Anti-il-31 antibody, pharmaceutical composition thereof and use thereof

Abstract

The present invention belongs to the field of biomedicine, and relates to an anti-IL-31 antibody, a pharmaceutical composition thereof and the use thereof. Specifically, the present invention relates to an anti-IL-31 monoclonal antibody or an antigen-binding fragment thereof. The antibody of the present invention has a high affinity for human IL-31 and cynomolgus monkey IL-31, can effectively block the binding of IL-31 to the IL-31RA receptor thereof, can reduce or eliminate intracellular downstream STAT3 phosphorylation signals and significantly inhibit pruritus caused by IL-31, and has good application prospects.

Description

Anti-il-31 antibodies, pharmaceutical compositions thereof, and uses thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to an anti-IL-31 antibody, a pharmaceutical composition thereof and uses thereof. Specifically, the present application relates to a monoclonal antibody or an antigen-binding fragment thereof against IL-31. The present application also relates to a nucleic acid molecule encoding the antibody or the antigen-binding fragment thereof and a host cell comprising the same. BACKGROUND

[0002] IL-31 is a four-helix bundle cytokine with 2 long helices and 2 short helices, belonging to the glycoprotein 130 (gp130) / IL-6 cytokine family, which was initially shown to be produced by activated helper T cells, especially Th2 cells, mast cells, macrophages and dendritic cells, in addition to the expression of IL-31 in eosinophils, basophils, etc. IL-31RA binds with OSMRβ to form a functional receptor complex, mediates IL-31 signaling, stimulates the phosphorylation of downstream signaling molecules STAT3, STAT5 and STAT1 (to a lesser extent), and plays a leading role in mediating inflammatory pruritus. In mice, overexpression of IL-31 can lead to similar clinical and histological features as atopic dermatitis (Saleem MD, Oussedik E, D'Amber V, Feldman SR. Interleukin-31 pathway and its role in atopic dermatitis: a systematic review. J Dermatolog Treat. 2017 Nov; 28(7): 591-599.); IL-31 is highly expressed in patients with atopic dermatitis, nodular prurigo and chronic spontaneous urticaria (Chaowattanapanit S, Choonhakarn C et al. Increased serum IL-31 levels in chronic spontaneous urticaria and psoriasis with pruritic symptoms. Heliyon. 2020 Dec 1; 6(12): e05621.).In IL-31RA knock-out mice, IL-31 was unable to induce a scratching response in mice (Dillon SR, Sprecher C, Hammond A et al. Interleukin 31, a cytokine produced by activated T cells, induces dermatitis in mice. Nat Immunol 2004; 5:752-760.); in dogs, blocking IL-31 significantly inhibited IL-31 -induced pruritus, experiments showed that a single subcutaneous injection of 2 mg / kg lokivetmab can significantly inhibit pruritus starting 3 hours after treatment and lasting for 42 days (Fleck TJ, Norris LR, Mahabir S, et al. Onset and duration of action of lokivetmab in a canine model of IL-31 induced pruritus. Vet Dermatol. 2021 Dec; 32(6):681-e182.); in monkeys, treatment with IL-31 mAb inhibited IL-31 -mediated scratching response in a dose-dependent manner (Lewis KE, Holdren MS, Maurer MF, et al. Interleukin (IL) 31 induces in cynomolgus monkeys a rapid and intense itch response that can be inhibited by an IL-31 neutralizing antibody. J Eur Acad Dermatol Venereol. 2017; 31:142-150.). These results suggest that IL-31 can be functionally related to skin pruritus, suggesting the important role of IL-31 in mediating pruritus.

[0003] BMS-981164 is a monoclonal antibody targeting IL-31 developed by BMS, which relieves itching by blocking the binding of IL-31 to IL-31RA. Preclinical experimental results show that injecting cynomolgus IL-31 into the body through intravenous, intradermal and subcutaneous injection can immediately cause itching, and the itching lasts for at least 3 hours, while BMS-981164 can inhibit the itching caused by cynomolgus IL-31 in a dose-dependent manner (Lewis KE, Holdren MS, Maurer MF, et al. Interleukin (IL) 31 induces in cynomolgus monkeys a rapid and intense itch response that can be inhibited by an IL-31 neutralizing antibody. J Eur Acad Dermatol Venereol. 2017; 31: 142-150.). BMS-981164 has completed clinical phase I to explore its safety and pharmacokinetic characteristics.

[0004] Nemolizumab developed by Maruho is a monoclonal antibody targeting IL-31RA, which was approved for marketing in Japan in 2022 for the treatment of refractory atopic dermatitis-related itching. It is the first and only antibody targeting the IL-31 pathway approved for marketing worldwide. The antibody relieves itching by blocking the binding of IL-31 to IL-31RA. Clinical results show that the itching of atopic dermatitis and nodular prurigo patients treated with Nemolizumab is rapidly and significantly reduced, and the sleep and quality of life of patients are significantly improved (Kabashima K, Matsumura T, Komazaki H, et al. Trial of Nemolizumab and Topical Agents for Atopic Dermatitis with Pruritus. N Engl J Med. 2020 Jul 9; 383(2): 141-150. Kwatra SG, Yosipovitch G, Legat FJ, et al. Phase 3 Trial of Nemolizumab in Patients with Prurigo Nodularis. N Engl J Med. 2023 Oct 26; 389(17): 1579-1589.).

[0005] In view of the fact that IL-31 can cause pruritus effect in various autoimmune diseases, IL-31 is considered to be an extremely attractive target for inhibiting pruritus effect in autoimmune diseases.

[0006] There is still a need to develop new IL-31 targeting antibodies for the treatment of autoimmune diseases, especially pruritus related treatment. SUMMARY

[0007] The present inventors have made a specific antibody targeting interleukin-31 (IL-31) through in-depth research and creative labor. The present inventors have surprisingly found that the antibody of the present application has high affinity to human IL-31 and cynomolgus IL-31, and can effectively block the binding of IL-31 to its receptor IL-31RA, reduce or eliminate the downstream STAT3 phosphorylation signal in cells, and significantly inhibit the pruritus caused by IL-31 when the antibody of the present application is administered in animal models.

[0008] Thus, the following invention is provided:

[0009] One aspect of the present application relates to an anti-IL-31 antibody or an antigen binding fragment thereof, wherein the anti-IL-31 antibody comprises a heavy chain variable region comprising complementarity determining regions HCDR1 to HCDR3, and a light chain variable region comprising complementarity determining regions LCDR1 to LCDR3, wherein:

[0010] the amino acid sequence of HCDR1 is set forth in SEQ ID NO: 1;

[0011] the amino acid sequence of HCDR2 is set forth in SEQ ID NO: 2;

[0012] the amino acid sequence of HCDR3 is set forth in SEQ ID NO: 3;

[0013] the amino acid sequence of LCDR1 is set forth in SEQ ID NO: 5;

[0014] the amino acid sequence of LCDR2 is set forth in SEQ ID NO: 6; and

[0015] the amino acid sequence of LCDR3 is selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19.

[0016] In the present application, the complementarity determining regions (CDRs) HCDR1 to HCDR3 and LCDR1 to LCDR3 are defined according to the IMGT numbering system if not otherwise specified.

[0017] In some embodiments of the present application, the anti-IL-31 antibody or antigen-binding fragment thereof, wherein,

[0018] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 4 or SEQ ID NO: 9; and

[0019] the amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20.

[0020] In some embodiments of the present application, the anti-IL-31 antibody or antigen-binding fragment thereof, wherein, the heavy chain variable region and the light chain variable region of the anti-IL-31 antibody are selected from the group consisting of (1) to (14) as follows:

[0021] (1)

[0022] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 4; and

[0023] the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 8;

[0024] (2)

[0025] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 4; and

[0026] the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 11;

[0027] (3)

[0028] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 4; and

[0029] the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 13;

[0030] (4)

[0031] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 4; and

[0032] the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 14;

[0033] (5)

[0034] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 4; and

[0035] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 4; and

[0036] (6)

[0037] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 4; and

[0038] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 4; and

[0039] (7)

[0040] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 4; and

[0041] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 4; and

[0042] (8)

[0043] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9; and

[0044] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9; and

[0045] (9)

[0046] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9; and

[0047] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9; and

[0048] (10)

[0049] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9; and

[0050] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9; and

[0051] (11)

[0052] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9; and

[0053] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9; and

[0054] (12)

[0055] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9; and

[0056] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9; and

[0057] (13)

[0058] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9; and

[0059] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9; and

[0060] (14)

[0061] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9; and

[0062] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9; and

[0063] In some embodiments of the present application, the anti-IL-31 antibody or antigen binding fragment thereof, wherein,

[0064] The antibody comprises non-CDR regions, and the non-CDR regions are from a species other than murine, for example from a human antibody.

[0065] In some embodiments of the present application, the anti-IL-31 antibody is a fully human antibody.

[0066] In some embodiments of the present application, the anti-IL-31 antibody is a fully human monoclonal antibody.

[0067] In some embodiments of the present application, the anti-IL-31 antibody or antigen binding fragment thereof, wherein,

[0068] The anti-IL-31 antibody is of IgGl, IgG2, IgG3 or IgG4 subtype;

[0069] Preferably, the heavy chain constant region of the anti-IL-31 antibody is human Ig gamma-1 chain C region (e.g. SEQ ID NO: 21) or human Ig gamma-4 chain C region; and the light chain constant region is human Ig kappa chain C region (e.g. SEQ ID NO: 22);

[0070] Preferably, the anti-IL-31 antibody is of IgGl, and its heavy chain constant region contains L234A and L235A mutations according to the EU numbering system;

[0071] Preferably, the amino acid sequence of the heavy chain constant region of the anti-IL-31 antibody is shown as SEQ ID NO: 29.

[0072] In the present application, the point mutation of leucine at position 234 to alanine (L234A) and the point mutation of leucine at position 235 to alanine (L235A) contained in the heavy chain constant region according to the EU numbering system is also referred to as LALA mutation.

[0073] In some embodiments of the present application, the anti-IL-31 antibody or antigen binding fragment thereof, wherein the anti-IL-31 antibody comprises a heavy chain and a light chain, and is selected from 1) to 7) as follows:

[0074] 1)

[0075] the amino acid sequence of the heavy chain is shown as SEQ ID NO: 30; and

[0076] the amino acid sequence of the light chain is shown as SEQ ID NO: 31;

[0077] 2)

[0078] the amino acid sequence of the heavy chain is shown as SEQ ID NO: 32; and

[0079] the amino acid sequence of the light chain is shown as SEQ ID NO: 33;

[0080] 3)

[0081] the amino acid sequence of the heavy chain is shown as SEQ ID NO: 32; and

[0082] the amino acid sequence of the light chain is shown as SEQ ID NO: 34;

[0083] 4)

[0084] the amino acid sequence of the heavy chain is shown as SEQ ID NO: 32; and

[0085] the amino acid sequence of the light chain is shown as SEQ ID NO: 35;

[0086] 5)

[0087] the amino acid sequence of the heavy chain is shown as SEQ ID NO: 32; and

[0088] the amino acid sequence of the light chain is shown as SEQ ID NO: 36;

[0089] 6)

[0090] the amino acid sequence of the heavy chain is shown as SEQ ID NO: 32; and

[0091] the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 32; and

[0092] 7)

[0093] the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 32; and

[0094] the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 32; and

[0095] In some embodiments of the present application, the anti-IL-31 antibody or antigen binding fragment thereof, wherein the anti-IL-31 antibody or antigen binding fragment thereof is selected from a Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single chain antibody, humanized antibody, or chimeric antibody.

[0096] In some embodiments of the present application, the anti-IL-31 antibody or antigen binding fragment thereof, wherein,

[0097] the anti-IL-31 antibody or antigen binding fragment thereof binds to human IL-31 protein with an EC 50 less than or equal to 0.2 nM, less than or equal to 0.15 nM, less than or equal to 0.12 nM, less than or equal to 0.06 nM, less than or equal to 0.05 nM, less than or equal to 0.04 nM, or less than or equal to 0.03 nM;

[0098] preferably, the anti-IL-31 antibody or antigen binding fragment thereof binds to human IL-31 protein with an EC 50 less than the EC of BMS-981164 binding to human IL-31 protein; 50 ;

[0099] preferably, the EC 50 is measured by an ELISA method.

[0100] In some embodiments of the present application, the anti-IL-31 antibody or antigen binding fragment thereof, wherein,

[0101] the anti-IL-31 antibody or antigen binding fragment thereof binds to cynomolgus IL-31 protein with an EC 50 less than or equal to 0.08 nM, less than or equal to 0.07 nM, less than or equal to 0.06 nM, or less than or equal to 0.05 nM;

[0102] preferably, the anti-IL-31 antibody or antigen binding fragment thereof binds to cynomolgus IL-31 protein with an EC 50 less than the EC of BMS-981164 binding to cynomolgus IL-31 protein.50 ;

[0103] Preferably, the EC 50 as measured by an ELISA method.

[0104] In some embodiments of the present application, the anti-IL-31 antibody or antigen binding fragment thereof, wherein,

[0105] the anti-IL-31 antibody or antigen binding fragment thereof has a Tm value of greater than or equal to 60°C, greater than or equal to 61°C, greater than or equal to 62°C, greater than or equal to 63°C, greater than or equal to 64°C, or greater than or equal to 65°C; preferably, the Tm value is measured by differential scanning calorimetry.

[0106] In some embodiments of the present application, the anti-IL-31 antibody or antigen binding fragment thereof, wherein,

[0107] the anti-IL-31 antibody or antigen binding fragment thereof has a half-life in mice of greater than or equal to 180 hours, greater than or equal to 190 hours, greater than or equal to 200 hours, greater than or equal to 210 hours, greater than or equal to 220 hours, greater than or equal to 230 hours, or 180-230 hours.

[0108] In some embodiments of the present application, the anti-IL-31 antibody or antigen binding fragment thereof is capable of specifically binding to both human IL-31 protein and cynomolgus IL-31 protein.

[0109] In some embodiments of the present application, the anti-IL-31 antibody is the anti-IL-31 monoclonal antibody.

[0110] In some embodiments of the present application, the anti-IL-31 antibody or antigen binding fragment thereof, the anti-IL-31 antibody comprises a heavy chain variable region comprising complementarity determining regions HCDR1 to HCDR3, and a light chain variable region comprising complementarity determining regions LCDR1 to LCDR3, wherein:

[0111] the amino acid sequence of HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of HCDR2 is set forth in SEQ ID NO: 2, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO: 3, and the amino acid sequence of LCDR1 is set forth in SEQ ID NO: 5, the amino acid sequence of LCDR2 is set forth in SEQ ID NO: 6, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO: 10; and

[0112] the heavy chain constant region of the anti-IL-31 antibody is set forth in SEQ ID NO: 21 or SEQ ID NO: 29, and the light chain constant region is set forth in SEQ ID NO: 22.

[0113] In some embodiments of the present application, the anti-IL-31 antibody or antigen binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region of the anti-IL-31 antibody is set forth in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the anti-IL-31 antibody is set forth in SEQ ID NO: 11; and

[0114] the heavy chain constant region of the anti-IL-31 antibody is set forth in SEQ ID NO: 21 or SEQ ID NO: 29, and the light chain constant region is set forth in SEQ ID NO: 22.

[0115] In some embodiments of the present application, the anti-IL-31 antibody or antigen binding fragment thereof, wherein the antibody is the antibody numbered 106, 106-1, 106-2, 106-4, 106-9, 106-12, or 106-13 of the present application.

[0116] The anti-IL-31 antibody or antigen binding fragment thereof according to any one of the present application for use in the treatment or prevention of a disease caused by IL-31 being higher than normal;

[0117] Preferably, the disease is one or more selected from the group consisting of pruritus, chronic spontaneous urticaria, atopic dermatitis, nodular prurigo, contact dermatitis, eczema, bullous pemphigoid, drug-induced delayed-type cutaneous hypersensitivity reactions, alopecia areata, rhinophyma, rosacea, and psoriasis.

[0118] Preferably, the pruritus is intractable atopic dermatitis-associated pruritus, virus-associated pruritus, or lymphoma neurotic pruritus.

[0119] Another aspect of the present application relates to an isolated nucleic acid molecule encoding the anti-IL-31 antibody or antigen binding fragment thereof according to any one of the present application.

[0120] Still another aspect of the present application relates to a recombinant vector comprising the isolated nucleic acid molecule of the present application.

[0121] Still another aspect of the present application relates to a host cell comprising the isolated nucleic acid molecule of the present application, or comprising the recombinant vector of the present application.

[0122] Yet another aspect of the present application relates to an antibody drug conjugate comprising an antibody or antigen binding fragment thereof and a small molecule drug, wherein the antibody or antigen binding fragment thereof is an anti-IL-31 antibody or antigen binding fragment thereof according to any one of the present application; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is a tumor chemotherapeutic drug.

[0123] In some embodiments of the present application, the antibody drug conjugate, wherein the antibody or antigen binding fragment thereof is linked to the small molecule drug via a linker; for example, the linker is a hydrazone bond, a disulfide bond or a peptide bond.

[0124] Preferably, the molar ratio of the antibody or antigen binding fragment thereof to the small molecule drug is 1:(2-4), 1:2, 1:3 or 1:4.

[0125] Yet another aspect of the present application relates to a pharmaceutical composition comprising an effective amount of an anti-IL-31 antibody or antigen binding fragment thereof according to any one of the present application or an antibody drug conjugate according to any one of the present application; optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0126] Yet another aspect of the present application relates to the use of an anti-IL-31 antibody or antigen binding fragment thereof according to any one of the present application in the manufacture of a medicament for treating or preventing a disease caused by elevated IL-31 levels.

[0127] Preferably, the disease is one or more selected from the group consisting of pruritus, chronic spontaneous urticaria, atopic dermatitis, nodular prurigo, contact dermatitis, eczema, bullous pemphigoid, drug-induced delayed-type hypersensitivity reactions, alopecia areata, rhinophyma, rosacea and psoriasis.

[0128] Preferably, the pruritus is refractory atopic dermatitis-associated pruritus, virus-associated pruritus or lymphoma neurotic pruritus.

[0129] Yet another aspect of the present application relates to a method of treating or preventing a disease caused by elevated IL-31 levels, comprising the step of administering to a subject in need thereof an effective amount of an anti-IL-31 antibody or antigen binding fragment thereof according to any one of the present application.

[0130] Preferably, the disease is one or more selected from the group consisting of pruritus, chronic spontaneous urticaria, atopic dermatitis, nodular prurigo, contact dermatitis, eczema, bullous pemphigoid, drug-induced delayed-type hypersensitivity reactions, alopecia areata, rhinophyma, rosacea and psoriasis.

[0131] Preferably, the pruritus is refractory atopic dermatitis-associated pruritus, virus-associated pruritus or lymphoma neurotic pruritus.

[0132] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. Also, the cell culture, molecular genetics, nucleic acid chemistry, immunological laboratory procedures described herein are in accordance with conventional techniques of the respective fields. In addition, the following terms are defined and explained as follows in order to better understand the present application.

[0133] As used herein, the term EC 50 refers to the concentration for 50% of maximal effect, i.e., the concentration that elicits 50% of maximal effect.

[0134] As used herein, the term IC 50 refers to the half maximal inhibitory concentration, i.e., the drug concentration that inhibits 50% of the activity of a biological process or of a component of that process (e.g., an enzyme, a receptor, a cell, etc.) under given conditions.

[0135] As used herein, the term "antibody" refers to an immunoglobulin molecule that is generally composed of two pairs of polypeptide chains (each pair having one "light" (L) chain and one "heavy" (H) chain). Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define a different class of antibodies as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (V H ) and a heavy chain constant region (C H ). The heavy chain constant region is comprised of three domains, C H1 , C H2 , and C H3 . Each light chain is comprised of a light chain variable region (V L ) and a light chain constant region (C L ). The light chain constant region is comprised of one domain, C L . The constant regions of antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. V H and V L regions can also be subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each V H and V LThe three CDRs and four FRs are arranged from the amino-terminus to the carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region (V H and V L ) of each heavy / light pair respectively forms an antigen binding site. The assignment of amino acids to each region or domain follows the definition of the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda M.d. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196:901-917; Chothia et al. Nature 1989; 342:878-883 or the IMGT numbering system definition, see Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. "IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF." Nucleic acids research 2009; 38(suppl_1):D301-D307. The term "antibody" is not restricted by any particular method of producing the antibody. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be an antibody of different isotype, for example, an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

[0136] As used herein, the terms "monoclonal" and "monoclonal antibody" refer to one antibody or one fragment of an antibody from a population of highly homogenous antibody molecules, i.e., a population of antibody molecules that are identical except for naturally-occurring mutations that can arise during production. A monoclonal antibody has high specificity for a single epitope on an antigen. Polyclonal antibodies are in contrast to monoclonal antibodies and generally comprise at least 2 or more different antibodies, which generally recognize different epitopes on an antigen. Monoclonal antibodies can generally be obtained using the hybridoma technology first reported by Kohler et al. G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. nature, 1975; 256(5517): 495), but can also be obtained using recombinant DNA technology (see, e.g., U.S. Patent 4,816,567).

[0137] As used herein, the term "humanized antibody" refers to antibodies or antibody fragments in which all or a portion of the CDR regions of a human immunoglobulin (recipient antibody) are replaced by CDR regions of a non-human antibody (donor antibody), which can be a non-human (e.g., mouse, rat, or rabbit) antibody having the desired specificity, affinity, or reactivity. Furthermore, some of the amino acid residues in the framework regions (FRs) of the recipient antibody can be replaced by amino acid residues from corresponding sites in the non-human antibody, or by amino acid residues from other antibodies, to further refine or optimize antibody performance. For more details on humanized antibodies, see, e.g., Jones et al., Nature 1986; 321 :522-525; Reichmann et al., Nature 1988; 332:323-329; Presta, Curr. Op. Struct. Biol., 1992; 2:593-596; and Clark M. Antibody humanization: a case of the 'Emperor's new clothes'? [J]. Immunol. Today, 2000; 21 (8):397-402.

[0138] As used herein, the terms "antibody full length chain", "full length antibody", "intact antibody", and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody or having a heavy chain with an Fc region as defined herein.

[0139] As used herein, the term "light chain" includes full length light chains and fragments thereof having sufficient variable region sequence to confer binding specificity. Full length light chains include a variable region domain VL and a constant region domain CL. The variable region domain of a light chain is at the amino terminal end of the polypeptide. Light chains include kappa chains and lambda chains.

[0140] As used herein, the term "heavy chain" includes full-length heavy chains and fragments thereof having sufficient variable region sequence to confer binding specificity. Full-length heavy chains include a variable region domain VH and three constant region domains CHI, CH2 and CH3. The VH domain is at the amino terminal end of the polypeptide and the CH domains are at the carboxy terminal end, with CH3 closest to the carboxy terminal end of the polypeptide. Heavy chains can be of any isotype, including IgG (including IgGl, IgG2, IgG3 and IgG4 subtypes), IgA (including IgAl and IgA2 subtypes), IgM and IgE.

[0141] As used herein, the term "antigen binding fragment" of an antibody refers to a polypeptide that comprises a fragment of a full-length antibody that retains the ability to specifically bind the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nded. Raven Press, N.Y. (1989)), which is incorporated herein by reference in its entirety for all purposes. Antigen binding fragments of an antibody can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding capacity to the polypeptide.

[0142] As used herein, the term "Fab fragment" consists of one light chain and CHI and the variable region of one heavy chain. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0143] As used herein, the term "Fc region" is an antibody fragment formed by disulfide bond linkage of the second, third constant regions of the first heavy chain with the second, third constant regions of the second heavy chain of an antibody.

[0144] As used herein, the term "Fab' fragment" contains one light chain and a portion of one heavy chain containing the VH domain and CHI domain and also a portion of the region between the CHI and CH2 domains so that an interchain disulfide bond can form between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.

[0145] As used herein, the term "F(ab')2 fragment" contains two light chains and two heavy chains containing portions of the constant region between the CHI and CH2 domains, so as to form interchain disulfide bonds between the two heavy chains. The F(ab')2 fragment thus consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

[0146] As used herein, the term "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0147] As used herein, the term "Fd" fragment means an antibody fragment consisting of the VH and CHI domains (Ward et al., Nature 341 :544-546 (1989)).

[0148] As used herein, the term "dAb" fragment (Ward et al., Nature 341 :544-546 (1989)) consists of the VH domain.

[0149] As used herein, the term "isolated" or "separated" means obtained by artificial means from a naturally occurring state. If a certain "isolated" substance or component appears in nature, it can be that the natural environment of the substance has been changed, or the substance has been separated from the natural environment, or both. For example, a certain polynucleotide or polypeptide naturally exists in a living organism without being isolated, but a high-purity same polynucleotide or polypeptide isolated from such a natural state is called isolated. The term "isolated" or "separated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impurities that do not affect the activity of the substance.

[0150] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of the inserted polynucleotide, the vector is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection and direct the expression of elements carried by the vector in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1 -derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), pox viruses, baculoviruses, papillomaviruses, papova viruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to, promoter sequences, transcriptional start sequences, enhancer sequences, selection elements and reporter genes. In addition, a vector can contain a replication origin.

[0151] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector and includes, but is not limited to, prokaryotic cells such as E. coli or B. subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, GS cells, BHK cells, HEK 293 cells or human cells.

[0152] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. In certain embodiments, an antibody that specifically binds to (or has specificity for) an antigen refers to an antibody that binds to the antigen with an affinity of less than about 10 -5 M, for example less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less. D ) to the antigen.

[0153] As used herein, the term "K D " refers to the dissociation equilibrium constant of a particular antibody-antigen interaction and is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, an antibody binds to an antigen with an affinity of less than about 10 -5 M, for example less than about 10 -6M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller dissociation equilibrium constant (K D ) binds to an antigen (e.g., IL-31 protein). K D , for example, can be determined using a Fortebio molecular interaction instrument.

[0154] As used herein, the terms "monoclonal antibody" and "monoclonal" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also in the present application, amino acids are generally represented by the one-letter and three-letter abbreviations well-known in the art. For example, alanine can be represented by A or Ala.

[0155] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are compatible, in a pharmacological and / or physiological sense, with the subject and the active ingredient, which are well-known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers. For example, pH adjusting agents include, but are not limited to, phosphate buffer; surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

[0156] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve a desired effect. For example, a prophylactically effective amount refers to an amount sufficient to prevent, arrest, or delay the onset of a disease; a therapeutically effective amount refers to an amount sufficient to cure or at least partially arrest the disease and its complications in an already afflicted patient.

[0157] Advantages of the Invention

[0158] The anti-IL-31 antibody or antigen-binding fragment thereof of the present application achieves one or more of the following technical effects (1) to (4):

[0159] (1) capable of specifically binding to human IL-31 and cynomolgus monkey IL-31, and has high affinity;

[0160] (2) can effectively block the binding of human IL-31 and IL-31 receptor, especially IL-31RA;

[0161] (3) can reduce or eliminate the downstream STAT3 phosphorylation signal in cells;

[0162] (4) can significantly inhibit the itching caused by IL-31 or IL-31 overexpression (e.g., higher than normal levels). BRIEF DESCRIPTION OF DRAWINGS

[0163] Figure 1: Binding curve of anti-IL-31 monoclonal antibody to human IL-31 protein.

[0164] Figure 2: Binding curve of anti-IL-31 monoclonal antibody to cynomolgus IL-31 protein.

[0165] Figure 3: Curve of anti-IL-31 monoclonal antibody blocking the binding of human IL-31 protein to CHO-huIL-31RA cells.

[0166] Figure 4: Binding curve of anti-IL-31 monoclonal antibody after affinity maturation to human IL-31 protein.

[0167] Figure 5: Binding curve of anti-IL-31 monoclonal antibody after affinity maturation to cynomolgus IL-31 protein.

[0168] Figure 6: Curve of anti-IL-31 monoclonal antibody after affinity maturation blocking the binding of human IL-31 protein to CHO-huIL-31RA cells.

[0169] Figure 7: Curve of anti-IL-31 monoclonal antibody after affinity maturation blocking the phosphorylation signal of STAT3 in 293T-huIL-31RA / OSMRβ cells induced by human IL-31.

[0170] Figure 8: Anti-IL-31 monoclonal antibody after affinity maturation blocking BEAS-2B cell expression of CCL2 induced by human IL-31. Wherein Nemolizumab analogue is Nemolizumab, "+" represents adding the corresponding protein or antibody, "-" means not adding the corresponding protein or antibody.

[0171] Figure 9: Curve of anti-IL-31 monoclonal antibody after affinity maturation in vivo half-life in mice.

[0172] Figures 10A to 10C: Pharmacodynamic diagram of anti-IL-31 monoclonal antibody after affinity maturation on the number of scratches in human IL-31-induced mouse itching model.

[0173] Figure 10D to Figure 10F: Pharmacodynamic plot of the effect of nemolizumab monoclonal antibody on the number of scratches in a mouse model of IL-31 -induced pruritus.

[0174] Part of the sequence information involved in the present application is shown in Table 1 below. The CDRs are numbered according to the IMGT numbering system.

[0175] Table 1: Part of the sequence information involved in the present application DETAILED DESCRIPTION

[0176] Embodiments of the present application will be described in detail with reference to the drawings, of which the following examples are provided by way of illustration only. As would be obvious to one of skill in the art, the following examples are provided for the purpose of illustration only, and should not be considered as limiting the scope of the present application. Unless otherwise indicated, the conditions in the examples are conventional conditions or those recommended by the manufacturer. Unless otherwise indicated, the reagents or instruments used are conventional products available on the market.

[0177] Example 1: Construction of antibody library

[0178] The lmg human interleukin-31 (IL-31) (purchased from AcroBiosystems) as an antigen was mixed with an equal volume of Freund's adjuvant, and 5 fully humanized mice (purchased from Alloy, USA) were immunized once a week for a total of 4 times, to stimulate B cells to express antibodies specific to the antigen. After the 4 immunizations, the spleen of the mice was removed, and total RNA was extracted using Trizol (purchased from Invitrogen). The total cDNA of the fully humanized mice was obtained by reverse transcription using a cDNA synthesis kit (purchased from Invitrogen). IgG1 sequences were amplified from the cDNA by first-round PCR. The PCR products were subjected to agarose gel electrophoresis, and the target fragments of 400-500 bp were recovered by gel excision. The first-round PCR products were used as templates for second-round sequence amplification to add homologous arms to the heavy and light chain genes. Finally, the target fragments were recovered using a PCR purification kit (purchased from QIAGEN).

[0179] The linearized yeast display vector and the second-round PCR products were mixed and then electroporated into S. cerevisiae (purchased from ATCC) to construct a library of fully human anti-IL-31 antibodies from the five animals and determine the library size, which was 3 x 107 .

[0180] Example 2: Screening of anti-IL-31 monoclonal antibodies

[0181] Dissolve human IL-31 protein (purchased from AcroBiosystems) in an appropriate volume of double-distilled water, and mix with the biotin solution according to the product instructions of the biotin labeling kit (purchased from Thermo). Incubate at 4°C for 2 hours. Remove the excess biotin using a desalting column (purchased from Thermo). The pretreatment of the desalting column and the collection of the sample are performed according to the product instructions.

[0182] Inoculate the antibody library constructed in Example 1 into SD-CAA amplification medium (1L SD-CAA amplification medium containing 6.7g YNB, 5g tyrosine, 13.62g Na2HPO4·12H2O, 7.44g NaH2PO4, and 2% glucose), and incubate at 30°C, 225rpm overnight. Centrifuge an appropriate amount of yeast cells at 3000rpm for 5 minutes (the centrifugation operation is the same below) to remove the culture medium, resuspend the yeast cells in SD-CAA induction medium, and induce overnight. Determine the concentration of the library after induction, centrifuge an appropriate amount of yeast cells to remove the culture medium. Resuspend the yeast cells in 50mL PBS, centrifuge to remove the supernatant. Resuspend the yeast cells in 10mL PBS.

[0183] Add biotin-labeled human IL-31 protein (final concentration 100nM), and incubate at room temperature for 30 minutes. Centrifuge to collect the yeast cells, and wash the yeast cells with 50mL PBS for 3 times. Resuspend the yeast cells in 5mL washing solution, and add 200μL SA magnetic beads (purchased from Meitenyi). Incubate for 10 minutes by inversion. Wash the yeast and magnetic bead mixture with PBS for 3 times, and add the mixture to an LS purification column (purchased from Meitenyi). Place the LS purification column on a magnetic stand, and wash with PBS to remove non-specifically bound yeast cells. Remove the purification column from the magnetic stand, and add PBS to elute the yeast. Centrifuge the eluted yeast, and transfer to SD-CAA amplification medium for amplification.

[0184] The yeast cells enriched by MACS (Magnetic Activated Cell Sorting) were inoculated in the SD-CAA expansion medium. The 30°C, 225 rpm shake flask culture was cultured overnight. The yeast cells were resuspended with the SD-CAA induction medium and induced overnight. The anti-c-Myc mouse antibody (purchased from Thermo) and 100 nM biotin-labeled IL-31 antigen were added and incubated for 10 minutes. The yeast cells were washed with PBS for 3 times, and the goat anti-mouse IgG (H+L) Alexa Fluor Plus 488 fluorescent antibody (purchased from Invitrogen) and streptavidin APC conjugate fluorescent antibody (purchased from Invitrogen) were added and incubated for 15 minutes. The cells were resuspended with PBS, and the BD Aria III instrument was used for sorting to obtain the yeast cells with high binding capacity to the IL-31 antigen.

[0185] The yeast cells with high binding capacity to the human IL-31 antigen obtained by MACS and FACS enrichment were coated on the solid culture plate of the SD-CAA, and then the single colonies were picked and cultured in the SD-CAA expansion medium at 30°C, 225 rpm overnight. The expanded single colonies were treated with 0.1% SDS, centrifuged, and the supernatant was used as the template for PCR amplification. The PCR product was sent for sequencing to obtain the gene sequence.

[0186] Example 3: Construction and expression and purification of anti-IL-31 monoclonal antibody

[0187] The heavy chain variable region gene sequence was connected with the nucleotide sequence of the human IgG1 constant region (SEQ ID NO: 21), and a homologous recombinase (purchased from Vazyme) was used to construct into the EcoR I / Not I double enzyme linearized pCDNA3.1 vector; the light chain variable region gene sequence was connected with the nucleotide sequence encoding the human kappa light chain constant region (SEQ ID NO: 22), and was constructed into the EcoR I / Xhol I double enzyme linearized pCDNA3.1 vector, and the process was according to the product instructions. The homologous recombination product was transformed into Top10 competent cells, coated on ampicillin resistance plates, and cultured at 37°C overnight. The single colonies were sequenced and the plasmid was extracted.

[0188] HEK293 cells were used for transfection and expression. The cell density was adjusted to 2.5×10 6 cells / ml the day before transfection, and diluted to 3.0×10 6Fab antibody structure, the cells were cultured for 5 days, and the supernatant was collected and purified by a Protein A column. 10x column volume of 1x PBS was added to the column to balance the column, and the cell supernatant was added to the column under gravity flow. After the sample was added, 20x column volume of 1x PBS was used to wash away the unbound impurities. After no liquid flowed out, the column was placed in a collection tube with pre-added neutralization buffer (1M Tris, pH 8.54), and 3-5 times column volume of elution buffer (0.1M sodium citrate, pH 3.2) was used to elute the target protein, including the antibody 106 of the application. The amino acid sequences of the complementarity determining regions, the heavy chain variable region and the light chain variable region of the antibody 106 are shown in Table 1.

[0189] Example 4: Determination of the affinity of the anti-IL-31 monoclonal antibody

[0190] The ForteBio affinity assay was performed according to the existing method (Estep, P, et al. Determination of antibody-antigen affinity and epitope binding based on high-throughput methods. MAbs, 2013. 5(2): p 270-278). Briefly, the sensor was equilibrated in the analysis buffer for 30 minutes, and then the baseline was established by detecting for 60 seconds. The purified antibody obtained as described above was loaded onto the AHQ sensor. The sensor was then placed in 100nM HSA antigen for 5 minutes, and then transferred to PBS for dissociation for 5 minutes. The kinetics were analyzed using a 1:1 binding model. The experiment detected the cross-reaction of the candidate molecules with different species proteins, including human and cynomolgus monkey-derived IL-31 proteins (purchased from AcroBiosystems).

[0191] Table 2: Affinity of the anti-IL-31 monoclonal antibody to human and cynomolgus IL-31 proteins

[0192] The results are shown in Table 2. The anti-IL-31 monoclonal antibody 106 of the application has good monovalent affinity to human IL-31 protein and cynomolgus IL-31 protein.

[0193] Example 5: Determination of the purity and thermal stability of the anti-IL-31 monoclonal antibody

[0194] Mobile phase: 150mM Na2HPO4·12H2O, pH 7.0.

[0195] Chromatographic conditions: detection wavelength 280 nm, column temperature 25 °C, flow rate 0.35 mL / min, detection time 20 min, Zenix-C SEC-300 column (SEPAX 4.6 x 300 mm, 3 μm). The results are shown in Table 3.

[0196] The thermal stability of the antibody was detected by differential scanning calorimetry (DSC). The sample was concentrated and diluted with PBS to 1 mg / mL; 5000x fluorescent colorant Cypro Orange (purchased from Bio-Rad) was diluted 50 times with ultrapure water to obtain 100x fluorescent colorant Sypro Orange. 50 μL of 1 mg / mL sample was added to 10 μL of 100x fluorescent colorant Sypro Orange, 40 μL of ultrapure water, mixed well, and then 30 μL was added to a 96-well PCR plate, 3 replicates were made for each sample, and then placed in a PCR instrument, and the temperature program was set as follows: 25 °C for 5 min, and then heated to 99 °C at a rate of 0.5 °C / min. After the program ended, the temperature value of the lowest point of the curve was read in the "Melt Curve" graph, which was the Tm value of the sample. The specific results are shown in Table 3 below:

[0197] Table 3: Purity and thermal stability detection results of anti-IL-31 monoclonal antibodies

[0198] Example 6: ELISA method for determining the binding activity of anti-IL-31 monoclonal antibodies to human IL-31 protein and cynomolgus IL-31 protein

[0199] Specifically, the human IL-31 protein sample and the cynomolgus IL-31 protein sample were diluted to 1 μg / mL respectively with 1x ELISA coating working solution, 100 μL / well was used to coat a 96-well ELISA plate, and the plate was incubated at 4°C overnight. After the coating was completed, the ELISA plate was taken out, the coating solution was discarded, 250 μL / well of ELISA washing solution was added, and the plate was allowed to stand for 30 seconds, the washing solution was discarded, and the above steps were repeated for 3 times, 200 μL / well of ELISA blocking solution was added, and the plate was incubated at room temperature for 2 hours. The antibody was diluted with ELISA working solution, and 3-fold gradient dilution was performed from 100 nM, 100 μL / well was added to the blocked ELISA plate, and the plate was allowed to react at room temperature for 2 hours. HRP anti-huIgG was diluted with ELISA working solution to HRP working solution, and was prepared for use. After the combination reaction was completed, the ELISA plate was taken out, the reaction solution was discarded, 250 μL / well of ELISA washing solution was added, and the plate was allowed to stand for 30 seconds, the washing solution was discarded, and the above steps were repeated for 3 times, 100 μL / well of HRP working solution was added, and the plate was incubated at room temperature for 1 hour. After the reaction with the HRP working solution was completed, the ELISA plate was taken out, the reaction solution was discarded, 250 μL / well of ELISA washing solution was added, and the plate was allowed to stand for 30 hours, the washing solution was discarded, and the above steps were repeated for 3 times, 100 μL / well of TMB single-component color developing solution was added, and the plate was allowed to react at room temperature for 3 minutes. After the color development was completed, 50 μL / well of ELISA termination solution was added to the ELISA plate, and the reaction was terminated. Within 5 minutes after the termination was completed, the absorbance value at 450 nm of the ELISA plate was read, and the value was recorded for data processing.

[0200] The results are shown in Table 4 and Figs. 1-2.

[0201] The results show that the antibody 106 of the present application has binding activity to both human IL-31 protein and cynomolgus IL-31 protein, and the affinity of the antibody 106 of the present application to human IL-31 protein is superior to that of the control molecule BMS-981164.

[0202] Example 7: Blocking activity of anti-IL-31 monoclonal antibody to the binding of human IL-31 to IL-31RA overexpressed CHO cells

[0203] The activity of anti-IL-31 monoclonal antibody in blocking huIL-31 / huIL-31RA binding was detected by flow cytometry. Specifically, the nucleotide sequence encoding the full-length human IL-31RA amino acid sequence (SEQ ID NO: 27) was inserted into the multiple cloning site of the PLVX-puro vector (purchased from Addgene) by EcoR I and Hind III double enzyme digestion, respectively. The 293T cells were used to package the lentivirus supernatant and infect CHO and 293T cells. The transfected cells were selected by adding puromycin (purchased from Invitrogen) to obtain stable overexpression of human IL-31RA CHO cells (CHO-huIL-31RA cells) and human IL-31RA 293T cells (293T-huIL-31RA cells). The purified antibody was diluted with 1xPBS, 400nM as the starting point, 3-fold gradient dilution for a total of 12 points, and 60μL / well of the diluted sample was added to a new 96-well flow plate. Then 60μL / well of biotinylated human IL-31 protein (Biotheus) diluted to the appropriate concentration with 1xPBS was added, mixed well, and incubated at 4°C for 30 minutes. The expanded CHO-huIL-31RA cells were adjusted to a density of 2x10 6 / mL, 100μL / well was added to a 96-well flow plate, and after centrifugation, the supernatant was removed. 100μL / well of the incubated sample was added to the centrifuged cell wells, mixed well, and incubated at 4°C for 30 minutes. Washed twice with 1xPBS, 100μL / well of SAPE antibody diluted 150 times with 1xPBS was added, and incubated at 4°C for 30 minutes. Washed twice with 1xPBS, 100μL / well of 1xPBS was added to resuspend the cells, and detected on a CytoFlex flow cytometer and calculated the corresponding mean fluorescence intensity (Mean Fluorescence Intensity, MFI).

[0204] The results are shown in Table 4 and Figure 3.

[0205] Table 4: Summary of anti-IL-31 monoclonal antibody binding blocking activity with human and cynomolgus IL-31

[0206] The results show that the antibody 106 of the present application and the control molecule can completely block the binding of human IL-31 protein to the surface of CHO cells overexpressing human IL-31RA protein.

[0207] Example 8: Affinity maturation of anti-IL-31 monoclonal antibody

[0208] Taking antibody 106 as a candidate molecule, random mutations were made to the heavy chain CDR region and the light chain CDR region to construct an antibody library, and then high-affinity antibodies were screened from the antibody library by yeast display technology. The specific steps are as follows:

[0209] The heavy chain gene segment VH and the light chain gene segment VK of the synthetic antibody 106 were used as templates to design degenerate primers for CDR2, CDR3 of VH and CDR3 of VK, respectively. To ensure that each amino acid could be mutated into any of the 20 amino acids, NNK was used as the mutation base form for PCR amplification. The PCR purification kit (purchased from QIAGEN) was used to recover the target fragments. The linearized yeast display vector and the PCR products of VH and VK were mixed and then electroporated into Saccharomyces cerevisiae, respectively, to construct the affinity maturation library of the heavy chain mutation and the light chain mutation and determine the library capacity.

[0210] The screening method of the affinity matured anti-IL-31 monoclonal antibody was the same as that in Example 2, and the yeast with the strongest binding to IL-31 antigen was obtained when using the BD Aria III instrument for sorting.

[0211] The construction and expression and purification method of the affinity matured anti-IL-31 monoclonal antibody were the same as those in Example 3.

[0212] The obtained affinity matured anti-IL-31 monoclonal antibodies were named 106-1, 106-2, 106-4, 106-9, 106-12 and 106-13, respectively. The amino acid sequences of the complementarity determining regions, the heavy chain variable region and the light chain variable region of these antibodies are shown in Table 1.

[0213] Example 9: Affinity determination of the affinity matured anti-IL-31 monoclonal antibody

[0214] The ForteBio affinity determination was performed according to the existing method (Estep, P, et al. Determination of antibody-antigen affinity and epitope binding based on high-throughput method. MAbs, 2013. 5(2): p. 270-8). Briefly, the sensor was equilibrated in the analysis buffer for 30 minutes, then the baseline was established by detecting on-line for 60 seconds, and the purified antibody obtained as described above was loaded on the AHQ sensor. Then the sensor was placed in 100 nM HSA antigen for 5 minutes, and then the sensor was transferred to PBS for dissociation for 5 minutes. The kinetics was analyzed using a 1:1 binding model. The results are shown in Table 5, in which 106-1 showed better affinity.

[0215] Table 5: Affinity of the affinity matured anti-IL-31 monoclonal antibody to human IL-31

[0216] Example 10: Purity and thermal stability determination of the affinity matured anti-IL-31 monoclonal antibody

[0217] The purity of the obtained protein was detected by HPLC. The HPLC method was as follows:

[0218] Mobile phase: 150 mM Na2HP04*12H20, pH 7.0.

[0219] Chromatographic conditions: detection wavelength 280 nm, column temperature 25 °C, flow rate 0.35 ml / min, detection time 20 min, Zenix-C SEC-300 column (SEPAX 4.6 x 300 mm, 3 μm).

[0220] The results are shown in Table 6. The results show that the purity of all the antibodies is high.

[0221] Table 6: Purity detection results of the affinity matured anti-IL-31 monoclonal antibodies

[0222] The thermal stability detection method of the affinity matured IL-31 monoclonal antibodies is the same as in Example 5. The results are shown in Table 7. The results show that all the antibodies exhibit good thermal stability.

[0223] Table 7: Tm values of the affinity matured anti-IL-31 monoclonal antibodies

[0224] Example 11: Binding activity of the affinity matured anti-IL-31 monoclonal antibodies to human IL-31 protein and cynomolgus IL-31 protein

[0225] The specific method is the same as in Example 6.

[0226] The results are shown in Table 8 and Figures 4 to 5.

[0227] The results show that all the anti-IL-31 monoclonal antibodies after affinity maturation have strong binding activity to human IL-31 protein and cynomolgus IL-31 protein. The results also show that the affinity of the anti-IL-31 monoclonal antibodies of the present application to cynomolgus IL-31 protein is better than that of the control molecule BMS-981164.

[0228] Example 12: Blocking activity of the affinity matured anti-IL-31 monoclonal antibodies to the binding of human IL-31 to IL-31RA overexpressing CHO cells

[0229] The affinity matured anti-IL-31 monoclonal antibodies are verified to have strong affinity to human IL-31, and further flow cytometry is used to detect the blocking activity of the affinity matured anti-IL-31 monoclonal antibodies to the binding of huIL-31 / huIL-31RA cells (the same as the CHO-huIL-31RA cells in Example 7).

[0230] The method is the same as in Example 7.

[0231] The results are shown in Table 8 and Figure 6.

[0232] The results show that most of the affinity matured anti-IL-31 monoclonal antibodies are able to completely block the binding of human IL-31 protein to CHO cells overexpressing human IL-31RA on the cell surface, and most of the affinity matured anti-IL-31 monoclonal antibodies are superior to the 106 molecules before affinity maturation.

[0233] Example 13: Affinity matured anti-IL-31 monoclonal antibodies block human IL-31 induced STAT3 phosphorylation in 293T-huIL-31RA / OSMRβ cells

[0234] Having verified that the affinity matured anti-IL-31 monoclonal antibodies block the binding of human IL-31 to IL-31RA at the cellular level, the blocking activity of the antibodies was further analyzed functionally. Specifically, the nucleotide sequence encoding the full-length human OSMRβ amino acid sequence (SEQ ID NO: 28) was inserted into the multiple cloning site of the PMS061-2-h-hygro vector using AgeI and NheI double enzyme digestion, respectively. The 293T cells were used to package the lentivirus supernatant and infect 293T-huIL-31RA cells, and the transfected cells were subjected to pressure screening by adding hygromycin (purchased from Invivogen) to obtain 293T cells stably overexpressing human IL-31RA / OSMRβ (293T-huIL-31RA / OSMRβ cells). When the cells are stimulated by IL-31, the STAT3 phosphorylation signal downstream of the receptor is activated. Flow cytometry was used to determine the inhibitory activity of the affinity matured anti-IL-31 monoclonal antibodies on human IL-31 induced STAT3 phosphorylation in 293T-huIL-31RA / OSMRβ cells.

[0235] Specifically, the purified antibodies were diluted with 1xPBS, starting at 400nM and 3-fold dilution for a total of 12 points, and then 1xPBS diluted human IL-31 protein (Kactus) was added to the above plate, and incubated at 4°C for a total of 30 minutes. The 293T-huIL-31RA / OSMRβ cells were removed, and the cell concentration was adjusted to 2.0x10 6Cells were harvested by trypsinization and resuspended in ice 1x wash buffer at a density of 1*10 cells / mL, 100 μL / well added to 96-well flow plate, centrifuged for later use. 100 μL / well of the incubated sample was added to the above 96-well flow plate with cells, 4°C for 10 minutes. Ice 1x PBS washed once, 100 μL / well of 4% paraformaldehyde was added, 4°C for 10 minutes. Ice 1x PBS washed once, 100 μL / well of ice methanol was added, 4°C for 30 minutes. 200 μL / well of ice 1x wash buffer was added twice, 100 μL / well of ice 1x wash buffer diluted pSTAT3 flow antibody (1:200) was added, 4°C for 20 minutes. 200 μL / well of ice 1x wash buffer was added twice, 100 μL / well of ice 1x wash buffer was added to resuspend the cells, and the cells were detected on a CytoFlex flow cytometer and the corresponding MFI was calculated.

[0236] The results, as shown in Table 8 and Figure 7, most of the antibodies can completely inhibit IL-31 induced phosphorylation of STAT3 in 293T-huIL-31RA / OSMRβ cells, wherein the affinity matured 106-1 molecule is superior to the affinity matured 106 molecule, and the blocking activity is substantially equivalent to the control antibody BMS-981164.

[0237] Table 8: Anti-IL-31 monoclonal antibody binding blocking activity to human and cynomolgus IL-31

[0238] Example 14: Affinity matured anti-IL-31 monoclonal antibody blocks IL-4 and IL-31 induced BEAS-2B cell CCL2 expression

[0239] BEAS-2B cells are healthy adult human bronchial epithelial cells, which express a variety of receptors on the surface, and human IL-4, IL-31 can induce the expression of CCL2. The ability of 106-1 antibody to block IL-31 mediated signaling pathway was verified by detecting the expression of CCL2 in BEAS-2B cells by ELISA method.

[0240] Specifically, BEAS-2B cells (ATCC) were trypsinized, the cells were resuspended with culture medium, and the cell density was adjusted to 3*10 5Add 100 μL / well to a 96-well cell culture plate and incubate overnight. Wash the cell culture plate twice with PBS and set aside. Dilute the antibody to 40 nM with culture medium and add 100 μL / well to the same 96-well plate, in triplicate. Dilute IL-4 and IL-31 to 4 ng / mL and 50 ng / mL respectively with culture medium and add 100 μL / well to each well, in triplicate. Include Blank and positive control wells. Incubate at 37°C, 5% CO2 for 24 hours. Detect the CCL2 content in the supernatant using a human CCL2 ELISA kit.

[0241] The results are shown in Figure 8. The results show that the ability of the 106-1 molecule to inhibit CCL2 expression in BEAS-2B cells is comparable to that of the control monoclonal antibody Nemolizumab.

[0242] Example 15: Half-life of anti-IL-31 monoclonal antibody in mice

[0243] Based on the in vitro activity data of the 106-1 molecule, the half-life of the 106-1 molecule in mice was determined by ELISA using tail vein injection and blood samples collected at different time points. Specifically, nine male and nine female Balb / c mice were used in the experiment, with 12 / 12-hour light / dark cycles, a temperature of 24±2℃, and a humidity of 40-70%, and free access to water and food. On the day of the experiment, Balb / c mice were injected with the monoclonal antibody molecule via a single tail vein injection at a dose of 10 mg / kg.

[0244] Blood collection time points: Blood was collected from the orbital region of mice at 5 minutes, 0.5 hours, 2 hours, 6 hours, 24 hours, 48 ​​hours, 96 hours, 168 hours, 336 hours, and 504 hours after drug administration. Whole blood samples were incubated at 2-8℃ for 30 minutes, centrifuged at 12,000 rpm for 5 minutes to collect serum. The obtained serum was then centrifuged again at 2-8℃ at 12,000 rpm for 5 minutes and stored at -80℃. The content of monoclonal antibody molecules in the serum was detected by ELISA.

[0245] The results are shown in Figure 9. The results show that the half-life of the 106-1 molecule in mice is approximately 232 hours.

[0246] Example 16: In vivo efficacy of anti-IL-31 monoclonal antibody in IL-31 / IL-31RA / OSMRβKI C57 mice

[0247] A human IL-31-induced mouse pruritus model was constructed using B-hIL-31 / hIL-31RA / OSMRβKI C57BL / 6 mice (mice with human IL-31 / IL-31RA / OSMRβ knock-in, purchased from Biocytogen) to evaluate the efficacy of anti-IL-31 monoclonal antibodies.

[0248] The specific steps are as follows:

[0249] On the first day, the mice were intradermally injected with PBS, and the scratching frequency and round number of the mice were recorded for 3 hours using the scratching monitoring device as a baseline. On the second day, the mice were intradermally injected with human IL-31 protein, and the IL-31 protein-mediated scratching frequency and round number of the mice were recorded for 3 hours using the scratching monitoring device. After the recording was completed, the mice were intraperitoneally injected with the anti-IL-31 monoclonal antibody or the control antibody. On the third day, the mice were intradermally injected with human IL-31 protein, and the scratching frequency and round number of the mice were recorded for 30 minutes using the scratching monitoring device before the injection of human IL-31 protein to exclude abnormal itching of the mice. Then, the scratching frequency and round number of the mice after the injection of human IL-31 protein were recorded for 3 hours using the scratching monitoring device. The data were then collated and statistically analyzed to reflect the itching inhibition effect of the antibody. The dosages and modes of administration are shown in Table 9. There were 3 mice in each group.

[0250] Table 9: Experimental scheme for inhibition of IL-31-mediated itching by 106-1

[0251] The experimental results are shown in FIGS. 10A to 10F.

[0252] As shown in FIGS. 10A to 10F, the number of mouse scratches was significantly increased in the subcutaneous injection of human IL-31 protein modeling group compared with the subcutaneous injection of blank PBS baseline, indicating that the human IL-31 protein-induced B-hIL-31 / hIL-31RA / OSMRβ KI C57BL / 6 mouse itching model was successfully modeled.

[0253] As shown in the results of the scratching frequency of the mice within 3 hours after the injection of human IL-31 protein in FIGS. 10A to 10C, the number of mouse scratches was significantly reduced in the 106-1 antibody administration group compared with the modeling group, and the itching improvement could last for at least 6 days, and the degree of itching improvement was comparable to that of the control antibody Nemolizumab (as shown in FIGS. 10D to 10F).

[0254] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details in accordance with all the teachings disclosed herein, and such changes are within the scope of protection of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. An anti-IL-31 antibody or antigen-binding fragment thereof, wherein, The anti-IL-31 antibody comprises a heavy chain variable region comprising complementarity determining regions HCDR1 to HCDR3 and a light chain variable region comprising complementarity determining regions LCDR1 to LCDR3, wherein: the amino acid sequence of HCDR1 is set forth in SEQ ID NO: 1; the amino acid sequence of HCDR2 is set forth in SEQ ID NO: 2; the amino acid sequence of HCDR3 is set forth in SEQ ID NO: 3; the amino acid sequence of LCDR1 is set forth in SEQ ID NO: 5; the amino acid sequence of LCDR2 is set forth in SEQ ID NO: 6; and the amino acid sequence of LCDR3 is selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO:

19.

2. The anti-IL-31 antibody or antigen-binding fragment thereof of claim 1, wherein, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9 or SEQ ID NO: 4; and the amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO:

20.

3. The anti-IL-31 antibody or antigen-binding fragment thereof of any one of claims 1-2, wherein, The heavy chain variable region and the light chain variable region of the anti-IL-31 antibody are selected from the group consisting of (1) to (14) as follows: (1) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 4; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 8; (2) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 4; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 11; (3) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 4; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 13; (4) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 4; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 14; (5) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 4; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 16; (6) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 4; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 18; (7) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 4; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 20; (8) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 8; (9) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 11; (10) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 13; (11) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 14; (12) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 16; (13) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 18; and (14) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:

20.

4. The anti-IL-31 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein, the antibody comprises non-CDR regions, and the non-CDR regions are from a species other than murine, for example from a human antibody; Preferably, the anti-IL-31 antibody is a fully human antibody. Preferably, the anti-IL-31 antibody is a fully human monoclonal antibody.

5. The anti-IL-31 antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein, the anti-IL-31 antibody is of IgGl, IgG2, IgG3 or IgG4 subtype; Preferably, the heavy chain constant region of the anti-IL-31 antibody is human Ig gamma-1 chain C region or human Ig gamma-4 chain C region; and the light chain constant region is human Ig kappa chain C region. Alternatively, the anti-IL-31 antibody is of IgGl, and its heavy chain constant region contains L234A and L235A mutations according to the EU numbering system. Preferably, the amino acid sequence of the heavy chain constant region of the anti-IL-31 antibody is set forth in SEQ ID NO: 21 or SEQ ID NO: 29, and / or the amino acid sequence of the light chain constant region is set forth in SEQ ID NO:

22.

6. The anti-IL-31 antibody or antigen-binding fragment thereof of any one of claims 1 to 5, wherein, the anti-IL-31 antibody comprises a heavy chain and a light chain, and is selected from 1) to 7) as follows: 1) the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 30; and the amino acid sequence of the light chain is set forth in SEQ ID NO: 31; 2) the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 32; and the amino acid sequence of the light chain is set forth in SEQ ID NO: 33; 3) the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 32; and the amino acid sequence of the light chain is set forth in SEQ ID NO: 34; 4) the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 32; and the amino acid sequence of the light chain is set forth in SEQ ID NO: 35; 5) the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 32; and the amino acid sequence of the light chain is set forth in SEQ ID NO: 36; 6) the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 32; and the amino acid sequence of the light chain is set forth in SEQ ID NO: 37; 7) the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 32; and the amino acid sequence of the light chain is set forth in SEQ ID NO:

38.

7. The anti-IL-31 antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein, the anti-IL-31 antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single-chain antibody, humanized antibody or chimeric antibody.

8. The anti-IL-31 antibody or antigen-binding fragment thereof of any one of claims 1 to 7, wherein, EC50of the anti-IL-31 antibody or antigen binding fragment thereof binding to human IL-31 protein 50 less than or equal to 0.2 nM, less than or equal to 0.15 nM, less than or equal to 0.12 nM, less than or equal to 0.06 nM, less than or equal to 0.05 nM, less than or equal to 0.04 nM, or less than or equal to 0.03 nM; Preferably, the anti-IL-31 antibody or its antigen-binding fragment binds to the human IL-31 protein in an EC. 50 ECs smaller than BMS-981164 that bind to human IL-31 protein 50 ; Preferably, the EC 50 is measured by the ELISA method.

9. The anti-IL-31 antibody or antigen-binding fragment thereof of any one of claims 1 to 8, wherein, EC50of the anti-IL-31 antibody or antigen binding fragment thereof binding to cynomolgus IL-31 protein 50 less than or equal to 0.08 nM, less than or equal to 0.07 nM, less than or equal to 0.06 nM, or less than or equal to 0.05 nM; Preferably, the anti-IL-31 antibody or its antigen-binding fragment binds to the cynomolgus monkey IL-31 protein in the EC. 50 ECs smaller than BMS-981164 that bind to cynomolgus monkey IL-31 protein 50 ; Preferably, the EC 50 is measured by the ELISA method.

10. The anti-IL-31 antibody or antigen-binding fragment thereof of any one of claims 1 to 9, wherein, the Tm value of the anti-IL-31 antibody or antigen-binding fragment thereof is greater than or equal to 60°C, greater than or equal to 61°C, greater than or equal to 62°C, greater than or equal to 63°C, greater than or equal to 64°C or greater than or equal to 65°C; preferably, the Tm value is measured by differential scanning calorimetry.

11. The anti-IL-31 antibody or antigen-binding fragment thereof of any one of claims 1 to 10, wherein, the half-life of the anti-IL-31 antibody or antigen-binding fragment thereof in a mouse in vivo is greater than or equal to 180 hours, greater than or equal to 190 hours, greater than or equal to 200 hours, greater than or equal to 210 hours, greater than or equal to 220 hours, greater than or equal to 230 hours or 180-230 hours.

12. An isolated nucleic acid molecule encoding the anti-IL-31 antibody or antigen-binding fragment thereof of any one of claims 1 to 11.

13. A recombinant vector comprising the isolated nucleic acid molecule of claim 12.

14. A host cell comprising the isolated nucleic acid molecule of claim 12, or comprising the recombinant vector of claim 13.

15. An antibody drug conjugate comprising an antibody or antigen binding fragment thereof and a small molecule drug, wherein, the antibody or antigen-binding fragment thereof is the anti-IL-31 antibody or antigen-binding fragment thereof of any one of claims 1 to 11; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is a tumor chemotherapeutic drug.

16. The antibody drug conjugate of claim 15, wherein, the antibody or antigen-binding fragment thereof is linked to the small molecule drug via a linker; for example, the linker is a hydrazone bond, a disulfide bond or a peptide bond; preferably, the molar ratio of the antibody or antigen-binding fragment thereof to the small molecule drug is 1:(2-4).

17. A pharmaceutical composition comprising an effective amount of the anti-IL-31 antibody or antigen-binding fragment thereof of any one of claims 1 to 11 or the antibody-drug conjugate of any one of claims 15 to 16; optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

18. Use of the anti-IL-31 antibody or antigen-binding fragment thereof of any one of claims 1 to 11 in the manufacture of a medicament for treating or preventing a disease caused by elevated levels of IL-31; preferably, the disease is one or more selected from the group consisting of pruritus, chronic spontaneous urticaria, atopic dermatitis, nodular prurigo, contact dermatitis, eczema, bullous pemphigoid, drug-induced delayed-type hypersensitivity reactions, alopecia areata, rhinophyma, rosacea and psoriasis; preferably, the pruritus is refractory atopic dermatitis-associated pruritus, virus-associated pruritus or lymphoma neurotic pruritus.

19. The anti-IL-31 antibody or antigen-binding fragment thereof of any one of claims 1 to 11 for use in treating or preventing a disease caused by elevated levels of IL-31; preferably, the disease is one or more selected from the group consisting of pruritus, chronic spontaneous urticaria, atopic dermatitis, nodular prurigo, contact dermatitis, eczema, bullous pemphigoid, drug-induced delayed-type hypersensitivity reactions, alopecia areata, rhinophyma, rosacea and psoriasis; preferably, the pruritus is refractory atopic dermatitis-associated pruritus, virus-associated pruritus or lymphoma neurotic pruritus.

20. A method of treating or preventing a disease caused by elevated levels of IL-31, comprising the step of administering to a subject in need thereof an effective amount of the anti-IL-31 antibody or antigen-binding fragment thereof of any one of claims 1 to 11; preferably, the disease is one or more selected from the group consisting of pruritus, chronic spontaneous urticaria, atopic dermatitis, nodular prurigo, contact dermatitis, eczema, bullous pemphigoid, drug-induced delayed-type hypersensitivity reactions, alopecia areata, rhinophyma, rosacea and psoriasis; preferably, the pruritus is refractory atopic dermatitis-associated pruritus, virus-associated pruritus or lymphoma neurotic pruritus.

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