Anti-IL-31 antibodies, pharmaceutical compositions and uses thereof

Abstract

The invention belongs to the field of biological medicine, and relates to an anti-IL-31 antibody, and a pharmaceutical composition and application thereof. Specifically, the invention relates to an anti-IL-31 monoclonal antibody or an antigen binding fragment thereof. The antibody has high affinity with human IL-31 and cynomolgus monkey IL-31, can effectively block the combination of IL-31 and its receptor IL-31RA, reduces or eliminates downstream STAT3 phosphorylation signals in cells, significantly inhibits pruritus caused by IL-31, and has good application prospects.

Description

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 use. 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] The present application provides the following invention:

[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 shown as SEQ ID NO: 1;

[0011] the amino acid sequence of HCDR2 is shown as SEQ ID NO: 2;

[0012] the amino acid sequence of HCDR3 is shown as SEQ ID NO: 3;

[0013] the amino acid sequence of LCDR1 is shown as SEQ ID NO: 5;

[0014] the amino acid sequence of LCDR2 is shown as 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; and

[0019] the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 8.

[0020] In some embodiments of the present application, the anti-IL-31 antibody or antigen-binding fragment thereof, wherein, the anti-IL-31 antibody is selected from (1) to (14) as follows: (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. (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. (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. (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. (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 light chain variable region is set forth in SEQ ID NO: 16. (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 (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 (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 (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 (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 (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 (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 (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 (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, such as 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 set forth in SEQ ID NO: 29.

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

[0073] In some embodiments of the 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.

[0074] In some embodiments of the 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.

[0075] 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;

[0076] 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 ;

[0077] The EC 50 is measured by an ELISA method.

[0078] In some embodiments of the 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.

[0079] 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;

[0080] 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 ;

[0081] The EC 50 is measured by an ELISA method.

[0082] In some embodiments of the 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.

[0083] 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.

[0084] In some embodiments of the present application, the anti-IL-31 antibody or antigen-binding fragment thereof, wherein,

[0085] 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.

[0086] 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.

[0087] In some embodiments of the present application, the anti-IL-31 antibody is the anti-IL-31 monoclonal antibody.

[0088] 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:

[0089] 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

[0090] 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.

[0091] 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.

[0092] The heavy chain constant region of the anti-IL-31 antibody is as shown in SEQ ID NO: 21 or SEQ ID NO: 29, and the light chain constant region is as shown in SEQ ID NO: 22.

[0093] In some embodiments of the present application, the anti-IL-31 antibody or antigen binding fragment thereof, wherein the antibody is antibody No. 106, 106-1, 106-2, 106-4, 106-9, 106-12 or 106-13 of the present application.

[0094] The anti-IL-31 antibody or antigen binding fragment thereof according to any one of the present application is used for treating or preventing a disease caused by IL-31 being higher than normal;

[0095] Preferably, the disease is one or more selected from the group consisting of itch, chronic spontaneous urticaria, atopic dermatitis, nodular prurigo, contact dermatitis, eczema, bullous pemphigoid, drug-induced delayed cutaneous hypersensitivity, alopecia areata, rhinophyma, rosacea and psoriasis.

[0096] Preferably, the itch is intractable atopic dermatitis-associated itch, virus-associated itch or lymphoma neurotic itch.

[0097] 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.

[0098] Still another aspect of the present application relates to a recombinant vector comprising the isolated nucleic acid molecule of the present application.

[0099] 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.

[0100] Still 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 the 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.

[0101] In some embodiments of the present application, the antibody drug conjugate, wherein the antibody or antigen binding fragment thereof is connected to the small molecule drug through a linker; for example, the linker is a hydrazone bond, a disulfide bond or a peptide bond.

[0102] 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.

[0103] Yet another aspect of the present application relates to a pharmaceutical composition comprising an effective amount of the anti-IL-31 antibody or antigen-binding fragment thereof of any one of the present application or the antibody-drug conjugate of any one of the present application; optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0104] Yet another aspect of the present application relates to use of the anti-IL-31 antibody or antigen-binding fragment thereof of any one of the present application in the manufacture of a medicament for treating or preventing a disease caused by IL-31 at a level higher than normal;

[0105] 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 reaction, alopecia areata, rhinophyma, rosacea and psoriasis.

[0106] Preferably, the pruritus is refractory atopic dermatitis-associated pruritus, virus-associated pruritus or lymphoma neurotic pruritus.

[0107] Yet another aspect of the present application relates to a method for treating or preventing a disease caused by IL-31 at a level higher than normal, 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 the present application;

[0108] 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 reaction, alopecia areata, rhinophyma, rosacea and psoriasis.

[0109] Preferably, the pruritus is refractory atopic dermatitis-associated pruritus, virus-associated pruritus or lymphoma neurotic pruritus.

[0110] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, the cell culture, molecular genetics, nucleic acid chemistry, immunological laboratory procedures used herein are in accordance with conventional techniques of the respective fields. Meanwhile, in order to better understand the present application, the definitions and explanations of the relevant terms are provided below.

[0111] As used herein, the term EC 50 refers to the concentration for 50% of maximal effect, i.e., the concentration that elicits 50% of the maximum effect.

[0112] As used herein, the term IC50 half maximal inhibitory concentration, refers to the concentration of a drug at which a biological process, or a component of that process (e.g., an enzyme, receptor, cell, etc.), is inhibited by 50% under defined conditions.

[0113] 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 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 L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy chain / light chain pair (V H and V L) respectively form antigen binding sites. The assignment of amino acids to each region or domain follows the definitions of 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 limited by any particular method of producing the antibody. For example, it includes, inter alia, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be an antibody of different isotype, e.g., an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

[0114] 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. Monoclonal antibodies have 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 that generally recognize different epitopes on an antigen. Monoclonal antibodies can generally be obtained using the hybridoma technology first reported by Kohler et al. (Kohler, 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). 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).

[0115] 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 framework region (FR) amino acid residues of the recipient antibody can be replaced by corresponding amino acid residues of the non-human antibody, or by amino acid residues of other antibodies, to further refine or optimize antibody performance. For further details of 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.

[0116] 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.

[0117] 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.

[0118] 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 CH1, 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 IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0119] 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 instances, 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 to the polypeptide.

[0120] As used herein, the term "Fab fragment" consists of one light chain and CH1 and the variable region of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0121] As used herein, the term "Fc region" is an antibody fragment formed by disulfide bonds between the second, third constant regions of the first heavy chain and the second, third constant regions of the second heavy chain of an antibody.

[0122] As used herein, the term "Fab' fragment" contains one light chain and a portion of one heavy chain that contains the VH domain and CH1 domain and also a portion of the region between the CH1 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.

[0123] As used herein, the term "F(ab')2 fragment" contains two light chains and two heavy chains that contain a portion of the constant region between the CH1 and CH2 domains, so that an interchain disulfide bond forms between the two heavy chains. The F(ab')2 fragment thus consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0124] As used herein, the term "Fv region" comprises the variable regions from the heavy and light chains, but lacks the constant regions.

[0125] As used herein, the term "Fd" fragment means an antibody fragment consisting of the VH and CH1 domains (Ward et al., Nature 341 :544-546 (1989)).

[0126] As used herein, the term "dAb" fragment (Ward et al., Nature 341 :544-546 (1989)) consists of a VH domain.

[0127] As used herein, the term "isolated" or "isolated" refers to an artificially obtained state from the natural state. If a certain "isolated" substance or component appears in nature, it is possible that the natural environment has been changed, or the substance has been separated from the natural environment, or both. For example, a certain polynucleotide or polypeptide naturally present in a certain living animal is not isolated, but a high-purity same polynucleotide or polypeptide isolated from such a natural state is isolated. The term "isolated" or "isolated" does not exclude the mixing of artificial or synthetic substances, nor does it exclude the presence of other impurities that do not affect the activity of the substance.

[0128] As used herein, the term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When the vector enables the expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by the vector are expressed in the host cell. The vector is well known to those skilled in the art, including but not limited to: plasmid; phagemid; cosmid; artificial chromosome, such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. 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, papovaviruses (such as SV40). A vector can contain multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector can also contain a replication initiation site.

[0129] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as E. coli or Bacillus 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, etc.

[0130] 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 an antibody specific for) an antigen means that the antibody 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.

[0131] As used herein, the term "K D " refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which 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 (e.g., an IL-31 protein) with a dissociation equilibrium constant (K -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. K D determinations can be made using methods known to those skilled in the art, for example using a Fortebio Octet®molecular interaction instrument. D

[0132] As used herein, the terms "monoclonal antibody" and "monoclonal" are used interchangeably and have the same meaning; the terms "polyclonal antibody" and "polyclonal" are used interchangeably and have the same meaning; the terms "polypeptide" and "protein" are used interchangeably and have the same meaning. Also in the present application, amino acids are generally represented by their commonly known single and three letter codes. For example, alanine can be represented by A or Ala.

[0133] ​As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are compatible, in pharmacology and / or physiology, 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, ion 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; ion strength enhancers include, but are not limited to, sodium chloride.

[0134] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain 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.

[0135] Advantages of the Invention

[0136] The anti-IL-31 antibody or antigen-binding fragment thereof of the present invention achieves one or more of the following technical effects (1) to (4):

[0137] (1) capable of specifically binding to human IL-31 and cynomolgus monkey IL-31, and has high affinity;

[0138] (2) capable of effectively blocking the binding of human IL-31 and IL-31 receptor, especially IL-31RA;

[0139] (3) capable of reducing or eliminating the downstream STAT3 phosphorylation signal in cells;

[0140] (4) capable of significantly inhibiting the itching caused by IL-31 or IL-31 overexpression (e.g., higher than normal levels). BRIEF DESCRIPTION OF DRAWINGS

[0141] Figure 1 : Binding curve of anti-IL-31 monoclonal antibody to human IL-31 protein.

[0142] Figure 2 : Binding curve of anti-IL-31 monoclonal antibody to cynomolgus monkey IL-31 protein.

[0143] Figure 3Figure 6: Binding curves of anti-IL-31 monoclonal antibodies to human IL-31 protein after affinity maturation.

[0144] Figure 4 Figure 7: Binding curves of anti-IL-31 monoclonal antibodies to cynomolgus IL-31 protein after affinity maturation.

[0145] Figure 5 Figure 8: Binding curves of anti-IL-31 monoclonal antibodies to CHO-huIL-31RA cells of human IL-31 protein after affinity maturation.

[0146] Figure 6 Figure 9: Binding curves of anti-IL-31 monoclonal antibodies to human IL-31 induced phosphorylation signal of STAT3 in 293T-huIL-31RA / OSMRβ cells after affinity maturation.

[0147] Figure 7 Figure 10: Anti-IL-31 monoclonal antibodies after affinity maturation block human IL-31 induced CCL2 expression in BEAS-2B cells. Nemolizumab analogue is Nemolizumab. "+" represents the addition of the corresponding protein or antibody, and "-" represents the absence of the corresponding protein or antibody.

[0148] Figure 8 Figure 11: Anti-IL-31 monoclonal antibodies after affinity maturation in mice in vivo half-life curves.

[0149] Figure 9 Figure 12: Anti-IL-31 monoclonal antibodies after affinity maturation in human IL-31 induced mouse scratching model of the number of scratching times effect of efficacy chart.

[0150] Figures 10A to 10C Figure 13: Nemolizumab monoclonal antibody in human IL-31 induced mouse scratching model of the number of scratching times effect of efficacy chart.

[0151] Figures 10D to 10F Table 1: Part of the sequence involved in the present application

[0152] Table 1: Part of the sequence involved in the present application

[0153] Table 1: Part of the sequence involved in the present application

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162] Detailed Implementation

[0163] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0164] Example 1: Construction of antibody library

[0165] Five fully humanized mice (purchased from Alloy, USA) were immunized with an equal volume of 1 mg of human interleukin-31 (IL-31) (purchased from AcroBiosystems) as antigen and Freund's adjuvant. Immunization was performed weekly for a total of four weeks to stimulate B cells to express antigen-specific antibodies. After the four immunizations, the spleens of the mice were harvested, and total RNA was extracted using Trizol RNA extraction reagent (purchased from Invitrogen). Total cDNA from the fully humanized mice was obtained by reverse transcription using a cDNA synthesis kit (purchased from Invitrogen). The IgG1 sequence was amplified from the cDNA using a first-round PCR. The PCR products were subjected to agarose gel electrophoresis, and the target fragment at 400-500 bp was recovered by gel excision. A second-round sequence amplification was performed using the first-round PCR product as a template to add homologous arms to the heavy and light chain genes. Finally, the target fragment was recovered using a PCR purification kit (purchased from QIAGEN).

[0166] The linearized yeast display vector and the second-round PCR products were mixed and electroporated into *Saccharomyces cerevisiae* (purchased from ATCC) to construct a fully human anti-IL-31 antibody library from five animals. The library size was determined to be 3 × 10⁻⁶. 7 .

[0167] Example 2: Screening of anti-IL-31 monoclonal antibodies

[0168] Dissolve the human IL-31 protein (purchased from AcroBiosystems) in a proper volume of double distilled water, and mix with the biotin solution according to the product instruction of the biotin labeling kit (purchased from Thermo). Incubate at 4℃ for 2 hours. Remove the excess biotin by desalting column (purchased from Thermo). The pretreatment of the desalting column and the collection of the sample are both according to the product instruction.

[0169] The antibody library constructed in Example 1 was inoculated in SD-CAA amplification medium (1 L SD-CAA amplification medium containing 6.7 g YNB, 5 g tyrosine, 13.62 g Na2HPO4·12H2O, 7.44 g NaH2PO4 and 2% glucose), and cultured at 30℃, 225 rpm overnight. A proper amount of yeast cells were centrifuged at 3000 rpm for 5 minutes to remove the culture medium, and resuspended in SD-CAA induction medium. The concentration of the induced library was determined, and a proper amount of yeast cells were centrifuged to remove the culture medium. The yeast cells were resuspended in 50 mL PBS, and centrifuged to remove the supernatant. The yeast cells were resuspended in 10 mL PBS.

[0170] Add biotin-labeled human IL-31 protein (final concentration 100 nM), and incubate at room temperature for 30 minutes. Centrifuge to collect the yeast cells, and wash the yeast cells with 50 mL PBS for 3 times. Resuspend the yeast cells in 5 mL washing solution, and add 200 μL SA magnetic beads (purchased from American Tianyan). Incubate for 10 minutes by inversion. Wash the mixture of yeast cells and magnetic beads with PBS for 3 times, and add the mixture to the LS purification column (purchased from American Tianyan). Place the LS purification column on the magnetic stand, and wash with PBS to remove the non-specifically bound yeast cells. Take the purification column from the magnetic stand, and add PBS to elute the yeast cells. Centrifuge the eluted yeast cells, and transfer to the SD-CAA amplification medium for amplification.

[0171] 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 with high binding capacity to the IL-31 antigen.

[0172] The yeast liquid with high binding capacity to the human IL-31 antigen obtained by MACS and FACS enrichment was coated on the solid culture plate of the SD-CAA, and then the single clone was picked and cultured in the SD-CAA expansion medium at 30 °C, 225 rpm overnight. The expanded single clone was 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.

[0173] Example 3: Construction and expression purification of anti-IL-31 monoclonal antibodies

[0174] The nucleotide sequence of the heavy chain variable region gene sequence and the human IgG1 constant region (SEQ ID NO: 21) was connected, and a homologous recombinase (purchased from Vazyme) was used to construct into the EcoR I / Not I double enzyme linearized pCDNA3.1 vector; the nucleotide sequence of the light chain variable region gene sequence and the human kappa light chain constant region (SEQ ID NO: 22) was connected, and constructed into the EcoR I / Xhol I double enzyme linearized pCDNA3.1 vector, and the process was according to the product instruction. The homologous recombination product was transformed into Top10 competent cells, coated on ampicillin resistance plates, and cultured at 37 °C overnight. The single clone was sequenced and the plasmid was extracted.

[0175] The HEK293 cells were used for transfection 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 using a Protein A column. 10x column volume of 1x PBS was added to the column to equilibrate 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 (1 M Tris, pH 8.54), and 3-5 times column volume of elution buffer (0.1 M 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, heavy chain variable region, and light chain variable region of the antibody 106 are shown in Table 1.

[0176] Example 4: Determination of affinity of anti-IL-31 monoclonal antibodies

[0177] 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 100 nM 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).

[0178] Table 2: Affinity of anti-IL-31 monoclonal antibodies to human and cynomolgus IL-31 proteins

[0179]

[0180] 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.

[0181] Example 5: Determination of purity and thermal stability of anti-IL-31 monoclonal antibodies The purity of the anti-IL-31 monoclonal antibody was detected by HPLC. The HPLC method is as follows:

[0182] Mobile phase: 150 mM Na2HPO4·12H2O, pH 7.0.

[0183] 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 detection results are shown in Table 3.

[0184] The thermal stability of the antibody was detected by differential scanning calorimetry (DSC). The sample was concentrated and then diluted with PBS to 1 mg / mL; 5000x fluorescence developer Cypro Orange (purchased from Bio-Rad) was diluted 50 times with ultrapure water to obtain 100x fluorescence developer Sypro Orange. 50 μL of 1 mg / mL sample was added to 10 μL of 100x fluorescence developer Sypro Orange, 40 μL of ultrapure water, mixed well, and then 30 μL was added to a 96-well PCR plate, each sample was made in triplicate, and then placed in a PCR instrument. The temperature program was set as follows: 25 °C for 5 min, and then increased 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 in the "Melt Curve" graph was read, which was the Tm value of the sample. The specific results are shown in Table 3.

[0185] Table 3: Purity and thermal stability detection results of anti-IL-31 monoclonal antibodies

[0186] Antibody No. Monomer ratio (%) Tm (°C) 106 99.5 65.2

[0187] Example 6: Determination of binding activity of anti-IL-31 monoclonal antibodies to human IL-31 protein and cynomolgus IL-31 protein by ELISA method Figures 1 to 2

[0188] 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 three 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 100 nM was diluted by 3-fold gradient dilution for a total of 12 points, 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 ready for use. After the binding 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 three 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 three 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. The ELISA plate after the termination reaction was read at 450 nm within 5 minutes after the termination was completed, and the value was recorded for data processing.

[0189] The results are shown in Table 4 and Example 7: Blocking activity of anti-IL-31 monoclonal antibodies to binding of human IL-31 to IL-31RA overexpressed CHO cells

[0190] 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 better than that of the control molecule BMS-981164.

[0191] Figure 3

[0192] ​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 resuspended cells was added, and detected on a CytoFlex flow cytometer and calculated the corresponding mean fluorescence intensity (Mean Fluorescence Intensity, MFI).

[0193] The results are shown in Table 4 and Example 8: Affinity maturation of anti-IL-31 monoclonal antibodies

[0194] Table 4: Summary of anti-IL-31 monoclonal antibody binding blocking activity with human and cynomolgus IL-31

[0195]

[0196] 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.

[0197] Example 9: Affinity determination of affinity-matured anti-IL-31 monoclonal antibodies

[0198] ​Using antibody 106 as a candidate molecule, the heavy chain CDR region and the light chain CDR region were randomly mutated 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:

[0199] The heavy chain gene segment VH and the light chain gene segment VK of the synthesized antibody 106 were used as templates, and degenerate primers were designed for CDR2, CDR3 of VH and CDR3 of VK, respectively. In order to ensure that each amino acid can be mutated into any one 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.

[0200] The screening method of the affinity matured anti-IL-31 monoclonal antibody is the same as that in Example 2, and the yeast with the strongest binding to IL-31 antigen is obtained when using the BD Aria III instrument for sorting.

[0201] The construction and expression and purification method of the affinity matured anti-IL-31 monoclonal antibody is the same as that in Example 3.

[0202] The obtained affinity matured anti-IL-31 monoclonal antibodies are 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.

[0203] Example 10: Determination of purity and thermal stability of affinity-matured anti-IL-31 monoclonal antibodies

[0204] 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. 270-8). Briefly, the sensor was equilibrated in the analysis buffer for 30 minutes, and then the baseline was established by detecting for 60 seconds on line, and the purified antibody obtained as described above was loaded on the AHQ sensor on line. 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 shows better affinity.

[0205] Table 5: Affinity of affinity matured anti-IL-31 monoclonal antibodies to human IL-31

[0206]

[0207]

[0208] Antibody No.

[0209] The purity of the protein was determined by HPLC. The HPLC method was as follows:

[0210] Mobile phase: 150 mM Na2HPO4-12H2O, pH 7.0.

[0211] Chromatographic conditions: detection wavelength 280 nm, column temperature 25 °C, flow rate 0.35 ml / min, detection time 20 min, Zenix-CSEC-300 column (SEPAX 4.6 x 300 mm, 3 μm).

[0212] The results are shown in Table 6. The results show that the purity of all the antibodies is high.

[0213] Table 6: Purity determination results of the affinity matured anti-IL-31 monoclonal antibodies

[0214] Monomer ratio (%) Example 11: Binding activity of affinity-matured anti-IL-31 monoclonal antibodies to human IL-31 protein and cynomolgus IL-31 protein 106-1 97.70 106-2 98.10 106-4 98.60 106-9 98.40 106-12 99.10 106-13 98.20

[0215] The method for determining the thermal stability of the affinity matured IL-31 monoclonal antibodies was the same as in Example 5. The results are shown in Table 7. The results show that all the antibodies exhibited good thermal stability.

[0216] Table 7: Tm values of the affinity matured anti-IL-31 monoclonal antibodies

[0217]

[0218]

[0219] Figures 4 to 5 Example 12: Blocking activity of affinity-matured anti-IL-31 monoclonal antibodies to binding of human IL-31 to IL-31RA overexpressed CHO cells

[0220] The specific method was the same as in Example 6.

[0221] The results are shown in Table 8 and Figure 6 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.

[0222]

[0223] Example 13: Blocking activity of affinity-matured anti-IL-31 monoclonal antibodies to human IL-31-induced Figure 7

[0224] ​The affinity of the affinity matured anti-IL-31 monoclonal antibodies to human IL-31 was verified. The blocking activity of the affinity matured anti-IL-31 monoclonal antibodies on the binding of huIL-31 / huIL-31RA cells (CHO-huIL-31RA cells as in Example 7) was further detected by flow cytometry.

[0225] The method was the same as in Example 7.

[0226] The results are shown in Table 8 and Example 14: Blocking activity of affinity-matured anti-IL-31 monoclonal antibodies to IL-4 and IL-31-induced BEAS-2B cell CCL2 expression

[0227] The results showed that most of the affinity matured anti-IL-31 monoclonal antibodies could 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 were superior to the 106 molecules before affinity maturation.

[0228] Figure 8 STAT3 phosphorylation of 293T-huIL-31RA / OSMRβ cells

[0229] The blocking activity of the affinity matured anti-IL-31 monoclonal antibodies on the binding of human IL-31 to IL-31RA has been verified at the cellular level, and 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 by AgeI and NheI double enzyme digestion. The 293T-huIL-31RA cells were infected with the lentivirus supernatant packaged by 293T cells, and the transfected cells were selected by adding hygromycin (purchased from Invivogen) to obtain 293T cells stably overexpressing human IL-31RA / OSMRβ (293T-huIL-31RA / OSMRβ cells). When the cells were stimulated by IL-31, the downstream STAT3 phosphorylation signal of the receptor was activated. The inhibitory activity of the affinity matured anti-IL-31 monoclonal antibodies on the STAT3 phosphorylation of 293T-huIL-31RA / OSMRβ cells induced by human IL-31 was determined by flow cytometry.

[0230] Specifically, the purified antibodies were diluted with 1xPBS, 3-fold dilution from 400nM 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 30 minutes. The 293T-huIL-31RA / OSMRβ cells were removed, and the cell concentration was adjusted to 2.0x10 6 ​Cells were harvested by trypsinization and resuspended in ice 1x wash buffer at 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 96-well flow plate with cells above, 4°C incubated for 10 minutes. Ice 1x PBS washed once, 100 μL / well of 4% paraformaldehyde was added, 4°C incubated for 10 minutes. Ice 1x PBS washed once, 100 μL / well of ice methanol was added, 4°C incubated for 30 minutes. 200 μL / well of ice 1x wash buffer was added to wash twice, 100 μL / well of ice 1x wash buffer diluted pSTAT3 flow antibody (1:200) was added, 4°C incubated for 20 minutes. 200 μL / well of ice 1x wash buffer was added to wash 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.

[0231] The results are shown in Table 8 and Example 15: In vivo half-life of anti-IL-31 monoclonal antibodies in mice Table 8: Anti-IL-31 monoclonal antibodies binding blocking activity to human and cynomolgus IL-31

[0232] Table 8: Anti-IL-31 monoclonal antibodies binding blocking activity to human and cynomolgus IL-31

[0233]

[0234]

[0235] Figure 9 In vivo efficacy in C57 mice

[0236] 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 pathways was verified by detecting the expression of CCL2 in BEAS-2B cells by ELISA method.

[0237] Specifically, the BEAS-2B cells (ATCC) were trypsinized, the cells were resuspended with culture medium, and the cell density was adjusted to 3*10 5The cells were seeded in 96-well cell culture plates at a density of 50,000 cells / mL, 100 μL / well overnight. The cell plates were washed twice with PBS and then 100 μL / well of the antibody diluted in culture medium to 40 nM was added to the 96-well cell plates in triplicate. 100 μL / well of IL-4 and IL-31 diluted in culture medium to 4 ng / mL and 50 ng / mL, respectively, was added to the 96-well cell plates in triplicate. Blank and positive control wells were also set up. The plates were incubated at 37°C in a 5% CO2 incubator for 24 hours. The supernatant was collected and the CCL2 content was determined using a human CCL2 ELISA kit.

[0238] The results are shown in Table 2. Group The results show that the 106-1 molecule has an inhibitory effect on the expression of CCL2 in BEAS-2B cells that is comparable to that of the control monoclonal antibody Nemolizumab.

[0239] huIL-31 dose

[0240] 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 tail vein injection, blood sampling at different time points, and ELISA. Specifically, Balb / c mice, 9 males and 9 females, were used in the experiment, and were adjusted to 12 / 12 hours of light / dark, 24±2°C of temperature, 40-70% of humidity, and free water intake. On the experimental day, the Balb / c mice were injected with a single dose of 10 mg / kg of the monoclonal antibody molecule via tail vein.

[0241] Blood sampling time points: blood was collected from the mice's eye sockets at 5 minutes, 0.5 hours, 2 hours, 6 hours, 24 hours, 48 hours, 96 hours, 168 hours, 336 hours, and 504 hours after administration. The whole blood samples were placed at 2-8°C for 30 minutes, centrifuged at 12,000 rpm for 5 minutes to collect the serum, and then the serum was centrifuged again at 12,000 rpm for 5 minutes at 2-8°C, and stored at -80°C. The monoclonal antibody molecule content in the serum was determined by ELISA.

[0242] The results are shown in Table 2. Dose amount The results show that the half-life of the 106-1 molecule in mice is about 232 hours.

[0243] Example 16: Anti-IL-31 monoclonal antibody in IL-31 / IL-31RA / OSMRβ KI Dose frequency

[0244] A human IL-31-induced mouse pruritus model was constructed using B-hIL-31 / hIL-31RA / OSMRβ KI C57BL / 6 mice (human IL-31 / IL-31RA / OSMRβ knockin mice, purchased from Bao Saite Company) to evaluate the efficacy of the anti-IL-31 monoclonal antibody.

[0245] The specific steps are as follows:

[0246] 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 half an hour 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 sorted 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.

[0247] Table 9: Experimental scheme for inhibition of IL-31-mediated itching by 106-1

[0248] 1 μg / 50 μL 40 mg / kg Nemolizumab 1 μg / 50 μL 106-1 40 mg / kg Figures 10A to 10F 1 Figures 10A to 10F Figures 10A to 10C Figures 10D to 10F 1

[0249] The experimental results are shown in ​ .

[0250] As shown in ​ , compared with the baseline of subcutaneous injection of blank PBS, the scratching frequency of the mice in the model group subcutaneously injected with human IL-31 protein was significantly increased, indicating that the B-hIL-31 / hIL-31RA / OSMRβ KI C57BL / 6 mouse itching model induced by human IL-31 protein was successfully established.

[0251] As shown in ​ , compared with the model group, the scratching frequency of the mice in the 106-1 antibody administration group was significantly reduced within 3 hours after the injection of human IL-31 protein, and the itching improvement degree was comparable to that of the control antibody Nemolizumab (as shown in ​ ).

[0252] 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. Anti-IL-31 antibody or its antigen-binding fragment, wherein, The anti-IL-31 antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region includes complementarity-determining regions HCDR1 to HCDR3, and the light chain variable region includes complementarity-determining regions LCDR1 to LCDR3, wherein: The amino acid sequence of HCDR1 is shown in SEQ ID NO:1; The amino acid sequence of HCDR2 is shown in SEQ ID NO:2; The amino acid sequence of HCDR3 is shown in SEQ ID NO:3; The amino acid sequence of LCDR1 is shown in SEQ ID NO:5; The amino acid sequence of LCDR2 is shown in SEQ ID NO:6; and The amino acid sequence of LCDR3 is selected from 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.

2. The anti-IL-31 antibody or its antigen-binding fragment according to claim 1, wherein, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:4 or SEQ ID NO:9; and The amino acid sequence of the light chain variable region is selected from 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.

3. The anti-IL-31 antibody or its antigen-binding fragment according to any one of claims 1 to 2, wherein, The anti-IL-31 antibody is selected from (1) to (14) below: (1) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:4; and The amino acid sequence of the light chain variable region is shown in SEQ ID NO:8; (2) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:4; and The amino acid sequence of the light chain variable region is shown in SEQ ID NO:11; (3) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:4; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:13; (4) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:4; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:14; (5) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:4; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

16. (6) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:4; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:18; (7) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:4; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

20. (8) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:9; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8; (9) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:9; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:11; (10) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:9; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:13; (11) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:9; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:14; (12) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:9; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:16; (13) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:9; and The amino acid sequence of the light chain variable region is shown in SEQ ID NO:18; and (14) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:9; and The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:

20.

4. The anti-IL-31 antibody or its antigen-binding fragment according to any one of claims 1 to 3, wherein, The antibody includes a non-CDR region, and the non-CDR region is derived from a species other than rodents, such as from human antibodies; 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 its antigen-binding fragment according to any one of claims 1 to 4, wherein, The anti-IL-31 antibody is an IgG1, IgG2, IgG3, or IgG4 subtype; Preferably, the heavy chain constant region of the anti-IL-31 antibody is the human Ig gamma-1 chain C region or the human Ig ggamma-4 chain C region; the light chain constant region is the human Ig kappa chain C region. Optionally, the anti-IL-31 antibody is IgG1, and according to the EU numbering system, its heavy chain constant region contains L234A and L235A mutations; Preferably, the amino acid sequence of the heavy chain constant region of the anti-IL-31 antibody is as shown in SEQ ID NO:21 or SEQ ID NO:29, and / or the light chain constant region is as shown in SEQ ID NO:

22.

6. The anti-IL-31 antibody or its antigen-binding fragment according to any one of claims 1 to 5, wherein, The anti-IL-31 antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, humanized antibody or chimeric antibody.

7. The anti-IL-31 antibody or its antigen-binding fragment according to any one of claims 1 to 6, wherein, The anti-IL-31 antibody or its antigen-binding fragment binds to human IL-31 protein in 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; 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 This was measured using the ELISA method.

8. The anti-IL-31 antibody or its antigen-binding fragment according to any one of claims 1 to 7, wherein, The anti-IL-31 antibody or its antigen-binding fragment binds to the cynomolgus monkey IL-31 protein in 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; 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 This was measured using the ELISA method.

9. The anti-IL-31 antibody or its antigen-binding fragment according to any one of claims 1 to 8, wherein, The Tm value of the anti-IL-31 antibody or its antigen-binding fragment 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 determined by differential scanning calorimetry.

10. The anti-IL-31 antibody or its antigen-binding fragment according to any one of claims 1 to 9, wherein, The half-life of the anti-IL-31 antibody or its antigen-binding fragment in mice 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.

11. An isolated nucleic acid molecule encoding the anti-IL-31 antibody or its antigen-binding fragment as described in any one of claims 1 to 10.

12. A recombinant vector comprising the isolated nucleic acid molecule of claim 11.

13. A host cell comprising the isolated nucleic acid molecule of claim 11, or comprising the recombinant vector of claim 12.

14. Antibody-drug conjugates, comprising an antibody or its antigen-binding fragment and a small molecule drug, wherein, The antibody or its antigen-binding fragment is the anti-IL-31 antibody or its antigen-binding fragment as described in any one of claims 1 to 10; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is a tumor chemotherapy drug.

15. The antibody-drug conjugate according to claim 14, wherein, The antibody or its antigen-binding fragment is linked to a 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 its antigen-binding fragment to the small molecule drug is 1:(2-4).

16. A pharmaceutical composition comprising an effective amount of the anti-IL-31 antibody or its antigen-binding fragment as claimed in any one of claims 1 to 10, or the antibody-drug conjugate as claimed in any one of claims 14 to 15; optionally, the pharmaceutical composition further comprising one or more pharmaceutically acceptable excipients.

17. Use of the anti-IL-31 antibody or its antigen-binding fragment as described in any one of claims 1 to 10 in the preparation of a medicament for treating or preventing diseases caused by IL-31 levels higher than normal; Preferably, the disease is selected from one or more of the following: pruritus, chronic spontaneous urticaria, atopic dermatitis, nodular prurigo, contact dermatitis, eczema, bullous pemphigus, drug-induced delayed-type hypersensitivity reaction, alopecia areata, rosacea, rosacea, and psoriasis. Preferably, the itching is pruritus associated with refractory atopic dermatitis, virus-associated pruritus, or lymphoma-related neuropathic pruritus.

18. The anti-IL-31 antibody or its antigen-binding fragment according to any one of claims 1 to 10, for the treatment or prevention of diseases caused by IL-31 levels higher than normal; Preferably, the disease is selected from one or more of the following: pruritus, chronic spontaneous urticaria, atopic dermatitis, nodular prurigo, contact dermatitis, eczema, bullous pemphigus, drug-induced delayed-type hypersensitivity reaction, alopecia areata, rosacea, rosacea, and psoriasis. Preferably, the itching is pruritus associated with refractory atopic dermatitis, virus-associated pruritus, or lymphoma-related neuropathic pruritus.

19. A method for treating or preventing disease caused by IL-31 being above normal levels, comprising administering to a subject in need an effective amount of the anti-IL-31 antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 10; Preferably, the disease is selected from one or more of the following: pruritus, chronic spontaneous urticaria, atopic dermatitis, nodular prurigo, contact dermatitis, eczema, bullous pemphigus, drug-induced delayed-type hypersensitivity reaction, alopecia areata, rosacea, rosacea, and psoriasis. Preferably, the itching is pruritus associated with refractory atopic dermatitis, virus-associated pruritus, or lymphoma-related neuropathic pruritus.

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