount will vary depending on the specific subject and disease to be treated, the subject's weight and age, the severity of the disease symptoms, the specific compound selected, the dosing regimen to be followed, the time of administration, the mode of administration, and the like, all of which can be readily determined by one of ordinary skill in the art.

[0069] As used herein, "therapeutic" encompasses the full range of treatments for a disease or condition. The "therapeutic" agents of the present invention may act in a preventative or prophylactic manner, including procedures designed to target animals that can be identified as at risk (pharmacogenetics); or in an ameliorative 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 being treated. "Treatment," "treating," and the like refer to both therapeutic treatment and prophylactic or preventative measures. Animals in need of treatment include those already suffering from the condition as well as those in which the condition is to be prevented. The terms "treating" or "treating" a disease or condition include preventing or protecting against the disease or condition (i.e., so that clinical symptoms do not develop); inhibiting the disease or condition (i.e., preventing or inhibiting the development of clinical symptoms); and / or alleviating the disease or condition (i.e., causing clinical symptoms to subside). As will be understood, it is not always possible to distinguish between "preventing" and "inhibiting" a disease or condition, as the ultimate precipitating event may be unknown or latent. Thus, the term "prophylaxis" should be understood to constitute a type of treatment that encompasses "preventing" and "suppressing." The term "treatment" thus includes "prevention."

[0070] As used herein, the term "allergic disease" is defined herein as a condition or disease caused by the interaction between the immune system and an external substance. Such foreign substances are referred to as "allergens." Common allergens include aeroallergens such as pollen, dust, mold, dust mite proteins, saliva injected by insect bites, and the like. Examples of allergic diseases include, but are not limited to, allergic dermatitis, summer eczema, urticaria, equine wheezing, inflammatory airway diseases, recurrent airway obstruction, airway hyperresponsiveness, chronic obstructive pulmonary disease, and autoimmune inflammatory processes such as irritable bowel syndrome (IBS).

[0071] As used herein, the terms "itch" and "pruritus" are defined herein as a disease or condition characterized by an intense itchy sensation that produces an urge to rub or scratch the skin for relief. Examples of pruritus include, but are not limited to, the following: atopic dermatitis, eczema, psoriasis, scleroderma, and pruritus.

[0072] II. Detailed description of specific implementation plan

[0073] In one aspect, the present 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:

[0074] The heavy chain variable region comprises: a HCDR1 having an amino acid sequence as shown in any one of SEQ ID NOs: 1-10 or any variant thereof, a HCDR2 having an amino acid sequence as shown in any one of SEQ ID NOs: 11-20 or any variant thereof, and a HCDR3 having an amino acid sequence as shown in any one of SEQ ID NOs: 21-30 or any variant thereof;

[0075] The light chain variable region comprises: LCDR1 having an amino acid sequence shown in any one of SEQ ID NOs: 31-40 or any variant thereof, LCDR2 having an amino acid sequence shown in any one of SEQ ID NOs: 41-50 or any variant thereof, and LCDR3 having an amino acid sequence shown in any one of SEQ ID NOs: 51-60 or any variant thereof.

[0076] In some embodiments, the antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises:

[0077] (1) HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 11, HCDR3 shown in SEQ ID NO: 21; or,

[0078] (2) HCDR1 shown in SEQ ID NO: 2, HCDR2 shown in SEQ ID NO: 12, HCDR3 shown in SEQ ID NO: 22; or

[0079] (3) HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 13, HCDR3 shown in SEQ ID NO: 23; or

[0080] (4) HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 14, HCDR3 shown in SEQ ID NO: 24; or

[0081] (5) HCDR1 shown in SEQ ID NO: 5, HCDR2 shown in SEQ ID NO: 15, HCDR3 shown in SEQ ID NO: 25; or

[0082] (6) HCDR1 shown in SEQ ID NO: 6, HCDR2 shown in SEQ ID NO: 16, HCDR3 shown in SEQ ID NO: 26; or

[0083] (7) HCDR1 shown in SEQ ID NO: 7, HCDR2 shown in SEQ ID NO: 17, HCDR3 shown in SEQ ID NO: 27; or

[0084] (8) HCDR1 shown in SEQ ID NO: 8, HCDR2 shown in SEQ ID NO: 18, HCDR3 shown in SEQ ID NO: 28; or

[0085] (9) HCDR1 shown in SEQ ID NO: 9, HCDR2 shown in SEQ ID NO: 19, HCDR3 shown in SEQ ID NO: 29; or

[0086] (10) HCDR1 shown in SEQ ID NO: 10, HCDR2 shown in SEQ ID NO: 20, and HCDR3 shown in SEQ ID NO: 30;

[0087] The light chain variable region comprises:

[0088] (1) LCDR1 shown in SEQ ID NO:31, LCDR2 shown in SEQ ID NO:41, LCDR3 shown in SEQ ID NO:51; or

[0089] (2) LCDR1 shown in SEQ ID NO: 32, LCDR2 shown in SEQ ID NO: 42, LCDR3 shown in SEQ ID NO: 52; or

[0090] (3) LCDR1 shown in SEQ ID NO: 33, LCDR2 shown in SEQ ID NO: 43, LCDR3 shown in SEQ ID NO: 53; or

[0091] (4) LCDR1 shown in SEQ ID NO: 34, LCDR2 shown in SEQ ID NO: 44, LCDR3 shown in SEQ ID NO: 54; or

[0092] (5) LCDR1 shown in SEQ ID NO: 35, LCDR2 shown in SEQ ID NO: 45, LCDR3 shown in SEQ ID NO: 55; or

[0093] (6) LCDR1 shown in SEQ ID NO: 36, LCDR2 shown in SEQ ID NO: 46, LCDR3 shown in SEQ ID NO: 56; or

[0094] (7) LCDR1 shown in SEQ ID NO: 37, LCDR2 shown in SEQ ID NO: 47, LCDR3 shown in SEQ ID NO: 57; or

[0095] (8) LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 48, LCDR3 shown in SEQ ID NO: 58; or

[0096] (9) LCDR1 shown in SEQ ID NO: 39, LCDR2 shown in SEQ ID NO: 49, LCDR3 shown in SEQ ID NO: 59; or

[0097] (10) LCDR1 shown in SEQ ID NO:40, LCDR2 shown in SEQ ID NO:50, and LCDR3 shown in SEQ ID NO:60.

[0098] In some embodiments, the antibody or antigen-binding fragment thereof,

[0099] (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,

[0100] (2) the heavy chain variable region comprises 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 comprises LCDR1 shown in SEQ ID NO: 32, LCDR2 shown in SEQ ID NO: 42, and LCDR3 shown in SEQ ID NO: 52; or,

[0101] (3) the heavy chain variable region comprises 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 comprises LCDR1 shown in SEQ ID NO: 33, LCDR2 shown in SEQ ID NO: 43, and LCDR3 shown in SEQ ID NO: 53; or,

[0102] (4) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 14, and HCDR3 shown in SEQ ID NO: 24; the light chain variable region comprises LCDR1 shown in SEQ ID NO: 34, LCDR2 shown in SEQ ID NO: 44, and LCDR3 shown in SEQ ID NO: 54; or,

[0103] (5) the heavy chain variable region comprises 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 comprises LCDR1 shown in SEQ ID NO: 35, LCDR2 shown in SEQ ID NO: 45, and LCDR3 shown in SEQ ID NO: 55; or,

[0104] (6) the heavy chain variable region comprises HCDR1 of SEQ ID NO: 6, HCDR2 of SEQ ID NO: 16, and HCDR3 of SEQ ID NO: 26; the light chain variable region comprises LCDR1 of SEQ ID NO: 36, LCDR2 of SEQ ID NO: 46, and LCDR3 of SEQ ID NO: 56; or

[0105] (7) the heavy chain variable region comprises HCDR1 of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 17, and HCDR3 of SEQ ID NO: 27; the light chain variable region comprises LCDR1 of SEQ ID NO: 37, LCDR2 of SEQ ID NO: 47, and LCDR3 of SEQ ID NO: 57; or,

[0106] (8) the heavy chain variable region comprises HCDR1 of SEQ ID NO: 8, HCDR2 of SEQ ID NO: 18, and HCDR3 of SEQ ID NO: 28; the light chain variable region comprises LCDR1 of SEQ ID NO: 38, LCDR2 of SEQ ID NO: 48, and LCDR3 of SEQ ID NO: 58; or,

[0107] (9) the heavy chain variable region comprises 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 comprises LCDR1 shown in SEQ ID NO: 39, LCDR2 shown in SEQ ID NO: 49, and LCDR3 shown in SEQ ID NO: 59; or,

[0108] (10) The heavy chain variable region comprises 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 comprises LCDR1 shown in SEQ ID NO: 40, LCDR2 shown in SEQ ID NO: 50, and LCDR3 shown in SEQ ID NO: 60.

[0109] In some embodiments, in the antibody or antigen-binding fragment thereof, (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,

[0110] (2) the heavy chain variable region comprises 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 comprises LCDR1 shown in SEQ ID NO: 32, LCDR2 shown in SEQ ID NO: 42, and LCDR3 shown in SEQ ID NO: 52; or,

[0111] (3) The heavy chain variable region comprises 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 comprises LCDR1 shown in SEQ ID NO: 33, LCDR2 shown in SEQ ID NO: 43, and LCDR3 shown in SEQ ID NO: 53.

[0112] In some embodiments, the antibody or antigen-binding fragment thereof is selected from any one of a rabbit antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, or a caninized antibody or antigen-binding fragment thereof.

[0113] In some embodiments, the antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof that specifically binds to canine IL-31 comprises a heavy chain selected from the group consisting of the heavy chains shown in the following sequences, or a heavy chain having at least 80%, 85%, 90%, 95% or 99% identity compared to the following sequences: SEQ ID NO: 61-70, 163-165, 175-180; and / or

[0114] The invention also comprises a light chain selected from the group consisting of the light chains shown in the following sequences, or a light chain having at least 80%, 85%, 90%, 95% or 99% identity compared to the following sequences: SEQ ID NO: 71-80, 166-168, 181-186.

[0115] In some embodiments, the antibody or antigen-binding fragment thereof, wherein

[0116] (1) the heavy chain comprises the amino acid sequence of SEQ ID NO: 61 or any variant thereof; the light chain comprises the amino acid sequence of SEQ ID NO: 71 or any variant thereof; or

[0117] (2) the heavy chain comprises the amino acid sequence of SEQ ID NO: 62 or any variant thereof; the light chain comprises the amino acid sequence of SEQ ID NO: 72 or any variant thereof; or

[0118] (3) the heavy chain comprises the amino acid sequence of SEQ ID NO: 63 or any variant thereof; the light chain comprises the amino acid sequence of SEQ ID NO: 73 or any variant thereof; or

[0119] (4) the heavy chain comprises the amino acid sequence of SEQ ID NO: 64 or any variant thereof; the light chain comprises the amino acid sequence of SEQ ID NO: 74 or any variant thereof; or

[0120] (5) the heavy chain comprises the amino acid sequence of SEQ ID NO: 65 or any variant thereof; the light chain comprises the amino acid sequence of SEQ ID NO: 75 or any variant thereof; or

[0121] (6) the heavy chain comprises the amino acid sequence of SEQ ID NO: 66 or any variant thereof; the light chain comprises the amino acid sequence of SEQ ID NO: 76 or any variant thereof; or

[0122] (7) the heavy chain comprises the amino acid sequence of SEQ ID NO: 67 or any variant thereof; the light chain comprises the amino acid sequence of SEQ ID NO: 77 or any variant thereof; or

[0123] (8) the heavy chain comprises the amino acid sequence of SEQ ID NO: 68 or any variant thereof; the light chain comprises the amino acid sequence of SEQ ID NO: 78 or any variant thereof; or

[0124] (9) the heavy chain comprises the amino acid sequence of SEQ ID NO: 69 or any variant thereof; the light chain comprises the amino acid sequence of SEQ ID NO: 79 or any variant thereof; or

[0125] (10) the heavy chain comprises the amino acid sequence of SEQ ID NO: 70 or any variant thereof; the light chain comprises the amino acid sequence of SEQ ID NO: 80 or any variant thereof; or

[0126] (11) the heavy chain comprises the amino acid sequence of SEQ ID NO: 163 or any variant thereof; the light chain comprises the amino acid sequence of SEQ ID NO: 166 or any variant thereof; or

[0127] (12) the heavy chain comprises the amino acid sequence of SEQ ID NO: 164 or any variant thereof; the light chain comprises the amino acid sequence of SEQ ID NO: 167 or any variant thereof; or

[0128] (13) the heavy chain comprises the amino acid sequence of SEQ ID NO: 165 or any variant thereof; the light chain comprises the amino acid sequence of SEQ ID NO: 168 or any variant thereof; or

[0129] (14) the heavy chain comprises the amino acid sequence of SEQ ID NO: 175 or any variant thereof; the light chain comprises the amino acid sequence of SEQ ID NO: 181 or any variant thereof; or

[0130] (15) the heavy chain comprises the amino acid sequence of SEQ ID NO: 176 or any variant thereof; the light chain comprises the amino acid sequence of SEQ ID NO: 182 or any variant thereof; or

[0131] (16) the heavy chain comprises the amino acid sequence of SEQ ID NO: 177 or any variant thereof; the light chain comprises the amino acid sequence of SEQ ID NO: 183 or any variant thereof; or

[0132] (17) the heavy chain comprises the amino acid sequence of SEQ ID NO: 178 or any variant thereof; the light chain comprises the amino acid sequence of SEQ ID NO: 184 or any variant thereof; or

[0133] (18) the heavy chain comprises the amino acid sequence of SEQ ID NO: 179 or any variant thereof; the light chain comprises the amino acid sequence of SEQ ID NO: 185 or any variant thereof; or

[0134] (19) The heavy chain comprises the amino acid sequence described in SEQ ID NO: 180 or any variant thereof; and the light chain comprises the amino acid sequence described in SEQ ID NO: 186 or any variant thereof.

[0135] In another aspect, the present disclosure provides a biomaterial selected from any one of the following:

[0136] (1) a polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6; or

[0137] (2) an expression vector containing the polynucleotide described in (1); or

[0138] (3) A host cell containing the polynucleotide described in (1) or the expression vector described in (2).

[0139] In another aspect, the present disclosure provides a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to canine IL-31, comprising the steps of culturing the aforementioned host cell, isolating the antibody from the culture medium, and purifying the antibody.

[0140] In another aspect, the present disclosure provides a pharmaceutical composition comprising the aforementioned antibody or antigen-binding fragment thereof, and a pharmaceutically acceptable excipient, diluent, or carrier.

[0141] In another aspect, the present disclosure provides a use of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned biomaterial, the aforementioned pharmaceutical composition, and the aforementioned kit in preparing a medicament for treating pruritus or allergy in dogs.

[0142] In some embodiments, the use comprises alleviating, inhibiting, 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 use comprises alleviating the clinical manifestations of atopic dermatitis. In some embodiments, the clinical manifestation of atopic dermatitis is pruritus.

[0143] On the other hand, the present disclosure provides a use of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned biomaterial, the aforementioned pharmaceutical composition, and the aforementioned kit in the preparation of a drug for reducing, inhibiting, or neutralizing IL-31 activity in dogs.

[0144] In another aspect, the present disclosure provides a method for treating canine pruritus or allergy, characterized in that a therapeutically effective amount of the aforementioned antibody or antigen-binding fragment thereof or the aforementioned pharmaceutical composition is used to reduce, inhibit or neutralize IL-31 activity in the dog.

[0145] Example

[0146] A further understanding of the present disclosure may be obtained by reference to some of the specific examples provided herein, which are intended to illustrate the present disclosure only and are not intended to limit the scope of the present disclosure in any way. Obviously, various modifications and variations may be made to the present disclosure without departing from the essence of the present disclosure, and therefore, such modifications and variations are also within the scope of protection claimed in this application.

[0147] Example 1: Preparation of rabbit anti-canine IL-31 monoclonal antibody

[0148] 1. Immunogen Preparation and Identification

[0149] The nucleotide sequence (Gene ID: 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 produced in CHO cells using a transient transfection system to produce canine IL-31 protein. The recombinant canine IL-31 protein was subsequently used as an immunogen. The recombinantly expressed canine IL-31 protein was analyzed for purity and molecular weight by SDS-PAGE, and activity was determined by ELISA.

[0150] SDS-PAGE assay

[0151] The specific experimental process is as follows:

[0152] (1) Installing the gel sheets: Remove the comb, place the gel sheets (in pairs) into the mold, and then place the whole sheet in the electrophoresis tank. For the reduced gel sheets, the bottom sealing film needs to be removed.

[0153] (2) Sample treatment: Non-reduced samples: Mix an appropriate amount of sample with non-reduced 5× loading buffer (4:1) and boil in a boiling water bath for 5 min. Reduced samples: Mix an appropriate amount of sample with reduced 5× loading buffer (4:1) and boil in a boiling water bath for 5 min.

[0154] (3) Sample addition: Fill the inner and outer chambers of the electrophoresis tank with electrophoresis buffer, then add the sample and marker. Close the tank lid, making sure the red color corresponds to the positive electrode and the black color corresponds to the negative electrode.

[0155] (4) Electrophoresis: Turn on the power and start electrophoresis. When using gradient gel, set the initial voltage to 90V, then 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.

[0156] (5) Staining: Place the gel in a culture dish containing staining solution and stain on a shaker for about 15 minutes.

[0157] (6) Decolorization: Take the gel out of the staining solution, immerse it in the decolorizing solution, and place it on a shaker until the base color becomes colorless.

[0158] (7) Use BioRad chemiluminescence imager to photograph the sample and mark the information.

[0159] Protein electrophoresis results are shown in Figure 1 , where M is the protein marker column (Bio-rad, Cat. no. 16103573S). The molecular weight can be referred to the annotation on the left. R is the reaction result of the reduced sample, and NR is the reaction result of the non-reduced sample. Figure 1 The results showed that the expression purity of the prepared recombinant canine IL-31 protein met the requirements and the molecular weight was 16.1965 kDa.

[0160] ELISA assay

[0161] 1 μg / mL of certosamin (canized monoclonal antibody to IL-31) was added to a 96-well plate, and 100 μL / well was coated at 4°C overnight; the plate was washed three times with 300 μL / well of 0.1% PBST washing solution, and then incubated with blocking solution at 37°C for 1 hour; after washing the plate three times, a gradient dilution of IL-31 was added and incubated at 37°C for 1 hour; after washing the plate three times, HRP-labeled 6xHis-IgG (H+L) (Abcam) was added at 1000, 5000 and 10000 times dilution for testing, and incubated at 37°C for 1 hour; after washing the plate three times, TMB color development solution was used for color development, 1 M H2SO4 solution was added to terminate the reaction, and the absorbance of the well plate at a wavelength of 450 nm was read using a microplate reader.

[0162] The results of ELISA binding activity are shown in Table 2. The results in Table 2 indicate that the recombinant canine IL-31 protein has good binding activity with ceramide.

[0163] Table 2 Canine IL-31 ELISA binding activity test

[0164]

[0165] 2. Animal immunization

[0166] In this example, a rapid immunization protocol (Phoenix MonRab) was used to immunize four New Zealand rabbits (named R10265, R10266, R10267, and R10268) three times with recombinant canine IL-31 protein. Seven days after the third immunization, the sera of the immunized animals were tested by indirect ELISA to determine the level of immune response. The ELISA detection process can be referred to the experimental steps in 1.2.

[0167] The test results are shown in Table 3. Finally, two rabbits, R10266 and R10267, were selected for B cell screening.

[0168] Table 3 Indirect ELISA detection of animal serum titers

[0169]

[0170]

[0171] Note: When (signal / blank)>=2.1, the titer is the highest, and the blank OD 450 Values are the mean of two replicates. A represents canine IL31, and B represents an unrelated negative protein with a His tag.

[0172] 3. B cell clone screening

[0173] PBMC collection and B cell screening

[0174] Blood was collected from rabbits with high immune response levels to collect PBMCs to obtain antigen-positive B cells, and the antigen-positive B cells were plated on 96-well plates for screening.

[0175] 3.2. Initial screening

[0176] The cell culture supernatants were collected and screened by indirect ELISA to select the supernatants that were positive for canine IL-31 protein.

[0177] 3.3. Confirmation screening

[0178] For all positive B cells obtained in the initial screening phase, the supernatants were screened with recombinant canine IL-31 protein using an indirect ELISA. Counterscreening was then performed with an unrelated protein containing a His-tag to identify clones specific to Group A (canine IL-31 protein-positive) and Group B (unrelated His-tag negative protein). Table 4 shows the results of indirect ELISA screening of B cell supernatants. Cell clones with high binding affinity to Group A and low binding affinity to Group B were used for subsequent experiments.

[0179] Table 4 Indirect ELISA screening of B cell supernatants

[0180]

[0181]

[0182] 3.4. Epitope Confirmation

[0183] B cell culture supernatant was co-incubated with ceramide (positive control) and a competitive ELISA was used to confirm whether the selected positive clones shared the same epitope with ceramide. The results of epitope confirmation in B cell culture supernatant are shown in Tables 5-1 and 5-2. R10268#1:100 represents a 100-fold dilution of the highest titer rabbit serum selected as a positive clone control.

[0184] The calculation formula of inhibition rate is: inhibition rate (%) = 100% - (OD 450 / blank medium OD 450 )*100%

[0185] Table 5-1B cell supernatant epitope confirmation

[0186]

[0187]

[0188] Table 5-2B cell supernatant epitope confirmation

[0189]

[0190]

[0191] As shown in Table 5, 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 good binding ability to canine IL-31 protein.

[0192] Depend on Figure 2It can be seen 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 epitope as the positive control.

[0193] ELISA assay

[0194] ELISA assay was performed on the 10 B cell clones with good binding ability to canine IL-31 screened above. The specific operation steps were 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; after washing the plate three times with 300 μl / well of 0.1% PBST washing solution, the plate was incubated with blocking solution at 37°C for 1 hour; after washing the plate three times, the plate was incubated with B cell culture supernatant at 37°C for 1 hour; after washing the plate three times, the plate was incubated with Goat-anti-Canine-IgG (H+L)-HRP at 37°C for 1 hour; after washing the plate three times, the plate was developed with TMB colorimetric solution, terminated with 1 M H2SO4 solution, and the absorbance value was read at a wavelength of 450 nm using an enzyme-labeled plate.

[0195] The binding activity of different cell samples is shown in Table 6. As can be seen from the results in Table 6, all cell types exhibited good binding activity. The binding activity of the ten cell types was ranked, and among them, B cell clone 19D1-1 had the strongest binding activity.

[0196] Table 6 ELISA test results

[0197] Experiment number sample <![CDATA[EC 50 Value / (ng / ml)]]> Binding 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

[0198] 3.6. Cell biological activity assay

[0199] As previously mentioned, the proinflammatory cytokine interleukin 31 (IL-31) is produced by activated T lymphocytes from various species and contributes to pruritus on canine skin. In canine atopic dermatitis, when dogs are exposed to allergens, skin helper T cells (Th2) produce the cytokine IL-31. IL-31 binds to a co-receptor consisting of IL-31 receptor A (IL-31RA) and oncostatin M receptor (OSMR), activating the STAT signaling cascade through Janus kinase phosphorylation and leading to upregulation of target genes, triggering pruritic behavior in dogs. Anti-IL-31 monoclonal antibodies can neutralize IL-31, thereby achieving an antipruritic effect.

[0200] In order to evaluate whether the 10 B cell clones screened can affect the ability of IL-31 to mediate pSTAT signal transduction, a cell biological activity assay was established in this example. The method was pre-treated with 10 ng / mL of canine-γ interferon (R&D Systems, 781-CG-050) for 24 hours and serum starved for 2 hours before IL-31 treatment to increase IL-31 receptor expression and pSTAT signal transduction. After pretreatment, recombinant canine IL-31 was added for 5 minutes at 1 μg / mL, and the ability of STAT phosphorylation was evaluated using HTRF technology (PerkinElmer, 62AT3PET). The ability of mAb to inhibit STAT phosphorylation was qualitatively measured after B cell supernatant was incubated with canine IL-31 for 1 hour.

[0201] The specific experimental steps are as follows:

[0202] (1) Cell sensitization: DH82 (canine macrophages / dog kidney malignant histiocytosis cells) (Cell Bank of the Chinese Academy of Sciences, catalog number TCO 3) were cultured at 1×10 5 cells / 100 μL were plated in a 96-well cell culture plate and sensitized by adding 10 ng / mL canine interferon-γ for 24 hours in MEM medium containing 15% FBS, 2 mmol / L GlutaMax, and 1 mmol / L sodium pyruvate at 37°C and 5% CO2;

[0203] (2) Washing: Remove the culture medium and wash the cells once with 200 μL / well of 1xPBS;

[0204] (3) Serum starvation: replace with serum-free medium 100 μl / well, starvation treatment for 2 h;

[0205] (4) Sample processing: After IL-31 cytokine was diluted to 2 μg / ml in serum-free medium, equal volumes of B cell culture supernatant and IL-31 were mixed and incubated for one hour. Canine IL-31 containing 1 μg / ml was used as a negative control;

[0206] (5) Add inhibitors and stimulators: 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 CO2 incubator for 1 h;

[0207] (6) Washing: Remove the culture medium and wash the cells once with 200 μl / well of 1xPBS;

[0208] (7) Cell lysis: Add 30 μL of 1x cell lysis buffer to each well, incubate at 100 rpm for 30 min, and the sample preparation is complete.

[0209] (8) Add samples: Add 16 μL of cell lysate supernatant and 4 μL of premixed antibodies to the 96-well plate provided with the HTRF. Incubate overnight at room temperature and read the sample. Add 16 μL of phosphorylation control and negative control. Cover the 96-well plate with a sealing film.

[0210] (9) Incubation: Incubate overnight at room temperature.

[0211] (10) Reading: Set up the Eu*Crypatate reader and read the fluorescence emission at two different wavelengths (665 nm and 620 nm) in a compatible HTRF reader to calculate the inhibition rate of the antibody.

[0212] (11) Data processing: Ratio = (665 nm signal / 620 nm signal) * 10000

[0213] Inhibition rate = 100% - (sample ratio / canine IL-31 negative control ratio) * 100%

[0214] The test was established when the ratio of phosphorylation control to negative control was >2.

[0215] (12) Results Analysis

[0216] After calculation, the value of the phosphorylation control ratio / negative control ratio was 3.8, and the experiment was established.

[0217] The results of the cell activity assay are shown in Table 7. As shown in Table 7, the B cell clones with inhibition rates above 20% include 38E2-1, 47C1-1, and 35C9-1.

[0218] Table 7 Cell biological activity assay

[0219] Serial number Clone number Inhibition rate (%) Sorting 1 5H4-2 8.7 10 2 18E8-1 19.5 5 3 19D1-1 19.8 4 4 19C7-1 13.4 8 5 30G5-1 12 9 6 35C9-1 24.6 3 7 38E2-1 29.4 1 8 44B4-1 19.5 6 9 47C1-1 28 2 10 30A11-1 13.5 7

[0220] 4. Antibody Sequencing

[0221] The 10 B cell clones that were positive by ELISA were sequenced in the V region by Sanger sequencing. The amino acid sequence numbers of the 10 antibodies are shown in Table 8, and the nucleotide sequence numbers are shown in 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 Determination

[0227] 5.1. Transient Expression of Rabbit Anti-IL-31 Monoclonal Antibody

[0228] (1) Gene sequence design

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

[0230] (2) Sequence synthesis and expression vector

[0231] The heavy and light chains of five molecules, 38E2-1, 47C1-1, 35C9-1, 19D1-1, and 5H4-2, were synthesized separately after fusion. The genes were first amplified by PCR, then linearized in the pcDNA3.1 vector using NheI and ApaI restriction enzymes. Finally, the genes were ligated into the NheI and ApaI restriction sites of the pcDNA3.1 vector using seamless cloning. The ligation products were transformed into competent Escherichia coli Top10 cells, plated, and single colonies were picked for sequencing verification. Successful transformation was then expanded to extract the endotoxin-free plasmid, which was then integrated into the pcDNA3.1 plasmid. Ultimately, the 38E2-1-pcDNA3.1, 47C1-1-pcDNA3.1, 35C9-1-pcDNA3.1, 19D1-1-pcDNA3.1, and 5H4-2-pcDNA3.1 plasmids were obtained.

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

[0233] 1) CHO-s cell culture: Under conventional culture conditions in 125 mL shake flasks, when the cell density reaches 1*10 7 When 2×10 5 The initial density of 10 / mL was inoculated into a new 125mL shake flask and cultured at 37℃ and 5% CO2. After 2 days, when the density reached 5×10 5 When the number of cells / mL was reached, transient transfection was started according to the operating instructions of the transfection reagent lipo2000;

[0234] 2) After 7 days, the cell supernatant was collected, centrifuged at 5000 rpm for 30 min to remove cells and debris, filtered through 0.45 μm, and stored at 4°C for subsequent antibody purification.

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

[0236] Protein A affinity chromatography was used to purify the cell culture supernatant. The specific operation steps were as follows: first, the supernatant was equilibrated with affinity chromatography equilibration solution (20mM PB, 0.15M NaCl pH 7.2), the clarified filtered liquid was loaded, and then the supernatant was equilibrated with affinity chromatography equilibration solution (20mM PB, 0.15M NaCl pH 7.2), then the supernatant was washed with affinity chromatography eluent (20mM PB, 1M NaCl pH 7.2), and then eluted with eluent (0.1M glycine-HCl, pH 3.0). The protein electrophoresis results are shown in Figure 2. Figure 3 ,Depend on Figure 3 It can be seen that the purity of the eluted sample by SDS-PAGE is more than 95%, and the target molecular weight is around 150KD. The results of the rabbit antibody candidate assay by Western Blot are shown in Figure 4 ,Depend on Figure 4 It can be seen that all rabbit antibody candidates can bind to canine IL-31.

[0237] 5.3. Affinity determination

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

[0239] The affinity test results are shown in Table 10. As shown in Table 10, the affinity of three clones is greater than that of the positive control ceramide (Canine IgG), namely 38E2-1, 47C1-1 and 35C9-1. The affinity of 38E2-1 is the best, with an affinity of 2.547E-09. Figure 5 The results were consistent with those of cell biological activity assay.

[0240] Table 10 BLI affinity determination

[0241]

[0242]

[0243] Example 2: Establishment of canine pruritus model

[0244] 1. Preliminary establishment of canine pruritus model

[0245] To confirm that the inhibition of IL-31-mediated cell signaling observed in the DH82 (canine renal malignant histiocytosis) cell assay correlates with the inhibition of IL-31-mediated pruritus in dogs, the applicant, referencing 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 inoculated intravenously into test dogs at a low dose of 3 μg / kg and a high dose of 8 μg / kg. Sterile PBS served as a negative control, with five dogs selected for 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. The frequency of pruritus in both animals was recorded. The 30-minute pre-inoculation clinical observation was used to establish a baseline. During the clinical observation period, any behaviors such as licking, scratching, biting, rubbing, frequent head shaking, and digging were considered scratching behaviors. Specifically, at consecutive 1-minute intervals, a "yes / no" judgment was made as to whether each dog displayed pruritus behavior. The display of pruritus behavior, such as licking / chewing the paws, flanks and / or anal area, scratching the flanks, neck and / or floor, shaking the head and rubbing their arms on the cage floor was sufficient to elicit a "yes" response during the specified time interval. At the end of the period, the number of "yes" responses was added together to obtain a cumulative pruritus score index (PSI). The PSI results for 30 minutes before injection and 2 hours after injection were statistically analyzed, and the significant differences in PSI between each dose group and the negative control group were compared using a one-way analysis of variance using SPSS software. When P>0.05, it indicates that the PSI levels of each dose group were not significantly different from those of the control group; if P<0.05, it indicates that the PSI levels of each dose group were significantly different from those of the control group.

[0246] Statistical analysis Figure 6 ,Depend on Figure 6 It can be seen that the PSI results of the high-dose group and the low-dose group half an hour after injection for 2 hours were significantly different from those of the negative control, and there was no significant difference in the PSI results between the high-dose group and the low-dose group. The 3μg / kg dosage was initially selected to establish a canine pruritus model.

[0247] 2. Itch Model Confirmation Experiment

[0248] After determining the intravenous dose of canine IL-31, we further validated the model. On Day 7, the test dogs were inoculated intravenously with 3 μg / kg of canine IL-31. Sterile PBS was used as a negative control, and 10 test dogs with significant differences compared to the negative control were selected. On Day 0, the treatment group (5 dogs / group) received a subcutaneous injection of 2 mg / kg of ceramide via the neck, while the control group (5 dogs / group) received an equal volume of sterile PBS via the neck. On Day 8, the test dogs were inoculated intravenously with 3 μg / kg of canine IL-31. Clinical observation began 30 minutes before inoculation and was recorded continuously for 30 minutes. Clinical observation began 30 minutes after inoculation and was recorded continuously for 2 hours. The frequency of pruritus was recorded twice. The clinical observation 30 minutes before inoculation was used to establish a baseline. The PSI results were calculated for 30 minutes before injection and 2 hours after injection, and the PSI of the treatment and control groups were compared for significant differences using one-way analysis of variance using SPSS software. When P>0.05, it means that the PSI levels of the treatment group and the control group are not significant; if P<0.05, it means that the PSI levels of the treatment group and the control group are significantly different.

[0249] The statistical results are shown in 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 D8, indicating that chloramphenicol can inhibit the pruritus symptoms mediated by IL-31 in dogs after 8 days of administration.

[0250] Finally, a canine pruritus model was successfully established by intravenous administration of canine IL-31, and the intravenous dose of canine IL-31 was determined to be 3 μg / kg.

[0251] Example 3: In vivo efficacy study of rabbit-derived anti-IL-31 monoclonal antibodies

[0252] After the canine pruritus model was established, the applicant further studied the in vivo efficacy of rabbit anti-L-31 monoclonal antibodies. A certain amount of rabbit antibody 38E2-1, 47C1-1 and 35C9-1 samples were transiently expressed, with chlorambucil 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 eliminated through the canine pruritus model. On D0, the experimental dogs in the treatment group (5 / group) were subcutaneously injected with rabbit antibody 38E2-1, rabbit antibody 47C1-1 and rabbit antibody 35C9-1 through the neck, with an injection dose of 2 mg / kg. The experimental dogs in the positive control group (5 / group) were subcutaneously injected with 2 mg / kg chlorambucil through the neck, and the experimental dogs in the negative control group (5 / group) were subcutaneously injected with an equal volume of sterile PBS through the neck. Referring to relevant literature, 2.5 hours later, the test dogs were inoculated intravenously with 3 μg / kg canine IL-31. 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. The frequency of pruritus was recorded 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 calculated. One-way analysis of variance using SPSS software was used to compare the PSI between the treatment and control groups. A P > 0.05 indicated that the PSI levels in the treatment and control groups were not significantly different; a P < 0.05 indicated that the PSI levels in the treatment and control groups were significantly different.

[0253] The experimental results are shown in Figure 8 ,Depend on Figure 8 Significant differences were observed between the treatment group (half an hour after injection and cumulatively for 2 hours) and the negative control group (sterile PBS). There was no significant difference between rabbit antibody 38E2-1 and the positive control group (Cetirizine) (P>0.05). There was no significant difference between rabbit antibody 47C1-1 and the positive control group (P>0.05). There was a significant difference between rabbit antibody 35C9-1 and the positive control group (P=0.00014). Of the three rabbit antibodies, 38E2-1 showed the best in vivo activity, followed by 47C1-1 and 35C9-1.

[0254] Example 4: Caninized Antibody Transformation Strategy

[0255] The production of anti-drug antibodies (ADA) can be associated with loss of efficacy of any biotherapeutic protein, including monoclonal antibodies. To help mitigate the risks associated with the formation of ADA to the rabbit anti-IL-31 monoclonal antibodies provided herein, a caninization strategy was employed to construct chimeric antibodies. Figure 9This caninization strategy is based on identifying the most suitable canine germline antibody sequence for CDR grafting. After extensive analysis of the heavy and light chains of all available canine germline sequences, candidate germlines are selected based on their homology to the rabbit mAbs. The specific steps involve finding a caninization template on the IMGT website that best matches the rabbit antibody FV region. The CDR regions in the caninization template are then replaced with rabbit CDR regions. The sequence is analyzed using bioinformatics software, and several amino acid backmutations are performed, with the goal of obtaining several caninized antibodies with comparable affinity and activity to the parental antibody.

[0256] Example 5: Construction and preparation of 38E2-1, 47C1-1 and 35C9-1 chimeric antibodies

[0257] 1. Construction and transient expression of 38E2-1, 47C1-1, and 35C9-1 chimeric antibodies

[0258] According to the caninization 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 sequences of the heavy and light chains of these three molecules. The amino acid sequences and DNA sequences of the heavy and light chains of canine IgGB can be obtained from the GenBank database. The accession number for the amino acid sequence of the IgGB heavy chain is AAL35302.1 (SEQ ID NO: 161), and the accession number for the DNA sequence is AF354265.1 (SEQ ID NO: 162); the accession number for the amino acid sequence of the canine antibody κ light chain is ABY 57289.1, and the accession number for the DNA sequence is EU305402.1. The amino acid sequence and nucleotide sequence encoding information of the chimeric antibody are shown in Table 11.

[0259] Table 11 Sequence coding information of chimeric antibodies

[0260]

[0261]

[0262] Sequence synthesis and CHO cell transient transfection expression were performed according to the method of Example 1.5, and the purity of the one-step affinity chimeric antibody was evaluated. The experimental results are shown in Figure 10 ,Depend on Figure 10 It can be seen that the purity of SDS-PAGE is more than 95%, the target molecular weight is about 150KD, and the results of protein immunoblotting are shown in Figure 11 ,Depend on Figure 11 It was found that all the chimeric antibody candidates were able to bind to canine IL-31.

[0263] 2. Chimeric Antibody Affinity Evaluation

[0264] 2.1 ELISA assay

[0265] 1 μg / mL canine IL-31 (100 μL / well) 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 solution, and then incubated with blocking solution at 37°C for 1 hour; after washing the plate three times, a serially diluted chimeric antibody was added and incubated at 37°C for 1 hour; after washing the plate three times, Goat-anti-Canine-IgG (H+L)-HRP was added and incubated at 37°C for 1 hour; after washing the plate three times, the plate was developed with TMB colorimetric solution, terminated with 1 M H2SO4 solution, and the absorbance was read at a wavelength of 450 nm using an enzyme-labeled plate.

[0266] 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, with 38E2-1 being the best, followed by 47C1-1, and 35C9-1 being the worst.

[0267] Table 12 Chimeric antibody ELISA assay

[0268] 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 Cetirizine 125.70

[0269] 2.2 BLI affinity determination

[0270] The purified antibodies were immobilized on a ProA biosensor and BLI affinity assays were performed using canine IL-31 protein as the mobile phase. Table 13 shows that the affinity of the chimeric antibodies was either similar to or slightly lower (within 3-fold) than that of the corresponding rabbit antibodies, and both exhibited higher affinity than metformin.

[0271] Table 13 BLI affinity determination of chimeric antibodies

[0272] 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 antibody 35C9-1 1.27E-08 Chimeric antibody 35C91 1.19E-08 Cetirizine 3.19E-08

[0273] 3. Evaluation of Cell Biological Activity of Chimeric Antibodies

[0274] 3.1 In vitro activity evaluation

[0275] The purified chimeric antibody sample was diluted to a starting concentration of 10 μg / mL, and then a 1.75-fold gradient dilution was performed to obtain a total of 8 concentration points. Each sample after the gradient dilution was mixed with an equal volume of IL-31 protein and incubated for 1 hour. In vitro activity evaluation was performed according to the cell biological activity assay in Example 1.6. The test results are shown in Tables 14 and Figure 13 From the above results, it can be seen that the three chimeric antibodies have in vitro activity, among which the IC 50 The smaller the value, the higher the inhibition rate and the best in vitro activity.

[0276] Table 14 Chimeric antibody cell biological activity assay

[0277] 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 Cetirizine 1548

[0278] Example 6: In vivo efficacy evaluation of chimeric antibodies

[0279] The in vivo efficacy evaluation of the chimeric antibodies was performed according to the method of Example 3. The applicant transiently expressed a certain amount of sample for evaluating the in vivo efficacy of the chimeric antibodies 38E2-1, 47C1-1 and 35C9-1, using chlorambucil as a positive control and sterile PBS as a negative control. Five dogs were selected for each group, for a total of 25 test dogs. Abnormal dogs were eliminated using the canine pruritus model. On D0, the test dogs in the treatment group (5 / group) were subcutaneously injected with chimeric antibody 38E2-1, chimeric antibody 47C1-1 and chimeric antibody 35C9-1 through the neck, respectively, with an injection dose of 2 mg / kg. The test dogs in the positive control group (5 / group) were subcutaneously injected with 2 mg / kg chlorambucil through the neck, and the test dogs in the negative control group (5 / group) were subcutaneously injected with an equal volume of sterile PBS through the neck. Referring to relevant literature, 2.5 hours later, the test dogs were inoculated intravenously with 3 μg / kg canine IL-31. 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. The frequency of pruritus was recorded 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 calculated. The PSI of the treatment group and the control group were compared for significance using a one-way analysis of variance using SPSS software. A P > 0.05 indicated that the PSI level in the treatment group was not significantly different from that in the control group; a P < 0.05 indicated that the PSI level in the treatment group was significantly different from that in the control group.

[0280] The experimental results are statistically shown as follows Figure 14 As shown by Figure 14 Significant differences were observed 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 38E2-1 showed the best in vivo activity, followed by chimeric 47C1-1 and chimeric 35C9-1.

[0281] Example 7: Construction and preparation of caninized antibodies

[0282] 1. Construction and transient expression of caninized antibodies 38E2-1, 47C1-1, and 35C9-1

[0283] 38E2-1, 47C1-1, and 35C9-1 were caninized according to the caninization strategy of Example 4, and two caninized antibodies were obtained for each parent antibody. The sequence encoding information of the caninized antibodies is shown in Table 15.

[0284] Table 15 Sequence coding information of caninized antibodies

[0285]

[0286]

[0287] Sequence synthesis and CHO cell transient transfection expression were performed according to the method of Example 1.5, and the purity of the one-step affinity chimeric antibody was evaluated. The experimental results are shown in Figure 15 ,Depend on Figure 15 It can be seen that the purity of SDS-PAGE is more than 95%, the target molecular weight is about 150KD, and the results of protein immunoblotting (Western Blot) are shown in Figure 16 ,Depend on Figure 16 It can be seen that all caninized antibody candidates can bind to canine IL-31.

[0288] 2. Affinity Evaluation of Caninized Antibodies

[0289] (1) ELISA assay

[0290] The caninized antibodies were determined by ELISA according to Example 4.2. Figure 17 As shown in Table 16, the above results show that the binding activity of the six caninized antibodies to canine IL-31 is lower than that of the corresponding chimeric antibodies. The binding activity of the caninized antibodies 38E2-1 and 47C1-1 is not much different from that of the positive control ceramide. The binding activity of the caninized antibodies 35C9-1-H1L1 and 35C9-1-H1L2 is not as good as that of the positive control ceramide.

[0291] Table 16 Caninized Antibody ELISA Determination

[0292] Sample name <![CDATA[ELISA(EC 50 Value ng / ml)]]> Chimeric antibody 38E2-1 92.85 Caninized antibody 38E2-1-H3L3 93.41 Caninized antibody 38E2-1-H3L2 97.75 Chimeric antibody 47C1-1 102.2 Caninized antibody 47C1-1-H2L2 113.4 Caninized antibody 47C1-1-H3L3 105.1 Chimeric antibody 35C9-1 102.7 Caninized antibody 35C9-1-H1L1 189.7 Caninized antibody 35C9-1-H1L2 157.6 Cetirizine 123.8

[0293] (2) BLI affinity determination

[0294] The caninized antibodies were assayed for affinity using the BLI method described in Example 4.2. The results in Table 17 indicate that the affinity of the caninized antibodies is one order of magnitude lower than that of the rabbit antibodies. However, the affinity of the modified antibodies 38E2-1 and 47C1-1 remains higher than that of the positive control, ceramide, while the affinity of the modified antibody 35C9-1 is lower than that of the positive control, ceramide.

[0295] Table 17 Caninized Antibody Affinity Determination

[0296]

[0297]

[0298] 3. Evaluation of Cellular Biological Activity of Caninized Antibodies

[0299] (1) In vitro activity evaluation

[0300] The purified caninized samples were evaluated for in vitro activity according to Example 4.3. The results are shown in Tables 18 and Figure 18 The above results show that the caninized 38E2-1 and 47C1-1 cells showed little difference from the corresponding chimeric antibodies and chloramphenicol, while the cell activity of the modified 35C9-1 cells decreased and was inferior to chloramphenicol.

[0301] Table 18 Results of cell biological activity assay of caninized antibodies

[0302] Sample name <![CDATA[In vitro activity (IC 50 value ng / ml)]]> Chimeric antibody 38E2 1296 Caninized antibody 38E2-1-H3L3 1397 Caninized antibody 38E2-1-H3L2 1500 Chimeric antibody 47C1 1558 Caninized antibody 47C1-1-H2L2 1448 Caninized antibody 47C1-1-H3L3 1612 Chimeric antibody 35C9 1711 Caninized antibody 35C9-1-H1L1 2256 Caninized antibody 35C9-1-H1L2 2045 Cetirizine 1508

[0303] Example 8: In vivo efficacy evaluation of caninized antibodies

[0304] 1. Evaluation of in vivo activity of caninized antibodies

[0305] The in vivo efficacy of caninized antibodies was evaluated according to the method of Example 3. A certain amount of sample was transiently expressed to evaluate the in vivo efficacy of caninized antibodies 38E2-1, 47C1-1, and 35C9-1. Cetirizine was used as a positive control, and sterile PBS was used as a negative control. Five dogs were selected for each group, for a total of 40 test dogs. Abnormal dogs were removed using a canine pruritus model. On D0, dogs in the treatment group (5 / group) were subcutaneously injected with caninized antibodies 38E2-1-H3L3, 38E2-1-H3L2, 47C1-1-H2L2, 47C1-1-H3L3, 35C9-1-H1L1, and 35C9-1-H1L2, respectively, at a dose of 2 mg / kg. Dogs in the positive control group (5 / group) were subcutaneously injected with 2 mg / kg Cetirizine, and dogs in the negative control group (5 / group) were subcutaneously injected with an equal volume of sterile PBS. Referring to relevant literature, 2.5 hours later, the test dogs were inoculated intravenously with 3 μg / kg canine IL-31. 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. The frequency of pruritus was recorded 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 calculated. The PSI of the treatment group and the control group were compared for significance using a one-way analysis of variance using SPSS software. A P > 0.05 indicated that the PSI level in the treatment group was not significantly different from that in the control group; a P < 0.05 indicated that the PSI level in the treatment group was significantly different from that in the control group.

[0306] The experimental results are statistically shown as follows Figure 19 As shown by Figure 19 It can be seen that there are significant differences between the treatment group and the negative control group, and the caninized antibodies 38E2-1-H3L3, 38E2-1-H3L2, and 47C1-1-H2L2 have equivalent efficacy to certosamol; the caninized 47C1-1-H3L3 has significant differences from certosamol, and the caninized antibodies 35C9-1-H1L1 and 35C9-1-H1L2 have extremely significant differences, and their efficacy is not as good as certosamol.

[0307] 2. Efficacy Study of Caninized Antibody 38E2-1-H3L3

[0308] To determine the minimum dosage and duration of administration of the caninized antibody 38E2-1-H3L3, an efficacy study of the antibody was conducted according to the method of Example 3. The experimental groups received 0.5 mg / kg, 1 mg / kg, and 2 mg / kg of the caninized antibody 38E2-1-H3L3, respectively. Cetirizine was used as a positive control, and sterile PBS was used as a negative control. Five dogs were selected per group, for a total of 25 experimental dogs. Abnormal dogs were removed using a canine pruritus model. On D0, dogs in the treatment groups (5 / group) received subcutaneous injections of 0.5 mg / kg, 1 mg / kg, and 2 mg / kg of the caninized antibody 38E2-1-H3L3, respectively. Dogs in the positive control group (5 / group) received subcutaneous injections of 2 mg / kg of Cetirizine. Dogs in the negative control group (5 / group) received subcutaneous injections of an equal volume of sterile PBS. Referring to relevant literature, dogs were inoculated intravenously with 3 μg / kg canine IL-31 on days 1, 7, 14, 21, and 28. Clinical observation began 30 minutes before each inoculation and was recorded continuously for 30 minutes. Clinical observation also began 30 minutes after each inoculation and was recorded continuously for 2 hours. The frequency of pruritus during clinical observation was recorded. The clinical observation 30 minutes before D1 inoculation was used to establish a baseline. PSI values were calculated for the cumulative 30 minutes before D1 injection and for the cumulative 2 hours after D1, D7, D14, D21, and D28 injections. One-way analysis of variance (ANOVA) using SPSS software was used to compare the PSI levels between the treatment and control groups. A P value greater than 0.05 indicated no significant difference in PSI between the treatment and control groups; a P value less than 0.05 indicated a significant difference in PSI between the treatment and control groups.

[0309] The 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 caninized antibody 38E2-1-H3L3 at doses of 0.5 mg / kg, 1 mg / kg and 2 mg / kg was equivalent to ceramide on the 28th day after administration, indicating that the minimum dosage of the caninized antibody 38E2-1-H3L3 is 0.5 mg / kg and the drug duration is at least 28 days.

[0310] Example 9: Evaluation of the immunogenicity of caninized antibody 38E2-1-H3L3

[0311] The production of anti-drug antibodies (ADAs) can affect drug activity and half-life. To evaluate the immunogenicity of the caninized antibody 38E2-1-H3L3, 10 healthy dogs were selected and divided into a test group and a control group, based on relevant patents and literature. The test group (5 dogs / group) received a subcutaneous injection of 9 mg / kg of the caninized antibody 38E2-1-H3L3 via the neck on days 0, 14, and 28. The negative control group (5 dogs / group) received an equal volume of sterile PBS via the neck on days 0, 14, and 28. Blood was collected on days 0, 7, 14, 21, and 28 for serum drug concentration and anti-drug antibody testing.

[0312] 1. Blood drug concentration determination

[0313] 100 μL / well of 1 μg / mL canine IL-31 was added to a 96-well plate and coated overnight at 4°C; after washing the plate three times with 300 μL / well of 0.1% PBST washing solution, a blocking solution containing 5% skim milk powder was added and incubated at 37°C for 1 hour; after washing the plate three times, the negative serum was diluted 200 times with PBS containing 1% skim milk powder as a diluent, and then a gradient dilution of canine antibody was added and incubated at 37°C for 1 hour; after washing the plate three times, Goat-anti-Canine-IgG (H+L)-HRP 1:2000 was added and incubated at 37°C for 1 hour; after washing the plate three times, the plate was developed with TMB colorimetric solution, terminated with 1M H2SO4 solution, and the absorbance was read at a wavelength of 450 nm using an enzyme-labeled plate.

[0314] Depend on Figure 21 It can be seen that the drug metabolism of the experimental groups is basically the same.

[0315] 2. Preparation of ADA-positive serum

[0316] (1) The caninized antibody 38E2-1-H3L3 was diluted to 1 mg / mL with physiological saline, mixed with an equal volume of adjuvant (Biolong QuickAntibody-Mouse5W), and then injected intramuscularly at a volume of 100 μL per mouse to immunize five mice (Balb / c female mice, 6 weeks old).

[0317] (2) Three weeks later, the caninized antibody 38E2-1-H3L3 was diluted to 1 mg / mL with physiological saline, mixed with an equal volume of adjuvant, and then injected intramuscularly at a volume of 100 μL / mouse to immunize 5 mice again.

[0318] (3) After about two weeks, blood was collected from five mice and centrifuged at 3000 rpm for 5 minutes to obtain serum. The titer of the serum from the immunized mice was determined by ELISA.

[0319] 3. ELISA determination of positive serum titer

[0320] 1 μg / mL canine antibody 38E2-1-H3L3 (100 μl / well) 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 solution and incubated with blocking solution at 37°C for 1 hour. After washing the plate three times, the sera of five immune mice were diluted 100-fold with blocking solution, then diluted 3-fold in a 10-point gradient, and loaded onto a 96-well plate at 100 μL / well and incubated at 37°C for 1 hour. Blocking solution was used as a blank control. After washing the plate three times, Rabbit-anti-mouse-IgG (H+L)-HRP was added and incubated at 37°C for 1 hour. After washing the plate three times, the plate was developed with TMB colorimetric solution, terminated with 1MH2SO4 solution, and the absorbance was read at 450 nm using an enzyme-labeled plate.

[0321] From the results in Table 19, it can be seen that the average measured value of the blank control group is about 0.45. Taking the detection value twice of 0.45 (i.e. greater than 0.9) as the basis for positive judgment, the serum titers of immunized mice 1-5 are: 1:24300, 1:72900, 1:72900, 1:218700, and 1:72900, respectively.

[0322] Table 19 Positive serum titer determination

[0323]

[0324] 3. Anti-drug antibody assay

[0325] 100 μL / well of 1 μg / mL caninized antibody was added to a 96-well plate and coated overnight at 4°C; after washing the plate three times with 300 μL / well of 0.1% PBST washing solution, a blocking solution containing 5% skim milk powder was added and incubated at 37°C for 1 hour; after washing the plate three times, PBS containing 1% skim milk powder was used as a diluent to dilute the mouse polyclonal positive serum containing anti-drug antibodies (serum titer 1:218700) 10-fold and then 2-fold serial dilutions of 10 points; the serum to be tested and the canine negative serum were all diluted 10-fold with the sample diluent and incubated at 37°C for 1 hour; after washing the plate three times, biotin-labeled caninized antibody was added; after washing the plate three times, Streptavidin-HRP was added; after washing the plate three times, TMB color development solution was used, 1M H2SO4 solution was used for termination, and the absorbance value was read at a wavelength of 450 nm using an enzyme-labeled plate.

[0326] The results of anti-drug antibody assays for positive serum are shown in Table 20, and those for the experimental and negative control groups are shown in Table 21. As can be seen from the table above, no anti-drug antibodies were detected in any of the tested samples, indicating that the immunogenicity risk of the caninized antibody 38E2-1-H3L3 is very low.

[0327] Table 20 Results of positive serum antidrug antibody assay

[0328] potency 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

[0329] Table 21 Antidrug antibody determination results of the experimental group and negative control group

[0330]

[0331]

[0332] In summary, the anti-canine IL-31 monoclonal antibodies of the present invention can effectively alleviate the clinical symptoms associated with canine atopic dermatitis, and the immunogenicity risk of the caninized antibodies is very low.

[0333] The above description is only an embodiment of the present invention and does not limit the present invention in any form or substance. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention. Any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the spirit and scope of the present invention by using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. 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: The heavy chain variable region comprises 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 comprises LCDR1 shown in SEQ ID NO: 33, LCDR2 shown in SEQ ID NO: 43, and LCDR3 shown in SEQ ID NO:

53.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof is selected from any one of a rabbit antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, or a caninized antibody or antigen-binding fragment thereof.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof that specifically binds to canine IL-31 comprises a heavy chain selected from the group consisting of the heavy chain shown in the following sequence, or a heavy chain having at least 80% identity with the following sequence: SEQ ID NO: 63, 163, 175 or 176; and / or The invention also comprises a light chain selected from the group consisting of SEQ ID NO: 73, 166, 181 or 182, or a light chain having at least 80% identity thereto.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof that specifically binds to canine IL-31 comprises a heavy chain selected from the group consisting of the heavy chain shown in the following sequence, or a heavy chain having at least 85% identity with the following sequence: SEQ ID NO: 63, 163, 175 or 176; and / or The invention also comprises a light chain selected from the group consisting of SEQ ID NO: 73, 166, 181 or 182, or a light chain having at least 85% identity thereto.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof that specifically binds to canine IL-31 comprises a heavy chain selected from the group consisting of the heavy chain shown in the following sequence, or a heavy chain having at least 90% identity with the following sequence: SEQ ID NO: 63, 163, 175 or 176; and / or The invention also comprises a light chain selected from the group consisting of SEQ ID NO: 73, 166, 181 or 182, or a light chain having at least 90% identity thereto.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof that specifically binds to canine IL-31 comprises a heavy chain selected from the group consisting of the heavy chain shown in the following sequence, or a heavy chain having at least 95% identity with the following sequence: SEQ ID NO: 63, 163, 175 or 176; and / or The invention also comprises a light chain selected from the group consisting of SEQ ID NO: 73, 166, 181 or 182, or a light chain having at least 95% identity thereto.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof that specifically binds to canine IL-31 comprises a heavy chain selected from the group consisting of the heavy chain shown in the following sequence, or a heavy chain having at least 99% identity with the following sequence: SEQ ID NO: 63, 163, 175 or 176; and / or The invention also comprises a light chain selected from the group consisting of SEQ ID NO: 73, 166, 181 or 182, or a light chain having at least 99% identity thereto.

8. The antibody or antigen-binding fragment thereof according to claim 3, wherein (1) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 63 or any variant thereof; the light chain comprises the amino acid sequence shown in SEQ ID NO: 73 or any variant thereof; or (2) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 163 or any variant thereof; the light chain comprises the amino acid sequence shown in SEQ ID NO: 166 or any variant thereof; or (3) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 175 or any variant thereof; the light chain comprises the amino acid sequence shown in SEQ ID NO: 181 or any variant thereof; or (4) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 176 or any variant thereof; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 182 or any variant thereof.

9. A biomaterial selected from any one of the following: (1) a polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8; 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).

10. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, and a pharmaceutically acceptable excipient, diluent or carrier.

11. A kit for detecting or quantifying canine IL-31 protein in a clinical or biological sample, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, and optionally further comprising one or more reagents for detecting binding of the antibody or antigen-binding fragment thereof to canine IL-31 or an epitope thereof.

12. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the biomaterial according to claim 9, the pharmaceutical composition according to claim 10, or the kit according to claim 11 in the preparation of a medicament for treating pruritus or allergy in dogs.

13. The use according to claim 12, wherein The pruritus is atopic dermatitis, eczema, psoriasis, or scleroderma.

14. The use according to claim 12, wherein The pruritus is atopic dermatitis.

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