Antibody or antigen binding fragment of targeting IL-31RA and application of antibody or antigen binding fragment

Abstract

The invention discloses an antibody targeting IL-31RA or an antigen binding fragment thereof and application of the antibody or the antigen binding fragment. The antibody or the antigen binding fragment thereof provided by the invention can be specifically bound with IL-31RA, and competitively inhibit a downstream signal channel mediated by a cytokine IL-31, so that inflammatory response can be effectively reduced, and a pharmaceutical composition containing the anti-IL-31RA antibody can be used for treating or preventing atopic dermatitis, pruritus, prurigo nodularis or systemic sclerosis.

Description

[0001] This application claims priority to Chinese patent application No. 202410175637.8, filed on February 7, 2024. This application incorporates the entirety of the aforementioned Chinese patent application. Technical Field

[0002] The present invention belongs to the field of biomedicine, and specifically relates to an antibody or antigen-binding fragment thereof targeting IL-31RA and applications thereof. Background Art

[0003] IL-31 is a tetrahelical cytokine discovered by Dillon et al. in 2004. It is composed of 141 amino acids and belongs to the IL-6 cytokine family. However, it has a low homology with the IL-6 cytokine family and is mainly produced by activated CD4 + Secreted by T lymphocytes, particularly 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. IL-31's primary targets 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.

[0004] The IL-31 functional receptor is a heterodimeric complex composed of two subunits, IL-31RA and OSMR. The IL-31 receptor is primarily distributed in epithelial cells and lymphocytes. IL-31RA and OSMR expression can be detected in the dorsal ganglion, and these two receptors are also expressed in epidermal cells. IL-31 binds to the heterodimeric complex of IL-31RA and OSMR, activating signaling pathways such as JAK-STAT, MAPK, and PI3K / AKT. While the biological role of IL-31 is not yet fully understood, studies suggest that IL-31 may play an important role in atopic dermatitis, pruritus, and other conditions.

[0005] Atopic dermatitis is a chronic inflammatory skin disease caused by an immune response to antigens, irritants, and mechanical stimulation, often associated with a personal or family history of type I allergies, allergic rhinitis, or asthma. Itch often leads to persistent scratching, sleep disturbances, and worsening of the disease. Itch severely impacts patients' quality of life, with the severity of the itch directly impacting their psychosocial well-being. Some patients experience pruritus. Although other symptoms are well controlled, and topical corticosteroids and antihistamines are approved for the treatment of pruritus, their effectiveness in atopic dermatitis is limited or associated with long-term side effects. Therefore, the primary goals of treatment for patients with atopic dermatitis should be to relieve pruritus, improve dermatitis, and enhance quality of life. Treatments for atopic dermatitis, such as emollients, topical corticosteroids, and calcineurin inhibitors, are approved by the US Food and Drug Administration, but their efficacy is limited for patients with moderate to severe atopic dermatitis. Although oral antihistamines are frequently used for atopic dermatitis, they have little antipruritic effect. Therefore, there is an urgent need for effective treatment options with a good safety profile. The mechanism of pruritus has not yet been fully elucidated, but there is considerable 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 pruritus itself is the result of the dual effects of the immune and nervous systems. IL-31 plays a crucial role in atopic dermatitis and pruritus. Its primary mechanisms of action are as follows: Th2 cells activate and produce IL-31, which acts on immune cells such as innate lymphocytes, dendritic cells, monocytes, and eosinophils, further secreting cytokines and chemokines that trigger inflammation and promote IL-31 release. IL-31 acts on keratinocytes, reducing filaggrin expression and leading to skin barrier dysfunction. IL-31 acts on skin sensory neurons, promoting neuronal growth and increasing sensitivity to itch. It also releases BNP, leading to neuroinflammation. IL-31 acts on the central nervous system through the bloodstream, directly causing pruritus. Overall, the IL-31 / IL-31RA signaling axis is a potential therapeutic target, and blocking the IL-31 signaling pathway with neutralizing antibodies against IL-31 or IL-31RA may offer certain advantages.

[0007] Only two products targeting IL-31 have entered clinical trials. BMS-981164, an IL-31 monoclonal antibody, completed Phase I clinical trials in 2015, but no progress has been reported, and no results have been disclosed. Nemolizumab, a humanized IgG2κ monoclonal antibody developed by Chugai Pharmaceutical in Japan, binds to IL-31RA, competitively blocking the binding of the receptor to its ligand, thereby inhibiting the biological activity of IL-31. In July 2016, Galderma obtained exclusive global development rights from Chugai Pharmaceutical, excluding Japan and Taiwan Province of China, for nemolizumab. In September 2016, Maruho acquired rights to develop and commercialize nemolizumab for dermatological diseases in Japan. In December 2019, the US FDA granted Breakthrough Therapy designation to nemolizumab, a first-in-class IL-31RA monoclonal antibody developed by the company, for the treatment of prurigo nodularis (PN). On March 28, 2022, Maruho announced that Nemolizumab had received marketing approval from the Pharmaceuticals and Medical Devices Agency (PMDA) of Japan for the treatment of pruritus associated with atopic dermatitis. While there are currently marketed products targeting the IL31 target, these products primarily target the pruritus associated with atopic dermatitis and are insufficient in improving overall atopic dermatitis symptoms. Combination therapy with glucocorticoids is still required, and unexplained adverse reactions such as exacerbation of atopic dermatitis have been reported in clinical practice. The present invention aims to develop a novel IL-31RA antibody molecule with improved pharmacodynamics and safety, addressing the urgent need for overall symptom improvement in patients with atopic dermatitis. 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-31RA in the prior art and provide an antibody or antigen-binding fragment thereof that targets IL-31RA, specifically binding to IL-31RA and competitively inhibiting the downstream signaling pathway mediated by the cytokine IL-31. The IL-31RA antibody or antigen-binding fragment thereof provided by the present invention can be used to treat atopic dermatitis, pruritus, prurigo nodularis, or systemic sclerosis.

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

[0010] In a first aspect, the present invention provides an antibody or antigen-binding fragment thereof targeting IL-31RA, wherein 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, wherein the amino acid sequence of HCDR1 is as shown in SEQ ID NO:10, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:11, SEQ ID NO:65, SEQ ID NO:69, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID NO:115, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:12, SEQ ID NO:66, SEQ ID NO:70, SEQ ID NO:73, or SEQ ID NO:80; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is as shown in SEQ ID NO:14, SEQ ID NO:67, SEQ ID NO:71, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID NO:115. NO:116, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:15, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:77 or SEQ ID NO:81, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:16, SEQ ID NO:68, SEQ ID NO:75, or SEQ ID NO:78; or,

[0011] The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively; or,

[0012] The amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region are shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region are shown in SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, respectively.

[0013] In some specific embodiments of the present invention, the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown as SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown as SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; or,

[0014] The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 115 and SEQ ID NO: 12, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 116, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; or,

[0015] The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 116, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; or,

[0016] The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 115 and SEQ ID NO: 12, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; or,

[0017] The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 116, SEQ ID NO: 15 and SEQ ID NO: 117, respectively; or,

[0018] The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 65 and SEQ ID NO: 66, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 67, SEQ ID NO: 15 and SEQ ID NO: 68, respectively; or,

[0019] The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 69 and SEQ ID NO: 70, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 71, SEQ ID NO: 15 and SEQ ID NO: 68, respectively; or,

[0020] The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 72 and SEQ ID NO: 73, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 67, SEQ ID NO: 74 and SEQ ID NO: 75, respectively; or,

[0021] The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 76 and SEQ ID NO: 70, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 71, SEQ ID NO: 77 and SEQ ID NO: 78, respectively; or,

[0022] The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 79 and SEQ ID NO: 80, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 71, SEQ ID NO: 81 and SEQ ID NO: 68, respectively; or,

[0023] The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 65 and SEQ ID NO: 66, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 67, SEQ ID NO: 77 and SEQ ID NO: 68, respectively; or,

[0024] The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 82 and SEQ ID NO: 66, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 83, SEQ ID NO: 15 and SEQ ID NO: 68, respectively; or,

[0025] The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 84 and SEQ ID NO: 73, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 67, SEQ ID NO: 77 and SEQ ID NO: 75, respectively; or,

[0026] The amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 85 and SEQ ID NO: 73, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region are shown in SEQ ID NO: 67, SEQ ID NO: 74 and SEQ ID NO: 75, respectively.

[0027] In some specific embodiments of the present invention, the antibody or antigen-binding fragment thereof comprises the following (i) or (ii):

[0028] (i) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13; or,

[0029] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 21; or

[0030] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 24, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28; or

[0031] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 42, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 43; or

[0032] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 46, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 53; or

[0033] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 46, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 54; or

[0034] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 46, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 55; or

[0035] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 47, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 53; or

[0036] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 47, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 54; or

[0037] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 47, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 55; or

[0038] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 48, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 53; or

[0039] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 48, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 54; or

[0040] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 48, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 55; or

[0041] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 49, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 53; or

[0042] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 49, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 54; or

[0043] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 49, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 55; or

[0044] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 50, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 54; or

[0045] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 50, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 56; or

[0046] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 50, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 57; or

[0047] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 50, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 58; or

[0048] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 50, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 128; or

[0049] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 51, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 54; or

[0050] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 51, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 56; or

[0051] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 51, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 57; or

[0052] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 52, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 54; or

[0053] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 52, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 56; or

[0054] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 52, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 57; or

[0055] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 130, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 129; or

[0056] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 110, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 111; or

[0057] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 112, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 113; or

[0058] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 112, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 114; or

[0059] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 86, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 87; or

[0060] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 88, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 89; or

[0061] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 90, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 91; or

[0062] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 118, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 119; or

[0063] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 120, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 121; or

[0064] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 86, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 122; or

[0065] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 123, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 124; or

[0066] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 125, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 126; or

[0067] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 127, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 91; or

[0068] (ii) the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:9, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:13; or

[0069] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 17, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 21; or

[0070] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 24, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 28; or

[0071] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 42, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 43; or

[0072] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 46, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 53; or

[0073] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 46, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 54; or

[0074] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:46, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:55; or

[0075] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 47, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 53; or

[0076] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 47, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 54; or

[0077] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 47, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 55; or

[0078] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 48, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 53; or

[0079] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 48, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 54; or

[0080] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 48, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 55; or

[0081] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 49, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 53; or

[0082] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 49, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 54; or

[0083] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 49, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 55; or

[0084] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:50, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:54; or

[0085] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:50, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:56; or

[0086] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:50, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:57; or

[0087] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:50, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:58; or

[0088] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:50, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:128; or

[0089] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:51, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:54; or

[0090] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:51, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:56; or

[0091] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:51, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:57; or

[0092] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:52, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:54; or

[0093] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:52, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:56; or

[0094] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:52, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:57; or

[0095] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:52, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:57; or

[0096] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 110, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 111; or

[0097] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 112, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 113; or

[0098] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 112, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 114; or

[0099] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 86, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 87; or

[0100] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 88, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 89; or

[0101] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 90, and the light chain variable region comprises an amino acid sequence that is at least 80%, 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% identical to SEQ ID NO: 91; or

[0102] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 118, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 119; or

[0103] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 120, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 121; or

[0104] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 86, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 122; or

[0105] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 123, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 124; or

[0106] the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 125, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 126; or

[0107] The heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 127, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 91.

[0108] In the present invention, the sequence identity may be limited to the framework regions of the heavy and light chain variable regions. The antibodies or antigen-binding fragments thereof of the present invention target antigen amino acid sequences, and the heavy and light chain variable regions that have at least 80% sequence identity with the framework regions of the reference sequences of the heavy and light chain variable regions corresponding to each CDR retain antigen-binding activity.

[0109] In the present invention, the antibody or antigen-binding fragment thereof satisfies one or more of the following conditions:

[0110] (1) The antibody is a full-length antibody, and the antigen-binding fragment is Fab, Fab', F(ab')2 or scFv;

[0111] (2) The antibody or antigen-binding fragment thereof is a monospecific antibody, a bispecific antibody, or a multispecific antibody;

[0112] (3) The antibody or antigen-binding fragment thereof is a monoclonal antibody or a polyclonal antibody;

[0113] (4) The antibody or antigen-binding fragment thereof is a mouse antibody or a humanized antibody;

[0114] In some embodiments of the present invention, when the antibody is a full-length antibody, the full-length antibody comprises a heavy chain constant region and a light chain constant region; the heavy chain constant region is selected from the heavy chain constant region of IgG, IgA, IgM, IgE and IgD, preferably the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4; the light chain constant region is a κ chain or a λ chain.

[0115] In some preferred embodiments of the present invention, the heavy chain constant region is IgG4; or, the heavy chain constant region is IgG1; the light chain constant region is a κ chain;

[0116] In some more preferred embodiments of the present invention, the amino acid sequence of the heavy chain constant region is preferably as shown in SEQ ID NO:44, SEQ ID NO:104 or SEQ ID NO:105; or, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108 or SEQ ID NO:109; the amino acid sequence of the light chain constant region is preferably as shown in SEQ ID NO:45.

[0117] In some embodiments of the present invention, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:59, 61, 63, 92, 94, 96, 98, 100 or 102, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO:60, 62, 64, 93, 95, 97, 99, 101 or 103.

[0118] In some specific embodiments of the present invention, the amino acid sequence of the heavy chain of the antibody is as shown in SEQ ID NO: 59, and the amino acid sequence of the light chain of the antibody is as shown in SEQ ID NO: 60; or

[0119] The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 61, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 62; or,

[0120] The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 63, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 64; or,

[0121] The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 92, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 93; or,

[0122] The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 94, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 95; or,

[0123] The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 96, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 97; or,

[0124] The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 98, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 99; or,

[0125] The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 100, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 101; or,

[0126] The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 102, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 103.

[0127] The second aspect of the present invention provides an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof according to the first aspect.

[0128] The third aspect of the present invention provides a recombinant expression vector comprising the nucleic acid as described in the second aspect.

[0129] In some embodiments of the present invention, the backbone of the recombinant expression vector is a plasmid, cosmid, phage or viral vector.

[0130] The fourth aspect of the present invention provides a transformant, which comprises the recombinant expression vector as described in the third aspect.

[0131] In the present invention, the host cell is a prokaryotic cell or a eukaryotic cell.

[0132] In some embodiments of the present invention, the host cell is selected from yeast cells, mammalian cells or other cells suitable for producing antibodies or antigen-binding fragments thereof.

[0133] In some specific embodiments of the present invention, the mammalian cells are, for example, SP2 / 0 cells, HEK293 cells, or CHO cells such as CHO-K1 cells.

[0134] The fifth aspect of the present invention provides a method for preparing an antibody or an antigen-binding fragment thereof targeting IL-31RA, comprising the steps of culturing the transformant according to the fourth aspect, and obtaining the antibody or antigen-binding fragment thereof targeting IL-31RA from the culture.

[0135] A sixth aspect of the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of the first aspects, and a pharmaceutically acceptable carrier.

[0136] The seventh aspect of the present invention provides a kit, comprising the antibody or antigen-binding fragment thereof according to the first aspect, the nucleic acid according to the second aspect, the recombinant expression vector according to the third aspect, the transformant according to the fourth aspect, and / or the pharmaceutical composition according to the sixth aspect;

[0137] 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 described in the first aspect to IL-31RA.

[0138] An eighth aspect of the present invention provides a method for detecting IL-31RA, comprising the following steps:

[0139] (1) contacting the sample with the antibody or antigen-binding fragment thereof according to the first aspect, or the kit according to the seventh aspect;

[0140] (2) quantitatively or qualitatively determining the IL-31RA in the sample based on the binding between the antibody or antigen-binding fragment thereof, or the kit according to the seventh aspect and the sample;

[0141] The method is not for diagnostic or therapeutic purposes.

[0142] The ninth aspect of the present invention provides use of the antibody or antigen-binding fragment thereof according to the first aspect, the nucleic acid according to the second aspect, the recombinant expression vector according to the third aspect, or the transformant according to the fourth aspect in preparing a reagent for detecting IL-31RA.

[0143] The tenth aspect of the present invention provides use of the antibody or antigen-binding fragment thereof according to the first aspect, the nucleic acid according to the second aspect, the recombinant expression vector according to the third aspect or the transformant according to the fourth aspect, the pharmaceutical composition according to the sixth aspect and / or the kit according to the seventh aspect in the preparation of a medicament for diagnosing, preventing and / or treating IL-31RA-related diseases or symptoms.

[0144] In some embodiments of the present invention, the IL-31RA-related disease is an inflammatory disease.

[0145] In some embodiments of the present invention, the inflammatory disease is atopic dermatitis, prurigo nodularis, or systemic sclerosis.

[0146] The eleventh aspect of the present invention provides use of the antibody or antigen-binding fragment thereof according to the first aspect, the nucleic acid according to the second aspect, the recombinant expression vector according to the third aspect or the transformant according to the fourth aspect, the pharmaceutical composition according to the sixth aspect, and / or the kit according to the seventh aspect in the preparation of a medicament for alleviating or eliminating pruritus symptoms;

[0147] In some embodiments of the present invention, the pruritus symptom is pruritus associated with chronic kidney disease.

[0148] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0149] The reagents and raw materials used in the present invention are commercially available.

[0150] The positive progress of the present invention is that the antibodies or antigen-binding fragments thereof provided by the present invention can specifically bind to IL-31RA and competitively inhibit the downstream signaling pathway mediated by the cytokine IL-31, thereby effectively reducing the inflammatory response. The drug combination containing the anti-IL-31RA antibody can be used for the treatment or prevention of atopic dermatitis, pruritus, nodular prurigo or systemic sclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0151] Figure 1A This is a reporter gene system activity test for a mouse antibody (2F6E12-mIgG).

[0152] Figure 1B This is a reporter gene system activity test for mouse antibodies (1A1C6-mIgG and 2G1G1-mIgG).

[0153] Figure 2A ELISA was used to detect the binding activity of humanized antibodies to IL-31RA.

[0154] Figure 2B ELISA was used to detect the binding activity of humanized antibodies to IL-31RA.

[0155] Figure 3A FACS was used to detect the binding activity of anti-IL-31RA antibodies to human IL-31RA-overexpressing cells.

[0156] Figure 3B FACS was used to detect the binding activity of anti-IL-31RA antibodies to cynomolgus monkey IL-31RA-overexpressing cells.

[0157] Figure 4A FACS was used to detect the activity of anti-IL-31RA antibodies in blocking the binding of IL-31 to human IL-31RA-overexpressing cells.

[0158] Figure 4B FACS was used to detect the activity of anti-IL-31RA antibodies in blocking the binding of IL-31 to cynomolgus monkey IL-31RA-overexpressing cells.

[0159] Figure 5A Luciferase reporter gene assay was used to detect the activity of anti-IL-31RA antibody inhibition reporter gene system (human).

[0160] Figure 5B The luciferase reporter gene method was used to detect the activity of anti-IL-31RA antibody inhibition reporter gene system (cynomolgus monkey).

[0161] Figure 6 The anti-IL-31RA antibody inhibits IL-31-dependent proliferation of BaF3-IL31RA / OSMR cells.

[0162] Figure 7 Anti-IL-31RA antibody inhibits IL-31-induced STAT3 phosphorylation activity in U87MG cells.

[0163] Figure 8 Anti-IL-31RA antibody inhibits IL-31-induced IL-6 release activity of HaCat cells.

[0164] Figure 9A In vitro binding activity assay of mutants.

[0165] Figure 9B In vitro blocking activity assay of mutants.

[0166] Figure 9C In vitro reporter gene activity (human) assay for mutants.

[0167] Figure 9D In vitro reporter gene activity assay for mutants (cynomolgus monkey).

[0168] Figure 9E To detect the in vitro proliferation inhibitory activity of the mutant.

[0169] Figure 9F To detect the inhibitory activity of mutants on STAT3 phosphorylation in vitro.

[0170] Figure 10A Anti-IL-31RA antibodies (1A1C6-H5L2 and 2G1G1-H1L1) do not block the signaling pathway of the IL-6 cytokine.

[0171] Figure 10B Anti-IL-31RA antibodies (1A1C6-H5L9, 1A1C6-H5L9-AM20, and 1A1C6-H5L9-AM22) do not block the signaling pathway of the IL-6 cytokine.

[0172] Figure 10C Anti-IL-31RA antibodies (1A1C6-H5L2 and 2G1G1-H1L1) do not block the signaling pathway of OSM cytokines.

[0173] Figure 10D Anti-IL-31RA antibodies (1A1C6-H5L9-AM20 and 1A1C6-H5L9-AM22) do not block the signaling pathway of OSM cytokines.

[0174] Figure 11 The neutralizing activity of anti-IL-31RA antibodies with different constant regions.

[0175] Figure 12A The neutralizing activity of anti-IL-31RA antibody (1A1C6-H5L9-AM20) under different treatment conditions.

[0176] Figure 12B The neutralizing activity of anti-IL-31RA antibody (1A1C6-H5L9-AM22) under different treatment conditions.

[0177] Figure 12C The neutralizing activity of anti-IL-31RA antibody (Nemolizumab) under different treatment conditions.

[0178] Figure 13A The binding epitope (IL-31RA 20-225) of anti-IL-31RA antibodies (1A1C6-H5L2 and 2G1G1-H1L1) was identified.

[0179] Figure 13B Identification of the binding epitope (IL-31RA 20-315) of the anti-IL-31RA antibody (2G1G1-H1L1).

[0180] Figure 13C Identification of the binding epitope (IL-31RA 20-416) of the anti-IL-31RA antibody (2G1G1-H1L1).

[0181] Figure 13D The binding epitope (extracellular region of IL-31RA) of anti-IL-31RA antibodies (1A1C6-H5L9 and 2G1G1-H1L1) was identified.

[0182] Figure 14A FACS competition binding of anti-IL-31RA (1A1C6-H5L9) antibody.

[0183] Figure 14B FACS competition binding of anti-IL-31RA antibody (2G1G1-H1L1).

[0184] Figure 15A The effect of anti-IL-31RA antibodies (1A1C6-H5L2 and 2G1G1-H1L1) on the number of scratching in mice (within 7 days of administration).

[0185] Figure 15B This is the effect of anti-IL-31RA antibodies (1A1C6-H5L2 and 2G1G1-H1L1) on the number of scratching in mice (within 3 hours after injection of IL-31 protein).

[0186] Figure 16 The effect of anti-IL-31RA antibodies (1A1C6-H5L2 and 2G1G1-H1L1) on the scratching latency of mice.

[0187] Figure 17 This is the skin damage caused by anti-IL-31RA antibodies (1A1C6-H5L2 and 2G1G1-H1L1).

[0188] Figure 18 The effects of anti-IL-31RA antibodies (1A1C6-H5L2 and 2G1G1-H1L1) in a dermatitis model.

[0189] Figure 19 The effect of anti-IL-31RA antibodies (1A1C6-H5L9-AM07 and 1A1C6-H5L9-AM22) on the frequency of itch scratching in crab-eating monkeys.

[0190] Figure 20 The effect of anti-IL-31RA antibodies (1A1C6-H5L9-AM07 and 1A1C6-H5L9-AM22) on the inhibition rate of itch-scratching behavior in crab-eating monkeys. DETAILED DESCRIPTION

[0191] the term

[0192] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0193] The term "antibody" as used herein refers to an immunoglobulin molecule, which is typically a tetramer composed of two identical heavy chains and two identical light chains interconnected by disulfide bonds. Based on the conservative differences in amino acid sequences, the heavy and light chains are divided into a variable region (V) located at the amino terminus and a constant region (C) located at the carboxyl terminus. Within the variable regions of the heavy and light chains, there are three local areas with a higher degree of variation in amino acid composition and arrangement order, which are key locations for antibody binding to antigen and are therefore also called complementary determining regions (CDRs). Herein, the three heavy chain complementary determining regions are referred to as HCDR1, HCDR2, and HCDR3, and the three light chain complementary determining regions are referred to as LCDR1, LCDR2, and LCDR3. The variable regions of one heavy chain and one light chain interact to form the antigen binding site (Fv). Antibodies can be divided into different classes based on the amino acid sequence of their heavy chain constant regions. There are five major types of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses, e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The subunit structures and three-dimensional conformations of the different classes of immunoglobulins are known in the art. The present invention is intended to include antibodies of any of the aforementioned classes or subclasses.

[0194] As used herein, the term "antibody" is also intended to encompass digested fragments or functional variants thereof, e.g., antibody fragments capable of binding to IL-31RA or a portion thereof, including but not limited to Fab (e.g., obtained by papain digestion of an antibody), F(ab')2 (e.g., obtained by pepsin digestion), Fv, or scFv (e.g., obtained by molecular biology techniques).

[0195] The term "monoclonal antibody" as used herein refers to an antibody that is homogeneous and directed only against a specific antigenic epitope. Compared to conventional polyclonal antibody preparations that typically include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody is directed against a single antigenic determinant on the antigen. The modifier "monoclonal" indicates the homogeneous character of the antibody and is not to be construed as requiring the antibody to be produced by any particular method. The monoclonal antibodies of the present invention are preferably produced by recombinant DNA methods or obtained by screening methods described elsewhere herein.

[0196] As used herein, the term "isolated nucleic acid" refers to a polynucleotide that does not exist naturally in nature, including polynucleotides isolated from nature (including within an organism) through biological techniques, as well as artificially synthesized polynucleotides. An isolated polynucleotide can be genomic DNA, cDNA, mRNA, or other synthetic RNA, or a combination thereof. Provided herein are multiple nucleotide sequences encoding the heavy and light chain variable regions of anti-human IL-31RA protein monoclonal antibodies. It should be noted that, based on the amino acid sequences of the heavy and light chain variable regions provided herein and codon degeneracy, one skilled in the art can design nucleotide sequences that are not identical to the nucleotide sequences provided above, but that encode the same amino acid sequence. These modified nucleotide sequences are also encompassed within the scope of the present invention.

[0197] When referring to polynucleotides, the term "vector" as used herein refers to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer nucleotide encoding information into a host cell. The term "expression vector" or "expression cassette" refers to a vector suitable for expressing a target gene (nucleotide sequence to be expressed) in a host cell, typically comprising the target gene, a promoter, and a terminator.

[0198] As used herein, the term "host cell" refers to a cell that has been or is capable of being transformed with a nucleic acid sequence to express a selected gene of interest. The term includes the progeny of a parent cell, regardless of whether the progeny is identical to the original parent cell in morphology or genetic composition, as long as the progeny harbors the selected gene of interest. Commonly used host cells include bacteria, yeast, and mammalian cells.

[0199] As used herein, the term "antigen-binding fragment" refers to the region of an antibody that binds to an antigen. The term encompasses various types of antibody fragments, such as Fab, Fab', F(ab')2, or scFv. Each fragment comprises at least the variable regions of the heavy and light immunoglobulin chains (VH and VL, respectively), typically held together by disulfide bonds to retain the antibody binding site.

[0200] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0201] Example 1 Expression and purification of IL-31RA recombinant protein

[0202] For mouse immunization and hybridoma screening, sequences encoding His-tagged human IL-31RA and cynomolgus macaque IL-31RA were ligated into the pTT5 vector to construct expression plasmids. Expi293F cells (ThermoFisher, A14527) with a viability exceeding 95% were seeded at a density of 0.8E6 / mL in expression medium and cultured for 24 hours at 6% CO2, 130 rpm, and 37°C. The mass ratio of 1 mg / mL transfection reagent PEI (Polysciences, 24765) to plasmid was 2:1. For transfection of 200 mL of Expi293F cells, 10 mL of Opti-MEM (Gibco, 11058021) was mixed with 200 μg of plasmid and allowed to stand for 5 minutes. Another 10 mL of Opti-MEM was mixed with 400 μl of PEI and allowed to stand for 5 minutes. Mix the plasmid and PEI mixtures thoroughly and let them stand at room temperature for 15 minutes. Slowly add the plasmid and PEI mixture to 200 mL of Expi293F cells and incubate in a shaker at 6% CO2, 130 rpm, and 37°C. 18-24 hours after transfection, add 250 mM sodium valproate solution at 0.8% of the transfection volume. On days 1 and 3 of transfection, supplement with 5% of the feed medium. On day 5 of transfection, collect the cell supernatant, centrifuge at high speed, and filter to remove impurities for purification. Equilibrate the nickel column with 20 mM PB + 0.5 M NaCl solution and rinse 10 column volumes. The filtered supernatant sample was loaded onto the column and rinsed with a 20 mM PB + 0.5 M NaCl solution containing 20 mM imidazole until the A280 reading dropped to the baseline. The target protein was then eluted with a 20 mM PB + 0.5 M NaCl solution containing 250 mM imidazole. The elution peak was collected and replaced with PBS buffer. After SDS-PAGE identification, the protein was divided into several groups for use.

[0203] >Amino acid sequence of recombinant human IL-31RA protein with His tag (huIL31RA-His; SEQ ID NO: 1) (mature protein is underlined)

[0204] MPLLLLLPLLWAGALAM ALPAKPENISCVYYYRKNLTCTSWSPGKETSYTQYTVKRTYAFGEKHDNCTTN SSSTSENRASCSFFLPRITIPDNYTIEVEAENGDGVIKSHMTYWRLENIAKTEPPKIFRVKPVLGIKRMIQIEWIKPE LAPVSSDLKYTLRFRTVNSTSWMEVNFAKNRKDKNQTYNLTGLQPFTEYVIALRCAVKESKFWSDWSQEKMGMTEEE APCGLELWRVLKPAEADGRRPVRLLWKKARGAPVLEKTLGYNIWYYPESNTNLTETMNTTNQQLELHLGGESFWVSM ISYNSLGKSPVATLRIPAIQEKSFQCIEVMQACVAEDQLVVKWQSSALDVNTWMIEWFPDVDSEPTTLSWESVSQAT NWTIQQDKLKPFWCYNISVYPMLHDKVGEPYSIQAYAKEGVPSEGPETKVENIGVKTVTITWKEIPKSERKGIICNY TIFYQAEGGKGFSKTVNSSILQYGLESLKRKTSYIVQVMASTSAGGTNGTSINFKTLSFSVFEGGGGSHHHHHHHHH H

[0205] >Amino acid sequence of recombinant cynomolgus monkey IL-31RA protein with His tag (cynoIL31RA-His; SEQ ID NO: 2) (mature protein is underlined)

[0206] METDTLLLWVLLLWVPGSTG ALPAKPENISCVYYYRKNLTCTWSPGKETSYTQYTAKRTYAFGKKHDNC TTSSSTSENRASCSFFLPRITIPDNYTIEVEAENGDGVIKSDMTCWRLEDIAKTEPPEIFSVKPVLGIKRMIRIEWI KPELAPVSSDLKYALRFRTVNSTSWMEVNFAKNRKDTNQTYNLMGLQAFTEYVVALRCAVKESKFWSDWSQEKMGMT EEEAPCGLELWRVLKPTEVDGRRPVRLLWKKARGAPVLEKTLGYNIWYFPENNTNLTETVNTTNQQLELHLGGESYW VSMISYNSLGKSPVTTLRIPAIQEKSFRCIEVMQACLAEDQLVVKWQSSALDVNTWMIEWFPDMDSEHPTLSWESVS QATNWTIQQDKLKPFWCYNISVYPMLHDKVGEPYSIQAYAKEGIPSKGPETKVENIGVKTVTITWKEIPKSERKGII CNYTIFYQAEGGKGFSKTVNSSILQYGLESLKRKTSYTVRVMASTSAGGINGTSINFKTLSFSVFEHHHHHHHH

[0207] Example 2 Construction of IL-31 receptor overexpressing stable cell line

[0208] 1. Construction of cell lines overexpressing IL-31RA and OSMR

[0209] To screen for antibodies that can block the binding of IL-31 to the IL-31 receptor, HEK293 or CHO-K1 cell lines overexpressing both IL-31RA and OSMR were constructed. The target genes were cloned into HEK293 or CHO-K1 cell lines using non-liposomal transfection to generate stable, high-expressing cell lines. The full-length human 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 full-length human OSMR gene was cloned into the pCDNA3.1(+)-CMV-MCS-BSD vector to construct the pCDNA3.1(+)-CMV-MCS-BSD-huOSMR plasmid. Human IL-31RA and OSMR were then cloned into HEK293 or CHO-K1 cells using non-liposomal transfection using Fugene 6 (Promega, Madison, WI, USA). Cells were selected for two weeks under 1 mg / mL geneticin and 20 μg / mL blasticidin. Finally, HEK293 or CHO-K1 cell lines expressing both human IL-31RA and OSMR were identified by flow cytometry. To perform cross-species screening, HEK293 or CHO-K1 cell lines overexpressing cynomolgus macaque IL31RA and OSMR were constructed using the same method. The sequences are as follows:

[0210] >Human IL-31RA protein amino acid sequence (SEQ ID NO: 3; underlined extracellular region)

[0211] MMWTWALWMLPSLCKFSLA ALPAKPENISCVYYYRKNLTCTWSPGKETSYTQYTVKRTYAFGEKHDNCT TNSSTSENRASCSFFLPRITIPDNYTIEVEAENGDGVIKSHMTYWRLENIAKTEPPKIFRVKPVLGIKRMIQIEWIK PELAPVSSDLKYTLRFRTVNSTSWMEVNFAKNRKDKNQTYNLTGLQPFTEYVIALRCAVKESKFWSDWSQEKMGMTE EEAPCGLELWRVLKPAEADGRRPVRLLWKKARGAPVLEKTLGYNIWYYPESNTNLTETMNTTNQQLELHLGGESFWV SMISYNSLGKSPVATLRIPAIQEKSFQCIEVMQACVAEDQLVVKWQSSALDVNTWMIEWFPDVDSEPTTLSWESVSQ ATNWTIQQDKLKPFWCYNISVYPMLHDKVGEPYSIQAYAKEGVPSEGPETKVENIGVKTVTITWKEIPKSERKGIIC NYTIFYQAEGGKGFSKTVNSSILQYGLESLKRKTSYIVQVMASTSAGGTNGTSINFKTLSFSVFE IILITSLIGGGLLILIILTVAYGLKKPNKLTHLCWPTVPNPAESSIATWHGDDFKDKLNLKESDDSVNTEDRILKPCSTPSDKLVIDKLVVNFGNVLQEIFTDEAR TGQENNLGGEKNGYVTCPFRPDCPLGKSFEELPVSPEIPPRKSQYLRSRMPEGTRPEAKEQLLFSGQSLVPDHLCEEGAPNPYLKNSVTAREFLVSEKLPEHTKGEV

[0212] > Human OSMR protein amino acid sequence (SEQ ID NO: 4; underlined extracellular region)

[0213] MALFAVFQTTFFLTLLSLRTYQSEVLA ERLPLTPVSLKVSTNSTRQSLHLQWTVHNLPYHQELKMVFQI QISRIETSNVIWVGNYSTTVKWNQVLHWSWESELPLECATHFVRIKSLVDDAKFPEPNFWSNWSSWEEVSVQDSTGQ DILFVFPKDKLVEEGTNVTICYVSRNIQNNVSCYLEGKQIHGEQLDPHVTAFNLNSVPFIRNKGTNIYCEASQGNVS EGMKGIVLFVSKVLEEPKDFSCETEDFKTLHCTWDPGTDTALGWSKQPSQSYTLFESFSGEKKLCTHKNWCNWQITQ DSQETYNFTLIAENYLRKRSVNILFNLTHRVYLMNPFSVNFENVNATNAIMTWKVHSIRNNFTYLCQIELHGEGKMM QYNVSIKVNGEYFLSELEPATEYMARVRCADASHFWKWSEWSGQNFTTLEAAPSEAPDVWRIVSLEPGNHTVTLFWK PLSKLHANGKILFYNVVVENLDKPSSSELHSIPAPANSTKLILDRCSYQICVIANNSVGASPASVIVISADPENKEV EEERIAGTEGGFSLSWKPQPGDVIGYVVDWCDHTQDVLGDFQWKNVGPNTTSTVISTDAFRPGVRYDFRIYGLSTKR IACLLEKKTGYSQELAPSDNPHVLVDTLTSHSFTLSWKDYSTESQPGFIQGYHVYLKSKARQCHPRFEKAVLSDGSE CCKYKIDNPEEKALIVDNLKPESFYEFFITPFTSAGEGPSATFTKVTTPDEHSSM LIHILLPMVFCVLLIMVMCYLKSQWIKETCYPDIPDPYKSSILSLIKFKENPHLIIMNVSDCIPDAIEVVSKPEGTKIQFLGTRKSLTETELTKPNYLYLLPTEKNHSGPGPCICFENL TYNQAASDSGSCGHVPVSPKAPSMLGLMTSPENVLKALEKNYMNSLGEIPAGETSLNYVSQLASPMFGDKDSLPTNPVEAPHCSEYKMQMAVSLRLALPPPTENSSLSSITLLDPGEHYC

[0214] > Cynomolgus monkey IL-31RA protein amino acid sequence (SEQ ID NO: 5; underlined extracellular region)

[0215] MMWTWALWMFPLLCKFGLA ALPAKPENISCVYYYRKNLTCTWSPGKETSYTQYTAKRTYAFGKKHDNCT TSSSTSENRASCSFFLPRITIPDNYTIEVEAENGDGVIKSDMTCWRLEDIAKTEPPEIFSVKPVLGIKRMIRIEWIK PELAPVSSDLKYALRFRTVNSTSWMEVNFAKNRKDTNQTYNLMGLQAFTEYVVALRCAVKESKFWSDWSQEKMGMTE EEAPCGLELWRVLKPTEVDGRRPVRLLWKKARGAPVLEKTLGYNIWYFPENNTNLTETVNTTNQQLELHLGGESYWV SMISYNSLGKSPVTTLRIPAIQEKSFRCIEVMQACLAEDQLVVKWQSSALDVNTWMIEWFPDMDSEHPTLSWESVSQ ATNWTIQQDKLKPFWCYNISVYPMLHDKVGEPYSIQAYAKEGIPSKGPETKVENIGVKTVTITWKEIPKSERKGIIC NYTIFYQAEGGKGFSKTVNSSILQYGLESLKRKTSYTVRVMASTSAGGINGTSINFKTLSFSVFE IILITSLIGGGLLILIILTVAYGLKKPNKLTHLCWPSVPPNPAESSIATWRGDDFKDKLNLKESDDSVNTEDRILKPCSTPSDKLVIDKSVVNFGNVLQEMFTDEARTG QENNLGGEKNEYVTHPFRPDCPLGKSFEELPVSPEIPPRKSQYLRSRMPEGTCLEAEEQLLVSGQSLESLAPDHVWEAAAPNPYLKNSVTTREFLVSQKLPEHTKGEV

[0216] > Cynomolgus monkey OSMR protein amino acid sequence (SEQ ID NO: 6; underlined extracellular region)

[0217] MALFVVFQTTFFLTLLSLRTYQSEVLA ERLPLTPVSLKVSTNSIHQSLHLQWTVHNLPYHQELKMVFQI QISRIETSNVVWVGNYSTTVKWNQVLHWSWESELPLECATHFVRIKSVIDDASFPEPNFWSNWSSWEEVSVQDYLGR GTLFVFPKDKLVEEGSNVTICYVSRNIQNNVSCYLEGKQIHGEQLDPHVTAFNLNSVPFIRNRGTNIYCEASQGNVS KGIEGIVLFVSKVLEEPKDFSCESQDFKTLHCTWDPGTDTALGWSKQPSQSYTLFESFSGEKKLCTHKNWCNWQITQ DSQEMYNFTLIAENYLRKRSVNILFNLTHRVYLMNPFSVNFENVNATNAIMTWKVHSMRNNFTYLCQIELHGEGKMM QYDVSINVNGEYFLSELEPATEYMARVRCADASHFWKWTEWSGQNFTTLEAAPSEAPDVWRSVNSEPGNHTVTLFWK PLSKLHANGKILFYNVVVENLDKPSRSELRSIPAPANSTKLILDRCSYQICVTANNSVGASPASIIVISADPENKEV EEERIAGTEGGFSLSWKPQPGDVIGYVVDWCDHPQDVLQWKNVGPNTTSTVISTDAFRPGVRYDFRIYGLSTKRIAC LLEKKTGYSQELAPSDNPHVLVDMLTSHSFTLSWKDYSTESQPGFIQGYHVYLKSKARQCHPRFQKAVLSDGSECCK YKIDNPEEKALIVDNLKPESFYEFFVTPFTSAGEGPNATFTKVTTPDEHSS MLIRILLPMVFCVLLIMIVCYLKSQWIKETCYPDIPDPYKSSILSLIKFKENPHLTIMNVSDCIPDAIEVVSKPEGTKIQLLGTRKSLTETELTKPNYLYLLPTEKNHSGPGPCICFENF TYNQAASDAGSCGHVPVPPKAPPSMLGLMTSPENVLKALEKNYMNSLGEVPAGETSLNYVSQLASPMSGDKDSLPTNPVEPPHCSEYKMQMAVPLRLALPPPTENSSLSSITLLDPGEHYR

[0218] 2. Construction of BaF3 cells overexpressing IL-31RA and OSMR and reporter gene system

[0219] The target genes IL-31RA and OSMR were cloned into the mouse IL-3-dependent pro-B cell line BaF3 using lentiviral packaging to form a stable, highly expressing cell line. The full-length genes of human IL-31RA and human OSMR were constructed into the pLVX-IRES-Puro and pLVX-IRES-Neo vectors, respectively, to generate the pLVX-IL31RA-IRES-Puro and pLVX-OSMR-IRES-Neo plasmids. The human IL-31RA and OSMR genes were cloned into BaF3 cells via lentiviral transfection. After two to three weeks of culture under 10 μg / mL puromycin and 1 mg / mL G418 selection pressure, a BaF3 cell line overexpressing human IL-31RA and OSMR was obtained. A monoclonal cell line, designated BaF3-huIL31RA / OSMR, was isolated by limiting dilution screening. This cell line exhibited human IL-31-dependent proliferation in the absence of mouse IL-3 and could be used to evaluate the blocking ability of anti-IL-31RA antibodies. Separately, the cynomolgus macaque IL-31-dependent overexpressing cell line, BaF3-cynoIL31RA / OSMR, was constructed using the same lentiviral transfection method.

[0220] To screen for neutralizing antibodies that block 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. After selection and culture under 400 μg / mL hygromycin B pressure for two to three weeks, a BaF3 cell line with high expression of SIE-Luc2P was obtained. A monoclonal cell line was obtained using the limiting dilution method, and the clone with the highest luciferase response and lowest background was screened by the luciferase assay (see below), which was named BaF3-huIL31RA / OSMR / SIE. In addition, the BaF3-cynoIL31RA / OSMR / SIE cell line was obtained using the same construction method.

[0221] Recombinant IL-31-induced luciferase reporter gene activity can be measured as follows: Wash BaF3-huIL31RA / OSMR / SIE or BaF3-cynoIL31RA / OSMR / SIE cells twice in RPMI1640 (10% FBS) without mouse IL-3, resuspend at a cell density of >1E6 / mL, and incubate overnight at 37°C, 5% CO₂ for 19-20 hours. BaF3 cells overexpressing the pGL4.47 plasmid, not transfected, serve as a control and are treated identically. Recombinant IL-31 protein is diluted in RPMI1640 (10% FBS) to concentrations of 1000 ng / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 0.1 ng / mL, 0.01 ng / mL, and 0.001 ng / mL. The next day, adjust the cell density to 4E5 / mL. Add 50 μL of cells and 50 μL of serially diluted recombinant IL-31 protein to a 96-well opaque white plate. Incubate the plate at 37°C, 5% CO₂ for 5 hours. Bring the Promega ONE-Glo Luciferase Assay System reagent to room temperature in advance. Add 100 μL of the assay reagent to each well and incubate in the dark for 3 minutes. Detect the chemiluminescence of the substrate using a multi-function microplate reader.

[0222] >Amino acid sequence of recombinant human IL-31 protein with His tag (huIL31-His; SEQ ID NO: 7) (mature protein is underlined)

[0223] MASHSGPSTSVLFLFCCLGGWLA SHTLPVRLLRPSDDVQKIVEELQSLSKMLLKDVEEEKGVLVSQNYT LPCLSPDAQPPNNIHSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIFQDAPETNISVPTDTHECKRFILTISQQFSE CMDLALKSLTSGAQQATTHHHHHHHH

[0224] >Amino acid sequence of recombinant cynomolgus monkey IL-31 protein with His tag (cynoIL31-His; SEQ ID NO: 8) (mature protein is underlined)

[0225] MASHSGPATSVLFLLCCLGGWLTSHT LPVHFLQPSDIQKIVEELQSLSKMLLKDVKEDKGVLVSQNYTL PCLTPDAQPPNIIHSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIFQDAPETNISVPTDTHECKRFILTISQQFSEC MDLALKSLTSGAQQATTHHHHHHHH

[0226] Example 3 Screening of anti-IL-31RA monoclonal hybridoma cells

[0227] 1. Mouse Immunization

[0228] 1.1 Recombinant protein immunization

[0229] Balb / c mice (female, 6-8 weeks old, purchased from Hubei Provincial Laboratory Animal Research Center) were immunized with recombinant huIL31RA protein. Acclimated mice were immunized with recombinant huIL31RA-His protein and adjuvant. The first immunization was performed with a subcutaneous injection of 50 μg of fusion protein in Freund's complete adjuvant. Thereafter, booster immunizations were performed every two weeks with a subcutaneous injection of 50 μg of fusion protein in Freund's complete adjuvant. After the fourth to fifth immunization, mice with high serum antibody titers that had plateaued were selected for shock immunizations with 50 μg of fusion protein per mouse, using the unadjuvanted fusion protein as the immunogen.

[0230] 1.2 Overexpression of cellular immunity

[0231] Balb / c mice (female, 6-8 weeks old, purchased from the Hubei Provincial Laboratory Animal Research Center) were immunized with CHO-K1 cells overexpressing IL-31RA. After one week of acclimatization, the mice were harvested and the cell density of huIL-31RA-overexpressing CHO-K1 cells was adjusted to 1E7 cells / 200 μL. For the first immunization, 200 μL of cells were injected intraperitoneally into each mouse. Supplementary immunizations were performed every two weeks in the same manner. After three immunizations, serum antibodies were tested for binding to IL-31RA-overexpressing HEK293 cells using FACS. Mice with high serum titers were selected for pulse immunizations with 50 μg of unadjuvanted IL-31RA fusion protein injected intraperitoneally into each mouse.

[0232] 1.3 Protein Cell Cross-Immunity

[0233] Balb / c mice (female, 6-8 weeks old, purchased from the Hubei Provincial Laboratory Animal Research Center) were cross-immunized with recombinant IL-31RA protein and CHO-K1 cells overexpressing IL-31RA. For the initial immunization, CHO-K1 cells overexpressing huIL-31RA were harvested and adjusted to a cell density of 1E7 cells / 200 μL. 200 μL of cells were injected intraperitoneally into each mouse. Two weeks later, each mouse was boosted subcutaneously with 50 μg of huIL31RA-His protein in Freund's complete adjuvant. Subsequently, immunizations with overexpressing cells and recombinant protein were alternating at two-week intervals. After the fourth or fifth immunization, mice with high serum antibody titers that had plateaued were selected for a surge immunization with 50 μg of the fusion protein injected intraperitoneally into each mouse using unadjuvanted IL-31RA protein as the immunogen.

[0234] 2. Cell Fusion

[0235] Mice with high serum titers were sacrificed 48-72 hours after the shock immunization, and spleen cells were fused with myeloma cells at a ratio of 5:1. Hybridoma cells were generated by fusing splenic lymphocytes with myeloma Sp2 / 0 cells using an optimized PEG-mediated fusion method. Hybridoma cells were plated in 96-well plates at a density of 1E5 cells / well in 200 μL of culture medium and cultured statically at 37°C and 5% CO2.

[0236] 3. Hybridoma Screening

[0237] For the initial screening, positive clones were identified using an ELISA binding assay targeting human IL-31RA. An ELISA plate was coated overnight with 100 μL / well of 500 ng / mL huIL31RA-His protein. After blocking with BSA, 100 μL of hybridoma supernatant was added and the plate was incubated at 37°C for 1 hour. Conventional ELISA procedures were then followed for plate washing, incubation with mouse IgG secondary antibody, and color development. Finally, the absorbance at 450 nm was read using a microplate reader. The top 10% clones with the highest OD450 were selected for further FACS analysis to test the ability of anti-IL-31RA antibodies to block the binding of recombinant IL-31 protein to HEK293 cells overexpressing IL-31RA and OSMR. HEK293-huIL31RA / OSMR or HEK293 cynoIL31RA / OSMR cells were trypsinized and harvested. The cells were then resuspended in PBS + 2% FBS to adjust the cell density to 2E6 / mL, with approximately 1E5 cells per well. The ligand huIL31-His was diluted to 3 μg / mL. 50 μL of cells, 25 μL of hybridoma supernatant, and 25 μL of ligand were added to each well, followed by incubation 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 a 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 incubation, the 96-well plate was centrifuged at 1000 rpm for 5 minutes, washed once with PBS + 2% FBS, and cells were resuspended in 100 μL of PBS + 2% FBS per well. Signals were measured in the PE channel on a flow cytometer. The signal value measured for the hybridoma sample was designated A, the signal value measured for a blank control without huIL31-His was designated B, and the signal value measured with both negative hybridoma supernatant and huIL31-His was designated C. The blocking efficiency (%) for each hybridoma supernatant well was calculated as (CA) / (CB) × 100. Data for some representative clones are shown in Table 1.

[0238] Table 1 Some representative clone screening data

[0239]

[0240]

[0241] After repeated screening, positive clones with a blocking rate greater than 90% were obtained and subcloned twice using the limiting dilution method, ultimately obtaining monoclonal hybridoma cells. The culture supernatants of the monoclonal hybridoma cells were subjected to FACS analysis of HEK293 cells overexpressing human and monkey IL-31RA, as well as to luciferase reporter gene activity testing of human and monkey BaF3-IL31RA / OSMR cells. Ultimately, preferred monoclonal hybridoma cells with better activity were obtained. The data of some representative clones are shown in Table 2.

[0242] Table 2 Partial monoclonal screening data

[0243]

[0244] Three monoclonal strains, 1A1C6, 2F6E12, and 2G1G1, with knockdown rates greater than 90% for both human and monkey reporter genes, were selected. Hybridoma cells in logarithmic growth phase were harvested and RNA was extracted using NucleoZol (MN) and reverse transcribed using PrimeScript™ Reverse Transcriptase, Takara, cat# 2680A. The resulting cDNA was PCR amplified using Mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and subsequently sequenced by a sequencing company. The variable region amino acid sequences of the murine antibodies obtained by sequencing are shown below, and the CDR region sequences are shown in Table 3.

[0245] >1A1C6 murine heavy chain variable region amino acid sequence (1A1C6-VH; SEQ ID NO: 9)

[0246] QVQLQQSGAELVRPGSSVKISCKASGYAFS DDWMN WLKQRPGQGLEWIG QIYPGDGDTNYNGKFKG KATLTADKSSSTAYMQLSSLTSEDSAVYFCAR DEDYFGGSPWFAY WGQGTLVTVST

[0247] >1A1C6 murine light chain variable region amino acid sequence (1A1C6-VL; SEQ ID NO: 13)

[0248] DIQMTQSPASQSASLGESVTITC LASQTIGTWLA WYQQKPGKSPQLLIY AATRLA D GVPSRFSGSGSGTKFSFKISSLQAEDFVNYYC QQFYRTPLT FGSGTKLEIK

[0249] >2F6E12 murine heavy chain variable region amino acid sequence (2F6E12-VH; SEQ ID NO: 17)

[0250] EVQLQQSGPELVKPGASVKISCKASGYTFT DYYYMN WVKQSHGKSLEWIG YIYPNNGGTDFNQKFED KATLTVDKSSSTAYMELRRSLTSEDSAVYYCAR GPYDYDYYVMDY WGQGTSVTVSS

[0251] >2F6E12 murine light chain variable region amino acid sequence (2F6E12-VL; SEQ ID NO: 21)

[0252] DIVLTQSPASLAVSLGQRATISC RASESVDSYGRTFMH WYQQKPGQPPKLLIY RAS ILES GIPARFSGSGSRTDFTLTINPVEADDVATYYC HQSNEDPWT FGGGTKVEIK

[0253] >2G1G1 murine heavy chain variable region amino acid sequence (2G1G1-VH; SEQ ID NO: 24)

[0254] EVQLQQSGPELEKPGASVKISCKASGYSFT DYNMN WVKQTNGKSLEWIG NIDPYYGGATYNRKFKG RATLTVDKSSSTAYMQLKSLTSEDSAVYYCAK NENDYDVGAMDF WGQGTSVTVSS

[0255] >2G1G1 murine light chain variable region amino acid sequence (2G1G1-VL; SEQ ID NO: 28)

[0256] DIQMTQSPASLSVSVGETVTITC RTSENVYSYFT WYHQKQGKSPQVLVY NAKTLA E GVPLRFSGSGSGTQFSLKINNLQPEDFGSYYC QHHYGTPLT FGTGTKLELK

[0257] Table 3 CDR region sequences of mouse antibodies

[0258]

[0259] 4. Identification of Mouse Antibody Activity

[0260] The three hybridoma cells were expanded and inoculated into serum-free medium. The cell density was adjusted to 2E5 / mL and cultured continuously at 37°C and 200 rpm for 5-7 days. The supernatant was collected by centrifugation and purified using a Protein A / G column to obtain mouse antibodies. The mouse monoclonal antibodies derived from the three hybridomas were named 1A1C6-mIgG, 2F6E12-mIgG, and 2G1G1-mIgG, respectively. The activity of the mouse antibodies was evaluated using a luciferase reporter gene system, and the IC50 was calculated and compared with the positive antibodies. The results are shown in Figure 2. Figure 1A and Figure 1B As shown in the figure, the mouse antibodies screened through hybridomas all have good biological activity. Among them, the positive antibody Nemolizumab (i.e., CIM331) is a recombinant antibody prepared in the laboratory with the same sequence as the marketed drug. Its sequence is as follows:

[0261] >Nemolizumab heavy chain amino acid sequence (SEQ ID NO: 32)

[0262] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYIMNWVRQAPGQGLEWMGLINPYNGGTDYNPQFQDRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYDDGPYTLETW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERK SCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE KTISKTKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSP

[0263] >Nemolizumab light chain amino acid sequence (SEQ ID NO: 33)

[0264] DIQMTQSPSSSLSASVGDRVTITCQASEDIYSFVAWYQQKPGKAPKLLIYNAQTEAQGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYDSPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0265] Example 4 Preparation of humanized antibodies

[0266] The above three antibodies were all humanized using methods known in the industry. The present invention briefly describes the process of preparation and identification of humanized antibodies using 1A1C6 as an example.

[0267] 1. Select the framework sequence

[0268] The heavy and light chain variable regions of the mouse 1A1C6 antibody were aligned with the human germline gene sequences for homology (according to the Kabat numbering system), and the germline sequence with the highest homology was selected as the template sequence. The framework sequence (FR) selected for the 1A1C6 antibody is shown in Table 4.

[0269] Table 4 1A1C6 antibody framework region sequence

[0270]

[0271] The CDR region of 1A1C6 (as shown in Table 3) was transplanted onto the template sequence to form a CDR-grafted antibody, the amino acid sequence of which is as follows, where H0 is the heavy chain variable region sequence, L0 is the light chain variable region sequence, and so on.

[0272] >1A1C6 CDR-grafted antibody heavy chain variable region amino acid sequence (1A1C6-H0; SEQ ID NO: 42, underlined CDR region)

[0273] QVQLVQSGAEVKKPGSSVKVSCKASGYAFS DDWMN WVRQAPGQGLEWMG QIYPGDGDANYAQKFQG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR DEDYFGGSPWFAY WGQGTTVTVSS

[0274] >1A1C6 CDR-grafted antibody light chain variable region amino acid sequence (1A1C6-L0; SEQ ID NO: 43, underlined CDR region)

[0275] DIQMTQSPSSLSASVGDRVTITC RASQTIGTWLA WYQQKPGKAPKLLLY AATRLA DGVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQFYRTPLT FGQGTKLEIK

[0276] 2. Humanized Antibody Design

[0277] Based on the structural comparison of the variable regions of the CDR-grafted antibody and the murine parent antibody, the selection of the optimized framework region was comprehensively considered to complete multiple rounds of humanization design. The CDR sequences of the back mutations are shown in Table 5, and the variable region sequences of the back mutations are shown in Tables 6 and 7. The heavy and light chains are matched and combined to obtain the variable regions of the humanized antibodies, and the variable regions are combined with the human IgG constant regions to obtain full-length humanized antibodies. The human IgG heavy chain constant region can be selected from the IgG1, IgG2 or IgG4 subtype, and the light chain constant region can be selected from the Kappa or Lambda subtype, or it can be replaced with other constant regions known in the art. For example, the heavy chain constant region of the 1A1C6 humanized antibody selects IgG4 S228P, and the light chain selects the Kappa chain constant region. The full-length humanized antibody composed of the H0 and L0 variable regions and the constant region is labeled 1A1C6-H0L0, and so on.

[0278] >Human IgG4 S228P heavy chain constant region amino acid sequence (SEQ ID NO: 44)

[0279] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0280] >Human Kappa light chain constant region amino acid sequence (SEQ ID NO: 45)

[0281] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0282] Table 5 CDR sequences of the backmutated 1A1C6 humanized antibody

[0283]

[0284]

[0285] Table 6 Variable region sequences of the 1A1C6 humanized antibody with back mutations

[0286]

[0287] Table 7 Variable region sequences of backmutated humanized antibodies 2F6E12 and 2G1G1

[0288]

[0289] 3. Expression and Purification of Humanized Antibodies

[0290] The variable regions and constant regions of the above-mentioned different combinations were linked to obtain full-length antibody sequences, and humanized antibodies 2F6E12 and 2G1G1 were obtained using the same method. Exemplary full-length antibody amino acid sequences of 1A1C6-H5L9, 2F6E12-H5L3, and 2G1G1-H1L1 are as follows. The sequences encoding these antibodies were cloned into the pTT5 vector and expressed in cells according to the protocol 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 baseline, then the sample was loaded. The target protein was eluted with 100mM glycine (pH 3.0) and immediately neutralized by adding 1M Tris-HCl (pH 8.0). The purified antibody was then buffered with PBS, and the absorbance at 280nm was measured to calculate the antibody concentration.

[0291] >1A1C6-H5L9 heavy chain amino acid sequence (SEQ ID NO: 59)

[0292] QVQLVQSGAEVKKPGSSVKVSCKASGYAFSDDWMNWLKQRPGQGLEWMGQIYPGDGDTNYNGKFKGKVTLTADKSTSTAYMELSSLRSEDTAVYFCARDEDYFGGSPWFAYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0293] >1A1C6-H5L9 light chain amino acid sequence (SEQ ID NO: 60)

[0294] DIQMTQSPSSLSASVGDRVTITCRASQTIGTWLAWYQQKPGKSPQLLIYAATRLADAVPSRFSGSGSGTDFTFTISSLQPEDFATYYCQQFYRTPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0295] >2F6E12-H5L3 heavy chain amino acid sequence (SEQ ID NO: 61)

[0296] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYYMNWVKQAPGQGLEWMGYIYPNNGGTDFNQKFEDKVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGPYDYDYYVMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0297] >2F6E12-H5L3 light chain amino acid sequence (SEQ ID NO: 62)

[0298] DIVMTQSPASLAVSLGERATINCRASESVDSYGRTFMHWYQQKPGQPPKLLIYRASILESGIPARFSGSGSGTDFTLTINPVEAEDVAVYYCHQSNEDPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0299] >2G1G1-H1L1 heavy chain amino acid sequence (SEQ ID NO: 63)

[0300] QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYNMNWVRQAPGQGLEWMGNIDPYYGGATYNRKFKGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCAKNENDYDVGAMDFWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0301] >2G1G1-H1L1 light chain amino acid sequence (SEQ ID NO: 64)

[0302] DIQMTQSPSSLSASVGDRVTITCRTSENVYSYFTWYQQKPGKSPQLLIYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0303] 4. Activity identification of humanized antibody

[0304] 4.1 IL-31RA protein binding activity based on ELISA

[0305] The binding activity of the humanized antibody to the antigen protein IL31-RA was tested by ELISA as follows: recombinant human IL31RA-His protein was diluted to 0.5 μg / mL in PBS buffer (pH 7.4), coated onto a 96-well microtiter plate (Costar, 42592) at a volume of 100 μL / well, and incubated overnight at 4°C. The next day, the wells were discarded, and the plates were washed three times with PBST (300 μL / well). Then, 300 μL / well of blocking solution (PBST containing 3% BSA) was added 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). Then, 100 μL of serially diluted antibody sample (initial concentration was 10 μg / mL) was added and incubated at 37°C for 1 hour. After washing three times with PBST (300 μL / well), a 1:5000 diluted HRP-labeled Goat Anti-human IgG (H+L) secondary antibody was added and incubated at 37°C for 1 hour. After washing four times with PBST (300 μL / well), the color was developed and the absorbance at 450 nm was read on a microplate reader. The EC50 value of the binding of the IL-31RA antibody to the human IL-31RA protein was calculated (as shown in Table 8). Figure 2A and Figure 2B As shown, the antibodies disclosed herein have good binding activity to human IL-31RA.

[0306] Table 8 EC50 values of humanized antibodies binding to human IL-31RA protein

[0307] Samples EC50 (nM) Samples EC50 (nM) Nemolizumab 0.1064 Nemolizumab 0.1196 1A1C6-VHVL 0.1445 1A1C6-VHVL 0.1141 1A1C6-H1L1 0.1994 1A1C6-H3L1 0.1610 1A1C6-H1L2 0.1889 1A1C6-H3L2 0.1649 1A1C6-H1L3 0.1711 1A1C6-H3L3 0.1734 1A1C6-H2L1 0.1917 1A1C6-H4L1 0.1686 1A1C6-H2L2 0.1680 1A1C6-H4L2 0.1801 1A1C6-H2L3 0.1802 1A1C6-H4L3 0.1621

[0308] 4.2 Determination of antibody affinity constant

[0309] The affinity kinetics of the humanized antibodies were determined using biomembrane interferometry (BLI) using a Protein A chip (Gator) coupled with a label-free biomolecular analyzer. After baseline equilibration, the antibodies were captured using PBS prepared to a final concentration of 0.025% Tween 20 and 0.2% BSA (IgG-free, protease-free). After continuous sampling for 120 seconds, various concentrations of recombinant human IL-31RA protein were injected for 120 seconds and dissociation was monitored for 360 seconds. Finally, the probe was regenerated with glycine (pH 1.7). Kinetic parameters were calculated using GatorOne software. Affinity constants, expressed as KD, were calculated from the ratio of kd / ka (dissociation rate / association rate). As shown in Table 9, the affinities of some humanized antibodies and recombinant human IL-31RA protein were comparable to those of the control antibody.

[0310] Table 9 Humanized Antibody Affinity Detection

[0311]

[0312]

[0313] 4.3 Detection of Antibody Binding Activity to Overexpressing Cells

[0314] The binding activity of the anti-IL-31RA antibodies against HEK293 cells overexpressing IL-31RA and OSMR was evaluated by FACS. HEK293-huIL31RA / OSMR or HEK293cynoIL31RA / OSMR cells were trypsinized and harvested, then resuspended in PBS + 2% FBS to a cell density of 2E6 / mL, with approximately 1E5 cells per well. Antibody samples were diluted to 60 μg / mL (final concentration 30 μg / mL) in the wash buffer and serially diluted three-fold. Fifty μL of cells and 50 μL of antibody sample were added to each well, followed by incubation at 4°C for 30 minutes. The 96-well plate was centrifuged at 1000 rpm for 5 minutes, washed once with PBS + 2% FBS, and 100 μL of a 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 minutes. After incubation, the 96-well plate was centrifuged at 1000 rpm for 5 min, washed once with PBS + 2% FBS, and cells were resuspended in 100 μL of PBS + 2% FBS per well. Signal values were detected by flow cytometry and EC50 values were calculated (Table 10). Figure 3A and Figure 3B As shown, the antibodies of the present invention have good binding activity to cells overexpressing human or monkey IL-31RA.

[0315] Table 10 Antibody binding activity to overexpressing cells

[0316] Antibody Human EC50 (nM) Monkey EC50 (nM) Nemolizumab 0.37 0.41 2F6E12-H5L3 0.62 0.6 1A1C6-H5L2 0.39 0.43 1A1C6-H5L9 0.28 0.41 1A1C6-H5L11 0.22 0.45 1A1C6-H26L14 0.46 0.74 2G1G1-H1L1 0.33 0.36 2G1G1-H1L3 0.44 0.56

[0317] 4.4 Determination of Antibody Activity Blocking the Binding of IL-31 and IL-31RA

[0318] FACS was used to test the ability of anti-IL-31RA antibodies to block the binding of recombinant IL-31 protein to HEK293 cells overexpressing IL-31RA and OSMR. HEK293-huIL31RA / OSMR or HEK293 cynoIL31RA / OSMR cells were trypsinized and harvested, then resuspended in PBS + 2% FBS to a cell density of 2E6 / mL, with approximately 1E5 cells per well. Antibody samples were diluted in the wash buffer to 60 μg / mL (final concentration 15 μg / mL) and serially diluted threefold. 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 cells, 25 μL antibody sample, and 25 μL ligand were added to each well in sequence 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 was completed, 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 of 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 (Table 11). Figure 4A and Figure 4B The test results show that the antibodies of the present invention have good blocking activity in both human and monkey overexpressing cells, and the activity of some antibodies is better than that of the control antibody.

[0319] Table 11 Antibody activity in blocking the binding of IL-31 and IL-31RA

[0320] Antibody Human IC50 (nM) Monkey IC50 (nM) Nemolizumab 0.4 0.35 2F6E12-H5L3 1.14 1 1A1C6-H5L2 0.34 0.42 1A1C6-H5L9 0.25 0.32 1A1C6-H5L11 0.2 0.51 1A1C6-H26L14 0.34 0.48 2G1G1-H1L1 0.25 0.34 2G1G1-H1L3 0.26 0.35

[0321] 4.5 Antibodies inhibit luciferase reporter gene activity in BaF3-IL31RA / OSMR cells

[0322] The neutralizing activity of anti-IL-31RA antibodies can be measured using a luciferase reporter gene system. A sufficient number of BaF3-huIL31RA / OSMR / SIE cells were washed twice with RPMI1640 + 10% FBS and resuspended to a cell density greater than 1E6 / mL. The cells were cultured overnight at 37°C, 5% CO2 for 19-20 hours. The antibody sample was diluted to 30 μg / mL (final concentration 7.5 μg / mL) in a 5-fold serial dilution. The ligand huIL31-His was diluted to 2 ng / mL (final concentration 0.5 ng / mL). The next day, the cell density was adjusted to 1E6 / mL. 20 μL of cells, 10 μL of sample, and 10 μL of ligand were added to a 96-well opaque white plate in sequence and incubated at 37°C, 5% CO2 for 5 hours. Take out the Promega ONE-Glo Luciferase Assay System in advance, return it to room temperature, add 40 μL of detection reagent to each well, incubate in the dark for 3 minutes, and detect the chemiluminescence value with a multifunctional microplate reader. The same method can be used to measure the inhibition of IL-31RA antibody on the luciferase reporter gene activity of BaF3-cynoIL31RA / OSMR cells. Use GraphPad Prism software to draw the curve and calculate the IC50 value (Table 12). Figure 5A and Figure 5B

[0323] As shown, the antibodies of the present invention have good neutralizing activity in both human and monkey reporter gene systems, and the activity of some antibodies is better than that of the control antibody.

[0324] Table 12 Antibodies inhibit reporter gene activity

[0325] Antibody Human IC50 (nM) Monkey IC50 (nM) Nemolizumab 0.028 0.12 2F6E12-H5L3 0.04 0.2 1A1C6-H5L2 0.022 0.34 1A1C6-H5L9 0.016 0.24 1A1C6-H5L11 0.014 1.29 1A1C6-H26L14 0.027 0.59 2G1G1-H1L1 0.019 0.12 2G1G1-H1L3 0.02 0.12

[0326] 4.6 Antibody Inhibits IL-31-Dependent BaF3-IL31RA / OSMR Cell Proliferation

[0327] BaF3 cells overexpressing IL31RA and OSMR proteins exhibit IL-31-dependent proliferation in the absence of murine IL-3. This property can be exploited to evaluate the ability of anti-IL-31RA antibodies to inhibit cell proliferation. BaF3-huIL31RA / OSMR cells in logarithmic growth phase were harvested and centrifuged at 1000 rpm for 5 minutes. Cells were washed twice in RPMI 1640 medium supplemented with 10% FBS, resuspended at a concentration of 4E5 / mL, and seeded with 50 μL of cells per well of a 96-well cell culture plate. Test antibodies were diluted to 20 μg / mL in medium containing 1 ng / mL huIL-31. After three-fold serial dilutions, 50 μL of sample was added to each well. The cell culture plates were incubated at 37°C, 5% CO₂, for 48 hours. After the incubation period, the cell culture plate was removed from the incubator and equilibrated at room temperature for 30 min. 100 μL CellCounting-Lite reagent (Vazyme, DD1101) was added to each well and shaken at room temperature for 2-5 min to fully lyse the cells. The chemiluminescence value was detected by a multifunctional microplate reader. GraphPad Prism software was used to plot the curve and calculate the IC50 value (Table 13). Figure 6 As shown, the antibodies of the present invention have a proliferation inhibitory activity comparable to that of the control antibody.

[0328] Table 13 Antibody activity in inhibiting proliferation of overexpressing cells

[0329] Antibody IC50(nM) Nemolizumab 0.028 1A1C6-H5L9 0.112 2G1G1-H1L1 0.016

[0330] 4.7 Antibody Inhibits IL-31-Induced STAT3 Phosphorylation in U87MG Cells

[0331] U87MG cells, a human glioblastoma cell line, have been identified as expressing high levels of IL-31RA and OSMR proteins. IL-31 stimulation can activate downstream signaling pathways, making them suitable for evaluating the biological activity of anti-IL-31RA antibodies. U87MG cells were seeded in 24-well cell culture plates at 40,000 cells per well and incubated at 37°C, 5% CO₂ for 24 hours. The test antibody was diluted to 200 μg / mL in MEM+10% FBS medium and serially diluted sixfold for a total of eight dilutions. The ligand IL-31 was diluted to 300 ng / mL in culture medium. After incubation, the supernatant from the 24-well plate was discarded, and 100 μL of antibody sample and 100 μL of ligand were added to each well, followed by incubation at 4°C for 30 minutes. Add cell lysis buffer and lyse the cells on ice for 30 minutes. Centrifuge at 13000 rpm and 4°C for 25 minutes to collect the supernatant. Use Phospho-Stat3 (Tyr705) ELISA kit (CST, 7300C) to detect the STAT3 phosphorylation level in the supernatant. Figure 7 As shown, the antibody of the present invention has an activity of inhibiting STAT3 phosphorylation comparable to that of the control antibody.

[0332] 4.8 Antibody inhibits IL-31-induced IL-6 release from HaCat cells

[0333] HaCat cells are an immortalized keratinocyte cell line derived from adult humans. IL-31 has been shown to stimulate IL-6 production in HaCat cells in the presence of IFNγ. Therefore, the biological activity of anti-IL-31RA antibodies can be tested by inhibiting cytokine release in HaCat cells. HaCat cells are seeded in 96-well plates at 30,000 cells per well and cultured at 37°C, 5% CO₂ for 12 hours. Pretreatment with 10 ng / mL IFNγ is added and cultured for an additional 24 hours. The test antibody sample is diluted to 400 μg / mL in DMEM + 10% FBS medium and serially diluted 10-fold over a total of six dilutions. The ligand IL-31 is diluted to 3 μg / mL in culture medium. The cell culture plate is removed, and 50 μL of each dilution of the test antibody sample and 50 μL of the ligand are added to each well. Culture is continued for 24 hours. After the culture was completed, the cell culture supernatant was removed and ELISA 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 based on the standard curve. Figure 8 As shown, the activity of 1A1C6-H5L2 antibody in inhibiting cytokine release was comparable to that of the control antibody.

[0334] Example 5 Preparation of Antibodies with Improved Affinity

[0335] To further enhance the biological activity of anti-IL-31RA antibodies, affinity maturation of the CDR regions is performed to improve the affinity of the molecules for IL-31RA. The present invention briefly describes the modification and validation process using 1A1C6-H5L9 as an example. Similar work can be performed for other molecules by referring to the following steps.

[0336] 1. Identification of mutation hotspots

[0337] For each amino acid site in the CDR region, primers containing 17 mutant amino acids (excluding self, methionine, and cysteine) were designed and synthesized. After PCR amplification, 17 antibody sequences with single-point mutations at that site were obtained. These sequences were inserted into a vector to construct a single-point saturation mutation plasmid library at that site. Expression in a high-throughput mammalian system was used to obtain expression supernatants of the corresponding monoclonal mutant plasmids. The binding activity of the expression supernatant to the antigen was identified by ELISA, which is briefly described as follows: goat anti-human IgG (Fc) was coated on the ELISA plate at 4°C overnight; after washing, the plate was blocked with casein blocking solution at 25°C for 1 hour; after washing, the mutant antibody supernatant and the parent antibody supernatant to be tested were added and incubated at 37°C for 2 hours; after washing, the biotin-labeled antigen was added and incubated at 25°C for 5-8 minutes; 1xPBS was added and dissociated at 25°C for 30 minutes; after washing, SA-HRP was added and incubated at 25°C for 45 minutes; after washing, TMB color development solution was added and color was developed at 25°C in the dark for 5 minutes; finally, TMB color development stop solution was added to terminate the reaction and the OD450 value was read. The monoclonal mutant plasmids corresponding to the OD450 values significantly higher than the OD450 values of the parent antibody were sequenced to obtain mutation hotspots that can significantly improve the antibody affinity. Taking into account the PTM modifications in the CDR region, the identified hotspot amino acid sites are summarized in Table 14. The results showed that after the hotspot mutations were converted to specific amino acids, the specific binding ability to the antigen was significantly improved.

[0338] Table 14 Mutation hotspot sequencing and antigen binding test results

[0339]

[0340]

[0341] 2. Affinity Verification of Mutation Combinations

[0342] The above mutation hotspots were combined, distributing them across CDR1, CDR2, and CDR3, taking into account both the heavy and light chains, while maintaining a balanced hydrophilicity and hydrophobicity of the amino acid sequence. The resulting mutation combinations are shown in Table 15, and the variable regions obtained from these combinations are shown in Table 16. After candidate antibody sequences were generated by combining the selected mutation hotspots, antibodies were expressed in a high-throughput mammalian cell system. Affinity constants for IL-31RA were determined by surface plasmon resonance (SPR) using a Protein A chip (Cytiva, Cat. No. 29127556) coupled with a Biacore 8K instrument. Antibodies were injected at a flow rate of 10 μL / min for 12 seconds. Different concentrations of human or monkey recombinant IL-31RA protein were injected at a flow rate of 30 μL / min for 120 seconds, and dissociation was monitored for 360 seconds. Finally, the chip was regenerated by flowing glycine (pH 1.7) at a flow rate of 30 μL / min for 30 seconds. Kinetic parameters were calculated using the 1:1 binding model in Bia-evaluation analysis software. The affinity constant, expressed as KD, was calculated from the kD / ka (dissociation rate / association rate) ratio. The affinity test results are shown in Table 17. These results indicate that the affinity of the modified antibodies has been significantly improved, with some exhibiting nearly 10-fold higher affinity than the control antibody.

[0343] Table 15 Mutation hotspot combinations

[0344]

[0345]

[0346] Table 16 Mutant variable region sequences

[0347]

[0348]

[0349] Table 17 Mutant affinity detection

[0350]

[0351]

[0352] 3. Confirmation of Mutant Activity

[0353] For example, the variable region of the mutant was linked to the IgG constant region and purified antibodies were prepared (the variable region amino acid sequence, full-length nucleotide sequence and full-length amino acid sequence of the partial mutant are as follows), and the mutant antibodies were evaluated for in vitro activity with reference to the method in "4. Activity identification of humanized antibodies" in Example 4, including binding and blocking activity based on FACS, neutralization activity of the BAF3 reporter gene system, cell proliferation inhibition activity and activity inhibiting STAT3 phosphorylation, and the activity was compared with the parent antibody and the control antibody. Figures 9A-9F As can be seen, compared to the parent antibody, the activity of the mutants was significantly improved. In particular, the neutralization activity against the monkey reporter gene was comparable to that of the control antibody, indicating that cynomolgus macaques are a relevant animal species and can be used for subsequent safety evaluation. Furthermore, at the level of cell proliferation and phosphorylation, the mutant antibodies showed superior inhibitory activity compared to the control antibody, suggesting that antibodies with improved affinity may have better pharmacodynamics.

[0354] >1A1C6-H5L9-AM07 heavy chain nucleotide sequence (SEQ ID NO:92) (variable region underlined)

[0355] ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGC CCAGGTTCAGCTCGTC CAGAGTGGAGCAGAGGTCAAAAAGCCCGGCTCCTCCGTGAAGGTGTCCTGCAAGGCCTCCGGCTACGCCTTCTCCGA CGACTGGATGAACTGGCTGAAGCAGAGGCCCGGCCAGGGCCTGGAGTGGATGGGCCAGATCTACCCCGGCTCCGGCG ACACCAACTACAACAACAAGTTCAAGGGCAAGGTGACCCTGACCGCCGACAAGTCCACCTCCACCGCCTACATGGAG CTGTCCTCCCTGAGGTCCGAGGACACCGCCGTGTACTTCTGCGCCAGGGACGAGGACTACTTCGGCGGCGACCCCTG GTTCGCCTACTGGGGCCAGGGCACCACCGTGACCGTGTCCTCC GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGA

[0356] >1A1C6-H5L9-AM07 light chain nucleotide sequence (SEQ ID NO:93) (variable region underlined)

[0357] ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGC CGACATCCAGATGACC CAGTCCCCATCCTCCCTGTCTGCTTCTGTGGGAGACAGGGTGACCATCACCTGCAGAGCTTCCCAGTATATTGGCAC CTGGCTGGCCTGGTACCAGCAGAAGCCTGGAAAATCCCCCCAGCTGCTCATCTACGCCGCTACCAGGTTGGCTGACG CAGTTCCTTCCAGATTTAGCGGTTCCGGAAGCGGCACCGACTTCACCTTTACTATCTCCTCCCTGCAGCCCGAAGAT TTTGCTACCTACTACTGCTCCCAGTTCTACAGGCACCCCCTGACATTCGGCCAGGGAACCAAACTGGAGATCAAG CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGA

[0358] >1A1C6-H5L9-AM20 heavy chain nucleotide sequence (SEQ ID NO:94) (variable region is underlined)

[0359] ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCC CAGGTTCAGCTCGTC CAGAGTGGAGCAGAGGTCAAAAAGCCCGGCTCCTCCGTGAAGGTGTCCTGCAAGGCCTCCGGCTACGCCTTCTCCGA CGACTGGATGAACTGGCTGAAGCAGAGGCCCGGCCAGGGCCTGGAGTGGATGGGCCAGATCTACCCCGGCACCGGCG ACACCAACTACCAGGGCAAGTTCAAGGGCAAGGTGACCCTGACCGCCGACAAGTCCACCTCCACCGCCTACATGGAG CTGTCCTCCCTGAGGTCCGAGGACACCGCCGTGTACTTCTGCGCCAGGGACGAGGACTACTTCGGCGGCGACCCCTG GTTCGTGTACTGGGGCCAGGGCACCACCGTGACCGTGTCCTCC GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGA

[0360] >1A1C6-H5L9-AM20 light chain nucleotide sequence (SEQ ID NO:95) (variable region underlined)

[0361] ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGC CGACATCCAGATGACC CAGTCCCCATCCTCCCTGTCTGCTTCTGTGGGAGACAGGGTGACCATCACCTGCAGAGCTTCCCAGTGGATCGGAAC CTGGCTGGCTTGGTACCAGCAGAAACCAGGCAAATCCCCCCAGCTGCTCATCTACGCTGCCACCAGGTTGGCTGACG CAGTTCCTTCTCGCTTCTCCGGAAGCGGTAGCGGAACAGATTTTACCTTCACTATCTCCTCCCTGCAGCCTGAGGAC TTCGCTACTTACTACTGCTCCCAGTTCTACCGTCATCCCCTGACCTTCGGCCAGGGAACCAAATTGGAGATCAAG CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGA

[0362] >1A1C6-H5L9-AM22 heavy chain nucleotide sequence (SEQ ID NO:96) (variable region underlined)

[0363] ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGC CCAGGTTCAGCTCGTC CAGAGTGGAGCAGAGGTCAAAAAGCCCGGCTCCTCCGTGAAGGTGTCCTGCAAGGCCTCCGGCTACGCCTTCTCCGA CGACTGGATGAACTGGCTGAAGCAGAGGCCCGGCCAGGGCCTGGAGTGGATGGGCCAGATCTACCCCGGCACCGGCG ACACCAACTACAACGTGAAGTTCAAGGGCAAGGTGACCCTGACCGCCGACAAGTCCACCTCCACCGCCTACATGGAG CTGTCCTCCCTGAGGTCCGAGGACACCGCCGTGTACTTCTGCGCCAGGGACGAGGACTACTTCGGCGGCGACCCCTG GTTCGTGTACTGGGGCCAGGGCACCACCGTGACCGTGTCCTCC GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGA

[0364] >1A1C6-H5L9-AM22 light chain nucleotide sequence (SEQ ID NO:97) (variable region underlined)

[0365] ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGC CGACATCCAGATGACC CAGTCCCCATCCTCCCTGTCTGCTTCTGTGGGAGACAGGGTGACCATCACCTGCAGAGCTTCCCAGTGGATCGGAAC CTGGCTGGCTTGGTACCAGCAGAAACCAGGCAAATCCCCCCAGCTGCTCATCTACGCTGCCACCAGGTTGGACTGGG CAGTTCCTTCTCGCTTCTCCGGAAGCGGATCTGGAACCGATTTTACCTTCACCATCTCCTCCCTGCAGCCCGAAGAT TTTGCTACCTACTACTGCCAGCAGTTCTACCGGCACCCCTTGACATTCGGCCAGGGAACCAAACTGGAGATCAAG CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGA

[0366] >1A1C6-H5L9-AM07 heavy chain amino acid sequence (SEQ ID NO:98) (signal peptide is underlined)

[0367] MHSSALLCCLVLLTGVRAQVQLVQSGAEVKKPGSSVKVSCKASGYAFSDDWMNWLKQRPGQGLEWMGQIYPGSGDTNYNNKFKGKVTLTADKSTSTAYMELSSLRSEDTAVYFCARDEDYFGGDPWFAYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0368] >1A1C6-H5L9-AM07 light chain amino acid sequence (SEQ ID NO:99) (the underlined part is the signal peptide)

[0369] MHSSALLCCLVLLTGVRA DIQMTQSPSSLSASVGDRVTITCRASQYIGTWLAWYQQKPGKSPQLLIYAATRLADAVPSRFSGSGSGTDFTFTISSLQPEDFATYYCSQFYRHPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0370] >1A1C6-H5L9-AM20 heavy chain amino acid sequence (SEQ ID NO:100) (the underlined part is the signal peptide)

[0371] MHSSALLCCLVLLTGVRAQVQLVQSGAEVKKPGSSVKVSCKASGYAFSDDWMNWLKQRPGQGLEWMGQIYPGTGDTNYQGKFKGKVTLTADKSTSTAYMELSSLRSEDTAVYFCARDEDYFGGDPWFVYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0372] >1A1C6-H5L9-AM20 light chain amino acid sequence (SEQ ID NO:101) (Signal peptide underlined)

[0373] MHSSALLCCLVLLTGVRA DIQMTQSPSSLSASVGDRVTITCRASQWIGTWLAWYQQKPGKSPQLLIYAATRLADAVPSRFSGSGSGTDFTFTISSLQPEDFATYYCSQFYRHPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0374] >1A1C6-H5L9-AM22 heavy chain amino acid sequence (SEQ ID NO:102) (Signal peptide underlined)

[0375] MHSSALLCCLVLLTGVRAQVQLVQSGAEVKKPGSSVKVSCKASGYAFSDDWMNWLKQRPGQGLEWMGQIYPGTGDTNYNVKFKGKVTLTADKSTSTAYMELSSLRSEDTAVYFCARDEDYFGGDPWFVYW GQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY GPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0376] >1A1C6-H5L9-AM22 light chain amino acid sequence (SEQ ID NO: 103) (signal peptide is underlined)

[0377] MHSSALLCCLVLLTGVRA DIQMTQSPSSSLSASVGDRVTITCRASQWIGTWLAWYQQKPGKSPQLLIYAATRLDWAVPSRFSGSGSGTDFTFTISSLQPEDFATYYCQQFYRHPLTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0378] Example 6 Effect of anti-IL-31RA antibodies on IL-6 cytokine family signaling

[0379] IL-31 and OSM belong to the IL-6 cytokine family and share a receptor, OSMR. IL-6 or OSM can promote the proliferation of human erythroid leukemia cells, TF-1. To verify that anti-IL-31RA antibodies can selectively bind to IL-31RA without blocking the signaling pathways of OSM and IL-6 cytokines, we evaluated the cell proliferation inhibitory activity by adding anti-IL-31RA antibodies to the cell culture system. Briefly, TF-1 cells in the logarithmic growth phase were harvested and centrifuged at 1000 rpm for 5 minutes. The cells were washed twice with RPMI 1640 medium supplemented with 10% FBS, resuspended, and adjusted to a cell concentration of 5E4 / mL. The test antibody was diluted to 40 μg / mL in medium and serially diluted fivefold to prepare cytokine concentrations of 400 ng / mL for OSM and 40 ng / mL for IL-6. 50 μL of cells, 25 μL of sample, and 25 μL of cytokine were seeded into each well of a 96-well cell culture plate. The plates were then incubated at 37°C, 5% CO₂ for 72 hours. After incubation, the plates were removed from the incubator and equilibrated at room temperature for 30 minutes. 100 μL of CellCounting-Lite reagent (Vazyme, DD1101) was added to each well. The cells were shaken and mixed at room temperature for 2-5 minutes to fully lyse the cells. Chemiluminescence was measured using a multi-function microplate reader. Neutralizing antibodies against IL-6 and OSMR (purchased from Abinvivo) were used as positive controls, respectively. Figures 10A-10D As shown, the anti-IL-31RA antibody screened through hybridoma screening cannot block the signaling pathways mediated by IL-6 and OSM, which are both members of the IL-6 cytokine family, and its specific binding properties are not changed after affinity maturation.

[0380] Example 7 Antibody Constant Region Selection

[0381] 1. Preparation of antibodies with different constant regions

[0382] The optimal constant and variable region combinations were screened by linking the variable regions of anti-IL-31RA antibodies to different constant regions. The selected heavy chain constant regions included IgG4 S228P, IgG4 M1, IgG4 M2, IgG1, IgG1 M1, IgG1 M2, and IgG1 M3, whose sequences are shown below. The light chain constant regions were all kappa subtypes. A novel combination antibody was obtained by referring to the protocol in Example 4.3, illustratively labeled 1A1C6-H5L2-IgG4 M1.

[0383] >IgG4 M1 heavy chain constant region (SEQ ID NO: 104)

[0384] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0385] >IgG4 M2 heavy chain constant region (SEQ ID NO:105)

[0386] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLGK

[0387] >IgG1 M1 heavy chain constant region (SEQ ID NO:106)

[0388] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0389] >IgG1 M2 heavy chain constant region (SEQ ID NO:107)

[0390] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0391] >IgG1 M3 heavy chain constant region (SEQ ID NO:108)

[0392] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0393] >IgG1 heavy chain constant region (SEQ ID NO: 109)

[0394] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0395] 2. Neutralization Activity Detection

[0396] The neutralizing activity of antibodies with different constant regions was evaluated using the BAF3 reporter gene system, and the assay was performed according to the protocol described in "4. Activity identification of humanized antibodies" in Example 4. Figure 11 The results showed that the neutralizing activity of anti-IL-31RA antibodies was largely unaffected after replacement of different constant regions.

[0397] 3. Thermal stability test

[0398] Differential Scanning Fluorimetry (DSF) is used to evaluate protein thermal stability and screen for combination antibodies with higher Tm and relative stability. The sample was diluted to 0.5 mg / mL with sterile ultrapure water, and the reaction system was prepared as follows: 5 μL protein binding dye buffer, 2.5 μL 8× protein binding dye, and 12.5 μL antibody sample. The sample was placed on a fluorescent PCR instrument, and the experimental type was selected as the melting curve. The continuous mode was adopted, the scanning temperature was 25-95°C, and the equilibrium was 25°C for 5 minutes. The heating rate was 1%, the reporter group was ROX, and the quencher group was None. Protein Thermal Shift was used. TM Data were analyzed using ELISA Software v1.4. The temperature corresponding to the first peak and valley of the melting curve derivative was determined as the protein's denaturation temperature, Tm1. The temperature corresponding to the second peak and valley was determined as Tm2. The temperature corresponding to the third peak and valley was determined as Tm3. The Tm values for each antibody combination are shown in Table 18. The results show that the combination of different constant and variable regions significantly affects their thermal stability.

[0399] Table 18 Thermal stability of anti-IL-31RA antibodies

[0400] Samples Tm1(℃) Tm2(℃) 1A1C6-H5L2 65.5 73.9 1A1C6-H5L2-IgG4 M1 58.9 73.8 1A1C6-H5L2-IgG4 M2 64.4 73.6 1A1C6-H5L2-IgG1 70.2 NA 1A1C6-H5L2-IgG1 M3 57.9 74.3

[0401] 4. FcRn affinity detection

[0402] The affinity of anti-IL-31RA antibodies for FcRn is correlated with their in vivo pharmacokinetics. The affinity of different constant region antibodies for human FcRn was measured using SPR to screen for combinations with high binding activity. Detection was performed using an NTA chip (Cytiva, Cat. No. 28994951) coupled with a Biacore 8K instrument. 0.1 μg / mL human FcRn (Acrobiosystems, FCN-H52W7) was injected at a flow rate of 10 μL / min, and capture was achieved to a response of approximately 40 RU. Different concentrations of anti-IL-31RA antibodies were injected at a flow rate of 30 μL / min for 90 s, and dissociation was monitored for 210 s. Finally, the chip was regenerated by flowing glycine (pH 1.7) at a flow rate of 20 μL / min for 30 s. The kinetic parameters were calculated using Bia-evaluation analysis software as shown in Table 19. The results showed that the affinity of some combinations to human FcRn was significantly improved, suggesting that their pharmacokinetic characteristics in vivo may be improved.

[0403] Table 19 Affinity of anti-IL-31RA antibodies to human FcRn

[0404]

[0405] Example 8 Evaluation of Anti-IL-31RA Antibody Stability

[0406] The anti-IL-31RA antibody was tested for factors affecting high temperature, freeze-thaw, and low pH incubation, and the activity and critical quality attributes of the samples were analyzed and tested to assess its developability. The sample grouping and processing procedures are briefly described as follows: A. High-temperature group: samples were placed in a 40°C constant-temperature incubator for 10 days; B. Freeze-thaw group: samples were frozen in a -80°C ultra-low temperature freezer for 1 hour, removed and thawed in a 2-8°C refrigerator for 1 hour until solution, and frozen and thawed five times; C. Low-pH group: samples were adjusted to pH 3.5 with 1% HCl, kept at room temperature for 2.5 hours, and then adjusted to the initial pH with 1% NaOH; D. Control group: samples were stored at 2-8°C without treatment. The above-mentioned differently treated samples were subjected to size exclusion chromatography (SEC) for purity identification, and the column model was TSKgel super SW3000. At the same time, the thermal stability and neutralization activity were tested according to the schemes of "3. Thermal Stability Test" in Example 7 and "4. Activity Identification of Humanized Antibodies" in Example 4, respectively. The results are summarized in Tables 20 and Figures 12A-12C The test data show that under low pH incubation conditions, anti-IL-31RA antibodies are prone to aggregation, but their thermal stability and neutralization activity do not change significantly, indicating that anti-IL-31RA antibodies are relatively stable.

[0407] Table 20 Anti-IL-31RA Antibody Stability

[0408]

[0409]

[0410] Example 9 Anti-IL-31RA Antibody Binding Epitope Analysis

[0411] 1. Preparation of extracellular domain proteins containing different structural domains

[0412] The human IL-31RA protein extracellular region has five domains. To identify the epitopes bound by anti-IL-31RA antibodies, IL-31RA proteins with different domains were constructed. Referring to Example 1, IL-31RA sequences of varying lengths containing a His-tag were ligated into the pTT5 vector to construct expression plasmids. Transient expression was performed using the Expi 293F expression system, and protein samples were purified using a nickel column. The sequences are shown below.

[0413] >Human IL-31RA (20-225) amino acid sequence (SEQ ID NO: 131, signal peptide is underlined)

[0414] MGWSCIILFLVATATGVHS ALPAKPENISCVYYYRKNLTCTWSPGKETSYTQYTVKRTYAFGEKHDNCTTNSSTSENRASCSFFLPRITIPDNYTIEVEAENGDGVIKSHMTYWRLENIAKTEPP KIFRVKPVLGIKRMIQIEWIKPELAPVSSDLKYTLRFRTVNSTSWMEVNFAKNRKDKNQTYNLTGLQPFTEYVIALRCAVKESKFWSDWSQEKMGMTEEEHHHHHH

[0415] >Human IL-31RA (20-315) amino acid sequence (SEQ ID NO: 132, signal peptide is underlined)

[0416] MGWSCIILFLVATATGVHS ALPAKPENISCVYYYRKNLTCTWSPGKETSYTQYTVKRTYAFGEKHDNCTTNSSTSENRASCSFFLPRITIPDNYTIEVEAENGDGVIKSHMTYWRLENIAKTEPPKIFRVKPVLGIKRMIQIEWIKPELAPVSSDLKYTLRFRTVNSTSW MEVNFAKNRKDKNQTYNLTGLQPFTEYVIALRCAVKESKFWSDWSQEKMGMTEEEAPCGLELWRVLKPAEADGRRPVRLLWKKARGAPVLEKTLGYNIWYYPESNTNLTETMNTTNQQLELHLGGESFWVSMISYNSLGKSPVATHHHHHH

[0417] >Human IL-31RA (20-416) amino acid sequence (SEQ ID NO: 133, signal peptide is underlined)

[0418] MGWSCIILFLVATATGVHSALPAKPENISCVYYYRKNLTCTWSPGKETSYTQYTVKRTYAFGEKHDNCTTNSSTSENRASCSFFLPRITIPDNYTIEVEAENGDGVIKSHMTYWRLENIAKTEPPKIFRVKPVLGIKRMIQIEWIKPELAPVSSDLKYTLRFRTVNSTSWMEVNFAKNRKDKNQTYNLTGLQPFTEYVIALRCAVKESKFWSDWSQEKMG MTEEEAPCGLELWRVLKPAEAADGRRPVRLLWKKARGAPVLEKTLGYNIWYYPESNTNLTETMNTTNQQLELHLGGESFWVSMISYNSLGKSPVATLRIPAI QEKSFQCIEVMQACVAEDQLVVKWQSSALDVNTWMIEWFPDVDSEPTTLSWESVSQATNWTIQQDKLKPFWCYNISVYPMLHDKVGEPYSIQAYAHHHHHH

[0419] 2. Detection of the Binding Ability of Anti-IL-31RA Antibodies to Different Domain Antigens

[0420] Refer to Example 4, "4. Identification of Humanized Antibody Activity," and use ELISA to identify the binding activity of anti-IL-31RA antibodies to IL-31RA of different lengths. Figures 13A-13D As shown, the binding epitope of 1A1C6-H5L9 to IL-31RA is similar to that of the control antibody nemolizumab, both of which can bind to the cytokine binding domain (CBD) of IL-31RA. However, 2G1G1-H1L1 can only bind to the full-length extracellular domain of IL-31RA and has almost no binding ability to different truncated forms of IL-31RA. This suggests that 2G1G1-H1L1 may have a differential binding epitope from the control antibody. Considering that the purified protein may differ conformationally from the membrane protein receptor, the epitope was further identified by overexpression in cells.

[0421] 3. Competitive Binding of Anti-IL-31RA Antibody and Control Antibody

[0422] Using HEK293 cells overexpressing IL31RA / OSMR, flow cytometry was used to further determine whether the binding epitopes of the anti-IL-31RA antibody and the control antibody overlapped. Nemolizumab was labeled with a biotin labeling kit to obtain nemolizumab-Biotin. FACS was then used to assess the competitive binding activity of the anti-IL-31RA antibody and nemolizumab-Biotin on the overexpressing cells to determine the epitope differences between the anti-IL-31RA antibody and the control antibody. HEK293-huIL31RA / OSMR cells were trypsinized and harvested, and resuspended in PBS + 2% FBS to adjust the cell density to 2E6 / mL, with approximately 1E5 cells per well. The antibody sample was diluted to 120 μg / mL (final concentration 30 μg / mL) in the wash buffer and serially diluted threefold; nemolizumab-Biotin 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 sequence and incubated at 4°C for 30 minutes. The 96-well plate was centrifuged at 1000 rpm for 5 minutes, washed once with PBS + 2% FBS, and 100 μL of 1:100 diluted APC-SA flow cytometry antibody was added to each well. The cells were resuspended and mixed, and incubated at 4°C for 30 minutes. After incubation, the 96-well plate was centrifuged at 1000 rpm for 5 minutes, washed once with PBS + 2% FBS, and the cells were resuspended in 100 μL of PBS + 2% FBS wash solution per well. The signal value of the APC channel was detected on the flow cytometer. The test results are as follows: Figure 14A and Figure 14B As shown, the competitive inhibition rate of 2G1G1-H1L1 and the control antibody was about 88%, further indicating that there was a difference in the binding epitopes of 2G1G1-H1L1 and the control antibody.

[0423] Example 10 In vivo activity of anti-IL-31RA antibodies

[0424] 1. Role in IL-31-induced pruritus

[0425] Transgenic mice expressing human IL-31, IL-31RA, and OSMR (purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. by Junke Zhengyuan (Beijing) Pharmaceutical Research Co., Ltd.) were used to evaluate the in vivo activity of anti-IL-31RA antibodies (1A1C6-H5L2 and 2G1G1-H1L1). The pharmacological effects of anti-IL-31RA antibodies in pruritus were investigated using a human IL-31-induced itch model. Mice were acclimated in acrylic cages for at least 1 hour, then injected with 10 μg of human IL-31 protein via the tail vein. Their behavior was videotaped for 3 hours. The video recordings were replayed to determine the total number of scratches per designated time period. The scratching maneuver was described as follows: the mouse extended its hind paw toward the itchy area, tilted its head toward the hind paw, moved rapidly several times, and then was placed back on the ground. Each time the mouse completed this sequence of movements, it was counted as one scratching bout, and the number of scratches per hour was calculated. Anti-IL-31RA antibody was intravenously administered 1 h before IL-31 protein injection. The administration time was recorded as Day 1. Human IL-31 was injected on Day 1 and Day 7 to induce pruritus. The number of scratching and scratching latency of different groups were recorded as follows: Figure 15A 、 Figure 15B and Figure 16 As shown in the results, transgenic mice injected with human IL-31 protein showed a significant increase in itch-scratching behavior within 3 hours. Anti-IL-31RA antibodies can significantly inhibit itch behavior and quickly delay the scratching latency. Its efficacy is slightly better than that of the positive control antibody. Figure 17 As shown, obvious dermatitis symptoms were observed in some transgenic mice in the positive drug group 7 days after administration, which is consistent with the adverse reaction of atopic dermatitis aggravation in clinical practice. However, the hair of all mice in the test group was shiny, and no dermatitis symptoms were observed, suggesting that the anti-IL-31RA antibody disclosed in this patent may have better safety.

[0426] 2. Effects in the Dinitrofluorobenzene-induced Dermatitis Model

[0427] The efficacy of anti-IL-31RA antibodies (1A1C6-H5L2, 2G1G1-H1L1) in atopic dermatitis was investigated using a dinitrofluorobenzene-induced contact dermatitis model. A 1% dinitrofluorobenzene (DNFB) solution was prepared by dissolving dinitrofluorobenzene in anhydrous ethanol. Transgenic mice were anesthetized by inhalation and the nape of their necks shaved. From Day 1 to Day 3, 25 μL of 1% DNFB solution was evenly applied to the nape of each mouse's neck. Sensitization was completed after the solution dried. On Day 6, after inhalation anesthesia, 20 μL of DNFB solution was evenly applied to the front and back of the right ear of the mouse. Sensitization was completed after the solution dried. The left ear of the mouse served as a control, and bilateral auricular thickness was measured and recorded. Anti-IL-31RA antibody was intravenously injected twice on Day 5 and Day 9, and the thickness of the bilateral auricles was recorded daily from Day 7 to Day 14. Blood was collected at the end point and the blood drug concentration was measured using ELISA. Figure 18 As shown in the results, anti-IL-31RA antibodies can inhibit the DNFB-induced auricle thickening symptoms, and the inhibitory effect is slightly better than that of the positive control antibody, suggesting that anti-IL-31RA antibodies may have better pharmacodynamics.

[0428] 3. Efficacy study of IL-31 in cynomolgus monkey pruritus model

[0429] Intravenous injection of IL-31 induces pruritus in cynomolgus macaques (purchased from Laibin Xingui Biotechnology Co., Ltd., quality certificate number 2024002). This pharmacodynamic model was used to evaluate the efficacy of anti-IL-31RA antibodies (1A1C6-H5L9-AM07 and 1A1C6-H5L9-AM22) in cynomolgus macaques. On Day (-5), baseline scratching frequency was recorded for 1 hour (one scratch was counted as one scratch using the fore or hind legs anywhere on the body). Following intravenous injection of 1 μg / kg of the pruritus factor cynomolgus macaque IL-31 (amino acid sequence shown in SEQ ID NO:8), scratching frequency was recorded for 2 hours as the pre-dose scratching frequency. Animals were then divided into groups balanced according to pre-dose scratching frequency (2 animals per blank control and positive control group, 3 animals per group for the candidate molecule test product group). On Day 0, each group received a subcutaneous injection of 1 mg / kg anti-IL-31RA antibody. On Day 7, Day 14, and Day 28 after administration, 1 μg / kg of the pruritus factor cynomolgus monkey IL-31 was intravenously injected. The number of scratching times of the cynomolgus monkeys was recorded 2 hours after itch induction. The effects of cynomolgus monkey IL-31 injection at different time points after subcutaneous administration on the number of itch and inhibition rate in each group of cynomolgus monkeys are shown in Figure 2. Figure 19 and Figure 20 As shown, the anti-IL-31RA antibody can significantly inhibit the itch behavior of crab-eating macaques, and its efficacy is better than that of the positive control antibody after a longer period of time (≥14 days) of antibody injection.

Claims

1. An antibody or antigen-binding fragment thereof targeting IL-31RA, 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, the amino acid sequence of HCDR1 being as shown in SEQ ID NO: 10, the amino acid sequence of HCDR2 being as shown in SEQ ID NO: 11, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 115, and the amino acid sequence of HCDR3 being as shown in SEQ ID NO: 12, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 73 or SEQ ID NO: 80; the light chain variable region comprising LCDR1, LCDR2 and LCDR3, the amino acid sequence of LCDR1 being as shown in SEQ ID NO: 14, SEQ ID NO: 67, SEQ ID NO: 71 or SEQ ID NO: 83 or SEQ ID NO: 116, and the amino acid sequence of LCDR2 being as shown in SEQ ID NO: 13, SEQ ID NO: 147, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165 NO:15, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:77 or SEQ ID NO:81, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:16, SEQ ID NO:68, SEQ ID NO:75, SEQ ID NO:78; or, The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively; or, The amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region are shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region are shown in SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; or, The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 115 and SEQ ID NO: 12, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 116, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; or, The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 116, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; or, The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 115 and SEQ ID NO: 12, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; or, The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 116, SEQ ID NO: 15 and SEQ ID NO: 117, respectively; or, The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 65 and SEQ ID NO: 66, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 67, SEQ ID NO: 15 and SEQ ID NO: 68, respectively; or, The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 69 and SEQ ID NO: 70, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 71, SEQ ID NO: 15 and SEQ ID NO: 68, respectively; or, The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 72 and SEQ ID NO: 73, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 67, SEQ ID NO: 74 and SEQ ID NO: 75, respectively; or, The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 76 and SEQ ID NO: 70, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 71, SEQ ID NO: 77 and SEQ ID NO: 78, respectively; or, The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 79 and SEQ ID NO: 80, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 71, SEQ ID NO: 81 and SEQ ID NO: 68, respectively; or, The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 65 and SEQ ID NO: 66, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 67, SEQ ID NO: 77 and SEQ ID NO: 68, respectively; or, The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 82 and SEQ ID NO: 66, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 83, SEQ ID NO: 15 and SEQ ID NO: 68, respectively; or, The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 84 and SEQ ID NO: 73, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO: 67, SEQ ID NO: 77 and SEQ ID NO: 75, respectively; or, The amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 85 and SEQ ID NO: 73, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region are shown in SEQ ID NO: 67, SEQ ID NO: 74 and SEQ ID NO: 75, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein The antibody or antigen-binding fragment thereof comprises the following (i) or (ii): (i) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13; or, The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 21; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 24, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 42, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 43; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 46, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 53; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 46, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 54; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 46, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 55; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 47, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 53; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 47, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 54; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 47, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 55; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 48, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 53; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 48, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 54; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 48, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 55; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 49, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 53; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 49, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 54; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 49, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 55; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 50, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 54; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 50, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 56; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 50, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 57; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 50, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 58; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 50, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 128; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 51, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 54; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 51, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 56; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 51, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 57; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 52, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 54; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 52, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 56; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 52, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 57; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 130, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 129; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 110, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 111; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 112, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 113; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 112, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 114; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 86, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 87; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 88, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 89; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 90, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 91; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 118, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 119; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 120, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 121; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 86, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 122; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 123, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 124; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 125, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 126; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 127, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 91; or (ii) the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:9, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:13; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 17, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 21; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 24, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 28; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 42, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 43; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 46, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 53; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 46, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 54; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:46, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:55; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 47, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 53; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 47, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 54; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 47, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 55; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 48, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 53; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 48, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 54; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 48, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 55; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 49, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 53; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 49, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 54; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 49, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 55; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:50, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:54; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:50, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:56; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:50, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:57; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:50, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:58; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:50, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:128; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:51, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:54; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:51, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:56; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:51, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:57; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:52, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:54; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:52, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:56; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:52, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:57; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:52, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:57; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 110, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 111; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 112, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 113; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 112, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 114; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 86, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 87; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 88, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 89; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 90, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 91; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 118, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 119; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 120, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 121; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 86, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 122; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 123, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 124; or the heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 125, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 126; or The heavy chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 127, and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:

91.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein The antibody or antigen-binding fragment thereof satisfies one or more of the following: (1) The antibody is a full-length antibody, and the antigen-binding fragment is Fab, Fab', F(ab')2 or scFv; (2) The antibody or antigen-binding fragment thereof is a monospecific antibody, a bispecific antibody, or a multispecific antibody; (3) The antibody or antigen-binding fragment thereof is a monoclonal antibody or a polyclonal antibody; (4) The antibody or antigen-binding fragment thereof is a mouse antibody or a humanized antibody; Preferably, when the antibody is a full-length antibody, the full-length antibody comprises a heavy chain constant region and a light chain constant region; the heavy chain constant region is selected from the heavy chain constant region of IgG, IgA, IgM, IgE and IgD, preferably the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4; the light chain constant region is a κ chain or a λ chain; More preferably, the heavy chain constant region is IgG4, and the amino acid sequence of the heavy chain constant region is preferably shown in SEQ ID NO:44, SEQ ID NO:104 or SEQ ID NO:105; or, the heavy chain constant region is IgG1, and the amino acid sequence of the heavy chain constant region is preferably shown in SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108 or SEQ ID NO:109; The light chain constant region is a κ chain, and the amino acid sequence of the light chain constant region is preferably as shown in SEQ ID NO:

45.

5. 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 4.

6. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid according to claim 5; Preferably, the backbone of the recombinant expression vector is a plasmid, cosmid, phage or viral vector.

7. A transformant, characterized in that: The transformant comprises the recombinant expression vector according to claim 6; 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, SP2 / 0 cells, HEK293 cells or CHO cells such as CHO-K1 cells.

8. A method for preparing an antibody or an antigen-binding fragment thereof targeting IL-31RA, characterized in that: The method comprises the steps of culturing the transformant according to claim 7, and obtaining the antibody or antigen-binding fragment thereof targeting IL-31RA from the culture.

9. 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 4, and a pharmaceutically acceptable carrier.

10. A kit, characterized in that The kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the nucleic acid according to claim 5, the recombinant expression vector according to claim 6, the transformant according to claim 7 and / or the pharmaceutical composition according to claim 9; Preferably, the kit further comprises a reagent for detecting the binding of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 to IL-31RA.

11. A method for detecting IL-31RA, characterized in that: The method comprises the following steps: (1) contacting a sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the kit according to claim 10; (2) quantitatively or qualitatively determining the IL-31RA in the sample based on the binding of the antibody or antigen-binding fragment thereof, or the kit according to claim 10, to the sample; The method is not for diagnostic or therapeutic purposes.

12. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the nucleic acid according to claim 5, the recombinant expression vector according to claim 6, or the transformant according to claim 7 in preparing a reagent for detecting IL-31RA.

13. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the nucleic acid according to claim 5, the recombinant expression vector according to claim 6 or the transformant according to claim 7, the pharmaceutical composition according to claim 9 and / or the kit according to claim 10 in the preparation of a medicament for diagnosing, preventing and / or treating an IL-31RA-related disease; Preferably, the IL-31RA-related disease is an inflammatory disease such as atopic dermatitis, prurigo nodularis, or systemic sclerosis.

14. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the nucleic acid according to claim 5, the recombinant expression vector according to claim 6 or the transformant according to claim 7, the pharmaceutical composition according to claim 9 and / or the kit according to claim 10 in the preparation of a drug for relieving or eliminating pruritus; Preferably, the pruritus is pruritus associated with chronic kidney disease.

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