Antibody targeting OSMR and use thereof

Abstract

Disclosed are an antibody targeting OSMR and the use thereof. Specifically, disclosed is an antibody against the OSMR protein or an antigen-binding fragment thereof. The antibody against the OSMR protein or the antigen-binding fragment thereof is capable of specifically binding to the OSMR protein and effectively blocking cellular inflammatory responses, and has excellent pharmacokinetic properties.

Description

Antibodies targeting OSMR and their applications

[0001] This application claims priority to Chinese patent application 2025103735012, filed on March 27, 2025. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biomedicine, and more specifically to an antibody targeting OSMR and its applications. Background Technology

[0003] Oncostatin M (OSM) and interleukin-31 (IL-31) belong to the IL-6 cytokine family and share a single receptor, oncostatin M receptor beta (OSM receptor beta, OSMR). OSM signaling is transmitted via a heterologous receptor complex of OSMR and glycoprotein 130 (gp130), while IL-31, in conjunction with OSMR, transduces signals through interleukin-31 receptor α (IL-31 receptor α, IL-31RA), inducing activation of the JAK / STAT and MAPK pathways, and leading to the production of inflammatory chemokines, cytokines, leukocyte adhesion factors, and extracellular matrix components. OSMR is highly expressed in various non-hematopoietic mesenchymal cells, including fibroblasts, endothelial cells, smooth muscle cells, osteoblasts, and adipocytes, as well as hepatocytes, mesothelial cells, glial cells, and epithelial cells from multiple organs. OSM and IL-31 are mainly expressed by T lymphocytes, macrophages and neutrophils, and their expression is upregulated when there is inflammatory stimulation.

[0004] Existing research has shown that OSMR plays an important role in various inflammatory diseases, including atopic dermatitis, rheumatoid arthritis, pulmonary fibrosis, multiple sclerosis, and inflammatory bowel disease. OSM and IL-31 expression has been found in the skin of patients with psoriasis and atopic dermatitis. OSM and IL-31 induce various pathological responses through OSMR signaling, including inflammation, extracellular matrix remodeling, pain, and pruritus. Therefore, OSMR is a promising therapeutic target for treating pruritic skin diseases, rheumatoid arthritis, inflammatory bowel disease, and sclerosis. Blocking OSMR helps inhibit the release of chemokines and inflammatory factors, providing more possibilities for the treatment of atopic dermatitis, psoriasis, or other OSMR-related diseases.

[0005] Therefore, there is an urgent need in this field to develop highly specific and effective OSMR-targeting antibody drugs for the treatment of inflammatory diseases caused by OSMR. Summary of the Invention

[0006] The purpose of this invention is to provide an antibody targeting OSMR and its application.

[0007] In one aspect, the present invention provides an antibody against an OSMR protein or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and said light chain variable region comprising LCDR1, LCDR2, and LCDR3; wherein said heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO:26, 4, 6, 8, 10, 23, 24, 27, 28, 29, 39, 40, 41, 42, 43, 54, 56, 58, 61, or 63, or has at least 85% sequence identity with them respectively; and / or said light chain variable region comprises amino acid sequences of SEQ ID NO:26, 4, 6, 8, 10, 23, 24, 27, 28, 29, 39, 40, 41, 42, 43, 54, 56, 58, 61, or 63. LCDR1, LCDR2, and LCDR3 in the light chain variable region shown in NO:35, 5, 7, 9, 11, 33, 34, 47, 48, 49, 50, 52, 64, 65, 67, or 68, or having at least 85% sequence identity with them respectively.

[0008] In some embodiments, HCDR1 comprises an amino acid sequence as shown in SEQ ID NO:25, 14, 20 or 36; HCDR2 comprises an amino acid sequence as shown in SEQ ID NO:21, 15, 37, 53, 55, 57, 59, 62 or 69; HCDR3 comprises an amino acid sequence as shown in SEQ ID NO:22, 16, 38 or 60; LCDR1 comprises an amino acid sequence as shown in SEQ ID NO:30, 17, 44, 51 or 70; LCDR2 comprises an amino acid sequence as shown in SEQ ID NO:31, 18, 45 or 71; and / or LCDR3 comprises an amino acid sequence as shown in SEQ ID NO:32, 19, 46 or 66.

[0009] In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:25; HCDR2 comprises the amino acid sequence shown in SEQ ID NO:21; and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:22; or HCDR1 comprises the amino acid sequence shown in SEQ ID NO:14; HCDR2 comprises the amino acid sequence shown in SEQ ID NO:15; and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:16; or HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20; HCDR2 comprises the amino acid sequence shown in SEQ ID NO:69; and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:22; or HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20; HCDR2 comprises the amino acid sequence shown in SEQ ID NO:21; and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:22; or HCDR1 comprises the amino acid sequence shown in SEQ ID NO:36; HCDR2 comprises the amino acid sequence shown in SEQ ID NO:37; and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:38; or HCDR1 comprises the amino acid sequence shown in SEQ ID NO:25 ...25; and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:21; or HCDR3 comprises the amino acid sequence shown in SEQ ID NO:22; and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:22; and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:22; or HCDR1 comprises the amino acid sequence shown in SEQ ID NO:25; HCDR2 comprises the amino acid sequence shown in SEQ ID NO The amino acid sequence shown in SEQ ID NO:25; HCDR2 contains the amino acid sequence shown in SEQ ID NO:53; and HCDR3 contains the amino acid sequence shown in SEQ ID NO:22; or HCDR1 contains the amino acid sequence shown in SEQ ID NO:25; HCDR2 contains the amino acid sequence shown in SEQ ID NO:55; and HCDR3 contains the amino acid sequence shown in SEQ ID NO:22; or HCDR1 contains the amino acid sequence shown in SEQ ID NO:25; HCDR2 contains the amino acid sequence shown in SEQ ID NO:57; and HCDR3 contains the amino acid sequence shown in SEQ ID NO:22; or HCDR1 contains the amino acid sequence shown in SEQ ID NO:25; HCDR2 contains the amino acid sequence shown in SEQ ID NO:59; and HCDR3 contains the amino acid sequence shown in SEQ ID NO:60; or HCDR1 contains the amino acid sequence shown in SEQ ID NO:25; HCDR2 contains the amino acid sequence shown in SEQ ID NO:62; and HCDR3 contains the amino acid sequence shown in SEQ ID NO:60; and LCDR1 contains the amino acid sequence shown in SEQ ID NO:25; HCDR2 contains the amino acid sequence shown in SEQ ID NO:62; and HCDR3 contains the amino acid sequence shown in SEQ ID NO:60; and LCDR1 contains the amino acid sequence shown in SEQ ID NO:25. The amino acid sequence shown in NO:30; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:31;The LCDR3 contains the amino acid sequence shown in SEQ ID NO:32; or the LCDR1 contains the amino acid sequence shown in SEQ ID NO:17; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:19; or the LCDR1 contains the amino acid sequence shown in SEQ ID NO:70; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:71; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:32; or the LCDR1 contains the amino acid sequence shown in SEQ ID NO:44; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:45; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:46; or the LCDR1 contains the amino acid sequence shown in SEQ ID NO:51; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:46; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:29; or the LCDR1 contains the amino acid sequence shown in SEQ ID NO:30; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:31; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:19. The amino acid sequence shown at NO:66.

[0010] In some embodiments, wherein (i) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:25; HCDR2 comprises the amino acid sequence shown in SEQ ID NO:21; HCDR3 comprises the amino acid sequence shown in SEQ ID NO:22; LCDR1 comprises the amino acid sequence shown in SEQ ID NO:30; LCDR2 comprises the amino acid sequence shown in SEQ ID NO:31; and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:32; or (ii) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:14; HCDR2 comprises the amino acid sequence shown in SEQ ID NO:15; HCDR3 comprises the amino acid sequence shown in SEQ ID NO:16; LCDR1 comprises the amino acid sequence shown in SEQ ID NO:17; LCDR2 comprises the amino acid sequence shown in SEQ ID NO:18; and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:19; or (iii) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20; HCDR2 comprises the amino acid sequence shown in SEQ ID NO:21; and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:2 ...iv) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20; HCDR2 comprises the amino acid sequence shown in SEQ ID NO:21; and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:22; or (iv) HCDR3 comprises the amino acid sequence shown in SEQ ID NO:22; or (iv) The amino acid sequence shown in SEQ ID NO:22; LCDR1 contains the amino acid sequence shown in SEQ ID NO:30; LCDR2 contains the amino acid sequence shown in SEQ ID NO:31; and LCDR3 contains the amino acid sequence shown in SEQ ID NO:32; or (iv) HCDR1 contains the amino acid sequence shown in SEQ ID NO:20; HCDR2 contains the amino acid sequence shown in SEQ ID NO:69; HCDR3 contains the amino acid sequence shown in SEQ ID NO:22; LCDR1 contains the amino acid sequence shown in SEQ ID NO:70; LCDR2 contains the amino acid sequence shown in SEQ ID NO:71; and LCDR3 contains the amino acid sequence shown in SEQ ID NO:32; or (v) HCDR1 contains the amino acid sequence shown in SEQ ID NO:36; HCDR2 contains the amino acid sequence shown in SEQ ID NO:37; HCDR3 contains the amino acid sequence shown in SEQ ID NO:38; LCDR1 contains the amino acid sequence shown in SEQ ID NO:44; LCDR2 contains the amino acid sequence shown in SEQ ID NO:45; and LCDR3 contains the amino acid sequence shown in SEQ ID NO:32. (vi) The HCDR1 contains the amino acid sequence shown in SEQ ID NO:46;The HCDR2 contains the amino acid sequence shown in SEQ ID NO:37; the HCDR3 contains the amino acid sequence shown in SEQ ID NO:38; the LCDR1 contains the amino acid sequence shown in SEQ ID NO:51; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:45; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:46; or (vii) the HCDR1 contains the amino acid sequence shown in SEQ ID NO:25; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:53; the HCDR3 contains the amino acid sequence shown in SEQ ID NO:22; the LCDR1 contains the amino acid sequence shown in SEQ ID NO:30; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:31; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:32; or (viii) the HCDR1 contains the amino acid sequence shown in SEQ ID NO:25; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:55; the HCDR3 contains the amino acid sequence shown in SEQ ID NO:22; the LCDR1 contains the amino acid sequence shown in SEQ ID NO:30; the LCDR2 ...6; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:37; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:38; the LCDR1 contains the amino acid sequence shown in SEQ ID NO:39; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:30; the LCDR2 contains the amino acid The amino acid sequence shown in SEQ ID NO:31; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:32; or (ix) the HCDR1 contains the amino acid sequence shown in SEQ ID NO:25; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:57; the HCDR3 contains the amino acid sequence shown in SEQ ID NO:22; the LCDR1 contains the amino acid sequence shown in SEQ ID NO:30; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:31; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:32; or (x) the HCDR1 contains the amino acid sequence shown in SEQ ID NO:25; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:59; the HCDR3 contains the amino acid sequence shown in SEQ ID NO:60; the LCDR1 contains the amino acid sequence shown in SEQ ID NO:30; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:31; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:32; or (xi) the HCDR1 contains the amino acid sequence shown in SEQ ID NO:25; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:32 ... and the LCDR1 contains the amino acid sequence shown in SEQ ID NO:31; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:32; and the LCDR1 contains the amino acid sequence shown in SEQ ID NO:25; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:32; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:32; and the LCDR1 contains The amino acid sequence shown in NO:62; the HCDR3 contains the amino acid sequence shown in SEQ ID NO:60;The LCDR1 contains the amino acid sequence shown in SEQ ID NO:30; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:31; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:32; or (xii) the HCDR1 contains the amino acid sequence shown in SEQ ID NO:25; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:59; the HCDR3 contains the amino acid sequence shown in SEQ ID NO:60; the LCDR1 contains the amino acid sequence shown in SEQ ID NO:30; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:31; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:32.

[0011] In some embodiments, the frame region of the heavy chain variable region and / or the light chain variable region is a human-derived, mouse-derived, or rabbit-derived frame region.

[0012] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:26, 4, 6, 8, 10, 23, 24, 27, 28, 29, 39, 40, 41, 42, 43, 54, 56, 58, 61, or 63, or comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:26, 4, 6, 8, 10, 23, 24, 27, 28, 29, 39, 40, 41, 42, 43, 54, 56, 58, 61, or 63; and / or the light chain variable region comprises an amino acid sequence as .... The amino acid sequence shown in SEQ ID NO:35, 5, 7, 9, 11, 33, 34, 47, 48, 49, 50, 52, 64, 65, 67 or 68, or an amino acid sequence containing at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the sequence shown in SEQ ID NO:35, 5, 7, 9, 11, 33, 34, 47, 48, 49, 50, 52, 64, 65, 67 or 68.

[0013] In some embodiments, wherein (i) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:26 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:35; or (ii) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:4 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:5; or (iii) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:6 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:7; or (iv) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:9; or (v) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:10 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:11; or (vi) 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:52; or (vii) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:35. The variable region comprises the amino acid sequence shown in SEQ ID NO:54, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:64; or (viii) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:56, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:64; or (ix) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:58, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:64; or (x) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:61, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:65; or (xi) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:63, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:64; or (xii) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:61, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:64.

[0014] In some embodiments, the antibody or its antigen-binding fragment is a full-length antibody, Fab, Fab', F(ab')2, Fv, or scFv. In some embodiments, the antibody is a full-length antibody.

[0015] In some embodiments, the antibody further includes a constant region comprising a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region is a heavy chain constant region derived from human IgA, IgD, IgE, IgG, or IgM, and / or the light chain constant region is a light chain constant region derived from the κ chain or λ chain; more preferably, the heavy chain constant region is a heavy chain constant region derived from human IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

[0016] In some embodiments, the heavy chain constant region contains mutations at one or more amino acid residue sites selected from the group consisting of: S228, F234, L235, M252, S254, T256, K288, T307, M428, N424, and Y436, wherein the amino acid residue sites are numbered according to the EU numbering system. In some embodiments, the amino acid residues in the heavy chain constant region contain mutations at one or more of the group consisting of: S228P, F234A, L235A, T307H, N434A, M252Y, S254T, T256E, M428L, Y436T, and T307Q, wherein the amino acid residue sites are numbered according to the EU numbering system.

[0017] In some embodiments, the constant region includes a heavy chain constant region and a light chain constant region. The amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:73, 12, or 72, or contains an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:73, 12, or 72. The amino acid sequence of the light chain constant region is as shown in SEQ ID NO:13, or contains an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:13.

[0018] In one aspect, the present invention provides an isolated nucleic acid that encodes an antibody or an antigen-binding fragment thereof as described in the present invention.

[0019] In one aspect, the present invention provides a recombinant expression vector comprising isolated nucleic acids as described in the present invention.

[0020] In some embodiments, the recombinant expression vector is a plasmid, granule, bacteriophage, or viral vector, preferably a retroviral vector, lentiviral vector, adenovirus vector, or adeno-associated virus vector.

[0021] In one aspect, the present invention provides a transformant comprising a nucleic acid isolated as described in the present invention or a recombinant expression vector as described in the present invention, wherein the host cell of the transformant is a prokaryotic cell or a eukaryotic cell.

[0022] In some embodiments, the host cell is a yeast cell or a mammalian cell, such as HEK293 cells or CHO cells; more preferably, the host cell is a 293F cell.

[0023] In one aspect, the present invention provides a method for preparing an antibody against an OSMR protein or an antigen-binding fragment thereof, the method comprising the steps of: culturing a transformant as described in the present invention under suitable conditions, and obtaining the antibody from the culture.

[0024] In one aspect, the present invention provides an immunoconjugate comprising an antibody or an antigen-binding fragment thereof as described in the present invention and a conjugation portion.

[0025] In some embodiments, the coupling portion is selected from: detectable markers, drugs, cytokines, radionuclides, enzymes, gold nanoparticles / nanorods, magnetic nanoparticles, viral capsid proteins or VLPs, and combinations thereof.

[0026] In some embodiments, the radionuclide includes diagnostic isotopes and / or therapeutic isotopes. In some embodiments, the diagnostic isotopes are selected from: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, and combinations thereof; and / or, the therapeutic isotopes are selected from: Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co. -60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, and combinations thereof.

[0027] In one aspect, the present invention provides a bispecific antibody or a multispecific antibody comprising an antibody or an antigen-binding fragment thereof as described in the present invention.

[0028] In one aspect, the present invention provides a pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment thereof as described in the present invention or an immunoconjugate as described in the present invention; and (ii) a pharmaceutically acceptable carrier.

[0029] In one aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof described herein, isolated nucleic acid, recombinant expression vector and / or transformant, bispecific antibody or multispecific antibody or pharmaceutical composition for the preparation of a medicament for treating OSMR-related diseases or conditions.

[0030] In some embodiments, the OSMR-related disease or condition is a disease or condition caused by abnormalities in the OSM or IL-31 signaling pathway mediated by OSMR.

[0031] In some embodiments, the OSMR-related diseases or conditions are selected from: atopic dermatitis, rheumatoid arthritis, pulmonary fibrosis, multiple sclerosis, inflammatory bowel disease, pruritus, hepatitis, atherosclerosis, tumorigenesis, pathological cardiac hypertrophy, and heart failure.

[0032] In one aspect, the present invention provides a method for treating OSMR-related diseases or conditions, comprising administering to a subject in need an effective amount of the antibody or antigen-binding fragment thereof described in the present invention, isolated nucleic acid, recombinant expression vector and / or transformant, bispecific antibody or multispecific antibody or pharmaceutical composition.

[0033] In some embodiments, the OSMR-related disease or condition is a disease or condition caused by abnormalities in the OSM or IL-31 signaling pathway mediated by OSMR.

[0034] In some embodiments, the OSMR-related diseases or conditions are selected from: atopic dermatitis, rheumatoid arthritis, pulmonary fibrosis, multiple sclerosis, inflammatory bowel disease, pruritus, hepatitis, atherosclerosis, tumorigenesis, pathological cardiac hypertrophy, and heart failure.

[0035] In one aspect, the present invention provides antibodies or antigen-binding fragments thereof as described herein, isolated nucleic acids, recombinant expression vectors and / or transformants, bispecific antibodies or multispecific antibodies or pharmaceutical compositions for the treatment of OSMR-related diseases or conditions.

[0036] In some embodiments, the OSMR-related disease or condition is a disease or condition caused by abnormalities in the OSM or IL-31 signaling pathway mediated by OSMR.

[0037] In some embodiments, the OSMR-related diseases or conditions are selected from: atopic dermatitis, rheumatoid arthritis, pulmonary fibrosis, multiple sclerosis, inflammatory bowel disease, pruritus, hepatitis, atherosclerosis, tumorigenesis, pathological cardiac hypertrophy, and heart failure.

[0038] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0039] The reagents and raw materials used in this invention are all commercially available.

[0040] The significant advantages of this invention are as follows: Compared to known OSMR-targeting antibodies, the antibody of this invention possesses different OSMR-binding epitopes, exhibits stronger affinity for OSMR proteins, significantly blocks OSMR-mediated OSM or IL-31 signaling pathways, and significantly inhibits OSM-induced cytokine release. Furthermore, the antibody of this invention effectively treats pruritus symptoms in mouse models, demonstrating significantly superior therapeutic efficacy compared to other known antibodies in the art. In addition, the antibody of this invention exhibits excellent pharmacokinetic properties, enabling it to exert a sustained and stable effect in animals. Attached Figure Description

[0041] Figure 1 shows the results of a competitive binding experiment of the present invention's antibody 4A12B11 and the control antibody KPL-716 to OSMR protein.

[0042] Figure 2 shows the results of the competitive binding experiments of the present invention's antibodies 9B10F11, 11A5C7, and 12E2D11 and the control antibody KPL-716 to OSMR protein.

[0043] Figure 3 shows the affinity of the antibody for the OSMR protein as determined by flow cytometry.

[0044] Figure 4 shows the blocking effect of each antibody on OSM-induced reporter cell activity.

[0045] Figure 5 shows the blocking effect of each antibody on IL31-induced reporter cell activity.

[0046] Figure 6 shows the blocking effect of each antibody on the release of MCP-1 from OSM-induced HDF cells (Human Dermal Fibroblasts).

[0047] Figure 7 shows the cross-reactivity results of each antibody with OSMR in cynomolgus monkeys.

[0048] Figure 8 shows the binding specificity of antibody hu9B10 to hOSMR.

[0049] Figure 9 shows the binding specificity of antibody hu12E2-1 to hOSMR.

[0050] Figure 10 shows the changes in body weight of mice during treatment in a mouse pruritus model.

[0051] Figure 11 shows the number of itch occurrences in each treatment group recorded on day 13 of the mouse itch model treatment.

[0052] Figure 12 shows the number of itch occurrences recorded on day 1 of the mouse itch model treatment, with each treatment group recording the number of itch occurrences over 60 minutes.

[0053] Figure 13 shows the difference in the number of itch occurrences per 60 minutes between the treatment groups on day 1 and day 13 in the mouse itch model treatment.

[0054] Figure 14 shows the difference in the number of itches per 60 minutes between the G4, G5, and G6 treatment groups on day 1 and day 13 in the mouse itch model treatment.

[0055] Figure 15 shows the changes in blood concentration of hu9B10-1 in cynomolgus monkeys at different time points. Detailed Implementation

[0056] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational steps used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below:

[0057] In one aspect of the invention, an antibody or antigen-binding fragment thereof specifically targeting the OSMR protein is provided.

[0058] In this invention, the letters in the amino acid sequence represent single-letter abbreviations of amino acids known in the art, such as those described in J. Biol. Chem, 243, p3558 (1968): alanine: Ala-A, arginine: Arg-R, aspartic acid: Asp-D, cysteine: Cys-C, glutamine: Gln-Q, glutamic acid: Glu-E, histidine: His-H, glycine: Gly-G, asparagine: Asn-N, tyrosine: Tyr-Y, proline: Pro-P, serine: Ser-S, methionine: Met-M, lysine: Lys-K, valine: Val-V, isoleucine: Ile-I, phenylalanine: Phe-F, leucine: Leu-L, tryptophan: Trp-W, threonine: Thr-T.

[0059] In this invention, the amino acid sequences of the listed complementarity determining regions (CDRs) are all as defined according to the Kabat numbering rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as Chothia based on the antibody's three-dimensional structure and the topology of the CDR rings, Kabat, AbM, Contact, the international ImMunoGeneTics database (IMGT) based on antibody sequence variability, and the North CDR definition based on affinity propagation clustering of numerous crystal structures. Those skilled in the art should understand that, unless otherwise specified, the terms "CDR" and "complementarity determining region" for a given antibody or its regions (e.g., variable regions) should be understood to encompass complementarity determining regions defined as described in any of the known schemes described above.

[0060] Therefore, when referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations). Although the scope of protection claimed by this invention is based on the sequence defined according to the IMGT numbering rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this invention.

[0061] In this invention, the term "full-length antibody" is used interchangeably to refer to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this invention) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in this invention) and a light chain constant region (abbreviated as CL in this invention). The light chain constant region consists of one domain: CL. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as λ or κ. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are classified as immunoglobulins IgA, IgD, IgE, IgG, and IgM. A complete antibody forms a "Y" shape. The stem of the Y is formed by the second and third constant regions of the two heavy chains (and, for IgE and IgM, a fourth constant region) linked together, and disulfide bonds (interchain) are formed in the hinge. Heavy chains γ, α, and δ have constant regions consisting of three tandem (in a row) Ig domains and hinge regions for increased flexibility; heavy chains μ and ε have constant regions consisting of four immunoglobulin domains. The second and third constant regions are referred to as the "CH2 domain" and the "CH3 domain," respectively. Each arm of Y includes a variable region of a single heavy chain and a first constant region that binds to a variable and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

[0062] In this invention, a "Fab fragment" consists of a light chain and a heavy chain, comprising the CH1 domain and a variable region. The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fc" region contains two heavy chain fragments containing the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain. A "Fab' fragment" contains a portion of a light chain and a heavy chain containing the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains of the two Fab' fragments to form the F(ab')2 molecule. An "F(ab')2 fragment" contains two light chains and two heavy chains containing portions of the constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Therefore, the F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains. The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody, but lacking the constant region.

[0063] In this invention, scFv refers to a single-chain antibody fragment, which includes a heavy chain variable region, a light chain variable region, and a linker peptide of 15-20 amino acids. The VL and VH domains enable the linker peptides to pair and form monovalent molecules as single polypeptide chains. Such scFv molecules may have a general structure: NH2-VL-linker peptide-VH-COOH or NH2-VH-linker peptide-VL-COOH.

[0064] In one aspect of the invention, a nucleic acid molecule encoding an antibody of the invention or an antigen-binding fragment thereof is provided, a vector containing said nucleic acid molecule, and a host cell and method for expressing the antibody of the invention or an antigen-binding fragment thereof are provided.

[0065] In this invention, "nucleic acid" refers to a nucleotide chain of any length and includes DNA and RNA. A nucleotide can be a deoxyribonucleotide, ribonucleotide, modified nucleotide or base, and / or its analogues, or any substrate capable of being incorporated into the chain by DNA or RNA polymerase.

[0066] In this invention, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that, when the construct contains a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, and the vector is contacted with a cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed in the cell, permits the expression of the mRNA, protein, polypeptide, or peptide by the host cell. The vectors of this invention are generally not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vectors of this invention can contain any type of nucleotide, including but not limited to DNA and RNA that can be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural, or modified nucleotides. Recombinant expression vectors can contain naturally occurring or non-naturally occurring nucleotide linkages, or both. In an exemplary aspect, modified nucleotides or non-naturally occurring nucleotide linkages do not impede transcription or replication of the vector.

[0067] The recombinant expression vector of the present invention can be any suitable recombinant expression vector capable of being used to transform or transfect one or more genes or sequences of interest into any suitable host cell and preferably to express the genes or sequences in the host cell. Suitable vectors include those designed for amplification and expansion or for expression or both of the above, and examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0068] In this invention, the term "host cell" refers to any type of cell that may contain the nucleic acids or vectors described herein. In exemplary aspects, the host cell is a eukaryotic cell, such as a plant, animal, fungus, or algae; or it may be a prokaryotic cell, such as a bacterium or protozoan.

[0069] In another aspect of the invention, a pharmaceutical composition is also provided.

[0070] In one embodiment, the pharmaceutical composition of the present invention comprises the antibody or its active fragment or fusion protein of the present invention, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intraperitoneal, intravenous, or local administration.

[0071] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described antibody (or conjugate thereof) of the present invention and a pharmaceutically acceptable carrier or excipient.

[0072] Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or aqueous solutions containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under aseptic conditions.

[0073] When using the pharmaceutical composition, a safe and effective amount of the antibody of the present invention or its antigen-binding fragment immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight, preferably about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0074] Furthermore, the pharmaceutical compositions of the present invention can also be used with other therapeutic agents for treating diseases or conditions caused by OSMR overexpression.

[0075] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0076] Example 1: Discovery of monoclonal antibodies targeting OSMR

[0077] 1.1 Immunity and Fusion

[0078] Spleen cells were isolated from Balb / c mice (purchased from Vital River Pharmaceuticals, Beijing) immunized with OSMR protein to isolate cells that showed a strong antibody-specific response to OSMR antigen. Spleen cells and myeloma cells were mixed at a 4:1 ratio, preheated at 37°C, centrifuged, and the supernatant was discarded. The bottom of the tube was gently tapped to loosen and evenly distribute the precipitated cells, and the mixture was incubated in a 37°C water bath. 1 mL of PEG was added to the mixed cells over 1 minute, mixing gently while adding. 2 mL of preheated SFM medium was added over 60 seconds, followed by 3 mL, 5 mL, 10 mL, and finally 30 mL over 3 minutes. The mixture was incubated at 37°C for 10 minutes. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in MD2001 Clone Easy medium containing HAT and hybridoma culture additive. The resuspended cells were then added to a total volume of 240 mL of medium and mixed thoroughly. The mixture was aliquoted into 96-well cell culture plates, 0.2 mL per well. After 6 days, the original medium was replaced with HAT medium. Observe the growth of hybridoma cells, and when they grow to more than 1 / 10 of the bottom area of ​​the well, aspirate the supernatant for antibody detection.

[0079] The amino acid sequence of OSMR is as follows (SEQ ID NO:1):

[0080] 1.2 Preliminary screening of positive hybridoma cells using reporter gene assay

[0081] To evaluate how antibodies block intracellular signal transduction by inhibiting OSM / OSMR interactions, we constructed the HEK293-OSMR-gp130-STAT3-Luc reporter cell line. HEK293 cells are human embryonic kidney cells. After transfection with cells expressing OSMR and gp130, HEK293 cells were further transfected with the STAT3-luciferase reporter gene to obtain a STAT3-driven luciferase gene-transduced cell line. In the presence of OSM, this luciferase reporter gene transcription was induced.

[0082] HEK293-OSMR-gp130-STAT3-Luc reporter cells in logarithmic growth phase were collected, counted, and 30,000 cells were added to each well. Hybridoma supernatant was added to 96-well white plates and incubated at 37°C for 30 minutes. Diluted OSM protein was added, and the plates were incubated at 37°C for 4.5 hours. 50 μL / well of One-lite chemiluminescent substrate reagent was added, and the plates were incubated at room temperature in the dark for 5 minutes. Chemiluminescence was then read using a microplate reader. Wells with good blocking activity were selected for transfection based on RLU values. Specific results are shown in Tables 1 and 2.

[0083] Table 1 Results of the first hybridoma fusion cell supernatant reporter gene screening method

[0084] Table 2. Results of the second hybridoma fusion cell supernatant reporter gene screening method

[0085] 1.3 Reporter cell experiments to identify hybridoma cell subclones

[0086] Cells from 11 positive wells were selected using the reporter gene assay and subjected to the first subcloning using the limiting dilution method. Cells from the positive clone wells in a 96-well plate were homogenized and transferred to a 24-well plate containing 1 mL of transfer medium. 20 μL of the suspended cell culture medium was added to 80 μL of transfer medium and mixed well. Cells were then counted. Based on the count results, a dilution medium containing a total of 100 cells was added to 20 mL of transfer medium and mixed thoroughly. 200 μL of the medium was then spread to each well of the 96-well plate. After all positive clones were sequentially spread, the plate was placed in a cell culture incubator. After 4-5 days, the 96-well plate was observed under a microscope and the number of single clone wells was recorded. Subcloning was performed using a reporter cell assay on day 7.

[0087] Based on RLU readings and cell status, cells from wells 4A12B11, 10G1D1, 2H10B7, 5A7F8, 9B10F11, 10H8D10, 11A5C7, 11H10D11, 12E2D11, and 13D11C9 were selected for transfection. Once the cells in the 24-well plates reached the logarithmic growth phase, they were transferred to 10cm diameter dishes for further culture. When the cells reached over 80% confluence with the bottom of the dish, they were cryopreserved.

[0088] Example 2: Expression and purification of hybridoma antibodies

[0089] After freezing, continue culturing the cells in serum-free medium until the hybridoma cells reach more than 80% confluence with the bottom of the cell plate. Then, separate the cells into individual plates, co-culturing approximately 50 mL for each clone. After 5-7 days, harvest the supernatant for purification. Centrifuge the harvested hybridoma cell culture supernatant, filter to clarify, and place on ice as the sample for loading. Equilibrate the protein A column with equilibration buffer for 5-8 column volumes. Then, pass the filtered supernatant through the column and collect the flow-through. After loading the sample, wash the chromatography column with equilibration buffer for 15-20 column volumes, or until the A280 absorbance drops below 20 mAu. Elute the bound antibody with elution buffer to obtain approximately 2.5 mL of eluted protein. Immediately adjust the pH of the elution buffer to neutral, and use a nanodrop to detect the antibody concentration and record the results.

[0090] Example 3: Preparation of an OSMR-targeting monoclonal antibody against Kiniksa as a reference antibody

[0091] Kiniksa's anti-OSMR monoclonal antibody, Vixarelimab (KPL-716), has a sequence referenced from patent US10421813B2, and the specific amino acid sequence is shown below:

[0092] H chain (SEQ ID NO:2):

[0093] L-chain (SEQ ID NO:3):

[0094] Example 4: Detecting the blocking effect of candidate molecules in reporter gene experiments

[0095] To evaluate how antibodies block intracellular signal transduction by inhibiting the IL31 / OSMR interaction, we constructed the HEK293-OSMR-IL31RA-STAT3-Luc reporter cell line. HEK293 cells are human embryonic kidney cells. After transfection with cells expressing OSMR and IL31RA, HEK293 cells were further transfected with the STAT3-luciferase reporter gene to obtain a STAT3-driven luciferase gene-transduced cell line. In the presence of IL31, the transcription of the luciferase reporter gene was induced.

[0096] HEK293-OSMR-gp130-STAT3-Luc and HEK293-OSMR-IL31RA-STAT3-Luc reporter cells in logarithmic growth phase were collected, and 30,000 cells were added to each well. Hybridoma antibody was added to 96-well white plates, and the plates were incubated at 37°C for 30 minutes. Diluted OSM protein was added to the wells of HEK293-OSMR-gp130-STAT3-Luc cells, and diluted IL31 protein was added to the wells of HEK293-OSMR-IL31RA-STAT3-Luc cells. The plates were incubated at 37°C for 4.5 hours, and 50 μL / well of One-lite chemiluminescent substrate reagent was added. After incubation at room temperature in the dark for 5 minutes, the chemiluminescence was read using a microplate reader. The results are shown in Table 3. The results were analyzed based on whether the reporter gene activation activity could be completely blocked and the IC50 reading. 50 Four clones, 4A12B11, 9B10F11, 11A5C7, and 12E2D11, were selected for further chimeric antibody construction and activity testing.

[0097] Table 3. Blocking activity of hybridoma antibodies in reporter gene assays.

[0098] Example 5: Detection of competitive binding between candidate molecules and KPL-716 antibody

[0099] Add 1 μg / mL of OSMR protein diluted in PBS to each well of an appropriate amount of microplate, and coat overnight at 4°C. Wash the coated cell plate three times with PBST, then add blocking buffer (PBS + 2% BSA + 0.01% Tween 20) and block at room temperature for about 1 hour. Discard the blocking buffer. Then mix diluted KPL-716 antibody and serially diluted hybridoma antibody, adding 100 μL to each well and incubating at room temperature for 1 hour. Wash three times with PBST, add 100 μL of secondary antibody diluted 1:50000 with antibody dilution buffer (PBS + 0.5% BSA + 0.01% Tween 20), and incubate at room temperature for 1 hour. Wash three times with PBST, add 100 μL of TMB solution for color development for about 5 minutes, then add stop solution to stop the color development and read the OD on a microplate reader. 450 .

[0100] The results showed that 4A12B11 and 12E2D11 competed with KPL-716 for binding to OSMR protein (as shown in Figures 1 and 2), while 9B10F11 and 11A5C7 did not compete with the KPL-716 antibody for binding to OSMR protein (as shown in Figure 2).

[0101] Example 6: Cloning, sequencing, and construction of chimeric antibodies for hybridoma antibodies

[0102] 6.1 Cloning and sequencing of mouse antibodies 4A12B11, 9B10F11, 11A5C7, and 12E2D11

[0103] Hybridoma cells that have reached the logarithmic growth phase were counted, and 500,000 to 1,000,000 cells were centrifuged and the supernatant was discarded. The cells were then washed once with sterile PBS, and total RNA was extracted from the cells using a kit. The concentration and purity of the extracted RNA were determined. Then, cDNA was synthesized by reverse transcription using oligoDT primers and reverse transcriptase at 42°C for 45 minutes. The antibody variable region DNA fragment was amplified using antibody variable region light and heavy chain primers, and the annealing temperature was set to 55°C. PCR reaction was performed in a 30 μL system. 10 μL of PCR product was identified by gel electrophoresis. PCR products with the correct band size were ligated into the T vector at room temperature, reacted for 30 minutes, and then transformed into DH5α cells. The cells were cultured at 37°C with shaking for 30 minutes, then plated overnight for blue-white screening. Ten white spots were selected for sequencing. The sequencing results were analyzed and compared to obtain the heavy and light chain sequences of the candidate antibody. The relevant sequences are shown in Table 4.

[0104] Table 4. VH, VL, and CDR sequences of each mouse antibody.

[0105] 6.2 Construction of chimeric antibodies

[0106] The obtained mouse antibody variable region light and heavy chain sequences and human IgG4 constant region sequences were cloned into an expression vector and transfected into CHO cells to obtain chimeric antibodies.

[0107] Heavy chain constant region (SEQ ID NO:12):

[0108] Light chain constant region (SEQ ID NO:13):

[0109] Example 7: Detection of the blocking effect of chimeric antibodies in reporter gene assays

[0110] HEK293-OSMR-gp130-STAT3-Luc reporter cells and HEK293-OSMR-IL31RA-STAT3-Luc reporter cells in logarithmic growth phase were collected, and 30,000 cells were added to each well. Chimeric antibody was added to 96-well white plates and incubated at 37°C for 30 minutes. Diluted OSM protein was added to the wells of HEK293-OSMR-gp130-STAT3-Luc cells, and diluted IL31 protein was added to the wells of HEK293-OSMR-IL31RA-STAT3-Luc cells. The plates were incubated at 37°C for 4.5 hours. 50 μL of One-lite chemiluminescent substrate reagent was added to each well, and the plates were incubated at room temperature in the dark for 5 minutes. The chemiluminescence was then read using a microplate reader.

[0111] The results are shown in Table 5. The results indicate that all chimeric antibodies exhibited good blocking activity in the reporter gene assay.

[0112] Table 5. Blocking activity of chimeric antibodies in reporter gene assays.

[0113] Example 8: Detection of the blocking effect of chimeric antibody on OSM-induced MCP-1 release from HDF cells

[0114] HDF cells in logarithmic growth phase were collected, resuspended in complete culture medium, and counted. 4000 cells were added to each well and incubated at 37°C for 18 h. The complete culture medium was washed off, and serially diluted antibody (50 μg / mL starting, 5-fold serial dilutions, 9 gradients) was added. The cells were incubated at 37°C for 1 h. OSM protein diluted to 50 ng / mL was added, and the cells were incubated at 37°C. After 24 hours of incubation, the cells were centrifuged at 2000 rpm for 15 minutes, and the culture supernatant was collected. The MCP-1 content in the supernatant was detected using ELISA.

[0115] The results are shown in Table 6, based on whether cytokine release activity can be completely blocked and IC50. 50 Based on the values ​​and epitope competition relationships, 9B10-ch and 12E2-ch were selected as the final cell clones.

[0116] Table 6. Blocking activity of chimeric antibodies in cytokine assays.

[0117] Example 9: Construction of Humanized Antibodies

[0118] The obtained chimeric antibody sequence was humanized. First, the variable region sequence of the antibody was analyzed to determine the antibody CDR region and the framework region. Then, the framework region was compared with the human germline sequence. The variable region framework of the best matching human germline antibody sequence was used to replace the variable region framework of the chimeric antibody, resulting in a humanized antibody with the CDR region being mouse-derived and the other regions being human-derived sequences. Based on the literature (Hwang WY et al., (2005) Use of human germline genes in a cdr homology-based approach to antibody humanization; Tamura M et al., (2000) Structural correlates of an anticarcinoma antibody: Identification of specificity-determining residues (SDRs) and development of a minimally immunogenic antibody variant by retention of SDRs only; Morrow JK, Zhang S. (2012) Computational prediction of protein hot spot residues) and antibody spatial structure, key amino acid sites in the humanized framework region need to be mutated back to the amino acids in the original mouse antibody sequence. If the reversion mutation exceeds two amino acids, it is necessary to perform permutation and combination before expression detection, and synthesize genes based on the heavy and light chain sequences of the antibody after reversion mutation.

[0119] Then, the heavy and light chains were arranged and combined and transfected into CHO cells for expression. The cell supernatant after expression was used to detect the affinity of the humanized antibody. The chimeric antibody was used as a control, and the humanized light and heavy chain combination with the best affinity was selected as the final humanized antibody.

[0120] Subsequently, sequence alignment revealed that the W amino acid in the CDR1 region of the heavy chain variable region of the 9B10 antibody corresponds to the conserved amino acid Y in the CDR1 region of the heavy chain variable region of the human antibody. Therefore, to further improve the degree of humanization, a W33Y amino acid mutation was performed in the CDR1 region of the heavy chain variable region of the 9B10 antibody, resulting in antibody hu9B10 (the heavy chain variable region sequence corresponds to hu9B10 VH-2-2 shown in Table 7, and the light chain variable region sequence corresponds to hu9B10 VL-2 shown in Table 7). The affinity of the humanized hu12E2 antibody obtained through humanization construction is lower than that of the chimeric antibody, requiring further affinity maturation.

[0121] Example 10: Antibody Affinity Maturation

[0122] Affinity maturation was performed using a combination of CDR1 walking randomization and rational design-based methods. First, overlapping PCR was used to introduce random mutations in CDR-H1 and CDR-L1 using NNK degenerate codons. DNA sequencing was performed before panning to ensure the correct reading frames of the clones in the library, enabling normal transcription and translation expression. Second, solid-phase panning was used, with 0.5 μg / mL, 0.3 μg / mL, and 0.2 μg / mL of soluble OSMR-his onto ELISA plates, and three independent panning experiments were conducted. After three rounds of panning, amino acid sequence analysis was performed on the clones randomly selected in the previous round, yielding enriched clones from the third round. Sequence alignment revealed three amino acid sites with mutations in the light chain, including an amber stop codon. To obtain accurate sequences, this site was mutated to all 20 amino acids except the non-stop codon, resulting in 20 point-mutated light chain sequences. These sequences, along with the original heavy chain sequences, were co-transfected for transient expression and purification. The 20 purified 12E2 mutant antibodies were tested for affinity on a Gator. Finally, an antibody with high affinity was obtained, and Koff was better than the positive control antibody. The antibody hu12E2-1 was obtained (the heavy chain variable region sequence corresponds to hu12E12 VH-2-2 shown in Table 7, and the light chain variable region sequence corresponds to hu12E2 VL-4 shown in Table 7).

[0123] Table 7. VH, VL, and CDR sequences of each antibody after affinity maturation.

[0124] Example 11: Identifying the affinity of hu9B10 and hu12E2-1 for OSMR proteins

[0125] HEK293-hOSMR cells in logarithmic growth phase were collected, resuspended in staining buffer (PBS + 2% FBS) for cell counting, and added at 50,000 cells / well to 96-well V plates. Serially diluted test antibodies (starting at 30 μg / mL, 5-fold serial dilutions, 8 gradients) were added to the 96-well V plates and incubated at 4°C for 1 hour. After washing twice with staining buffer, FITC-labeled goat anti-human IgG secondary antibody was added, and the plates were incubated at 4°C for 1 hour. After washing twice with staining buffer, the cells were analyzed using a CytoFLEX flow cytometer. The results are shown in Figure 3, indicating the ECMO binding of hu9B10 and hu12E2-1 to hOSMR. 50 The ECGs of KPL-716 binding to hOSMR were 0.01717 μg / mL and 0.01079 μg / mL, respectively. 50 It is 0.04125 μg / mL.

[0126] The experimental results show that the antibodies hu9B10 and hu12E2-1 of this invention have a better affinity for OSMR proteins than the reference antibody KPL-716.

[0127] Example 12: Identification of the blocking effect of hu9B10 and hu12E2-1 on reporter cell activity

[0128] HEK293-OSMR-gp130-STAT3-Luc reporter cells and HEK293-OSMR-IL31RA-STAT3-Luc reporter cells in logarithmic growth phase were collected, and 30,000 cells were added to each well. Antibody was added to 96-well white plates and incubated at 37°C for 30 minutes. Diluted OSM protein was added to the wells of HEK293-OSMR-gp130-STAT3-Luc cells, and diluted IL31 protein was added to the wells of HEK293-OSMR-IL31RA-STAT3-Luc cells. The plates were incubated at 37°C for 4.5 hours, and 50 μL / well of One-lite chemiluminescent substrate reagent was added. The plates were incubated at room temperature in the dark for 5 minutes, and the chemiluminescence was read using a microplate reader.

[0129] The results are shown in Figures 4 and 5. hu9B10 and hu12E2-1 inhibited the IC50 of OSM-induced reporter cell activity. 50 The IC50 values ​​for KPL-716 were 0.4708 μg / mL and 0.1895 μg / mL, respectively. 50 The concentration was 0.2020 μg / mL; the IC50 values ​​of hu9B10 and hu12E2-1 for blocking IL31-induced reporter cell activity were 0.2020 μg / mL. 50 The IC50 values ​​for KPL-716 were 0.2037 μg / mL and 0.2174 μg / mL, respectively. 50The concentration was 0.2256 μg / mL. The results show that the blocking activity of the antibodies hu9B10 and hu12E2-1 against OSM or IL31-induced antibodies is comparable to that of the reference antibody KPL-716.

[0130] Example 13: Identification of the inhibitory effect of hu9B10 and hu12E2-1 on OSM-induced MCP-1 release from HDF cells

[0131] HDF cells in logarithmic growth phase were collected, resuspended in complete culture medium, and counted. 4000 cells were added to each well and incubated at 37°C for 18 h. The complete culture medium was washed off, and serially diluted antibody (50 μg / mL starting, 5-fold serial dilutions, 9 gradients) was added. The cells were incubated at 37°C for 1 h. OSM protein diluted to 50 ng / mL was added, and the cells were incubated at 37°C. After 24 hours of incubation, the cells were centrifuged at 2000 rpm for 15 minutes, and the culture supernatant was collected. The MCP-1 content in the supernatant was detected using ELISA.

[0132] The results are shown in Figure 6. hu9B10 and hu12E2-1 inhibited the release of IC50 from OSM-induced MCP-1. 50 The values ​​were 0.1539 μg / mL and 0.1811 μg / mL, respectively, and the IC50 of KPL-716 was... 50 The concentration was 0.1432 μg / mL. The results show that the blocking activity of the antibodies hu9B10 and hu12E2-1 in the present invention against the release of MCP-1 from OSM or IL31-induced HDF cells is comparable to that of the reference antibody KPL-716.

[0133] Example 14: Identification of the cross-reactivity of hu9B10 and hu12E2-1 to cynomolgus monkey OSMR

[0134] HEK293-cynoOSMR cells in logarithmic growth phase were collected, resuspended in staining buffer (PBS + 2% FBS), and counted at 50,000 cells / well in 96-well V plates. Serially diluted test antibodies (starting at 30 μg / mL, 5-fold serial dilutions, 8 gradients) were added to the 96-well V plates and incubated at 4°C for 1 hour. The cells were washed twice with staining buffer, then FITC-labeled goat anti-human IgG secondary antibody was added, and the cells were incubated at 4°C for 1 hour. After washing twice with staining buffer, the cells were analyzed using a CytoFLEX flow cytometer.

[0135] The results are shown in Figure 7, illustrating the ECG binding of hu9B10 to cynoOSMR. 50 The ECG binding of KPL-716 to cynoOSMR was 0.01919 μg / mL. 50 The concentration was 0.04846 μg / mL, and hu12E2-1 did not show significant binding to cynoOSMR.

[0136] Example 15: Identification of the binding specificity of hu9B10 and hu12E2-1 to hOSMR

[0137] Add 1 μg / mL of LIFR protein diluted in PBS to each well of an appropriate amount of microplate, and coat overnight at 4°C. Wash the coated cell plate three times with PBST, then add blocking buffer (PBS + 2% BSA + 0.01% Tween 20) and block at room temperature for about 1 hour. Discard the blocking buffer, then add 100 μL of diluted hu9B10 and hu12E2-1 antibodies to each well and incubate at room temperature for 1 hour. Wash three times with PBST, then add 100 μL of secondary antibody diluted 1:130000 with antibody dilution buffer (PBS + 0.5% BSA + 0.01% Tween 20) and incubate at room temperature for 1 hour. Wash three times with PBST, then add 100 μL of TMB solution for color development for about 5 minutes. Finally, add stop solution to stop the color development and read the OD on a microplate reader. 450 .

[0138] The results are shown in Figures 8 and 9. The results show that hu9B10 and hu12E2-1 do not bind to LIFR proteins of the same family. These results indicate that the antibodies hu9B10 and hu12E2-1 in this application exhibit high specificity for binding to OSMR proteins.

[0139] Example 16: Binding affinity of the antibody modified with the heavy chain constant region to FcRn

[0140] To enhance the affinity of the antibodies for human FcRn, we further engineered the heavy chain constant regions of hu9B10 and hu12E2-1 by replacing the heavy chain constant region sequence with the sequence shown in SEQ ID NO:73, resulting in antibodies hu9B10-1 and hu12E2-2.

[0141] Engineered IgG4 heavy chain constant region (SEQ ID NO:73):

[0142] The affinity of the antibody for human FcRn was detected using bio-layer interferometry (BLI). Using a Gator instrument, human FcRn protein was first immobilized onto an Anti-his probe, and then the antibody was diluted to 1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM. Antibody binding was detected using the Gator instrument, and the dissociation curve detection program is shown in Table 8. The dissociation constants of hu9B10 and hu12E2-1 antibodies before and after Fc modification with human FcRn are also shown in Table 9. NA represents no binding.

[0143] The results show that the FcRn affinity of antibodies hu9B10-1 and hu12E2-2 modified with heavy chain constant region in this invention is significantly improved.

[0144] Table 8. Procedure parameters for antibody-human FcRn dissociation curve detection.

[0145] Table 9. Dissociation constants of antibody-human FcRn binding.

[0146] Example 17: Construction of Antibody Stability Modification

[0147] The variable region amino acid sequence of the hu9B10 antibody contains a deamidation site NG. This non-enzymatic deamidation, as a special post-translational modification (PTM), can occur in vitro or in vivo, affecting protein structure, stability, and aggregation. Therefore, the NG site of the antibody was mutated to improve protein stability. The mutated antibody variable region sequence was linked to the human antibody constant region sequence and cloned into the expression vector. The constant region of the heavy chain was replaced with an engineered IgG4 heavy chain constant region sequence (SEQ ID NO:73). The vector was transfected into CHO cells for expression. After expression, the supernatant was collected for antibody purification. Gator verification showed that the affinity of the mutated antibody did not change significantly. Antibodies hu9B10-2 (heavy chain variable region and light chain variable region correspond to hu9B10 VH-5 and hu9B10 VL-2, respectively), hu9B10-3 (heavy chain variable region and light chain variable region correspond to hu9B10 VH-6 and hu9B10 VL-2, respectively), and hu9B10-4 (heavy chain variable region and light chain variable region correspond to hu9B10 VH-7 and hu9B10 VL-2, respectively) were obtained.

[0148] In addition, after comparing the amino acid sequences with IGKV1-9*01 and IGHV1-46*01, point mutations were performed on amino acid sites P46, F87, T93, and Y96 in the light chain variable region, and on amino acid sites E50, G56, N61, E62, K65, I66, and Y104 in the heavy chain variable region, with the aim of obtaining antibody clones with PTM sites removed, improved humanization, and enhanced stability.

[0149] Subsequently, a hu9B10 Fab phage display library was constructed, and three rounds of solid-phase panning were performed. The coating concentrations of the OSMR recombinant protein antigen were 10 μg / mL, 4 μg / mL, and 1 μg / mL, respectively. Clones that specifically bound the OSMR recombinant protein were enriched. Thirty clones were randomly selected for sequencing identification, revealing the enriched light and heavy chain mutation sites.

[0150] Based on the results of phage display library screening, multiple point-mutated hu9B10 light and heavy chain clones were constructed, transfected into CHO cells for expression, and the supernatant was collected for antibody purification to obtain antibodies hu9B10-5 (heavy chain variable region and light chain variable region correspond to hu9B10 VH-9 and hu9B10 VL-2-1, respectively), hu9B10-6 (heavy chain variable region and light chain variable region correspond to hu9B10 VH-9-1 and hu9B10 VL-2, respectively), and hu9B10-7 (heavy chain variable region and light chain variable region correspond to hu9B10 VH-9 and hu9B10 VL-2, respectively).

[0151] Table 10. VH, VL, and CDR sequences of each antibody after stability modification.

[0152] Example 18: Identification of the stability of the modified antibody under low pH incubation and thermal stability.

[0153] 18.1 pH Challenge Experiment

[0154] The stability of the stability-modified antibody under low pH conditions was assessed using a pH challenge experiment. The stability-modified antibody was treated with citrate buffer (pH 2.4–3.0) to achieve a pH of 3.5 ± 0.2, and incubated at room temperature for 0, 2, and 6 hours, respectively. The pH was then adjusted back to the normal range, and samples were collected for SEC-HPLC analysis to determine antibody purity and compositional changes. HPLC analysis was performed using a Waters Arc UHPLC system with a Waters BEH200 2.5µm gel column (7.8mm × 300mm). The mobile phase consisted of PBS + 250mM NaCl, pH 7.2 ± 0.2, a flow rate of 0.70 mL / min, a detection wavelength of 280 nm, and an injection volume of 50µg.

[0155] The stability-modified antibodies hu9B10-8 (with the heavy chain constant region replaced by a heavy chain constant region derived from IgG1 compared to hu9B10-2) and hu9B10-2 showed good low pH stability.

[0156] The amino acid sequence of the heavy chain constant region of hu9B10-8 (SEQ ID NO:72):

[0157] The results are shown in Table 11:

[0158] Table 11 Results of antibody stability testing under low pH after stability modification

[0159] 18.2 Thermal stability test

[0160] After mixing the stability-modified antibody with a fluorescent dye, the sample was slowly heated on a quantitative PCR instrument. During heating, the fluorescent dye bound to the structurally altered antibody, producing fluorescence. The TM value was calculated based on the fluorescence intensity-time curve. The Tm value is the temperature at which a protein unfolds and undergoes a conformational change during heating, and is used to evaluate the thermal stability of a protein.

[0161] Simultaneously, samples were taken for SEC-HPLC analysis to determine the purity and composition of the antibodies. HPLC analysis was performed using a Waters Arc UHPLC system with a Waters BEH200 2.5µm gel column (7.8mm × 300mm). The mobile phase consisted of PBS + 250mM NaCl, pH 7.2 ± 0.2, the flow rate was 0.70 mL / min, the detection wavelength was 280 nm, and the injection volume was 50 µg.

[0162] The thermostability of antibodies hu9B10-6 and hu9B10-7 was improved after stability modification. The results are shown in Table 12.

[0163] Table 12 Results of thermostability testing of antibodies after stability modification.

[0164] Example 19: Evaluation of the therapeutic activity of the antibody against an hIL31-induced mouse pruritus model

[0165] The therapeutic activity of the antibody against an hIL31-induced mouse pruritus model was evaluated using B-hIL31 / hIL31RA / hOSM / hOSMR mice (purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.). The modeling method is as follows:

[0166] Before the Day-13 experiment, the mice's cheeks were shaved, and 20 μL of PBS was injected intradermally into the left cheek. Itching videos were then recorded for one hour. Mice were grouped according to the total number of itching behaviors observed within one hour on Day-13. On Day 1, the blank control group (G1) received an intradermal injection of 20 μL of PBS into the right cheek, while the experimental model groups (G2-G6) received an intradermal injection (id) of 20 μL of 20 μg hIL31 solution into the right cheek to induce itching. Itching was recorded for each group in the afternoon of Day 1. Mice were allowed 30 minutes to acclimatize to the environment before recording. The video recording duration was one hour, with each recording session remaining relatively consistent. The recording environment was kept quiet and dark.

[0167] A total of 33 B-hIL31 / hIL31RA / hOSM / hOSMR mice were used for the experiment. There were 3 mice in the blank control group (G1) and 6 mice in each of the remaining experimental model groups (G2-G6). The animals were administered the drug intraperitoneally on Day 0, once, with a volume of 10 μL. Day 1 was the experimental endpoint. Specific grouping and administration protocols are shown in Table 13.

[0168] Table 13 Animal grouping and administration regimen for hIL31-induced mouse pruritus model

[0169] The experiment recorded and analyzed mouse weight and scratching behavior to evaluate the scratching status of each group of animals and the effect of the tested molecules on scratching. Scratching videos of mice were recorded at 17:00. Before recording, mice were allowed 30 minutes to acclimatize to the environment. The video recording duration was 1 hour, with each recording session maintaining a relatively consistent timeframe. The recording environment was kept quiet and dark. The standard for the number of scratching behaviors was defined as one or more scratches on the ear or back with the mouse's hind paw off the ground until the hind paw touched the ground.

[0170] The changes in body weight are shown in Figure 10. Compared with the blank control group (G1), there was no significant difference in body weight among the experimental model groups (G2-G6). Compared with the solvent model group (G2), there was no significant difference in body weight among the test sample treatment groups (G3-G6).

[0171] Changes in pruritus behavior are shown in Figures 11 to 14. Recordings were taken of all animals on Day 13, and the number of pruritus episodes in mice within one hour was counted. There was no significant difference in baseline pruritus levels among groups G1-G6. In the Day 1 experiment, compared to the blank control group (G1), the number of pruritus episodes in the solvent-based model group (G2) was significantly increased, indicating successful establishment of the pruritus model. Compared to the solvent-based model group G2, the number of pruritus episodes in the Nemolizumab (IL31RA antagonist) treatment group (G3) changed but was not significantly different. The number of pruritus episodes in the Vixarelimab (OSMR antagonist) treatment group (G4), the hu9B10-1 (50 mg / kg) treatment group, and the hu9B10-1 (25 mg / kg) treatment group (G6) was significantly reduced. Compared to the Vixarelimab treatment group (G4), the hu9B10-1 (50 mg / kg) treatment group (G5) significantly reduced the number of pruritus episodes in mice. Compared with the hu9B10-1 (25 mg / kg) treatment group (G6), the hu9B10-1 (50 mg / kg) treatment group (G5) showed a certain decreasing trend in reducing the number of times mice itch.

[0172] In conclusion, Nemolizumab, Vixarelimab, and hu9B10-1 all showed good therapeutic effects on hIL31-induced pruritus, with hu9B10-1 demonstrating superior therapeutic efficacy against hIL31-induced pruritus compared to Nemolizumab and Vixarelimab.

[0173] Example 20: Evaluation of antibody pharmacokinetics in cynomolgus monkeys

[0174] The pharmacokinetic characteristics of the antibody were evaluated using cynomolgus monkeys (purchased from Guangxi Fangchenggang Changchun Biotechnology Development Co., Ltd.) via a single intravenous infusion.

[0175] Three male cynomolgus monkeys were used for pharmacokinetic evaluation. Each monkey was administered 10 mg / mL hu9B10-1 intravenously at a volume of 5 mL / kg over 30 minutes, once in total. Whole blood was collected via an upper limb vein at 30 min, 2 h, 8 h, 24 h (Day 1), 48 h (Day 2), 72 h (Day 3), 168 h (Day 7), 240 h (Day 10), 336 h (Day 14), 408 h (Day 17), 504 h (Day 21), 672 h (Day 28), 840 h (Day 35), 1008 h (Day 42), 1176 h (Day 49), and 1344 h (Day 56) after administration. The whole blood was allowed to clot naturally before centrifugation to obtain serum for pharmacokinetic analysis. The method for detecting antibody drug concentration in serum is as follows: Take an appropriate amount of ELISA plate and add 1 μg / mL of hOSMR protein diluted in PBS to each well. Coat overnight at 4°C. Wash the coated cell plate three times with PBST, then add blocking buffer (PBS + 0.2% I-Block + 0.05% Tween 20) and block at room temperature for about 1 hour. Discard the blocking buffer, wash three times with PBST, add diluted monkey serum, and incubate at room temperature for 1.5 hours. Wash three times with PBST, add Goat Anti-Human IgG and Monkey Ads-HRP (Southernbiotech 2049-05, 1:10000 dilution), and incubate at room temperature for 1 hour. Wash three times with PBST, add 100 μL of TMB solution for color development for about 6 minutes, then add stop solution to stop the color development. Read OD450 and OD620 on an ELISA reader.

[0176] Figure 15 shows the changes in blood drug concentration of hu9B10-1 at different time points. Pharmacokinetic parameters were calculated using the Phoenix WinNonlin non-compartmental model, and the main pharmacokinetic parameters are shown in Table 14.

[0177] Table 14. Pharmacokinetic parameters of the antibody in cynomolgus monkeys.

Claims

1. An antibody against OSMR protein or an antigen-binding fragment thereof, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3. Wherein, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO:26, 4, 6, 8, 10, 23, 24, 27, 28, 29, 39, 40, 41, 42, 43, 54, 56, 58, 61, or 63, or has at least 85% sequence identity with each of them; and / or The light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO:35, 5, 7, 9, 11, 33, 34, 47, 48, 49, 50, 52, 64, 65, 67, or 68, or has at least 85% sequence identity with them.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, The HCDR1 contains an amino acid sequence as shown in SEQ ID NO:25, 14, 20, or 36; the HCDR2 contains an amino acid sequence as shown in SEQ ID NO:21, 15, 37, 53, 55, 57, 59, 62, or 69; the HCDR3 contains an amino acid sequence as shown in SEQ ID NO:22, 16, 38, or 60; the LCDR1 contains an amino acid sequence as shown in SEQ ID NO:30, 17, 44, 51, or 70; the LCDR2 contains an amino acid sequence as shown in SEQ ID NO:31, 18, 45, or 71; and / or the LCDR3 contains an amino acid sequence as shown in SEQ ID NO:32, 19, 46, or 66. Preferably, The HCDR1 contains the amino acid sequence shown in SEQ ID NO:25; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:21; and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:22; or The HCDR1 contains the amino acid sequence shown in SEQ ID NO:14; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:15; and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:16; or The HCDR1 contains the amino acid sequence shown in SEQ ID NO:20; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:69; and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:22; or The HCDR1 contains the amino acid sequence shown in SEQ ID NO:20; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:21; and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:22; or The HCDR1 contains the amino acid sequence shown in SEQ ID NO:36; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:37; and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:38; or The HCDR1 contains the amino acid sequence shown in SEQ ID NO:25; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:53; and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:22; or The HCDR1 contains the amino acid sequence shown in SEQ ID NO:25; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:55; and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:22; or The HCDR1 contains the amino acid sequence shown in SEQ ID NO:25; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:57; and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:22; or The HCDR1 contains the amino acid sequence shown in SEQ ID NO:25; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:59; and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:60; or The HCDR1 contains the amino acid sequence shown in SEQ ID NO:25; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:62; and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:

60. and, The LCDR1 contains the amino acid sequence shown in SEQ ID NO:30; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:31; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:32; or The LCDR1 contains the amino acid sequence shown in SEQ ID NO:17; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:19; or The LCDR1 contains the amino acid sequence shown in SEQ ID NO:70; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:71; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:32; or The LCDR1 contains the amino acid sequence shown in SEQ ID NO:44; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:45; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:46; or The LCDR1 contains the amino acid sequence shown in SEQ ID NO:51; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:46; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:29; or The LCDR1 contains the amino acid sequence shown in SEQ ID NO:30; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:31; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:66; More preferably, wherein (i) The HCDR1 contains the amino acid sequence shown in SEQ ID NO:25; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:21; the HCDR3 contains the amino acid sequence shown in SEQ ID NO:22; the LCDR1 contains the amino acid sequence shown in SEQ ID NO:30; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:31; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:32; or (ii) The HCDR1 contains the amino acid sequence shown in SEQ ID NO:14; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:15; the HCDR3 contains the amino acid sequence shown in SEQ ID NO:16; the LCDR1 contains the amino acid sequence shown in SEQ ID NO:17; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:19; or (iii) The HCDR1 contains the amino acid sequence shown in SEQ ID NO:20; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:21; the HCDR3 contains the amino acid sequence shown in SEQ ID NO:22; the LCDR1 contains the amino acid sequence shown in SEQ ID NO:30; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:31; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:32; or (iv) The HCDR1 contains the amino acid sequence shown in SEQ ID NO:20; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:69; the HCDR3 contains the amino acid sequence shown in SEQ ID NO:22; the LCDR1 contains the amino acid sequence shown in SEQ ID NO:70; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:71; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:32; or (v) The HCDR1 contains the amino acid sequence shown in SEQ ID NO:36; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:37; the HCDR3 contains the amino acid sequence shown in SEQ ID NO:38; the LCDR1 contains the amino acid sequence shown in SEQ ID NO:44; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:45; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:46; or (vi) The HCDR1 contains the amino acid sequence shown in SEQ ID NO:36; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:37; the HCDR3 contains the amino acid sequence shown in SEQ ID NO:38; the LCDR1 contains the amino acid sequence shown in SEQ ID NO:51; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:45; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:46; or (vii) The HCDR1 contains the amino acid sequence shown in SEQ ID NO:25; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:53; the HCDR3 contains the amino acid sequence shown in SEQ ID NO:22; the LCDR1 contains the amino acid sequence shown in SEQ ID NO:30; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:31; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:32; or (viii) The HCDR1 contains the amino acid sequence shown in SEQ ID NO:25; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:55; the HCDR3 contains the amino acid sequence shown in SEQ ID NO:22; the LCDR1 contains the amino acid sequence shown in SEQ ID NO:30; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:31; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:32; or (ix) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:25; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:57; the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:22; the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:30; the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:31; and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:32; or (x) The HCDR1 contains the amino acid sequence shown in SEQ ID NO:25; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:59; the HCDR3 contains the amino acid sequence shown in SEQ ID NO:60; the LCDR1 contains the amino acid sequence shown in SEQ ID NO:30; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:31; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:32; or (xi) The HCDR1 contains the amino acid sequence shown in SEQ ID NO:25; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:62; the HCDR3 contains the amino acid sequence shown in SEQ ID NO:60; the LCDR1 contains the amino acid sequence shown in SEQ ID NO:30; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:31; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:32; or (xii) The HCDR1 contains the amino acid sequence shown in SEQ ID NO:25; the HCDR2 contains the amino acid sequence shown in SEQ ID NO:59; the HCDR3 contains the amino acid sequence shown in SEQ ID NO:60; the LCDR1 contains the amino acid sequence shown in SEQ ID NO:30; the LCDR2 contains the amino acid sequence shown in SEQ ID NO:31; and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:

32.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein, The frame region of the heavy chain variable region and / or the light chain variable region is a human-derived, mouse-derived, or rabbit-derived frame region. Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:26, 4, 6, 8, 10, 23, 24, 27, 28, 29, 39, 40, 41, 42, 43, 54, 56, 58, 61 or 63, or contains an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:26, 4, 6, 8, 10, 23, 24, 27, 28, 29, 39, 40, 41, 42, 43, 54, 56, 58, 61 or 63; and / or The amino acid sequence of the light chain variable region is as shown in SEQ ID NO:35, 5, 7, 9, 11, 33, 34, 47, 48, 49, 50, 52, 64, 65, 67 or 68, or contains an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:35, 5, 7, 9, 11, 33, 34, 47, 48, 49, 50, 52, 64, 65, 67 or 68. Preferably, (i) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:26, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:35; or (ii) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:4, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:5; or (iii) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:6, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:7; or (iv) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:9; or (v) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:10, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:11; or (vi) 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:52; or (vii) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:54, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:64; or (viii) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:56, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:64; or (ix) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:58, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:64; or (x) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:61, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:65; or (xi) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:63, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:64; or (xii) The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:61, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:

64.

4. The antibody or antigen-binding fragment thereof as described in any one of claims 1-3, characterized in that, The antibody or its antigen-binding fragment is a full-length antibody, Fab, Fab', F(ab')2, Fv or scFv; Preferably, the antibody is a full-length antibody; More preferably, the antibody further includes a constant region comprising a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region is a heavy chain constant region derived from human IgA, IgD, IgE, IgG, or IgM, and / or the light chain constant region is a light chain constant region derived from the κ chain or λ chain; more preferably, the heavy chain constant region is a heavy chain constant region derived from human IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. More preferably, the heavy chain constant region has a mutation at one or more amino acid residue sites selected from the group consisting of: S228, F234, L235, M252, S254, T256, K288, T307, M428, N424, and Y436, wherein the amino acid residue sites are numbered according to the EU numbering system; preferably, the heavy chain constant region has one or more mutations of: S228P, F234A, L235A, T307H, N434A, M252Y, S254T, T256E, M428L, Y436T, and T307Q, wherein the amino acid residue sites are numbered according to the EU numbering system; For example, the constant region includes a heavy chain constant region and a light chain constant region, wherein the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:73, 12 or 72, or contains an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:73, 12 or 72; and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:13, or contains an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:

13.

5. An isolated nucleic acid, characterized in that, The isolated nucleic acid encodes the antibody or its antigen-binding fragment as described in any one of claims 1-4.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the isolated nucleic acid as described in claim 5; Preferably, the recombinant expression vector is a plasmid, granule, bacteriophage, or viral vector, and the viral vector is preferably a retroviral vector, lentiviral vector, adenovirus vector, or adeno-associated virus vector.

7. A transformant characterized in that, The transformant comprises the isolated nucleic acid as described in claim 5 or the recombinant expression vector as described in claim 4, and the host cell of the transformant is a prokaryotic cell or a eukaryotic cell; Preferably, the host cell is a yeast cell or a mammalian cell, such as HEK293 cells or CHO cells; more preferably, the host cell is a 293F cell.

8. A method of making an antibody or antigen-binding fragment thereof against OSMR protein, characterized in that, The method comprises the following steps: culturing the transformant as described in claim 7 under suitable conditions, and obtaining the antibody or its antigen-binding fragment from the culture.

9. An immunoconjugate, comprising, The immunoconjugate comprises an antibody or an antigen-binding fragment thereof and a conjugation portion as described in any one of claims 1-4; Preferably, the coupling portion is selected from: detectable markers, drugs, cytokines, radionuclides, enzymes, gold nanoparticles / nanorobars, magnetic nanoparticles, viral capsid proteins or VLPs, and combinations thereof; More preferably, the radionuclide includes diagnostic isotopes and / or therapeutic isotopes; More preferably, the diagnostic isotopes are selected from: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, and combinations thereof; and / or, the therapeutic isotopes are selected from: Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co- 60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, and combinations thereof.

10. A bispecific antibody or a multispecific antibody, characterized in that, The bispecific or multispecific antibody comprises the antibody or its antigen-binding fragment as described in any one of claims 1-4.

11. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (i) the antibody or antigen-binding fragment thereof as described in any one of claims 1-4, or the immunoconjugate as described in claim 9; and (ii) Pharmaceutically acceptable carriers.

12. Use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-4, the isolated nucleic acid as described in claim 5, the recombinant expression vector as described in claim 6 and / or the transformant as described in claim 7, the bispecific antibody or multispecific antibody as described in claim 10, or the pharmaceutical composition as described in claim 11, for the preparation of a medicament for treating OSMR-related diseases or conditions; Preferably, the OSMR-related disease or condition is a disease or condition caused by abnormalities in the OSM or IL-31 signaling pathway mediated by OSMR. More preferably, the OSMR-related diseases or conditions are selected from: atopic dermatitis, rheumatoid arthritis, pulmonary fibrosis, multiple sclerosis, inflammatory bowel disease, pruritus, hepatitis, atherosclerosis, tumorigenesis, pathological cardiac hypertrophy, and heart failure.

13. A method for treating OSMR-related diseases or conditions, characterized in that, This includes administering an effective amount of the antibody or antigen-binding fragment thereof as described in any one of claims 1-4, the isolated nucleic acid as described in claim 5, the recombinant expression vector as described in claim 6, the transformant as described in claim 7, the bispecific antibody or multispecific antibody as described in claim 10, and / or the pharmaceutical composition as described in claim 11 to a subject in need; Preferably, the OSMR-related disease or condition is a disease or condition caused by abnormalities in the OSM or IL-31 signaling pathway mediated by OSMR. More preferably, the OSMR-related diseases or conditions are selected from: atopic dermatitis, rheumatoid arthritis, pulmonary fibrosis, multiple sclerosis, inflammatory bowel disease, pruritus, hepatitis, atherosclerosis, tumorigenesis, pathological cardiac hypertrophy, and heart failure.

14. The antibody or antigen-binding fragment thereof as described in any one of claims 1-4, the isolated nucleic acid as described in claim 5, the recombinant expression vector as described in claim 6, the transformant as described in claim 7, the bispecific antibody or multispecific antibody as described in claim 10, or the pharmaceutical composition as described in claim 11, for the treatment of OSMR-related diseases or conditions; Preferably, the OSMR-related disease or condition is a disease or condition caused by abnormalities in the OSM or IL-31 signaling pathway mediated by OSMR. More preferably, the OSMR-related diseases or conditions are selected from: atopic dermatitis, rheumatoid arthritis, pulmonary fibrosis, multiple sclerosis, inflammatory bowel disease, pruritus, hepatitis, atherosclerosis, tumorigenesis, pathological cardiac hypertrophy, and heart failure.

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