Anti-IL-31 antibody and application thereof

Abstract

The invention discloses an anti-IL-31 antibody and an application thereof. The antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of which the amino acid sequences are respectively shown as SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34; the light chain variable region comprises an LCDR1, an LCDR2 and an LCDR3, wherein the amino acid sequences of the LCDR1, the LCDR2 and the LCDR3 are respectively shown as SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37. The antibody disclosed by the invention can be specifically combined with a cytokine IL-31, and block the combination of the IL-31 and a receptor IL-31RA, so that the activation of a downstream signal channel is further inhibited, and the inflammatory response can be effectively reduced. The pharmaceutical composition containing the anti-IL-31 antibody can be used for the treatment or prevention of atopic dermatitis, pruritus, prurigo nodularis and systemic sclerosis.

Description

[0001] This application claims the priority of Chinese patent application No. 202311289887.6 filed on September 28, 2023. This application cites the entire text of the above Chinese patent application. Technical Field

[0002] The present invention belongs to the field of antibodies, and specifically relates to an anti-IL-31 antibody and an application thereof. Background Art

[0003] IL-31 is a four-helix cytokine discovered by Dillon et al. in 2004. It is composed of 141 amino acids and belongs to the IL-6 cytokine family, but has low homology with the IL-6 cytokine family. It is mainly secreted by activated CD4+T lymphocytes, especially activated helper Th2 cells, mast cells, monocytes / macrophages and dendritic cells. In addition to IL-6 and IL-31, this family also includes IL-11, IL-27, oncostatin, leukemia inhibitory factor, ciliary neurotrophic factor and cardiotrophin-like factor. The main targets of IL-31 are located in the skin, lungs, intestines and nervous system. It is also expressed at low levels in the testis, bone marrow, thymus, spleen, kidney and skeletal muscle, and is widely involved in cell proliferation, differentiation and immune regulation activities.

[0004] The IL-31 functional receptor is a heterodimer complex composed of two subunits, IL-31RA and OSMR. The IL-31 receptor is mainly distributed in epithelial cells and lymphocytes. The expression of IL-31RA and OSMR can be detected in the dorsal ganglia. The same two receptors are also expressed in epidermal cells. IL-31 activates JAK-STAT, MAPK, PI3K / AKT and other signaling pathways to transmit signals by binding to the heterodimer complex composed of IL-31RA and OSMR. The biological role of IL-31 is not yet fully understood. Studies have shown that IL-31 may play an important role in atopic dermatitis, pruritus, etc.

[0005] Atopic dermatitis is a chronic inflammatory skin disease that is an immune response triggered by antigenic substances, irritants, and mechanical stimulation, usually associated with a personal or family history of type I allergies, allergic rhinitis, or asthma. Itch often causes patients to scratch their skin continuously, leading to sleep disorders and worsening of the disease. Itch seriously affects the quality of life of patients, and the degree of itching directly affects their psychosocial health. Some patients have itching symptoms. Although other symptoms are well controlled, and topical glucocorticoids and antihistamines have been approved for the treatment of itching, their role in atopic dermatitis is limited or has side effects when used for a long time. Therefore, in patients with atopic dermatitis, the main treatment goals should be to relieve itching, improve dermatitis, and improve quality of life. Treatments for atopic dermatitis, such as emollients, topical glucocorticoids, and calcineurin inhibitors have been approved by the U.S. Food and Drug Administration, but their efficacy is limited for patients with moderate to severe atopic dermatitis. Although oral antihistamines are often used for atopic dermatitis, such drugs have little antipruritic effect. Therefore, there is an urgent need for a treatment option with good safety and effectiveness. The mechanism of pruritus has not yet been fully elucidated, but there is a lot of evidence that the activation of cytokine receptors on peripheral nerve cells is closely related to pruritus.

[0006] IL-31 is a cytokine that connects the immune and nervous systems, and itching itself is the result of the dual effects of the immune and nervous systems. IL-31 plays an important role in atopic dermatitis and pruritus symptoms, and its main mechanism of action is as follows: Th2 cells are activated to produce IL-31, which acts on immune cells such as innate lymphocytes, DC cells, monocytes, and eosinophils, further secreting cytokines and chemokines to cause inflammation and promote the release of IL-31; IL-31 acts on keratinocytes, reduces the expression of filaggrin, and leads to skin barrier dysfunction; IL-31 acts on skin sensory neurons, promotes neuronal growth, is highly sensitive to itching, and releases BNP, leading to neuroinflammation; IL-31 acts on the central nervous system through blood circulation, directly causing itching. In general, the IL-31 / IL-31RA signaling axis is a potential therapeutic target, and blocking the IL-31 signaling pathway through neutralizing antibody drugs of IL-31 or IL-31RA may have certain application advantages.

[0007] Only two products targeting IL31 have entered clinical trials. Among them, IL31 monoclonal antibody BMS-981164 has not had any progress information or results disclosed after completing Phase I clinical trials in 2015; Nemolizumab, a humanized IgG2κ monoclonal antibody, competitively blocks the binding of the receptor to the ligand by binding to the IL-31 receptor α, inhibiting the biological activity of IL-31. The "first-in-class" IL-31 receptor α monoclonal antibody nemolizumab is used to treat nodular prurigo (prurigo nodularis, PN). Nemolizumab is used to treat pruritus associated with atopic dermatitis. Although there are marketed products targeting IL31, the existing products mainly target the pruritus associated with atopic dermatitis, and are insufficient in improving the overall symptoms of atopic dermatitis. They still need to be treated with combined glucocorticoids. There is a lack of new IL-31 antibody molecules with good pharmacodynamic effects to meet the urgent needs of patients with atopic dermatitis to improve their overall symptoms. Summary of the invention

[0008] The technical problem to be solved by the present invention is to overcome the lack of antibodies with good effects on IL-31 in the prior art, and to provide an antibody targeting IL-31 or an antigen-binding fragment thereof, which can specifically bind to human IL-31 and competitively inhibit the binding of IL-31 with its functional receptor, and is expected to be used for the treatment of atopic dermatitis, pruritus, nodular prurigo and systemic sclerosis.

[0009] The present invention solves the above technical problems through the following technical solutions:

[0010] The first aspect of the present invention provides an antibody targeting IL-31 or an antigen-binding fragment thereof, the antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37, respectively.

[0011] In some embodiments of the present invention, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; or,

[0012] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:7, SEQ ID NO:59 and SEQ ID NO:8, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively; or,

[0013] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:7, SEQ ID NO:2 and SEQ ID NO:12, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:13, SEQ ID NO:5 and SEQ ID NO:11, respectively.

[0014] In some specific embodiments of the present invention, the amino acid sequences of the framework sequences FR1, FR2, FR3 and FR4 contained in the heavy chain variable region are shown in SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41, respectively, and the amino acid sequences of the framework sequences FR1, FR2, FR3 and FR4 of the light chain variable region are shown in SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45, respectively.

[0015] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:26 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:26, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:27 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:27.

[0016] In other embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:28 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:28, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:29; or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:29.

[0017] In other embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:30 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:30, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:31 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:31.

[0018] In other embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:46 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:46, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:47 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:47.

[0019] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:18, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:18, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:19, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:19.

[0020] In other embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:18 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:18, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:50 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:50.

[0021] In other embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:18 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:18, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:51 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:51.

[0022] In other embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:18 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:18, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:52 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:52.

[0023] In other embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:48 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:48, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:19 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:19.

[0024] In other embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:48 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:48, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:50 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:50.

[0025] In other embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:48 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:48, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:51 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:51.

[0026] In other embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:48 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:48, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:52 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:52.

[0027] In other embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:49 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:49, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:19 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:19.

[0028] In other embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:49 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:49, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:50 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:50.

[0029] In other embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:49 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:49, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:51 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:51.

[0030] In other embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:49 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:49, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:52 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:52.

[0031] In other embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:14, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:14; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:15.

[0032] In other embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:16, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:16, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:17, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:17.

[0033] In the present invention, the sequence identity may be limited to the framework region of the heavy chain variable region and the light chain variable region. The antibody or antigen-binding fragment thereof of the present invention targets the antigen amino acid sequence, and the heavy chain variable region and the light chain variable region having at least 80% sequence identity with the framework region of the reference sequence of the heavy chain variable region and the light chain variable region corresponding to each CDR retain the binding activity to the antigen.

[0034] In the present invention, the antibody is a full-length antibody, Fab, Fab', F(ab')2, Fv or a multispecific antibody.

[0035] In some embodiments of the present invention, when the antibody is a full-length antibody, the heavy chain constant region of the full-length antibody is derived from the heavy chain of a human antibody or a variant thereof, and the light chain constant region of the full-length antibody is derived from the κ chain or λ chain of a human antibody or a variant thereof; when the antibody is Fv, the Fv is scFv.

[0036] In some specific embodiments of the present invention, when the antibody is a full-length antibody, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:53, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:54.

[0037] In other specific embodiments of the present invention, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:55, and the amino acid sequence of the light chain is shown in SEQ ID NO:56.

[0038] The second aspect of the present invention provides an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof as described in the first aspect.

[0039] The third aspect of the present invention provides a recombinant expression vector, which comprises the nucleic acid as described in the second aspect.

[0040] In some embodiments of the present invention, the recombinant expression vector is a plasmid, cosmid, phage or viral vector.

[0041] The fourth aspect of the present invention provides a transformant, which comprises the recombinant expression vector as described in the third aspect in a host cell.

[0042] In the present invention, the host cell is a prokaryotic cell or a eukaryotic cell.

[0043] In some embodiments of the present invention, the host cell is selected from yeast cells, mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments thereof.

[0044] In some specific embodiments of the present invention, the mammalian cell is a HEK293 cell or a CHO cell such as a CHO-K1 cell.

[0045] The fifth aspect of the present invention provides a method for preparing an antibody or an antigen-binding fragment thereof targeting IL-31, the method comprising culturing the transformant as described in the fourth aspect, and obtaining the antibody or an antigen-binding fragment thereof targeting IL-31 from the culture.

[0046] The sixth aspect of the present invention provides a pharmaceutical composition, comprising the antibody or antigen-binding fragment thereof as described in the first aspect, and a pharmaceutically acceptable carrier.

[0047] The seventh aspect of the present invention provides a kit, which comprises the antibody or antigen-binding fragment thereof as described in the first aspect, the nucleic acid as described in the second aspect, the recombinant expression vector as described in the third aspect, the transformant as described in the fourth aspect, or the pharmaceutical composition as described in the sixth aspect.

[0048] In some embodiments of the present invention, the kit further comprises a reagent for detecting the binding of the antibody or antigen-binding fragment thereof to IL-31.

[0049] The eighth aspect of the present invention provides an antibody or antigen-binding fragment thereof as described in the first aspect, the nucleic acid as described in the second aspect, the recombinant expression vector as described in the third aspect, the transformant as described in the fourth aspect, the pharmaceutical composition as described in the sixth aspect, or the kit as described in the seventh aspect for use in the preparation of a drug for diagnosing, preventing and / or treating atopic dermatitis, pruritus, nodular prurigo and systemic sclerosis.

[0050] In some embodiments of the present invention, the pruritus comprises chronic kidney disease-related pruritus.

[0051] A ninth aspect of the present invention provides a method for detecting IL-31 in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof as described in the first aspect, and determining whether the sample contains IL-31 based on the binding status of the antibody or antigen-binding fragment thereof with the sample.

[0052] In some preferred embodiments of the present invention, the method is for purposes other than disease diagnosis and / or treatment.

[0053] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0054] The reagents and raw materials used in the present invention are commercially available.

[0055] The positive and progressive effect of the present invention is that the above-mentioned humanized antibody sequence is constructed into an expression vector by conventional molecular biological methods, and a recombinant humanized anti-IL-31 antibody molecule is obtained by transient expression in CHO cells and one-step purification by Protein A. The antibody can specifically bind to the cytokine IL-31, and block the binding of IL-31 to the receptor IL-31RA, further inhibiting the activation of downstream signaling pathways, and can effectively reduce inflammatory responses. The drug combination containing the anti-IL-31 antibody can be used for the treatment or prevention of atopic dermatitis, pruritus, nodular prurigo, and systemic sclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 ELISA was used to detect the binding and blocking activity of mouse antibodies.

[0057] Figure 2 The activity of anti-IL-31 antibody in blocking the binding between IL-31 and IL-31RA was detected by ELISA.

[0058] Figure 3 FACS was used to detect the activity of anti-IL-31 antibodies in blocking the binding of human IL-31 and human IL-31RA receptor overexpressing cell lines.

[0059] Figure 4 FACS was used to detect the activity of anti-IL-31 antibodies in blocking the binding of monkey IL-31 to monkey IL-31RA receptor overexpressing cell lines.

[0060] Figure 5 The luciferase reporter gene method was used to detect the inhibition of human BaF3-IL31RA / OSMR cell activity by anti-IL-31 antibody.

[0061] Figure 6 The luciferase reporter gene method was used to detect the inhibition of monkey BaF3-IL31RA / OSMR cell activity by anti-IL-31 antibody.

[0062] Figure 7 The anti-IL-31 antibody inhibits IL-31-dependent BaF3-IL31RA / OSMR cell proliferation.

[0063] Figure 8 Anti-IL-31 antibody inhibits IL-31-induced STAT3 phosphorylation activity in U87MG cells.

[0064] Figure 9 The anti-IL-31 antibody inhibits the IL-6 release activity of HaCat cells induced by IL-31. DETAILED DESCRIPTION

[0065] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0066] In the present invention, antibodies can be prepared by immunizing Balb / c mice with purified recombinant human IL-31 protein. The amino acid sequence of human IL-31 is known, Uniprot number Q6EBC2, amino acids 1-23 (i.e., as underlined below) are signal peptides, and amino acids 24-164 are mature proteins.

[0067] >human IL-31 protein amino acid sequence

[0068] MASHSGPSTSVLFLFCCLGGWLA SHTLPVRLLRPSDDVQKIVEELQSLSKMLLKD VEEEKGVLVSQNYTLPCLSPDAQPPNNIHSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIF QDAPETNISVPTDTHECKRFILTISQQFSECMDLALKSLTSGAQQATT(SEQ ID NO:20)

[0069] Each mouse was subcutaneously injected with 50 μg of human IL-31 protein in Freund's complete adjuvant for the first immunization, and then subcutaneously injected with 25 μg of human IL-31 protein in Freund's incomplete adjuvant every two weeks for booster immunization. 7-10 days after the third booster immunization, the mice were bled and the serum was collected. The titer of immune serum binding to human IL-31 protein was detected by ELISA, and one or more mice with the best titer were selected and intraperitoneally injected with 50 μg of human IL-31 protein for shock immunization. Mouse spleen cells were collected 48-72 hours after shock immunization, and the mouse spleen cells were fused with mouse myeloma cells SP2 / 0 using the optimized PEG-mediated cell fusion protocol to obtain hybridoma cells. After the hybridoma cells were grown in a 96-well cell culture plate for 10 days, 500 ng / mL recombinant human IL-31 protein was used as a specific binding target by ELISA to screen positive hybridoma cell groups that could bind to human IL-31 protein. Next, the positive hybridoma cell groups were subjected to ELISA blocking detection to screen hybridoma groups that could block the binding of IL-31 to its receptor IL-31RA. The screened hybridoma groups were subjected to one or more rounds of subcloning by limiting dilution to obtain monoclonal hybridoma cells. The supernatant of the monoclonal hybridoma cells was confirmed by ELISA-based recombinant IL-31 protein binding and blocking activity, and the luciferase reporter gene activity of BaF3-IL31RA / OSMR cells was determined. The three clones with good activity were 20A7, 30F4 and 36C5, respectively. The hybridoma cells in the logarithmic growth phase were collected to extract RNA and reverse transcribed to obtain cDNA, which was amplified by PCR and sent to a sequencing company for sequencing to obtain the sequence of the mouse anti-IL-31 antibody.

[0070] The variable regions of the above-mentioned mouse antibodies can be used to make other forms of antigen-binding fragments, including but not limited to Fab, scFv and chimeric antibodies. Chimeric antibodies contain variable regions derived from mice and constant regions derived from humans, so that when administered to human subjects, the chimeric antibodies have a longer half-life and lower immunogenicity. The method for preparing chimeric antibodies is known in the art. The light and heavy chain variable regions of the above-mentioned mouse antibodies are connected to the light and heavy chain constant regions of human antibodies to form chimeric antibodies. The chimeric antibody corresponding to 20A7 is named 20A7-VHVL, and other antibodies are analogous. In order to further reduce immunogenicity, the CDR of the above-mentioned mouse antibody is transplanted into an arbitrarily selected human framework region to produce the desired humanized antibody. The method for making humanized antibodies is known in the art, and the specific scheme can be referred to Example 4. In brief, the human germline sequence was selected based on the homology between the variable regions of mouse antibodies and human antibodies, and the CDR region in the human germline sequence was replaced with the mouse CDR sequence. Then, based on the three-dimensional structure of the mouse antibody, in order to maintain the affinity and biological activity of the humanized antibody, key light and heavy chain amino acid residues were selected for back mutation, and the human amino acid residues were mutated into mouse amino acid residues, ultimately obtaining the humanized anti-IL-31 antibody sequence.

[0071] Example 1 Expression and purification of IL-31 recombinant protein

[0072] For mouse immunization and hybridoma screening, the sequences encoding human IL-31 (Uniprot No. Q6EBC2) and cynomolgus monkey IL-31 with His tag were connected to the pTT5 vector to construct expression plasmids. Expi293F cells with a viability of more than 95% were inoculated in expression medium (CD 293TGE Medium, Acrobiosystems, CM-1156-11) at a density of 0.8E6 / mL and placed in 6% CO2, 130rpm, 37°C for 24 hours. The mass ratio of 1mg / mL transfection reagent PEI and plasmid is 2:1. Taking the transfection of 200mL Expi293F cells as an example, 10mL of Opti-MEM and 200μg of plasmid were mixed and allowed to stand for 5min; another 10mL of Opti-MEM and 400μl PEI were mixed and allowed to stand for 5min. The plasmid mixture and PEI mixture were mixed and allowed to stand at room temperature for 15min. The plasmid and PEI mixture was slowly added to 200mL of Expi293F cells and cultured in a shaker at 6% CO2, 130rpm, and 37°C. 250mM sodium valproate solution was added at 0.8% of the transfection volume for 18-24h after transfection. On the 1st and 3rd days of transfection, 5% volume of feed medium (CD Feed XSupplement, Acrobiosystems, CF-1116-12) was added respectively. On the 5th day of transfection, the cell supernatant was collected, and the supernatant was filtered to remove impurities after high-speed centrifugation for purification. The nickel column was balanced with 20mM PB+0.5M NaCl solution and rinsed 10 times the column volume. The filtered supernatant sample was loaded onto the column and rinsed with 20mM PB + 0.5M NaCl solution containing 20mM imidazole until the A280 reading dropped to the baseline. The target protein was then eluted with 20mM PB + 0.5M NaCl solution containing 250mM imidazole. The elution peak was collected and replaced with PBS buffer. After SDS-PAGE identification, the samples were divided for use.

[0073] >Amino acid sequence of recombinant human IL-31 protein with His tag (huIL31-His) (underlined is mature protein) (SEQ ID NO: 20)

[0074] MASHSGPSTSVLFLFCCLGGWLA SHTLPVRLLRPSDDVQKIVEELQSLSKMLLKDVEEEKGVLVSQNYT LPCLSPDAQPPNNIHSPAAIRYLKTIRQLDNKSVIDEIIEHLDKLIFQDAPETNISVPTDTHECKRFILTISQQFSE CMDLALKSLTSGAQQATT HHHHHHHH

[0075] >Amino acid sequence of recombinant cynomolgus monkey IL-31 protein with His tag (cynoIL31-His) (mature protein underlined) (SEQ ID NO: 21)

[0076] MASHSGPATSVLFLLCCLGGWLTSHT LPVHFLQPSDIQKIVEELQSLSKMLLKDVKEDKGVLVSQNYTL PCLTPDAQPPNIIHSPAAIRYLKTIRQLDNKSVIDEIIEHLDKLIFQDAPETNISVPTDTHECKRFILTISQQFSEC MDLALKSLTSGAQQATT HHHHHHHH

[0077] Example 2 Construction of IL-31 receptor overexpressing stable cell line

[0078] 2.1 Construction of HEK293 cell lines overexpressing IL-31RA and OSMR

[0079] In order to screen antibodies that can block the binding of IL-31 and IL-31 receptor, a HEK293 cell line overexpressing IL-31RA and OSMR was constructed. The target gene was cloned into the HEK293 cell line by non-lipid transfection to form a stable high-expression cell line. The human full-length IL-31RA gene was cloned into the pCDNA3.1(+)-CMV-MCS-NEO vector to construct the pCDNA3.1(+)-CMV-MCS-NEO-huIL13RA plasmid, and the human full-length OSMR gene was cloned into the pCDNA3.1(+)-CMV-MCS-BSD vector to construct the pCDNA3.1(+)-CMV-MCS-BSD-huOSMR plasmid. Then, human IL31RA and OSMR were cloned into the HEK293 cell line by non-lipid transfection using fugene 6 (Promega, Madison, WI, USA), and selected and cultured for two weeks under 1 mg / mL geneticin + 20 μg / mL blasticidin selection pressure. Finally, the HEK293 monoclonal cell line that simultaneously highly expressed human IL31RA and OSMR was screened by flow cytometry. To perform cross-screening of different species, the same method was used to construct a HEK293 cell line overexpressing cynomolgus monkey IL31RA and OSMR. The sequence is as follows:

[0080] > Human IL-31RA protein amino acid sequence (underlined is the extracellular region) (SEQ ID NO: 22)

[0081] MMWTWALWMLPSLCKFSLA ALPAKPENISCVYYYRKNLTCWTSPGKETSYTQYTVKRTYAFGEKHDNCT TNSSTSENRASCSFFLPRITIPDNYTIEVEAENGDGVIKSMHMTYWRLENIAKTEPPKIFRVKPVLGIKRMIQIEWIK PELAPVSSDLKYTLRFRTVNSTSWMEVNFAKNRKDKNQTYNLTGLQPFTEYVIALRCAVKESKFWSDWSQEKMGMTE EEAPCGLELWRVLKPAEADGRRPVRLLWKKARGAPVLEKTLGYNIWYYPESNTNLTETMNTTNQQLELHLGGESFWV SMISYNSLGKSPVATLRIPAIQEKSFQCIEVMQACVAEDQLVVKWQSSALDVNTWMIEWFPDVDSEPTTLSWESVSQ ATNWTIQQDKLKPFWCYNISVYPMLHDKVGEPYSIQAYAKEGVPSEGPETKVENIGVKTVTITWKEIPKSERKGIIC NYTIFYQAEGGKGFSKTVNSSILQYGLESLKRKTSYIVQVMASTSAGGTNGTSINFKTLSFSVFEIILITSLIGGGLLILIILTVAYGLKKPNKLTHLCWPTVPNPAESSIATWHGDDFKDKLNLKESDDSVNTEDRILKPCSTPSDKLVIDKLVVNFGNVLQEIFTDEAR TGQENNLGGEKNGYVTCPFRPDCPLGKSFEELPVSPEIPPRKSQYLRSRMPEGTRPEAKEQLLFSGQSLVPDHLCEEGAPNPYLKNSVTAREFLVSEKLPEHTKGEV

[0082] > Human OSMR protein amino acid sequence (underlined extracellular region) (SEQ ID NO: 23)

[0083] MALFAVFQTTFFLTLLSLRTYQSEVLA ERLPLTPVSLKVSTNSTRQSLHLQWTVHNLPYHQELKMVFQI QISRIETSNVIWVGNYSTTVKWNQVLHWSWESELPLECATHFVRIKSLVDDAKFPEPNFWSNWSSWEEVSVQDSTGQ DILFVFPKDKLVEEGTNVTICYVSRNIQNNVSCYLEGKQIHGEQLDPHVTAFNLNSVPFIRNKGTNIYCEASQGNVS EGMKGIVLFVSKVLEEPKDFSCETEDFKTLHCTWDPGTDTALGWSKQPSQSYTLFESFSGEKKLCTHKNWCNWQITQ DSQETYNFTLIAENYLRKRSVNILFNLTHRVYLMNPFSVNFENVNATNAIMTWKVHSIRNNFTYLCQIELHGEGKMM QYNVSIKVNGEYFLSELEPATEYMARVRCADASHFWKWSEWSGQNFTTLEAAPSEAPDVWRIVSLEPGNHTVTLFWK PLSKLHANGKILFYNVVVENLDKPSSSELHSIPAPANSTKLILDRCSYQICVIANNSVGASPASVIVISADPENKEV EEERIAGTEGGFSLSWKPQPGDVIGYVVDWCDHTQDVLGDFQWKNVGPNTTSTVISTDAFRPGVRYDFRIYGLSTKR IACLLEKKTGYSQELAPSDNPHVLVDTLTSHSFTLSWKDYSTESQPGFIQGYHVYLKSKARQCHPRFEKAVLSDGSE CCKYKIDNPEEKALIVDNLKPESFYEFFITPFTSAGEGPSATFTKVTTPDEHSSM LIHILLPMVFCVLLIMVMCYLKSQWIKETCYPDIPDPYKSSILSLIKFKENPHLIIMNVSDCIPDAIEVVSKPEGTKIQFLGTRKSLTETELTKPNYLYLLPTEKNHSGPGPCICFENL TYNQAASDSGSCGHVPVSPKAPSMLGLMTSPENVLKALEKNYMNSLGEIPAGETSLNYVSQLASPMFGDKDSLPTNPVEAPHCSEYKMQMAVSLRLALPPPTENSSLSSITLLDPGEHYC

[0084] >Cynomolgus monkey IL-31RA protein amino acid sequence (underlined extracellular region) (SEQ ID NO: 24)

[0085] MMWTWALWMFPLLCKFGLA ALPAKPENISCVYYYRKNLTCTWSPGKETSYTQYTAKRTYAFGKKHDNCT TSSSTSENRASCSFFLPRITIPDNYTIEVEAENGDGVIKSDMTCWRLEDIAKTEPPEIFSVKPVLGIKRMIRIEWIK PELAPVSSDLKYALRFRTVNSTSWMEVNFAKNRKDTNQTYNLMGLQAFTEYVVALRCAVKESKFWSDWSQEKMGMTE EEAPCGLELWRVLKPTEVDGRRPVRLLWKKARGAPVLEKTLGYNIWYFPENNTNLTETVNTTNQQLELHLGGESYWV SMISYNSLGKSPVTTLRIPAIQEKSFRCIEVMQACLAEDQLVVKWQSSALDVNTWMIEWFPDMDSEHPTLSWESVSQ ATNWTIQQDKLKPFWCYNISVYPMLHDKVGEPYSIQAYAKEGIPSKGPETKVENIGVKTVTITWKEIPKSERKGIIC NYTIFYQAEGGKGFSKTVNSSILQYGLESLKRKTSYTVRVMASTSAGGINGTSINFKTLSFSVFEIILITSLIGGGLLILIILTVAYGLKKPNKLTHLCWPSVPNPAESSIATWRGDDFKDKLNLKESDDSVNTEDRILKPCSTPSDKLVIDKSVVNFGNVLQEMFTDEARTG QENNLGGEKNEYVTHPFRPDCPLGKSFEELPVSPEIPPRKSQYLRSRMPEGTCLEAEEQLLVSGQSLESLAPDHVWEAAAPNPYLKNSVTTREFLVSQKLPEHTKGEV

[0086] > Cynomolgus monkey OSMR protein amino acid sequence (underlined extracellular region) (SEQ ID NO: 25)

[0087] MALFVVFQTTFFLTLLSLRTYQSEVLA ERLPLTPVSLKVSTNSIHQSLHLQWTVHNLPYHQELKMVFQI QISRIETSNVVWVGNYSTTVKWNQVLHWSWESELPLECATHFVRIKSVIDDASFPEPNFWSNWSSWEEVSVQDYLGR GTLFVFPKDKLVEEGSNVTICYVSRNIQNNVSCYLEGKQIHGEQLDPHVTAFNLNSVPFIRNRGTNIYCEASQGNVS KGIEGIVLFVSKVLEEPKDFSCESQDFKTLHCTWDPGTDTALGWSKQPSQSYTLFESFSGEKKLCTHKNWCNWQITQ DSQEMYNFTLIAENYLRKRSVNILFNLTHRVYLMNPFSVNFENVNATNAIMTWKVHSMRNNFTYLCQIELHGEGKMM QYDVSINVNGEYFLSELEPATEYMARVRCADASHFWKWTEWSGQNFTTLEAAPSEAPDVWRSVNSEPGNHTVTLFWK PLSKLHANGKILFYNVVVENLDKPSRSELRSIPAPANSTKLILDRCSYQICVTANNSVGASPASIIVISADPENKEV EEERIAGTEGGFSLSWKPQPGDVIGYVVDWCDHPQDVLQWKNVGPNTTSTVISTDAFRPGVRYDFRIYGLSTKRIAC LLEKKTGYSQELAPSDNPHVLVDMLTSHSFTLSWKDYSTESQPGFIQGYHVYLKSKARQCHPRFQKAVLSDGSECCK YKIDNPEEKALIVDNLKPESFYEFFVTPFTSAGEGPNATFTKVTTPDEHSS MLIRILLPMVFCVLLIMIVCYLKSQWIKETCYPDIPDPYKSSILSLIKFKENPHLTIMNVSDCIPDAIEVVSKPEGTKIQLLGTRKSLTETELTKPNYLYLLPTEKNHSGPGPCICFENF TYNQAASDAGSCGHVPVPPKAPPSMLGLMTSPENVLKALEKNYMNSLGEVPAGETSLNYVSQLASPMSGDKDSLPTNPVEPPHCSEYKMQMAVPLRLALPPTENSSLSSITLLDPGEHYR

[0088] 2.2 Construction of BaF3 cells overexpressing IL-31RA and OSMR and reporter gene system

[0089] The target genes IL-31RA and OSMR were cloned into the mouse IL-3-dependent pro-B cell BaF3 cell line by lentiviral packaging to form a stable high-expression cell line. The full-length genes of human IL-31RA and human OSMR were constructed into pLVX-IRES-Puro and pLVX-IRES-Neo vectors, respectively, to obtain pLVX-IL31RA-IRES-Puro and pLVX-OSMR-IRES-Neo plasmids. Human IL-31RA and human OSMR genes were cloned into BaF3 cells by lentiviral transfection, and cultured under 10μg / mL puromycin and 1mg / mL G418 selection pressure for two to three weeks to obtain BaF3 cell lines overexpressing human IL-31RA and human OSMR. The monoclonal cell line was screened by limiting dilution and named BaF3-huIL31RA / OSMR. In the absence of mouse IL-3, the cell has the characteristics of human IL-31-dependent proliferation and can be used to evaluate the blocking ability of anti-IL-31 antibodies. In addition, the same lentiviral transfection method was used to construct the cynomolgus macaque IL-31-dependent overexpression cell line BaF3-cynoIL31RA / OSMR.

[0090] In order to screen out neutralizing antibodies with the function of blocking IL-31 and IL-31 receptor signaling pathways, a reporter gene system based on BaF3 overexpressing cells was constructed to evaluate neutralizing activity. The pGL4.47-luc2P-SIE-Hygro plasmid (Promega, E4041) was cloned into the BaF3 cell line by electroporation transfection, and selected and cultured for two to three weeks under 400μg / mL hygromycin B screening pressure to obtain a BaF3 cell line with high expression of SIE-Luc2P. A monoclonal cell line was obtained by the limiting dilution method, and a clone with the highest luciferase reaction and the lowest background was screened by the luciferase assay (see below), named BaF3-huIL31RA / OSMR / SIE. In addition, the BaF3-cynoIL31RA / OSMR / SIE cell line was obtained by the same construction method.

[0091] The luciferase reporter gene activity induced by recombinant IL-31 can be measured as follows: take enough BaF3-huIL31RA / OSMR / SIE or BaF3-cynoIL31RA / OSMR / SIE cells, wash them twice with RPMI1640+10% FBS medium without mouse IL-3, resuspend them, adjust the cell density to greater than 1E6 / mL, and culture them at 37°C, 5% CO2 overnight for 19-20h. BaF3 overexpressing cells not transfected with pGL4.47 plasmid are used as a control group and treated under the same conditions. Recombinant IL-31 protein is diluted to 1000ng / mL, 100ng / mL, 10ng / mL, 1ng / mL, 0.1ng / mL, 0.01ng / mL and 0.001ng / mL using RPMI1640+10% FBS medium. The next day, the cell density was adjusted to 4E5 / mL, and 50 μL of cells and 50 μL of gradient diluted recombinant IL-31 protein were added to a 96-well opaque white plate, and the plate was incubated for 5 hours at 37°C and 5% CO2. The Promega ONE-Glo Luciferase Assay System reagent was restored to room temperature in advance, 100 μL of the detection reagent was added to each well, incubated in the dark for 3 minutes, and the chemiluminescence value of the substrate was detected using a multifunctional microplate reader.

[0092] Example 3 Screening of anti-IL-31 monoclonal hybridoma cells

[0093] 3.1 Immunization of mice

[0094] Anti-human IL-31 monoclonal antibodies were produced by immunizing mice. Balb / c mice, female, 6-8 weeks old (purchased from Hubei Experimental Animal Research Center) were used in the experiment. The acclimatized mice were immunized with recombinant huIL31-His protein and adjuvant. For the first immunization, each mouse was subcutaneously injected with 50 μg of fusion protein plus Freund's complete adjuvant. Thereafter, booster immunizations were performed every two weeks, with each mouse being subcutaneously injected with 50 μg of fusion protein plus Freund's complete adjuvant. After the 4th to 5th immunization, mice with high antibody titers in serum and titers approaching a plateau were selected for shock immunization. The fusion protein without adjuvant was used as an immunogen, and each mouse was injected with 50 μg of fusion protein. After 3-4 days, the mice were killed to obtain spleen cells, which were fused with myeloma cells at a ratio of 5:1. The spleen lymphocytes and myeloma cells Sp2 / 0 were fused with an optimized PEG-mediated fusion method to obtain hybridoma cells. The hybridoma cells were plated into 96-well plates at a density of 1E5 cells / well / 200 μL of culture medium and cultured statically at 37° C. and 5% CO 2 .

[0095] 3.2 Hybridoma Screening

[0096] In the initial screening, positive clones were identified using an ELISA binding experiment for human IL-31. 100 μL / well of 500 ng / mL huIL31-His protein was coated on an ELISA plate overnight. After blocking with BSA, 100 μL of hybridoma supernatant was added and the ELISA plate was incubated in a 37°C incubator for 1 hour. Thereafter, the plate was washed, incubated with mouse IgG secondary antibody, and color was developed according to the conventional ELISA operating procedures. Finally, the absorbance at 450 nm was read using an ELISA reader. The top 10% clones with the highest OD450 were selected for ELISA blocking rescreening. The blocking screening method is briefly summarized as follows: 5 μg / mL huIL31RA-Fc (Acrobiosystems, ILA-H5256) was coated, and after blocking, 50 μL hybridoma supernatant and 50 μL 200 ng / mL huIL31-His were added in sequence. The secondary antibody was HRP-labeled His tag antibody. The OD450 measured by the hybridoma sample was recorded as A, and the OD450 value measured by the blank control without huIL31-His was recorded as B. The OD450 value measured by adding negative hybridoma supernatant and huIL31-His at the same time was recorded as C. The blocking rate (%) of each hybridoma supernatant sample well was calculated as (CA) / (CB)×100%. After repeated screening, positive clones with a cut rate greater than 90% were obtained, and two rounds of subcloning were performed using the limiting dilution method to finally obtain monoclonal hybridoma cells. The culture supernatant of the monoclonal hybridoma cells was subjected to the recombinant human monkey IL-31 protein ELISA binding and the human monkey BaF3-IL31RA / OSMR cell luciferase reporter gene activity detection, and finally the preferred monoclonal hybridoma cells with good activity were obtained. The hybridoma cells in the logarithmic growth phase were collected separately, and RNA was extracted with NucleoZol (MN) and reverse transcribed (PrimeScript TM Reverse Transcriptase, Takara, cat#2680A). The cDNA obtained by reverse transcription was PCR amplified using mouse Ig-Primer Set (Novagen, TB326Rev.B 0503) and then sent to a sequencing company for sequencing. The amino acid sequence of the variable region of the mouse antibody obtained by sequencing is as follows:

[0097] >20A7 murine heavy chain variable region amino acid sequence (20A7-VH) (SEQ ID NO: 26)

[0098] EVKVVESGGGLVKPGGSLKLSCVASGFTFSNSDMSWVRQTPEKRLEWVASSSRGGRTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDSAIYYCARGYYGGFFDVWGAGTTVTVSS

[0099] >20A7 murine light chain variable region amino acid sequence (20A7-VL) (SEQ ID NO: 27)

[0100] SIVMTQTPKFLLVSTGDRVTITCKASQSVSDDVAWYQQKPGQSPKLLIYYASNRYT GVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDFSSPPWTFGGGTKLEIK

[0101] >30F4 murine heavy chain variable region amino acid sequence (30F4-VH) (SEQ ID NO: 28)

[0102] EVKLVESGGDLVKSGGSLKVSCVASGFTFSSSDMSWVRQTPEKRLEWVASSSRAGRTYYPDSVKGRFTISRDNARNVLYLQMNSLRSDDTAMYYCARNLYGGFFDVWGAGTTVTVSS

[0103] >30F4 murine light chain variable region amino acid sequence (30F4-VL) (SEQ ID NO: 29)

[0104] SIVMTQTPKFLLVSPGDRVTITCKASQSVNDVVAWYQQKPGQSPRLLIYYASSRYA GVPDRFTGSGYGTDFTFTISTVQAEDLAIYFCHQDYSSPPWTFGGGTKLEIK

[0105] >36C5 murine heavy chain variable region amino acid sequence (36C5-VH) (SEQ ID NO: 30)

[0106] EVKLVESGGGLVKPGGSLKLSCVASGFTFSSSDMSWVRQTPEKRLEWVASSSRGGRTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAIYYCARIYYGGFFDVWGAGTAVTVSS

[0107] >36C5 murine light chain variable region amino acid sequence (36C5-VL) (SEQ ID NO: 31)

[0108] SIVMTQTPKFLLVSTGDRVTITCKASQSVSDDVAWYQQKPGQSPKLLIYYASNRYT GVPDRFTGSGYGTDFTFTITTVQAEDLALYFCHQDYSSPPWTFGGGTKLEIK

[0109] The amino acid sequences of the CDR regions of the anti-IL-31 antibodies are shown in Table 2, and specifically in Table 1.

[0110] Table 1 Anti-IL-31 antibody CDR region amino acid sequence

[0111]

[0112] Table 2 Anti-IL-31 antibody CDR region amino acid sequence

[0113]

[0114] X1=N / S; X2=G / N / I; X3=Y / L; X4=K / R; X5=N / S;

[0115] X 10 =D / V.

[0116] The variable regions of anti-IL-31 antibodies are shown in Table 3.

[0117] Table 3 Anti-IL-31 antibody variable regions

[0118]

[0119]

[0120] 3.3 Mouse antibody activity detection

[0121] The three hybridoma cells 20A7, 30F4 and 36C5 were expanded and cultured at 37°C, 5% CO2 and acclimated to serum-free medium. The culture supernatant was collected and purified using Protein A / G filler to obtain pure mouse antibodies labeled as 20A7-mIgG, 30F4-mIgG and 36C5-mIgG. The three mouse antibodies were tested for IL-31 binding and ligand blocking activity. The results are as follows Figure 1 As shown, the activities of the three mouse antibodies were comparable, and 36C5 antibody was preferred for subsequent humanization.

[0122] Example 4 Preparation of 36C5 humanized antibody

[0123] 4.1 Selected framework sequence

[0124] The homology of the heavy chain and light chain variable regions of the mouse 36C5 antibody with the human germline gene sequence (according to the Kabat numbering system) was compared respectively, and the germline sequence with the highest homology was selected as the template sequence. The framework sequence (Framework, FR) selected for the 36C5 antibody is shown in Table 4.

[0125] Table 4 Framework sequence of 36C5 antibody

[0126]

[0127] The CDR region of 36C5 was transplanted onto the template sequence to form a CDR-transplanted antibody, labeled 36C5-H0L0, with the following amino acid sequence, where H0 is the heavy chain variable region sequence, L0 is the light chain variable region sequence, and so on.

[0128] >36C5 CDR-grafted antibody heavy chain variable region amino acid sequence (36C5-H0)

[0129] EVQLVESGGGLVKPGGSLRLSCAASGFTFSSSDMSWVRQAPGKGLEWVSSSSRG GRTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARIYYGGFFDVWGQGT MVTVSS(SEQ ID NO:46)

[0130] >36C5 CDR-grafted antibody light chain variable region amino acid sequence (36C5-L0)

[0131] DIRMTQSPSSLSASTGDRVTITCRASQSVSDDVAWYQQKPGKAPKLLIYYASNRYT GVPSRFSGSGSGTDFTLTISCLQSEDFATYYCHQDYSSPPWTFGGGTKVEIK(SEQ ID NO:47)

[0132] 4.2 Humanized Antibody Design

[0133] Based on the structural comparison of the variable regions of 36C5-H0L0 and the murine parent antibody, the following back mutation sequences were designed as shown in Tables 5 and 6. The heavy chains and light chains were combined in 3×4 combinations to obtain variable regions of a total of 12 humanized antibodies such as H1L1 and H1L2. The variable regions were then combined with human IgG constant regions to obtain full-length humanized antibodies.

[0134] Table 5 Heavy chain reverse mutation sequence

[0135]

[0136] Table 6 Light chain reverse mutation sequence

[0137]

[0138] The human IgG heavy chain constant region can be selected from IgG1, IgG2 or IgG4 subtypes, and the light chain constant region can be selected from Kappa or lambda subtypes, or can be replaced with other constant regions known in the art. Exemplarily, the heavy chain constant region of 36C5 is selected from IgG4 S228P, and the light chain is selected from the Kappa chain constant region, and the constant region sequence is as follows.

[0139] > Human IgG4 S228P heavy chain constant region amino acid sequence

[0140] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:53)

[0141] > Human Kappa light chain constant region amino acid sequence

[0142] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:54)

[0143] 4.3 Expression and purification of humanized antibodies

[0144] The variable regions of the above different combinations were connected to the constant regions to obtain the full-length antibody sequence. The exemplary 36C5-H1L1 full-length antibody amino acid sequence is as follows. The sequence encoding the antibody was cloned into the pTT5 vector, and the cell expression was performed according to the scheme of Example 1. The recombinant antibody expression supernatant was collected and purified using a Protein A column. The column was rinsed with PBS buffer until the A280 reading dropped to the baseline, and then the sample was loaded. The target protein was eluted with 100mM glycine (pH3.0), and 1M Tris-HCl (pH8.0) was immediately added for neutralization. The collected purified antibody replaced the buffer with PBS, and the absorbance at 280nm was measured and the antibody concentration was calculated.

[0145] >36C5-H1L1 heavy chain amino acid sequence

[0146] EVQLVESGGGLVKPGGSLRLSCAASGFTFSSSDMSWVRQAPGKGLEWVASSSRGGRTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARIYYGGFFDVWGQGTM VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP CPAPEFLGPSVFLFPPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQID NO:55)

[0147] >36C5-H1L1 light chain amino acid sequence

[0148] DIRMTQSPSSLSASTGDRVTITCRASQSVSDDVAWYQQKPGQAPKLLIYYASNRYTGVPDRFSGSGYGTDFTLTISTLQSEDFATYYCHQDYSSPPWTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQID NO:56)

[0149] Example 5 Anti-IL-31 Antibody Affinity Determination

[0150] 5.1 IL-31 protein binding activity based on ELISA

[0151] The above humanized antibody was tested for binding activity with the antigen protein IL31 by ELISA, as follows: recombinant human IL-31-His protein or recombinant monkey IL-31-His protein was diluted to 0.5 μg / mL using PBS buffer at pH 7.4, coated on a 96-well ELISA plate (Costar, 42592) at a volume of 100 μL / well, and incubated overnight at 4°C. The next day, the liquid in the well was discarded, the plate was washed 3 times with PBST (300 μL / well), and then a blocking solution (PBST containing 3% BSA) was added at a volume of 300 μL / well, and blocked at 37°C for 2h. After blocking, the liquid in the wells was discarded, the plate was washed 3 times with PBST (300 μL / well), and then 100 μL of gradient diluted antibody sample (initial concentration was 10 μg / mL) was added, incubated at 37°C for 1 h, washed 3 times with PBST (300 μL / well), and then 1:5000 diluted HRP-labeled Goat Anti-human IgG (H+L) secondary antibody was added, incubated at 37°C for 1 h, washed 4 times with PBST (300 μL / well), and the color was developed and the absorbance at 450 nm was read on the microplate reader. The EC50 value of the binding of IL-31 antibody to human or monkey IL-31 protein was calculated. The results are shown in Table 7, showing that the antibodies disclosed in the present invention have good binding activity with human or monkey IL-31.

[0152] Table 7 Anti-IL-31 antibody affinity determination results

[0153] Antibody huIL31-His EC50 (nM) cynoIL31-His EC50 (nM) 36C5-H1L1 0.0489 0.0360 36C5-H1L2 0.0646 0.0382 36C5-H1L3 0.0444 0.0282 36C5-H1L4 0.1040 0.0713 36C5-H2L1 0.0749 0.0446 36C5-H2L2 0.0615 0.0403 36C5-H2L3 0.0735 0.0527 36C5-H2L4 0.1073 0.0709 36C5-H3L1 0.0585 0.0423 36C5-H3L2 0.0802 0.0532 36C5-H3L3 0.0842 0.0439 36C5-H3L4 0.1310 0.0843 BMS-981164 0.0967 0.0669

[0154] The BMS-981164 control antibody is a positive control antibody, and its sequence is as follows:

[0155] >BMS-981164 heavy chain amino acid sequence

[0156] QVQLVQSGAEVKKPGASVKVSCKASGYTLTRYWMQWVRQAPGQGLEWMGAIY PGDGDTRYSQKFQGRVTLTADKSTSTAYMELSSLRSEDTAVYYCAFPDGYYAAPYGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:57)

[0157] >Amino acid sequence of the light chain of BMS-981164

[0158] DIQMTQSPSSLSASVGDRVTITCRASGNIHNYLAWYQQKPGKVPKLLIYNAKTLADGVPSRFSGSRSETQYSLTISSLQPEDVATYYCQHFWSTPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO:58)

[0159] 5.2 Determination of antibody affinity constant

[0160] The affinity constants of the two humanized antibodies 36C5-H1L1 and 36C5-H1L2 for IL-31 were determined by surface plasmon resonance (SPR) using a Protein A chip (Cytiva, Cat. No.: 29127555) in conjunction with a Biacore 8K device. The antibody was injected at a flow rate of 10 μL / min and flowed through the instrument for 12 seconds. Different concentrations of human or monkey recombinant IL-31 protein were injected at a flow rate of 30 μL / min for 120 seconds and then the dissociation was monitored for 360 seconds. Finally, glycine (pH 1.7) was used to flow through the chip at a flow rate of 30 μL / min for 30 seconds for regeneration. The kinetic parameters were calculated using the 1:1 Binding model of the Bia-evaluation analysis software, and the affinity constant was expressed as KD, which was calculated from the kd / ka (dissociation rate / binding rate) ratio. The affinity determination results of the two antibodies 36C5-H1L1 and 36C5-H1L2 are shown in Table 8. Both antibodies have strong affinity to human or monkey IL-31 antigen.

[0161] Table 8 Affinity determination results of 36C5-H1L1 and 36C5-H1L2

[0162]

[0163] Example 6 Determination of the activity of anti-IL-31 antibodies in blocking the binding of IL-31 and IL-31RA

[0164] 6.1 ELISA-based blocking activity

[0165] The ELISA method was used to detect the activity of anti-IL-31 antibodies in blocking the binding of IL-31 to its receptor IL-31RA, as follows: recombinant human huIL31RA-Fc (Acrobiosystems, ILA-H5256) protein was diluted to 5 μg / mL with PBS buffer at pH 7.4, and coated on a 96-well ELISA plate (Costar, 42592) at a volume of 100 μL / well, and incubated overnight at 4°C. The next day, the liquid in the wells was discarded, and the plate was washed 3 times with PBST (300 μL / well), and then a blocking solution (PBST containing 3% BSA) was added at a volume of 300 μL / well, and blocked at 37°C for 2 hours. After blocking, the liquid in the wells was discarded, and the plate was washed three times with PBST (300 μL / well). HuIL31-His was diluted to 200 ng / mL in advance, and 50 μL of gradient diluted antibody sample (starting concentration was 20 μg / mL) and 50 μL of huIL31-His were added to each well in turn. The plates were incubated at 37°C for 1 hour. After washing three times with PBST (300 μL / well), a 1:5000 diluted HRP-labeled His tag secondary antibody was added. The plates were incubated at 37°C for 1 hour. After washing four times with PBST (300 μL / well), the plate was colored and the absorbance at 450 nm was read on an enzyme reader. The IC50 value of IL-31 antibody blocking the binding of human IL-31 to its receptor IL-31RA was calculated. The results are shown in Figure 2 shown.

[0166] 6.2 FACS-based blocking activity

[0167] The FACS method was further used to detect the activity of anti-IL-31 antibodies in blocking the binding of recombinant IL-31 protein to HEK293 cells overexpressing IL-31RA and OSMR. HEK293-huIL31RA / OSMR cells or HEK293cynoIL31RA / OSMR cells were trypsinized and collected, and the cells were resuspended in PBS + 2% FBS washing solution to adjust the cell density to 2E6 / mL, with about 1E5 cells per well. The antibody sample was diluted to 400μg / mL (final concentration 100μg / mL) with washing solution, and diluted 3 times;

[0168] The ligand huIL31-His was diluted to 3 μg / mL (final concentration 750 ng / mL) or cynoIL31-His was diluted to 2 μg / mL (final concentration 500 ng / mL). 50 μL of cells, 25 μL of antibody sample, and 25 μL of ligand were added to each well in turn, and incubated at 4°C for 30 min; the 96-well plate was centrifuged at 1000 rpm for 5 min, washed once with PBS + 2% FBS, and 100 μL of 1:100 diluted PE-Anti-His flow cytometry antibody was added to each well, the cells were resuspended and mixed, and incubated at 4°C for 30 min. After the incubation, the 96-well plate was centrifuged at 1000 rpm for 5 min, washed once with PBS + 2% FBS, and the cells were resuspended in 100 μL PBS + 2% FBS washing solution in each well. The signal value of the PE channel was detected on the flow cytometer, and the IC50 value was calculated, as shown in Table 9. According to the test results, the two antibodies have blocking activity equivalent to that of the control antibody. The results are shown in Table 9. Figure 3 and Figure 4 shown.

[0169] Table 9 Results of the activity assay of anti-IL-31 antibodies blocking the binding of IL-31 and IL-31RA

[0170] Antibody Human IC50(nM) Monkey IC50(nM) BMS-981164 7.753 5.517 36C5-H1L1 7.862 5.442 36C5-H1L2 7.509 4.538

[0171] Example 7 Anti-IL-31 antibody inhibits luciferase reporter gene activity in BaF3-IL31RA / OSMR cells

[0172] The biological activity of anti-IL-31 antibodies can be measured by the luciferase reporter gene system. A sufficient amount of BaF3-huIL31RA / OSMR / SIE cells were washed twice with RPMI1640+10% FBS and resuspended to adjust the cell density to greater than 1E6 / mL, and cultured overnight at 37°C and 5% CO2 for 19-20h; the antibody sample was diluted to 40μg / mL (final concentration 10μg / mL) and diluted 5 times; the ligand huIL31-His was diluted to 2ng / mL (final concentration 0.5ng / mL); the cell density was adjusted to 1E6 / mL the next day, 20μL cells, 10μL sample, and 10μL ligand were added to a 96-well opaque white plate in sequence, and incubated at 37°C and 5% CO2 for 5h. Take the Promega ONE-Glo Luciferase Assay System in advance, restore to room temperature, add 40 μL of detection reagent to each well, incubate in the dark for 3 minutes, detect the chemiluminescence value with a multifunctional microplate reader, and use GraphPad Prism software to draw a curve and calculate the IC50 value, as shown in Table 10. The results show that the activity of the two IL-31 antibodies is better than that of the control antibody. The same method can be used to measure the inhibition of the luciferase reporter gene activity of BaF3-cynoIL31RA / OSMR cells by IL-31 antibodies. The results are shown in Table 10. Figure 5 and Figure 6 As shown, the two antibodies had comparable or even superior reporter gene blocking activities to the control antibody.

[0173] Table 10 Results of anti-IL-31 antibody inhibiting luciferase reporter gene activity in BaF3-IL31RA / OSMR cells

[0174] Antibody Human IC50(nM) Monkey IC50(nM) BMS-981164 0.050 0.078 36C5-H1L1 0.033 0.044 36C5-H1L2 0.041 0.054

[0175] Example 8 Anti-IL-31 Antibodies Inhibit IL-31-Dependent BaF3-IL31RA / OSMR Cell Proliferation

[0176] In the absence of murine IL-3, BaF3 cells overexpressing IL31RA and OSMR proteins have the characteristics of IL-31-dependent proliferation, which can be used to evaluate the ability of IL-31 antibodies to inhibit cell proliferation. Collect BaF3-huIL31RA / OSMR cells in the logarithmic growth phase and centrifuge at 1000rpm for 5min, wash the cells twice with RPMI 1640+10% FBS medium, resuspend the cells and adjust the cell concentration to 4E5 / mL, and inoculate 50μL cells per well of a 96-well cell culture plate. The antibody to be tested is diluted to 20μg / mL with a medium containing 1ng / mL huIL-31. After 3-fold gradient dilution, 50μL of sample is added to each well, and the cell culture plate is cultured at 37°C and 5% CO2 for 48h. After the culture was completed, the cell culture plate was removed from the incubator and equilibrated at room temperature for 30 min. 100 μL of CellCounting-Lite reagent (Vazyme, DD1101) was added to each well and the cells were shaken and mixed at room temperature for 2-5 min to fully lyse the cells. The chemiluminescence value was detected by a multifunctional microplate reader, and the curve was drawn using GraphPad Prism software to calculate the IC50 value. Figure 7 As shown in Table 11, the two antibodies had superior cell proliferation inhibitory activity compared with the control antibody.

[0177] Table 11 Results of the inhibition of IL-31-dependent proliferation of BaF3-IL31RA / OSMR cells by anti-IL-31 antibodies

[0178] Antibody Cell proliferation IC50(nM) BMS-981164 1.051 36C5-H1L1 0.418 36C5-H1L2 0.335

[0179] Example 9 Anti-IL-31 antibody inhibits IL-31-induced STAT3 phosphorylation activity in U87MG cells

[0180] U87MG cells are a human malignant glioblastoma cell line. It has been identified that the cells highly express IL-31RA and OSMR proteins, and IL-31 stimulation can activate downstream signaling pathways, which can be used to evaluate the biological activity of anti-IL-31 antibodies. U87MG cells were inoculated in a 24-well cell culture plate, with 40,000 cells per well, and cultured at 37°C and 5% CO2 for 24 hours. The antibody to be tested was diluted to 200 μg / mL with MEM+10% FBS medium, 6-fold gradient dilution, a total of 8 dilutions, and the ligand IL-31 was diluted to 300 ng / mL with culture medium. After the culture was completed, the supernatant of the 24-well plate culture medium was discarded, and 100 μL of antibody sample and 100 μL of ligand were added to each well in turn, and incubated at 4°C for 30 minutes. Cell lysis buffer was added to lyse the cells on ice for 30 min, and the supernatant was collected by centrifugation at 13000 rpm and 4°C for 25 min. The STAT3 phosphorylation level in the supernatant was detected using the Phospho-Stat3 (Tyr705) ELISA kit (CST, 7300C). Figure 8 As shown, both antibodies had comparable STAT3 phosphorylation blocking activity to the control antibody BMS981164.

[0181] Example 10 Anti-IL-31 antibody inhibits IL-31-induced IL-6 release activity of HaCat cytokines

[0182] HaCat cells are a cell line of immortalized keratinocytes from adult humans. It has been identified that IL-31 can stimulate the production of IL-6 in HaCat under IFNγ conditions. Therefore, the biological activity of anti-IL-31 antibodies can also be tested by inhibiting the level of cytokine release in HaCat cells. HaCat cells (CCTCC) were inoculated in 96-well plates, with 30,000 cells per well, and cultured for 12 hours at 37°C and 5% CO2. 10ng / mL IFNγ was added for pretreatment and continued to be cultured for 24 hours. The antibody sample to be tested was diluted to 400μg / mL with DMEM+10% FBS culture medium, 10-fold gradient dilution, a total of 6 coefficients, and the ligand IL-31 was diluted to 3μg / mL with culture medium. Take out the cell culture plate, add 50μL of the antibody sample to be tested and 50μL of the ligand at each dilution to each well, and continue to culture for 24 hours. After the culture was completed, the cell culture supernatant was taken out and ELISA detection was performed using OptEIA Human IL-6 ELISA Kit II (BD, 550799). The concentration of IL-6 in the cell culture supernatant under each condition was calculated according to the standard curve. Fig. 9 As shown, the activity of 36C5-H1L1 antibody in inhibiting cytokine release was comparable to that of the control antibody BMS981164.

Claims

1. An anti-IL-31 antibody or an antigen-binding fragment thereof, characterized in that: The antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in amino acid sequences of SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in amino acid sequences of SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; or, The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:7, SEQ ID NO:59 and SEQ ID NO:8, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively; or, The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:7, SEQ ID NO:2 and SEQ ID NO:12, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:13, SEQ ID NO:5 and SEQ ID NO:11, respectively; Preferably, the amino acid sequences of the framework sequences FR1, FR2, FR3 and FR4 contained in the heavy chain variable region are shown as SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41, respectively, and the amino acid sequences of the framework sequences FR1, FR2, FR3 and FR4 contained in the light chain variable region are shown as SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:26, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:26; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:27, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:27; or The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:28, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO:28, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:29; or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO:29; or, The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:30, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:30; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:31, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:31; or The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:46, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:46; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:47, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:

47.

4. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:18, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:18, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:19, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:19; or The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:18, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:18; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:50, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:50; or The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:18, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:18; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:51, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:51; or The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:18, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:18; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:52, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:52; or The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:48, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:48, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:19, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:19; or The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:48, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:48; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:50, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:50; or The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:48, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:48; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:51, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:51; or The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:48, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:48; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:52, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:52; or The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:49, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO:49, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:19, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO:19; or The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:49, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:49; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:50, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:50; or The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:49, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:49; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:51, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:51; or The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:49, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:49; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:52, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:52; or The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:14, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:14; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:15; or The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:16, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:16; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:17, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:

17.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that: The antibody is a full-length antibody, Fab, Fab', F(ab')2, Fv or a multispecific antibody; Preferably, when the antibody is a full-length antibody, the heavy chain constant region of the full-length antibody is derived from the heavy chain of a human antibody or a variant thereof, and the light chain constant region of the full-length antibody is derived from the κ chain or λ chain of a human antibody or a variant thereof; When the antibody is an Fv, the Fv is a scFv; More preferably, when the antibody is a full-length antibody, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:53, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:54; Further more preferably, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:55, and the amino acid sequence of the light chain is shown in SEQ ID NO:

56.

6. An isolated nucleic acid, characterized in that The nucleic acid encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

7. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid according to claim 6; Preferably, the backbone of the recombinant expression vector is a plasmid, cosmid, phage or viral vector.

8. A transformant, characterized in that The transformant comprises the recombinant expression vector according to claim 7 in a host cell; Preferably, the host cell is a prokaryotic cell or a eukaryotic cell; More preferably, the host cell is selected from yeast cells, mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments thereof; the mammalian cells are, for example, HEK293 cells or CHO cells such as CHO-K1 cells.

9. A method for preparing an antibody or an antigen-binding fragment thereof targeting IL-31, characterized in that: The method comprises culturing the transformant according to claim 8, and obtaining an antibody or an antigen-binding fragment thereof targeting IL-31 from the culture.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, and a pharmaceutically acceptable carrier.

11. A kit, characterized in that: The kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the nucleic acid according to claim 6, the recombinant expression vector according to claim 7, the transformant according to claim 8 or the pharmaceutical composition according to claim 10; Preferably, the kit further comprises a reagent for detecting the binding of the antibody or antigen-binding fragment thereof to IL-31.

12. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the nucleic acid according to claim 6, the recombinant expression vector according to claim 7, the transformant according to claim 8, the pharmaceutical composition according to claim 10 or the kit according to claim 11 in the preparation of a drug for diagnosing, preventing and / or treating atopic dermatitis, pruritus, nodular prurigo and systemic sclerosis; Preferably, the pruritus comprises chronic kidney disease-related pruritus.

13. A method for detecting IL-31 in a sample, characterized in that contacting the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 with the sample, and determining whether the sample contains IL-31 based on the binding status of the antibody or antigen-binding fragment thereof with the sample; Preferably, the method is for non-disease diagnosis and / or treatment purposes.

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