Anti-canine il-31 antibodies and uses thereof

Abstract

The present invention relates to anti-canine IL-31 antibodies that specifically bind IL-31 and uses thereof. The antibodies, when used alone, inhibit IL-31 downstream signaling activation in canine macrophages mediated by canine IL-31. The antibodies can be used to treat IL-31 induced disorders in dogs.

Description

Technical Field

[0001] This invention belongs to the field of recombinant antibodies and relates to IL-31 antibodies, their antigen-binding fragments, their pharmaceutical compositions, and their medicinal uses, such as treating IL31-induced diseases in dogs or reducing cellular IL31 signaling. Background Technology

[0002] Atopic dermatitis (AD) is an inflammatory and pruritic skin disease characterized by antibodies or antigen-binding fragments with a genetic predisposition, and its clinical features vary (Halliwell R et al., Vet Immunol Immunopathol 2006; 114:207–208). One of the most common clinical features in dogs with AD is itching, which has a significant impact on the quality of life for both the pet and its owner.

[0003] Interleukin-31 (IL-31) is a cytokine known for its pruritus-inducing effects in various species. As early as 2004, scientists discovered that IL-31 belongs to the glycoprotein 130 / IL-6 cytokine family and proposed that IL-31 is a cytokine produced by T helper cells 2 (Th2) after Langerhans cells present allergens (McCandless EE et al., Immunol Immunopathol 2014; 157:42–48). Studies have also shown that activated macrophages, basophils, eosinophils, and keratinocytes also secrete this cytokine after exposure to allergens or after type 2 inflammation is induced in tissues (Bagci IS et al., J Allergy Clin Immunol 2018; 141:858–866). Research has shown that canine interleukin-31 (IL-31) can induce pruritus behavior in dogs. Canine IL-31 has been detected in most dogs with natural AD, suggesting that this cytokine may play an important role in pruritic allergic skin diseases such as atopic dermatitis in this species (Andrea J et al., Vet Dermatol 2013; 24:48–e12).

[0004] IL-31 binds to heterodimer receptors (IL-31 receptor A and oncostatin M receptor beta), activating intracellular signal transduction pathways. When IL-31 binds to heterodimer receptors, various signaling pathways, including Janus kinase (JAK) and activator of transcription (STAT), phosphatidylinositol 3-kinase (PI3K) / AKT, as well as different mitogen-activated protein kinase pathways such as extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase, are activated, leading to biological changes in the cell. These changes include immune cell chemotaxis, secretion of pro-inflammatory cytokines and chemokines, pruritus response, and skin barrier disruption due to changes in cell proliferation, differentiation, and barrier protein synthesis (Bagci IS et al., J Allergy Clin Immunol 2018; 141:858–866).

[0005] Lokivetmab (Cytopoint, Zoetis) is currently the only canine monoclonal antibody approved for the treatment of AD-related clinical symptoms and other forms of atopic dermatitis in dogs. It binds to and neutralizes canine IL-31 and is administered subcutaneously every four to eight weeks. Clinical trials have shown that lokivetmab significantly reduces itching symptoms in dogs on the first day of treatment and significantly improves skin condition at the first clinical efficacy assessment point (Michels GM et al., Vet Dermatol 2016; 27:478–e129). These results confirm the clinical value of IL-31 monoclonal antibodies in the treatment of canine atopic dermatitis.

[0006] Patent WO2013011407A1 discloses an anti-IL-31 antibody in the form of a veterinary composition for treating pruritus or allergies in dogs or cats. The disclosed antibody 34D03 specifically binds to canine or feline IL-31, and the antibody reduces, inhibits, or neutralizes the biological activity of IL-31 in dogs or cats. Patent WO2018156180A1 discloses the use of an anti-IL-31 antibody. The disclosed antibody binds to canine IL-31 and reduces IL-31-mediated signal transduction at cellular levels.

[0007] Although existing IL-31 antibodies exhibit some canine IL-31 inhibitory activity, the availability of such antibody products for canine atopic dermatitis (AD) treatment still does not fully meet clinical needs. Therefore, there is a need for IL-31 antibodies that can specifically bind to canine IL-31 and neutralize or interfere with IL-31-mediated biological activity for the treatment of canine AD and other forms of atopic dermatitis. Summary of the Invention

[0008] This invention provides a novel canine-derived anti-canine IL-31 antibody that specifically binds to IL-31. This antibody, used alone, inhibits IL-31-mediated activation of downstream IL-31 signaling in canine macrophages.

[0009] This invention uses recombinant IL-31 protein to immunize mice. Spleen cells from immunized mice are isolated and screened using single-B cell screening technology to obtain mouse single-B cells that meet the screening criteria. These cells secrete antibodies that recognize canine IL-31. Genetic engineering is used to obtain the VH heavy chain variable region and VL light chain variable region sequences of the IL-31 mouse monoclonal antibody from the single-B cells. The variable region sequences are then spliced ​​into the human immunoglobulin IgG1 framework and recombinantly expressed to obtain a human-mouse chimeric antibody for antibody identification. Using 3D modeling, the VH heavy chain variable region and VL light chain variable region sequences of the IL-31 mouse monoclonal antibody are compared with canine antibody sequences in a database. The mouse sequences are mutated into canine sequences and spliced ​​into the canine immunoglobulin IgG framework to obtain canine-derived IL-31 antibodies.

[0010] In one aspect, the present invention provides an isolated antibody or its antigen-binding fragment that binds interleukin-31 (IL-31), comprising the heavy chain CDR1 (HCDR1) shown in SEQ ID NO.1, the heavy chain CDR2 (HCDR2) shown in SEQ ID NO.2, and the heavy chain CDR3 (HCDR3) shown in SEQ ID NO.3, and the light chain CDR1 (LCDR1) shown in SEQ ID NO.6, the light chain CDR2 (LCDR2) shown in SEQ ID NO.7, and the light chain CDR3 (LCDR3) shown in SEQ ID NO.8.

[0011] In another aspect, the present invention provides a nucleic acid encoding the aforementioned antibody or its antigen-binding fragment.

[0012] In another aspect, the present invention provides an expression vector comprising the aforementioned nucleic acid.

[0013] In another aspect, the present invention provides a host cell comprising the aforementioned nucleic acid or expression vector.

[0014] In another aspect, the present invention provides a composition comprising the aforementioned antibody or its antigen-binding fragment, nucleic acid, expression vector and / or host cell, and a pharmaceutically acceptable carrier or diluent.

[0015] In another aspect, the present invention provides an immunoconjugate comprising the aforementioned antibody or its antigen-binding fragment conjugated with a cytotoxic agent.

[0016] In another aspect, the present invention provides a kit comprising the aforementioned antibody or its antigen-binding fragment.

[0017] In another aspect, the present invention provides a method for generating the aforementioned antibody or its antigen-binding fragment, the method comprising culturing a host cell containing a nucleic acid encoding claim 4 or an expression vector of claim 5.

[0018] In another aspect, the present invention provides a method for detecting IL-31 in a sample, comprising contacting the aforementioned antibody or its antigen-binding fragment with the sample and detecting the antibody bound to IL-31 in the sample.

[0019] In another aspect, the present invention provides the use of the aforementioned antibody or its antigen-binding fragment, nucleic acid, expression vector, host cell, composition or immunoconjugate in the preparation of pharmaceutical compositions for treating, preventing and / or alleviating IL-31-related diseases; or the use of the aforementioned antibody or its antigen-binding fragment or kit in the preparation of a detection kit for detecting IL-31.

[0020] In another aspect, the present invention provides a method for reducing the activation of IL-31 downstream transcriptional activator (STAT) signaling in canine cells, the method comprising exposing the cells to an anti-IL-31 antibody or the pharmaceutical composition described herein, provided that the aforementioned antibody is allowed to bind to extracellular IL-31. Experiments have demonstrated that the antibody of this application inhibits downstream STAT3 phosphorylation in canine IL-31-activated macrophages, thereby reducing the binding of IL-31 to its heterodimeric receptors (IL-31 receptor A and oncostatin M receptor beta) and interfering with downstream STAT signaling transduction leading to cellular biological changes. These changes include immune cell chemotaxis, secretion of pro-inflammatory cytokines and chemokines, and pruritus responses. The antibody of this application can be used to treat, prevent, and / or alleviate IL-31-induced diseases. Attached Figure Description

[0021] Figure 1 The binding activity of the anti-IL-31 canine antibody to canine IL-31 was demonstrated.

[0022] Figure 2 The effect of anti-IL-31 canine antibody on downstream activation signaling in canine IL-31-mediated macrophages was demonstrated. Specific Implementation

[0023] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0024] In this invention, "antibody" refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in its heavy chain, it can be further divided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ chain or a λ chain.

[0025] The antibody light chain of the present invention may further include a light chain constant region, wherein the light chain constant region comprises human, canine or mouse κ, λ chains or variants thereof.

[0026] The antibody heavy chain of the present invention may further include a heavy chain constant region, wherein the heavy chain constant region comprises human, canine or mouse IgG1, IgG2, IgG3, IgG4 or variants thereof.

[0027] The sequence of approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains varies considerably and is known as the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are called the constant region. The variable region includes three hypervariable regions (HVRs) and four relatively conserved backbone regions (FRs). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The CDR amino acid residues in the LCVR and HCVR regions of the antibody or antigen-binding fragments described in this invention conform to the known Kabat numbering rules (LCDR1-3, HCDR1-3) in terms of both number and position.

[0028] The antibodies of the present invention include murine antibodies, chimeric antibodies, and canine antibodies, with canine antibodies being preferred.

[0029] In this invention, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than the full length but contains at least a portion of the variable region of the antibody that binds to the antigen (e.g., one or more CDRs and / or one or more antibody binding sites), and thus retains binding specificity and at least a portion of the specific binding ability of the full-length antibody. Therefore, an antigen-binding fragment refers to an antibody fragment containing an antigen-binding portion that binds to the same antigen as the antibody fragment derived from the antibody. Antibody fragments include antibody derivatives produced by enzymatic treatment of a full-length antibody, as well as synthetically produced derivatives, such as recombinant derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, biantibodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, for example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragment may comprise multiple chains linked together, for example by disulfide bonds and / or by peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, upon insertion into an antibody framework (e.g., by replacing the corresponding region), acquires immune-specific binding (i.e., exhibits at least or at least about 10 amino acids). 7 -10 8 M -1 Antibodies against the Ka antigen. A “functional fragment” or “anti-Trop-2 antibody analog” is a fragment or analog that prevents or substantially reduces the ability of the receptor to bind ligands or initiate signal transduction. As used herein, a functional fragment generally has the same meaning as “antibody fragment”, and in the context of antibodies, it can refer to a fragment that prevents or substantially reduces the ability of the receptor to bind ligands or initiate signal transduction, such as Fv, Fab, F(ab')2, etc. An “Fv” fragment is a dimer (V) formed by the non-covalent binding of a variable domain of a heavy chain and a variable domain of a light chain. H -V L (Dimer) composition. In this configuration, the three CDRs of each variable domain interact to determine V. H -V L The target binding sites on the surface of the dimer are the same as in the case of the intact antibody. The six CDRs collectively confer target binding specificity to the intact antibody. However, even a single variable domain (or half the Fv of only including three target-specific CDRs) can still have the ability to recognize and bind to the target.

[0030] In this invention, "monoclonal antibody" or "monoclonal antibody" refers to a population of identical antibodies, meaning that each individual antibody molecule in a monoclonal antibody population is identical to the other antibody molecules. This characteristic contrasts with the characteristic of a polyclonal population of antibodies, which contains antibodies having a variety of different sequences. Monoclonal antibodies can be prepared by many known methods. For example, monoclonal antibodies can be prepared from immortalized B cells, such as by fusing with myeloma cells to generate hybridoma cell lines or by infecting B cells with a virus such as EBV. Recombinant technology can also be used to prepare antibodies in vitro from a clonal population of host cells by transforming host cells with plasmids carrying artificial sequences of nucleotides encoding antibodies.

[0031] In this invention, the full-length antibody is an antibody having two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region, such as antibodies naturally produced by antibody-secreting B cells and synthetically produced antibodies having the same structural domains.

[0032] The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a canine antibody and the constant region sequence is derived from a human antibody.

[0033] In this invention, the terms "specific binding" or "immune-specific binding" for antibodies or their antigen-binding fragments are used interchangeably and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with the same antigen through a non-covalent interaction between the antibody and the antigen's antibody-binding site. The antigen may be an isolated antigen or present in tumor cells. Typically, antibodies that immune-specifically bind (or specifically bind) antigens are present in quantities of about 1 × 10⁻⁶. 7 M -1 Or 1x10 8 M -1 Or a larger affinity constant Ka (or 1x10) -7 M or 1×10 -8 The affinity constant (M or lower, Kd) binds to the antigen. The affinity constant can be determined using standard kinetic methods of antibody reactions, such as immunoassay, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art. Instruments and methods for real-time detection and monitoring of the binding rate are known and commercially available.

[0034] In this invention, the terms "polynucleotide" and "nucleic acid molecule" refer to oligomers or polymers comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) typically linked together by phosphodiester bonds. As used herein, the term "nucleic acid molecule" is intended to include both DNA and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, and may be cDNA.

[0035] In this invention, the isolated nucleic acid molecule is a nucleic acid molecule isolated from other nucleic acid molecules present in natural sources of nucleic acid molecules. "Isolated" nucleic acid molecules, such as cDNA molecules, may be substantially free of other cellular material or culture medium when prepared by recombinant technology, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding provided antibody or antigen-binding fragments.

[0036] In this invention, "operably linked" in relation to nucleic acid sequences, regions, elements, or domains indicates that nucleic acid regions are functionally related to each other. For example, a promoter can be operably linked to a nucleic acid encoding a polypeptide, thereby the promoter regulating or mediating the transcription of the nucleic acid.

[0037] In this invention, "expression" refers to the process of producing polypeptides through the transcription and translation of polynucleotides. The expression level of a polypeptide can be evaluated using any method known in the art, including, for example, methods for determining the amount of polypeptide produced from host cells. Such methods may include, but are not limited to, quantifying polypeptides in cell lysates by ELISA, Coomassie blue staining following gel electrophoresis, Lowry protein assays, and Bradford protein assays.

[0038] In this invention, a "host cell" is a cell used to receive, maintain, replicate, and amplify the vector. The host cell can also be used to express the polypeptide encoded by the vector. When the host cell divides, the nucleic acids contained in the vector replicate, thereby amplifying the nucleic acids. The host cell can be a eukaryotic or prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, and HEK cells such as HEK 293 cells.

[0039] In this invention, a "vector" is a reproducible nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into a suitable host cell. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced via restriction enzyme digestion and ligation. Vectors also include those containing nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for amplification of nucleic acids or for expression / display of the polypeptide encoded by the nucleic acid. Vectors are typically kept free but can be designed to integrate genes or portions thereof into the chromosome of the genome. Vectors for artificial chromosomes are also considered, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vectors are well known to those skilled in the art.

[0040] In this invention, the vector also includes a "viral vector" or "vector of a virus". A viral vector is an engineered virus that is operatively linked to a foreign gene to transfer (as a medium or shuttle) the foreign gene into the cell.

[0041] In this invention, an "expression vector" includes a vector capable of expressing DNA, said DNA being operatively linked to a regulatory sequence, such as a promoter region, capable of influencing the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and optionally may include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, bacteriophage, recombinant virus, or other vector, which, when introduced into a suitable host cell, results in the expression of clonal DNA. Suitable expression vectors are well known to those skilled in the art and include reproducible expression vectors in eukaryotic and / or prokaryotic cells, as well as expression vectors that remain free or are integrated into the host cell genome.

[0042] In this invention, "treatment" of an individual suffering from a disease or disease condition means that the individual's symptoms are partially or completely relieved, or remain unchanged after treatment. Therefore, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing potential disease and / or preventing the worsening of symptoms or the development of disease. Treatment also includes any pharmaceutical use of the provided ADCs and the pharmaceutical compositions and preparations provided herein.

[0043] In this invention, "therapeutic effect" refers to the effect resulting from treatment of an individual, which alters, usually improves or enhances the symptoms of a disease or disease condition, or cures a disease or disease condition.

[0044] In this invention, "therapeutic effective amount" or "therapeutic effective dose" refers to the amount of a substance, compound, material, or composition containing a compound that, when applied to a subject, is at least sufficient to produce a therapeutic effect. Therefore, it is the amount necessary to prevent, cure, improve, block, or partially block the symptoms of a disease or condition.

[0045] In this invention, "preventative effective amount" or "preventative effective dose" refers to the amount of a substance, compound, material, or composition containing a compound that, when applied to a subject, would have the intended preventive effect, such as preventing or delaying the occurrence or recurrence of a disease or symptom, or reducing the likelihood of the occurrence or recurrence of a disease or symptom. A fully preventative effective dose does not necessarily occur through the application of a single dose and can occur only after the application of a series of doses. Therefore, a preventative effective amount can be applied in one or more administrations.

[0046] All references mentioned in this article are cited in their entirety in this article.

[0047] In one aspect, the present invention provides an isolated antibody or its antigen-binding fragment that binds interleukin-31 (IL-31), comprising the heavy chain CDR1 (HCDR1) shown in SEQ ID NO.1, the heavy chain CDR2 (HCDR2) shown in SEQ ID NO.2, and the heavy chain CDR3 (HCDR3) shown in SEQ ID NO.3, and the light chain CDR1 (LCDR1) shown in SEQ ID NO.6, the light chain CDR2 (LCDR2) shown in SEQ ID NO.7, and the light chain CDR3 (LCDR3) shown in SEQ ID NO.8.

[0048] In some embodiments, the antibody or its antigen-binding fragment comprises a combination of a heavy chain variable region and a light chain variable region selected from any of the following:

[0049] (1) The heavy chain variable region comprises or is composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO.4, and the light chain variable region comprises or is composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO.9;

[0050] (2) The heavy chain variable region comprises or is composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO. 5, and the light chain variable region comprises or is composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO. 10.

[0051] In some implementations, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.

[0052] In some implementations, the antibody is an IgG1 antibody.

[0053] In some embodiments, the constant region of the heavy chain comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO. 15 or 17. In some embodiments, the constant region of the heavy chain comprises the amino acid sequence shown in SEQ ID NO. 17.

[0054] In some embodiments, the constant region of the light chain comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO. 16 or 18. In some embodiments, the constant region of the light chain comprises the amino acid sequence shown in SEQ ID NO. 18.

[0055] In another aspect, the present invention provides a nucleic acid encoding the aforementioned antibody or its antigen-binding fragment.

[0056] In some embodiments, the nucleic acid molecule comprises an antibody heavy chain variable region nucleic acid sequence selected from any variant of SEQ ID NO. 11 or 13, and an antibody light chain variable region nucleic acid sequence selected from any variant of SEQ ID NO. 12 or 14.

[0057] In another aspect, the present invention provides an expression vector comprising the aforementioned nucleic acid.

[0058] In another aspect, the present invention provides a host cell comprising the aforementioned nucleic acid or expression vector.

[0059] In another aspect, the present invention provides a composition comprising the aforementioned antibody or its antigen-binding fragment, nucleic acid, expression vector and / or host cell, and a pharmaceutically acceptable carrier or diluent.

[0060] In another aspect, the present invention provides an immunoconjugate comprising the aforementioned antibody or its antigen-binding fragment conjugated with a cytotoxic agent.

[0061] In some embodiments, the cytotoxic agent is selected from chemotherapeutic agents, drugs, growth inhibitors, toxins (e.g., enzyme-active toxins or fragments thereof of bacterial, fungal, plant, or animal origin), or radioactive isotopes (i.e., radioconjugates).

[0062] In another aspect, the present invention provides a kit comprising the aforementioned antibody or its antigen-binding fragment.

[0063] In another aspect, the present invention provides a method for generating the aforementioned antibody or its antigen-binding fragment, the method comprising culturing a host cell containing a nucleic acid encoding claim 4 or an expression vector of claim 5.

[0064] In another aspect, the present invention provides a method for detecting IL-31 in a sample, comprising contacting the aforementioned antibody or its antigen-binding fragment with the sample and detecting the antibody bound to IL-31 in the sample.

[0065] In another aspect, the present invention provides the use of the aforementioned antibody or its antigen-binding fragment, nucleic acid, expression vector, host cell, composition or immunoconjugate in the preparation of pharmaceutical compositions for treating, preventing and / or alleviating IL-31-related diseases; or the use of the aforementioned antibody or its antigen-binding fragment or kit in the preparation of a detection kit for detecting IL-31.

[0066] In some implementations, the IL-31-related disease is selected from pruritus or allergy.

[0067] In some implementations, the pruritus is selected from atopic dermatitis, eczema, psoriasis, scleroderma, and pruritus.

[0068] In some implementations, the allergy is selected from allergic dermatitis, summer eczema, urticaria, airway inflammatory diseases, recurrent airway obstruction, airway hyperresponsiveness, chronic obstructive pulmonary disease, and autoimmune inflammatory processes.

[0069] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delayers compatible with drug administration. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, glucose solution, and 5% human serum albumin. Liposomes and non-aqueous carriers, such as immobilized oils, may also be used. The use of such media and reagents for pharmaceutically active substances is well known in the art. The intended use of any conventional media or reagent in the composition is contemplated, except that it may be incompatible with the antibody. The formulation intended for in vivo administration must be sterile. This can be readily achieved through filtration using a sterile filter membrane.

[0070] The pharmaceutical composition of the embodiments described herein is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., local), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: sterile diluents for injection such as water, saline solutions, fixative oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates; and osmotic pressure adjusting agents such as sodium chloride or dextran. pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0071] Table 1 provides a list of some of the sequences mentioned in this article.

[0072] Table 1. Sequence Description

[0073]

[0074]

[0075] Example

[0076] The present invention will be explained below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the following embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0077] Example 1. Production of mouse monoclonal antibody against canine IL-31

[0078] laboratory animalsAll animal experiments were conducted under the authorization of the Experimental Animal Center of Zhuhai Beston Biotechnology Co., Ltd., and BALB / c mice were purchased from the Experimental Animal Center of Zhuhai Beston Biotechnology Co., Ltd.

[0079] Monoclonal antibody production Female BALB / c mice were subcutaneously immunized three times with recombinant canine IL-31 (UniProt:C7G0W1) extracellular domain protein (Novus, #NBP2-59591) via subcutaneous injection (SC), with each injection spaced two weeks apart. One week after the third injection, blood samples were collected from the immunized mice, and serum was used for indirect ELISA to determine the immune response. Mouse spleens were used as the source tissue for single-cell B cells. After isolating spleen cells from the immunized mice, mouse B cells were enriched using a CD138 positively selected magnetic bead kit (STEMCELL). The enriched mouse B cells were introduced into a single-cell optical fiber microarray and separated into individual cells according to a standard procedure, then placed in individual compartments of the microarray. Antigen-antibody binding assays were performed on experimental microspheres modified with canine IL-31 recombinant protein within the microarray, and the binding of antibodies secreted by single B cells to different protein antigens on the experimental microspheres was monitored in real time. Single B cells that can secrete the target antibody (hereinafter referred to as 4F11) were identified by antigen-antibody binding assays performed on the chip. The target antibody can bind to the canine IL-31 recombinant protein.

[0080] Antibody sequences were obtained by single B-cell sequencing. Following standard procedures, monoclonal B cells capable of secreting the target antibody were lysed on a microarray, and their mRNA was captured using capture microspheres. The mRNA was then reverse transcribed into cDNA and exported. The cDNA was used as a template for PCR amplification followed by sequencing. Sequence analysis yielded the variable region sequence of the light and heavy chains of the mouse monoclonal antibody 4F11.

[0081] The amino acid sequence (SEQ ID NO.4) of the heavy chain variable region (VH) of antibody 4F11 is as follows:

[0082]

[0083] Note: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Based on the Kabat numbering system, the bolded and underlined parts are VHCDR1, VHCDR2, and VHCDR3, respectively.

[0084] The nucleotide sequence of the heavy chain variable region of antibody 4F11 (SEQ ID NO.11) is as follows:

[0085] CAGGTTCAGCTCCAGCAGTCAGGCGCAGCTCGTTAAACCCGGCGCATCTGTCAAACTCTCCTGCAAGGCCTCCGGCTACACATTCACCTCCAACTACATGTACTGGGTCAAACAGAGGCCCGGCCAGGGATTGGAGTGGATTGGAGAAATCAACCCCTCCAACGGGGGCACAAAT TTCAACGAGAAATTCAAGTCCAAGGCCACCCTGACAGTGGACAAGAGCAGTTCCACAGCATACATGCAGCTGAACAGCCTCGCCAGCGAAGATTCTGCTGTGTACTACTGTACCAGGTACGACGGGTACGACGGCTCCGCTTACTGGGGACAGGGAACACTGGTGACCGTTTCCGCC

[0086] The amino acid sequence (SEQ ID NO.9) of the light chain variable region (VL) of antibody 4F11 is as follows:

[0087]

[0088] Note: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Based on the Kabat numbering system, the bolded and underlined parts are VLCDR1, VLCDR2, and VLCDR3, respectively.

[0089] The nucleotide sequence of the light chain variable region of antibody 4F11 (SEQ ID NO.12) is as follows:

[0090] GACATCCAGATGACCCAGTCCTCCTCCTCCTTCTCCGTGTCTCTGGGAGACAGGGTGACCATCACCTGCAAGGCTTCCGAGGACATCTACAACCGGCTGGCTTGGTATCAGCAGAAACCCGGAAACGCCCCAGATTGCTCATCTCTGGCGCTTCTACCC TCGAAACCGGCGTTCCTTCCCGATTTTCCGGTTCCGGTTCCGGCAAGGACTACACACTGAGCATCACAACCCTGCAGACCGAGGATCTCGCCACATACTACTGCCAGCAGTACTGGCTGACCCCCTGGACCTTTGGTGGAGGAACTAAGCTCGAAATCAAG

[0091] Example 2. Affinity detection of anti-canine IL-31 chimeric antibody BLI

[0092] Chimeric antibody expression and purification The variable region sequence of a murine antibody was spliced ​​with the constant region gene of a human IgG antibody to obtain a chimeric antibody sequence. The control antibody 34D03 sequence was derived from the published patent WO2013011407A1. The antibody heavy chain adopted the CH1-Fc constant region of IgG1 (SEQ ID NO.15), and the light chain adopted the VL constant region of the kappa light chain (SEQ ID NO.16). The chimeric antibody molecule's light and heavy chain genes were inserted into the pcDNA3.4 vector via NotI and XbaI sites to construct an expression vector, which was then transiently transfected into HEK293 cells for expression. After collecting the expression supernatant, the human-mouse chimeric antibody was obtained by affinity purification with Protein A.

[0093] Biomembrane interferometry (BLI) for detecting antibody affinity The affinity of the anti-IL-31 chimeric antibody for canine IL-31 recombinant protein (Novus) was determined using Octet RED96e (ForteBio). Table 2 shows the detection results for 4F11. The results showed that the affinity of the IL-31 chimeric antibody 4F11 for canine IL-31 was slightly better than that of the control antibody 34D03.

[0094] Table 2. Affinity of anti-IL-31 chimeric antibodies to canine IL-31

[0095] 34D03 2.03E+05 8.18E-04 4.03E-09 4F11 5.12E+05 9.38E-04 1.83E-09

[0096] Example 3. Canine Derivatization of Mouse Antibody

[0097] The variable regions (VH) and light chain (VL) of the above-mentioned antibody were compared with the canine IgG gene sequence database to determine the best-matching canine IgG gene sequence. The preferred canine IgG heavy chain for 4F11 is IGHV1-30*01, and the preferred canine IgG light chain is IGKV3-18*01. The CDR region sequences from the variable regions of the 4F11 light and heavy chains were respectively transplanted into the framework genes of the matching canine light and heavy chain genes. The canine-derived molecules were named 303, and the VH, VL, and CDR region sequences of the resulting canine-derived antibody are shown below. The antibody heavy chain constant region (HC) and antibody light chain constant region (LC) are shown below. Those skilled in the art can clearly understand the complete amino acid sequences of the heavy and light chains of the 303 antibody based on the above disclosure information.

[0098] The amino acid sequence (SEQ ID NO.5) of the heavy chain variable region (VH) of antibody 303 is as follows:

[0099]

[0100] Note: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Based on the Kabat numbering system, the bolded and underlined parts are VHCDR1, VHCDR2, and VHCDR3, respectively.

[0101] The nucleotide sequence of the heavy chain variable region of antibody 303 (SEQ ID NO.13) is as follows:

[0102] GAAGTGCAGCTGGTGCAGAGCGGAGCAGAGGTTAAGAAGCCCGGAGCAAGCGTGAAGGTGAGCTGCAAGACAAGCGGATACACATTCACAAGCAACTACATGTACTGGGTGAGGCAGGCCCCAGGCCAGGGATTGGATTGGATCGGAGAGATCAACCCAGCAACGGAGGAACAAAC TTCAACGAGAAGTTCAAGAGCAGGGTCACACTGACCGTGGACAAGAGCACCAGCACAGCATACATGGAACTGAGCTCCCTGGCAGCCGAAGATGCTGCTGTGTACTATTGCGCCAGGTACGACGGCTACGACGGAAGTGCATATTGGGGCCAGGGAACACTGGTGACCGTGAGTTCT

[0103] The amino acid sequence of the light chain variable region (VL) of antibody 303 (SEQ ID NO.10) is as follows:

[0104]

[0105] Note: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Based on the Kabat numbering system, the bolded and underlined parts are VLCDR1, VLCDR2, and VLCDR3, respectively.

[0106] The nucleotide sequence of the light chain variable region of antibody 303 (SEQ ID NO.14) is as follows:

[0107] GAAATCGTGATGACCCAGTCCCCCGCCTCCTTGAGTCTGAGTCAGGAGGAGAAGGTGACCATCACCTGCAAGGCCTCCGAGGACATCTACAACAGGCTGGCCTGGTACCAGCAGAAGCCAGGACAGGCTCCCAAACTGCTCATTTCCGGCGCCTCCACCCTCGAAACAGGAGTTCCTTCCCGCTTCTCCGGTTCCGGTTCCGGCACAGATTTCACCTTCACTATTTCCTCCCTGGAGCCCGAGGATGTCGCAGTTTACTACTGCCAGCAGTACTGGCTCACCCCCTGGACATTCGGCCAGGGAACTAAGCTCGAAATCAAA

[0108] The amino acid sequence of the constant region of the heavy chain (HC) of antibody 303 (SEQ ID NO.17) is as follows:

[0109] ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGK.

[0110] The amino acid sequence of the constant region of the light chain (LC) of antibody 303 (SEQ ID NO.18) is as follows:

[0111] RNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSEC.

[0112] The canine-derived antibody molecule 303 light and heavy chain genes were inserted into pcDNA3.4 to construct an expression vector, which was then transiently transfected into HEK293 cells for expression. The expression supernatant was collected and purified using Protein A affinity purification to obtain purified antibodies for subsequent identification.

[0113] Example 4. Affinity detection of canine anti-canine IL-31 canine antibody SPR

[0114] The affinity of canine anti-IL-31 canine antibody 303 for canine recombinant IL-31 protein (Novus) was determined using a Biacore T200 (Cytiva) instrument and compared with that of the maternal chimeric antibody 4F11 in Example 2. Table 3 shows that the affinity of canine antibody 303 for canine recombinant IL-31 protein is comparable to that of maternal chimeric antibody 4F11.

[0115] Table 3. Affinity of anti-IL-31 canine antibody to canine IL-31

[0116] 4F11 1.60E+06 2.02E-03 1.26E-09 303 1.08E+06 2.94E-03 2.73E-09

[0117] Example 5. ELISA detection of binding activity of canine anti-canine IL-31 canine-derived antibody.

[0118] The binding activity of anti-IL-31 canine antibody 303 to canine IL-31 recombinant protein (Novus) was determined by ELISA and compared with control antibody 34D03 or isotype control antibody (canine IgGB, Bio-Tech, #LPDD3D003). Figure 1 The test results showed that the binding activity of canine antibody 303 to recombinant canine IL-31 protein was comparable to that of the control antibody 34D03 [IC]. 50 :0.22nM(303)vs.0.18nM(34D03)].

[0119] Example 6. Effects of canine anti-canine IL-31 canine-derived antibody on functional signal transduction in canine macrophages

[0120] Upon binding to its receptor, IL-31 activates JAK1 and JAK2 signaling, which in turn activates the phosphorylation of downstream signaling pathways STAT3 and STAT5. Anti-canine IL-31 neutralizing antibodies can block the binding of IL-31 to its receptor, thereby inhibiting IL-31-mediated STAT3 phosphorylation. Canine macrophages DH82 (ATCC, CRL-10389) were cultured in MEM complete medium (Gibco, #12561056) at 5% CO2 and 37°C, and induced with a stimulating factor (canine IFN-γ, R&D Systems, #781-CG). After 24 hours, the cells were starved in MEM basal medium for 2 hours. The successfully induced DH82 cells were then added with an anti-canine IL-31 antibody or isotype control antibody (canine IgGB, Baiying Biotechnology, #LPDD3D003) pre-incubated with the canine IL-31 fusion protein, and cultured at 5% CO2 and 37°C for 5 minutes. After washing cells with PBS, 100 μl of ice-cold cell lysis buffer was added according to the STAT3 phosphorylation detection kit (PathScan Phospho-stat3(Tyr705) sandwich ELISA kit, CST), and subsequent ELISA was performed to detect STAT3 phosphorylation levels. STAT3 phosphorylation levels in DH82 cells were expressed as OD values. Figure 2 This study demonstrates the effects of different IL-31 antibodies on downstream activation signaling in canine macrophages mediated by IL-31. The canine-derived antibody 303 showed comparable inhibitory activity against IL-31-activated STAT3 phosphorylation in canine macrophages to the control antibody 34D03 [IC50]. 50 :3.408nM(303)vs.1.195nM(34D03)].

Claims

1. An isolated antibody or its antigen-binding fragment thereof, which binds interleukin-31, comprising heavy chain CDR1 shown in SEQ ID NO. 1, heavy chain CDR2 shown in SEQ ID NO. 2 and heavy chain CDR3 shown in SEQ ID NO. 3, and light chain CDR1 shown in SEQ ID NO. 6, light chain CDR2 shown in SEQ ID NO. 7 and light chain CDR3 shown in SEQ ID NO.

8.

2. The antibody or its antigen-binding fragment according to claim 1, wherein, The antibody or its antigen-binding fragment comprises a combination of a heavy chain variable region and a light chain variable region selected from any of the following: (1) The amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO. 4, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO. 9; and (2) The amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO. 5, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO.

10.

3. The antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.

4. The antibody or its antigen-binding fragment according to claim 3, wherein, The antibody is an IgG1 type antibody.

5. The antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The amino acid sequence of the constant region of the heavy chain is the amino acid sequence shown in SEQ ID NO. 15, and the amino acid sequence of the constant region of the light chain is the amino acid sequence shown in SEQ ID NO.

16.

6. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein, The amino acid sequence of the constant region of the heavy chain is the amino acid sequence shown in SEQ ID NO. 17, and the amino acid sequence of the constant region of the light chain is the amino acid sequence shown in SEQ ID NO.

18.

7. A nucleic acid encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1-6.

8. The nucleic acid according to claim 7, comprising a combination of a heavy chain variable region nucleic acid sequence and a light chain variable region nucleic acid sequence selected from any one of the following: (1) The antibody heavy chain variable region nucleic acid sequence shown in SEQ ID NO. 11, and the antibody light chain variable region nucleic acid sequence shown in SEQ ID NO. 12; and (2) The antibody heavy chain variable region nucleic acid sequence shown in SEQ ID NO. 13 and the antibody light chain variable region nucleic acid sequence shown in SEQ ID NO.

14.

9. An expression vector comprising the nucleic acid of claim 7 or 8.

10. A host cell comprising the nucleic acid of claim 7 or 8 or the expression vector of claim 9.

11. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-6, the nucleic acid of claim 7 or 8, the expression vector of claim 9, and / or the host cell of claim 10, and a pharmaceutically acceptable carrier or diluent.

12. An immunoconjugate comprising an antibody or antigen-binding fragment thereof conjugated with any one of claims 1-6 of a cytotoxic agent; The cytotoxic agent is a radioactive isotope.

13. A kit comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-6.

14. A method for generating the antibody of claim 1 or an antigen-binding fragment thereof, the method comprising culturing the host cell of claim 10.

15. A method for detecting IL-31 in a sample in vitro, comprising contacting the sample with the antibody or antigen-binding fragment thereof as described in any one of claims 1-6, and detecting the antibody bound to IL-31 in the sample.

16. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-6, the nucleic acid of claim 7 or 8, the expression vector of claim 9, the host cell of claim 10, the composition of claim 11, or the immunoconjugate of claim 12 in the preparation of a pharmaceutical composition for treating, preventing, and / or alleviating IL-31-related diseases; The diseases associated with IL-31 are selected from pruritus or allergies.

17. The use as described in claim 16, wherein, The pruritus is selected from atopic dermatitis, eczema, psoriasis, scleroderma and pruritus.

18. The use as described in claim 16, wherein, The allergies mentioned are selected from allergic dermatitis, summer eczema, urticaria, airway inflammatory diseases, recurrent airway obstruction, airway hyperresponsiveness, chronic obstructive pulmonary disease, and inflammatory processes caused by autoimmunity.

19. Use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-6 in the preparation of a detection kit for detecting IL-31.

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