ST2 antigen-binding protein

Abstract

This invention provides an antibody used in a method to block the binding of IL33 to ST2 and inhibit the IL33 / ST2 signaling pathway and its induced inflammatory response. [Solution] The present invention provides ST2 antigen-binding proteins, such as mouse, human, chimeric, or humanized antibodies or their antigen-binding fragments that specifically bind to ST2, as well as nucleic acid molecules encoding the antibodies and their antigen-binding fragments, and expression vectors and host cells for expressing the antibodies or their antigen-binding fragments. Furthermore, the present invention provides methods for preparing and using the anti-antibodies and their antigen-binding fragments, the methods of use including the treatment and prevention of IL33 / ST2-mediated related diseases and conditions.

Description

[Technical Field]

[0001] Cross-references to related applications This publication claims priority based on patent application No. 202010397572.3 filed in China on May 12, 2020, and incorporates all of its contents in their entirety for all purposes.

[0002] Technical field This publication relates to isolated antibodies. Specifically, this publication provides mouse, chimeric, and humanized monoclonal antibodies or their antigen-binding fragments that specifically bind to ST2, as well as nucleic acids, expression vectors, and host cells for producing the antibodies or their antigen-binding fragments. This publication further provides polypeptide fusions, multispecific molecules, viral vectors, and pharmaceutical compositions containing the antibodies or their antigen-binding moieties, as well as diagnostic and therapeutic methods using the antibodies or their antigen-binding fragments. [Background technology]

[0003] Background technology Interleukin 33 (IL33) is a member of the IL-1 cytokine family and is expressed by tissue endothelial cells or epithelial cells. When cells are stimulated by stress, infection, or injury, IL33 is expressed as "alarmin" in the injured cells and transmits external antigen-stimulating signals to the Th2 pathway. The IL33 receptor consists of two proteins: IL-1 receptor-related proteins (IL-1RL1, ST2) and IL-1 receptor co-protein (IL-1RAP). With the involvement of IL-1RAP, the binding of IL33 and ST2 can induce signal transduction, but IL-1RAP cannot directly bind to IL33 or ST2 (Chackerian et al., (2007) J Immunol. 179:2551-2555).

[0004] ST2 belongs to the Toll / IL1 receptor family, which comprises three subtypes: transmembrane ST2L, soluble sST2, and mutant ST2V. ST2L primarily mediates the intracellular signaling response to IL33. ST2L is mainly expressed on the surface of Th2 cells, ILC2 cells, mast cells, and Treg cells, and is also expressed on the surface of NK cells, NKT cells, macrophages, eosinophils, and basophils.

[0005] IL33 binds to ST2L on the cell surface and recruits IL-1RAcP, thereby activating the MyD88 / NFκB cell pathway. This induces the secretion of inflammatory factors such as IL-5, IL-6, IL-13, TNF, and INF-γ, as well as chemokines such as CCL17, CCL22, and CXCL8, in cells such as mast cells, Th2 cells, Treg cells, and ILC2s, thereby triggering an inflammatory response. IL-33 expression is abnormally elevated in diseases of mucosal inflammation, arthritis, and chronic skin inflammation, such as allergic rhinitis (Kamekura R et al., (2012) Clin Exp Allergy. 42:218-28), rheumatoid arthritis, ankylosing spondylitis, atopic dermatitis (Savinko T et al., (2012) J Invest Dermatol. 132:1392-1400), psoriasis, and other conditions involving inflammation of mucous membranes, arthritis, and chronic skin inflammation. Furthermore, dysregulation of the IL33 / ST2 signaling pathway is associated with asthma, chronic obstructive pulmonary disease (Hacker, Lambers et al., (2009) J Clin Lab Anal. 23:372-9), bronchitis, and inflammatory bowel disease (Beltran CJ et al., (2010) Inflamm Bowel). Dis.16:1097-107), it is closely associated with immune-mediated diseases such as systemic lupus erythematosus, hepatic fibrosis, and systemic sclerosis. Genetic analysis has shown that the IL33 and ST2 genes are related to the number of basophils and atopic There are several single nucleotide polymorphism (SNP) sites closely associated with the development of inflammatory and immune-related diseases (Shimizu M et al., (2005) Hum Mol Genet. 14:2919-27; Gudbjartsson DF et al., (2009) Nat Genet. 41:342-7). Therefore, blocking the binding of IL33 to ST2 and inhibiting the IL33 / ST2 signaling pathway and its induced inflammatory response will be an important direction in the treatment of inflammatory immune-related diseases. [Overview of the project] [Problems that the invention aims to solve]

[0006] Summary of the Invention This publication provides an ST2 antibody with high affinity, high specificity, and high biological activity, suitable for the treatment of IL33 / ST2-mediated related diseases. [Means for solving the problem]

[0007] This publication provides isolated antibodies, such as mouse, human, chimeric or humanized monoclonal antibodies or antigen-binding fragments thereof, that bind to ST2 (e.g., human ST2 and monkey ST2).

[0008] The antibodies or their antigen-binding fragments disclosed herein have various applications, including the detection of the ST2 protein and the treatment and prevention of IL33 / ST2-mediated related diseases and conditions. These IL33 / ST2-mediated related diseases and conditions include asthma, allergic rhinitis, chronic obstructive pulmonary disease, eosinophilic bronchiolitis, eosinophilic esophagitis, atopic dermatitis, psoriasis, systemic lupus erythematosus, bullous pemphigoid, rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, pulmonary fibrosis, hepatic fibrosis, systemic sclerosis, sarcoidosis, graft-versus-host disease (GVHD), diabetic diseases, cardiovascular diseases, or combinations thereof.

[0009] In one aspect, this disclosure provides an isolated antibody or its antigen-binding fragment containing a heavy chain CDR. The heavy chain CDR comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3. Herein, (1) heavy chain CDR1 contains the sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and heavy chain CDR2 contains the sequence shown in SEQ ID NO: 15 or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, (1) The heavy chain CDR3 contains an amino acid sequence having 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; (2) The heavy chain CDR1 contains an amino acid sequence having 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; (3) The heavy chain CDR1 contains an amino acid sequence having 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto The heavy chain CDR2 contains an amino acid sequence having 2%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the heavy chain CDR2 contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and heavy (3) The CDR3 chain contains an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; (3) The CDR1 heavy chain contains an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 99% identical thereto; (4) The CDR1 heavy chain contains an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 99% identical thereto The heavy chain CDR2 contains an amino acid sequence having 4%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the heavy chain CDR2 contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and heavy chain CDR3 contains the sequence shown in SEQ ID NO: 22 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; (4) Heavy chain CDR1 contains the sequence shown in SEQ ID NO: 13 or an amino acid sequence having at least 80%, 81%, 82% identity thereto , 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequences, and the heavy chain CDR2 contains the sequence shown in SEQ ID NO: 18 or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% identical thereto. , comprising an amino acid sequence having 94%, 95%, 96%, 97%, 98%, or 99% identity, and heavy chain CDR3 comprising the sequence shown in SEQ ID NO: 23 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto;(5) Heavy chain CDR1 contains the sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and heavy chain CDR2 contains the sequence shown in SEQ ID NO: 19 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, The heavy chain CDR3 contains an amino acid sequence having 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or (6) Heavy chain CDR1 contains an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and heavy chain CDR2 contains an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, or 87% identical thereto It contains an amino acid sequence having 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, and the heavy chain CDR3 contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO: 24.

[0010] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2. Here, the amino acid sequence of SEQ ID NO: 17 is AIDPETGDTVYX1X2KFX3G, where X1=N or A, X2=Q, E or K, and X3=K or Q.

[0011] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2. Here, the amino acid sequence of SEQ ID NO: 17 is AIDPETGDTVYX1X2KFX3G, where X1 = N or A, X2 = Q, X3 = K or Q; X1 = N or A, X2 = E, X3 = K or Q; or X1 = N or A, X2 = K, X3 = K or Q.

[0012] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2. Here, the amino acid sequence of SEQ ID NO: 17 is AIDPETGDTVYX1X2KFX3G, where X1 = N, X2 = Q, X3 = K or Q; X1 = A, X2 = Q, X3 = K or Q; X1 = N, X2 = E, X3 = K or Q; X1 = A, X2 = E, X3 = K or Q; X1 = N, X2 = K, X3 = K or Q; or X1 = A, X2 = K, X3 = K or Q.

[0013] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2. Here, the amino acid sequence of SEQ ID NO: 17 is AIDPETGDTVYX1X2KFX3G, where X1 = N, X2 = Q, X3 = K; X1 = A, X2 = E, X3 = Q; or X1 = A, X2 = K, X3 = K.

[0014] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2. And it is a monoclonal antibody (e.g., a mouse, chimeric or humanized antibody).

[0015] In some embodiments, the isolated antibody or its antigen-binding fragment comprises a heavy chain variable region, the amino acid sequence of the heavy chain variable region comprising the sequence shown in SEQ ID NOs. 35, 37, 39, 41, 43, 45, 71, or 73; or comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NOs.

[0016] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2, which is a monoclonal antibody (e.g., a mouse, chimeric, or humanized antibody).

[0017] In some specific embodiments, the amino acid shown in SEQ ID NO: 71 may be encoded by the nucleic acid shown in SEQ ID NO: 72, and the amino acid shown in SEQ ID NO: 73 may be encoded by the nucleic acid shown in SEQ ID NO: 74.

[0018] In some embodiments, the isolated antibody or its antigen-binding fragment comprises a light chain CDR. The light chain CDR comprises light chain CDR1, light chain CDR2, and light chain CDR3. Herein, (1) light chain CDR1 comprises the sequence shown in SEQ ID NO: 25 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and light chain CDR2 comprises the sequence shown in SEQ ID NO: 29 or at least 80% It contains an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain CDR3 contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% identity thereto. (2) The light chain CDR1 contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. The light chain CDR2 contains an amino acid sequence having identity with the sequence shown in SEQ ID NO: 30 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, and the light chain CDR3 contains the sequence shown in SEQ ID NO: 34 or (3) The light chain CDR1 contains an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; (4) The light chain CDR1 contains an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; (5) The light chain CDR1 contains an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 99% identical thereto The light chain CDR2 contains an amino acid sequence having identity with the sequence shown in SEQ ID NO: 30 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and the light chain CDR3 contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 99% identity with the sequence shown in SEQ ID NO: 34 or (4) light chain CDR1 contains an amino acid sequence having 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or (4) light chain CDR1 contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and light chain CDR2 contains an amino acid sequence having at least 80%, The light chain CDR3 contains an amino acid sequence having 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain CDR3 contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0019] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2. Here, the amino acid sequence of SEQ ID NO: 30 is QX4SNLAS, X4=M or L.

[0020] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2, which is a monoclonal antibody (e.g., a mouse, chimeric, or humanized antibody).

[0021] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region. The amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NOs. 36, 38, 40, 42, 44, 46, or 79; or includes an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NOs.

[0022] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2, which is a monoclonal antibody (e.g., a mouse, chimeric, or humanized antibody).

[0023] In some specific embodiments, the amino acid shown in SEQ ID NO: 79 may be encoded by the nucleic acid shown in SEQ ID NO: 80.

[0024] In some embodiments, the isolated antibody or its antigen-binding fragment comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, and light chain CDR1, light chain CDR2 and light chain CDR3. Herein, (1) heavy chain CDR1 comprises the sequence shown in SEQ ID NO: 11 or therewith The heavy chain CDR2 contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the heavy chain CDR2 contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 9 The heavy chain CDR3 contains an amino acid sequence having 3%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO: 20 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, and the light chain CDR1 contains an amino acid sequence showing SEQ ID NO: 2 The sequence shown in 5 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therein, and the light chain CDR2 contains the sequence shown in SEQ ID NO: 29 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, The amino acid sequence contains 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, and the light chain CDR3 contains the sequence shown in SEQ ID NO: 32 or an amino acid sequence that contains at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto;(2) Heavy chain CDR1 contains the sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and heavy chain CDR2 contains the sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% identity thereto The heavy chain CDR3 contains an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO: 21 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. Light chain CDR1 contains an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and light chain CDR2 contains an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, or 8% identical thereto, and light chain CDR2 contains an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, or 8% identical thereto The amino acid sequence contains 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, and the light chain CDR3 contains the sequence shown in SEQ ID NO: 34 or an amino acid sequence containing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto;(3) Heavy chain CDR1 contains the sequence shown in SEQ ID NO: 12 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and heavy chain CDR2 contains the sequence shown in SEQ ID NO: 17 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, The heavy chain CDR3 contains an amino acid sequence having 98% or 99% identity with the sequence shown in SEQ ID NO: 22 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, and the light chain CDR1 contains the sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, or 92% identity with it; The light chain CDR2 contains an amino acid sequence having 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO: 30 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, and the light chain CDR3 contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO: 34 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it;(4) Heavy chain CDR1 contains the sequence shown in SEQ ID NO: 13 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and heavy chain CDR2 contains the sequence shown in SEQ ID NO: 18 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% identity thereto The heavy chain CDR3 contains an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO: 23 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. Light chain CDR1 contains an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and light chain CDR2 contains an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, or 8% identical thereto, and light chain CDR2 contains an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, or 8% identical thereto The amino acid sequence contains 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, and the light chain CDR3 contains the sequence shown in SEQ ID NO: 34 or an amino acid sequence containing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto;(5) Heavy chain CDR1 contains the sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and heavy chain CDR2 contains the sequence shown in SEQ ID NO: 19 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, The heavy chain CDR3 contains an amino acid sequence having 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO: 22 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, and the light chain CDR1 contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, or 88% identity with the sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, or 88% identity with it. , contains an amino acid sequence having 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and light chain CDR2 contains the sequence shown in SEQ ID NO: 30 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and light chain CDR3 contains the sequence shown in SEQ ID NO: 34 or at least 80%, (6) The heavy chain CDR1 contains an amino acid sequence having 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or (6) the heavy chain CDR1 contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; Heavy chain CDR2 contains the sequence shown in SEQ ID NO: 18 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and heavy chain CDR3 contains the sequence shown in SEQ ID NO: 24 or at least The amino acid sequence contains 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, and the light chain CDR1 contains the sequence shown in SEQ ID NO: 28 or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 8 The amino acid sequence contains 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, and the light chain CDR2 contains the sequence shown in SEQ ID NO: 31 or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96. The amino acid sequence comprises %, 97%, 98%, or 99% identical, and the light chain CDR3 comprises the sequence shown in SEQ ID NO: 33 or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0025] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2. Here, the amino acid sequence of SEQ ID NO: 17 is AIDPETGDTVYX1X2KFX3G, where X1=N or A, X2=Q, E or K, and X3=K or Q. The amino acid sequence of SEQ ID NO: 30 is QX4SNLAS, where X4=M or L.

[0026] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2. Here, the amino acid sequence of SEQ ID NO: 17 is AIDPETGDTVYX1X2KFX3G, where X1=N or A, X2=Q, X3=K or Q; X1=N or A, X2=E, X3=K or Q; X1=N or A, X2=K, X3=K or Q. And the amino acid sequence of SEQ ID NO: 30 is QX4SNLAS, where X4=M or L.

[0027] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2. Here, the amino acid sequence of SEQ ID NO: 17 is AIDPETGDTVYX1X2KFX3G, where X1=N, X2=Q, X3=K or Q;X1=A, X2=Q, X3=K or Q;X1=N, X2=E, X3=K or Q;X1=A, X2=E, X3=K or Q;X1=N, X2=K, X3=K or Q; or X1=A, X2=K, X3=K or Q. And the amino acid sequence of SEQ ID NO: 30 is QX4SNLAS, where X4=M or L.

[0028] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2. Here, the amino acid sequence of SEQ ID NO: 17 is AIDPETGDTVYX1X2KFX3G, where X1=N, X2=Q, X3=K; X1=A, X2=E, X3=Q; or X1=A, X2=K, X3=K; and the amino acid sequence of SEQ ID NO: 30 is QX4SNLAS, where X4=M or L.

[0029] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2, which is a monoclonal antibody (e.g., a mouse, chimeric, or humanized antibody).

[0030] In some embodiments, the isolated antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region. Herein, (1) the heavy chain variable region includes SEQ ID NO: 3 The sequence shown in 5 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region includes the sequence shown in SEQ ID NO: 36 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, (2) The heavy chain variable region includes an amino acid sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; (2) The heavy chain variable region includes an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region (3) The region contains the sequence shown in SEQ ID NO: 38 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; (3) The heavy chain variable region contains the sequence shown in SEQ ID NO: 39 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% identity thereto The light chain variable region includes an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 97%, 98%, or 99% identity thereto; and the light chain variable region includes an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto;(4) The heavy chain variable region includes the sequence shown in SEQ ID NO: 41 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region includes the sequence shown in SEQ ID NO: 42 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% identity thereto (5) The heavy chain variable region includes an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 97%, 98%, or 99% identity therewith; (6) The heavy chain variable region includes the sequence shown in SEQ ID NO: 43 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith (6) The light chain variable region includes an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; (6) The heavy chain variable region includes an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, or 8 The light chain variable region includes an amino acid sequence having 7%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; and the light chain variable region includes the sequence shown in SEQ ID NO: 46 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto;(7) The heavy chain variable region includes the sequence shown in SEQ ID NO: 71 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region includes the sequence shown in SEQ ID NO: 79 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89% identity thereto , comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or (8) the heavy chain variable region comprising the sequence shown in SEQ ID NO: 73 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region comprising; The amino acid sequence includes the sequence shown in Sequence ID No. 79 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0031] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2, which is a monoclonal antibody (e.g., a mouse, chimeric, or humanized antibody).

[0032] In some embodiments, the isolated antibody or its antigen-binding fragment comprises a heavy chain. The amino acid sequence of the heavy chain comprises the sequence shown in SEQ ID NOs. 47, 51, 55, 59, 63, 67, 75, or 77; or comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NOs.

[0033] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2, which is a monoclonal antibody (e.g., a mouse, chimeric, or humanized antibody).

[0034] In some specific embodiments, the amino acids represented by SEQ ID NOs: 47, 51, 55, 59, 63, 67, 75, and 77 may be encoded by nucleic acids represented by SEQ ID NOs: 48, 52, 56, 60, 64, 68, 76, and 78, respectively.

[0035] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain. The amino acid sequence of the light chain includes the sequence shown in SEQ ID NOs. 49, 53, 57, 61, 65, 69, or 81; or includes an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NOs.

[0036] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2, which is a monoclonal antibody (e.g., a mouse, chimeric, or humanized antibody).

[0037] In some specific embodiments, the amino acids represented by SEQ ID NOs: 49, 53, 57, 61, 65, 69, and 81 may be encoded by nucleic acids represented by SEQ ID NOs: 50, 54, 58, 62, 66, 70, and 82, respectively.

[0038] In some embodiments, the isolated antibody or its antigen-binding fragment comprises a heavy chain and a light chain, where (1) the heavy chain comprises the sequence shown in SEQ ID NO: 47 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain comprises the sequence shown in SEQ ID NO: 49 or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% (2) The heavy chain includes an amino acid sequence having 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith; (2) The heavy chain includes an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and (3) The light chain contains an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; (4) The heavy chain contains an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9 (4) The light chain contains an amino acid sequence having 6%, 97%, 98%, or 99% identity thereto; (5) The heavy chain contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; (6) The heavy chain contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, (5) The light chain contains an amino acid sequence having 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; (6) The heavy chain contains an amino acid sequence having 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; (7) The heavy chain contains an amino acid sequence having 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto It also contains an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto;(6) The heavy chain contains an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain contains an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 8 (7) The heavy chain includes an amino acid sequence having 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith; and (8) The light chain contains an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; or (8) the heavy chain contains an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, or 87% identical thereto , comprising an amino acid sequence having 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0039] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2, which is a monoclonal antibody (e.g., a mouse, chimeric, or humanized antibody).

[0040] In some embodiments, the isolated antibody (e.g., mouse, chimeric, or humanized antibody) or its antigen-binding fragment comprises a heavy chain and a light chain. The heavy chain includes a heavy chain variable region and The heavy chain constant region is included. The light chain includes a light chain variable region and a light chain constant region. Here, the heavy chain variable region and the light chain variable region include the above amino acid sequence. The heavy chain constant region has a human IgG1, IgG2, or IgG4 constant region, preferably a human IgG2 constant region. The light chain constant region has a human κ constant region or a λ constant region. Here, this antibody or antigen-binding fragment binds to ST2.

[0041] In some embodiments, the publicly disclosed antibody comprises or consists of two heavy chains and two light chains. The heavy chains and light chains are linked by disulfide bonds. Here, each heavy chain comprises a heavy chain constant region, a heavy chain variable region, or a CDR sequence, and each light chain comprises a light chain constant region, a light chain variable region, or a CDR sequence. Here, the C-terminus of the heavy chain variable region is ligated to the N-terminus of the heavy chain constant region, and the C-terminus of the light chain variable region is ligated to the N-terminus of the light chain constant region. The publicly disclosed antibody may be, for example, a full-length antibody of an IgG1, IgG2, or IgG4 isotype. In another embodiment, the publicly disclosed antibody may be a single-chain antibody (scFv), or an antibody fragment such as, for example, Fab, F(ab')2 fragment, Fd fragment, Fv fragment, dAb, or a separated CDR region.

[0042] In one aspect, this publication provides a polypeptide fusion comprising the antibody or its antigen-binding fragment, and other functional molecules. The other functional molecules may be peptides, proteins, or non-proteins. The polypeptide fusion has the function of binding to ST2, and one or more other functions. In one aspect, this publication further provides a multispecific molecule comprising the antibody or its antigen-binding fragment, and at least one other functional moiety different from the specificity of the antibody or antigen-binding fragment. The other functional moiety may be another peptide or protein, and the multispecific molecule can bind to ST2 and at least one other disease-related protein, such as IgE. On the other hand, the ST2 antibody or its antigen-binding fragment is encoded by or supported by a viral vector.

[0043] In one aspect, this disclosure further provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, the multispecific molecule, polypeptide fusion, or viral vector thereof, and a pharmaceutically acceptable excipient, diluent, or carrier.

[0044] In one aspect, this disclosure further provides isolated nucleic acid molecules encoding the antibody or its antigen-binding fragment, an expression vector containing the nucleic acid molecule, and a host cell containing the expression vector.

[0045] In one aspect, this publication further provides a method for preparing an ST2 antibody or its antigen-binding fragment. The method includes the following steps: (i) expressing the antibody or its antigen-binding fragment in host cells, and (ii) isolating the antibody or its antigen-binding fragment from host cells or its cell culture.

[0046] On the other hand, this publication provides a method for detecting the amount of ST2 expression in a sample derived from a subject. This method includes a step of contacting the sample with the antibody or its antigen-binding fragment under conditions that enable binding of the antibody or its antigen-binding fragment to ST2 (or conditions that enable complex formation with ST2).

[0047] On the other hand, this disclosure provides a method for treating or achieving remission of IL33 / ST2-mediated related diseases and conditions in subjects who require it. These IL33 / ST2-mediated related diseases and conditions include asthma, allergic rhinitis, chronic obstructive pulmonary disease, eosinophilic bronchiolitis, eosinophilic esophagitis, atopic dermatitis, psoriasis, systemic lupus erythematosus, bullous pemphigoid, rheumatoid arthritis, and ankylosing spine. This includes, but is not limited to, inflammation, inflammatory bowel disease, pulmonary fibrosis, hepatic fibrosis, systemic sclerosis, sarcoidosis, graft-versus-host disease (GVHD), diabetic disease, cardiovascular disease, or combinations thereof. The method involves administering to the subject a therapeutically effective amount of the publicly disclosed antibody or its antigen-binding fragment, or a pharmaceutical composition thereof. In some embodiments, the method involves administering to the subject a therapeutically effective amount of a nucleic acid molecule encoding the publicly disclosed antibody or its antigen-binding fragment. In some embodiments, the method involves administering to the subject a therapeutically effective amount of the publicly disclosed polypeptide fusion, multispecific molecule, viral vector, or pharmaceutical composition thereof. In some embodiments, the multispecific molecule can bind to ST2 and at least one other disease-related protein, such as TSLP, IgE, IL4, IL13, or IL-5. In some embodiments, at least one other antibody is used together with the publicly disclosed antibody or its antigen-binding fragment, such as a TSLP antibody, a TSLPR antibody, an IL4 antibody, an IL4R antibody, or an IgE antibody. In some embodiments, at least one other pharmaceutical agent is used in conjunction with the antibody or its antigen-binding fragment described herein. For example, an anti-asthmatic agent, an anti-chronic obstructive pulmonary disease agent, an anti-ulcerative colitis agent, an anti-abnormal dermatitis agent, or an anti-psoriasis agent.

[0048] Other characteristics and advantages of this disclosure will become clearer based on the following specific descriptions and embodiments, and the specific descriptions and embodiments should not be construed as restrictive. All documents, GenBank records, patents and previously published patent applications cited in this publication are expressly included in this publication by reference. [Brief explanation of the drawing]

[0049] [Figure 1] Figure 1: Binding of chimeric anti-ST2 antibodies to hST2 within a certain concentration range, as detected by ELISA. Figure 1 shows the binding of chimeric antibodies xi17E2, xi8F4, xi26A1, xi31C8, xi3C6, and xi7D1 to hST2, with RG6149 shown as a control. [Figure 2] Figure 2: Binding of chimeric anti-ST2 antibodies to cyno-ST2 within a certain concentration range, as detected by ELISA. Figure 2 shows the binding of chimeric antibodies xi17E2, xi8F4, xi26A1, xi31C8, xi3C6, and xi7D1 to cyno-ST2, with the binding of RG6149 shown as a control. [Figure 3] Figure 3: Inhibitory effect of chimeric anti-ST2 antibodies on IL33 / ST2 binding within a certain antibody concentration range as detected by ELISA. Figure 3 shows the inhibition of IL33 / ST2 binding by chimeric antibodies xi26A1, xi7D1, xi17E2, xi31C8, xi3C6, and xi8F4, with inhibition of RG6149 shown as a control. [Figure 4] Figure 4: Binding of humanized anti-ST2 antibodies to hST2 within a certain concentration range as detected by ELISA. Figure 4 shows the binding of humanized antibodies hz31C8-1.1 and hz31C8-1.2 to hST2, the binding of chimeric antibodies xi31C8, RG6149, and GSK3772847 as controls, and the binding of IgG2 as a negative control. [Figure 5] Figure 5: Binding of humanized anti-ST2 antibodies to cyno-ST2 within a certain concentration range as detected by ELISA. Figure 5 shows that humanized antibodies hz31C8-1.1 and hz31C8-1.2 bind to cyno-ST2, the binding of chimeric antibodies xi31C8, RG6149, and GSK3772847 is shown as controls, and the binding of IgG2 is shown as a negative control. [Figure 6]Figure 6: Inhibitory effect of humanized anti-ST2 antibodies on IL33 / ST2 binding within a certain antibody concentration range as detected by ELISA. Figure 6 shows the inhibition of IL33 / ST2 binding by humanized antibodies hz31C8-1.1 and hz31C8-1.2, and the inhibition of chimeric antibodies xi31C8, RG6149, and GSK3772847 is shown as controls. [Figure 7] Figure 7: Binding of chimeric anti-ST2 antibodies to hST2-overexpressing cells within a certain antibody concentration range detected by FACS. Figure 7 shows the binding of chimeric antibodies xi31C8, xi26A1, xi7D1, xi3C6, xi8F4, and xi17E2 to hST2-overexpressing HEK293T-hST2-NFκB-Luciferase cells, with RG6149 binding shown as a control and IgG4 binding as a negative control. [Figure 8] Figure 8: Binding of chimeric anti-ST2 antibodies to cyno-ST2 overexpressing cells within a certain antibody concentration range detected by FACS. Figure 8 shows the binding of chimeric antibodies xi31C8, xi26A1, xi7D1, xi3C6, xi8F4, and xi17E2 to cyno-ST2 overexpressing HEK293T-Cyno-ST2-NFκB-Luciferase cells, with RG6149 binding shown as a control and IgG4 binding as a negative control. [Figure 9] Figure 9: Inhibitory effect of chimeric anti-ST2 antibodies on IL33 / ST2 binding within a certain antibody concentration range, as detected by cell activity. Figure 9 shows the inhibition of the interaction between IL33 protein and hST2-expressing HEK293T-hST2-NFκB-Luciferase cells by chimeric antibodies xi31C8, xi26A1, xi7D1, and xi3C6, with inhibition of RG6149 shown as a control. [Figure 10]Figure 10: Binding of humanized anti-ST2 antibodies to hST2-overexpressing cells within a certain antibody concentration range detected by FACS. Figure 10 shows the binding of humanized antibodies hz31C8-1.1 and hz31C8-1.2 to hST2-overexpressing HEK293T-hST2-NFκB-Luciferase cells, with the binding of chimeric antibodies xi31C8, RG6149, and GSK3772847 shown as controls, and the binding of IgG2 shown as a negative control. [Figure 11] Figure 11: Inhibitory effect of humanized anti-ST2 antibodies on IL33 / ST2 binding within a certain antibody concentration range, as detected by cell activity. Figure 11 shows the inhibition of the interaction between IL33 protein and hST2-expressing HEK293T-hST2-NFκB-Luciferase cells by humanized antibodies hz31C8-1.1 and hz31C8-1.2, with inhibition of chimeric antibodies xi31C8, RG6149, and GSK3772847 as controls, and inhibition of IgG2 as a negative control. [Figure 12] Figure 12: Inhibitory effect of humanized anti-ST2 antibodies on IL33 / ST2 binding within a certain antibody concentration range, as detected by cell activity. Figure 12 shows the inhibition of the interaction between IL33 protein and hST2-expressing KU812-NFκB-Luciferasae cells by humanized antibodies hz31C8-1.1 and hz31C8-1.2, with inhibition of chimeric antibodies xi31C8, RG6149, and GSK3772847 as controls, and inhibition of IgG2 as a negative control. [Figure 13] Figure 13: Inhibition of humanized anti-ST2 antibodies against IL33 / ST2-induced IL5 secretion on CD4+ T cells within a certain antibody concentration range as detected by cell activity. Figure 13 shows that humanized antibodies hz31C8-1.1 and hz31C8-1.2 inhibit IL33 / ST2-induced IL5 secretion on CD4+ T cells, with inhibition of chimeric antibodies xi31C8, RG6149, and GSK3772847 shown as controls. [Modes for carrying out the invention]

[0050] It should be understood that the terminology used in this publication is not intended to be restrictive, but rather to illustrate specific examples. Unless otherwise defined, all technical and scientific terms used in this publication have the same meaning as those commonly understood by those skilled in the art.

[0051] "ST2" includes ST2 variants, homologs, homologs, and orthologues. For example, in some embodiments, antibodies specific to the human ST2 protein may cross-react with ST2 proteins of other species (e.g., monkeys). In this embodiment, an antibody specific to human ST2 protein may not specifically cross-react with other species or other types of proteins, but may specifically cross-react with human ST2 protein, or it may not cross-react with ST2 proteins of any other species, but may cross-react with a specific species.

[0052] The terms "human ST2" or "hST2" are interchangeable in this disclosure and refer to a protein having a human ST2 amino acid sequence, for example, the human ST2 amino acid sequence shown in SEQ ID NO: 1. The protein is composed of several domains: amino acids 1-18 correspond to the leader sequence, amino acids 19-331 correspond to the extracellular domain, amino acids 332-350 correspond to the transmembrane domain, and amino acids 351-556 correspond to the intracellular domain. The terms "monkey ST2" or "cyno-ST2" are interchangeable in this disclosure and refer to a protein having a monkey ST2 amino acid sequence, for example, the monkey ST2 amino acid sequence shown in SEQ ID NO: 10.

[0053] The "antibodies" disclosed herein include full-length antibodies and any antigen-binding fragments (i.e., "antigen-binding portions") or single chains thereof. Full-length antibodies are glycoproteins containing two heavy (H) chains and two light (L) chains, linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) 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 (VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions are further divided into hypervariable regions, namely complementarity-determining regions (CDRs) and relative-conserved framework regions (FRs). Each VH and VL consists of three CDRs and four FRs. The amino end to the carboxyl end is designated as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, respectively. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors. The host tissues or factors include multiple cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0054] The "antigen-binding fragment" or "antibody-binding portion" of an antibody refers to one or more fragments of the antibody that retain the function of specific binding to an antigen (e.g., ST2 protein). It has already been confirmed that the antigen-binding function of an antibody is carried out through fragments of the full-length antibody. Examples of what is included in the term "antigen-binding portion / fragment" of an antibody include (i) Fab fragment: V L , V H(ii) Monovalent fragments consisting of CL and CH1 domains; (ii) F(ab')2 fragment: a bivalent fragment containing two Fab fragments linked by disulfide crosslinks in the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) Fv fragment consisting of VL and VH domains in one arm of the antibody; (v) dAb fragment consisting of the VH domain (see Ward et al., Nature. 341:544-546 (1989)); (vi) isolated complementarity-determining regions (CDRs); and (vii) nanoantibodies: heavy chain variable regions containing a single variable domain and two constant domains. In addition, although the two domains VL and VH of the Fv fragment are encoded by different genes, linkers can be synthesized using recombination methods to link VH and VL to a single protein chain. Here, VL and VH pair to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird et al., Science. 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. 85:5879-5883 (1988)). These single-chain antibodies are also included in the term antigen-binding moiety / fragment. These antibody fragments can be obtained by prior art known to those skilled in the art, and the fragments can be obtained by functional screening in the same manner as full-length antibodies.

[0055] "Isolated antibodies" refers to antibodies that do not contain other antibodies with substantially different antigen specificity. It is important to note that (for example, an isolated antibody that specifically binds to the ST2 protein is substantially free of proteins that specifically bind to antigens other than ST2). However, an isolated antibody that specifically binds to the human ST2 protein may have cross-binding properties with other antigens (e.g., ST2 proteins from other species). Also, the isolated antibody is substantially free of other cellular components and / or chemical substances.

[0056] "Mouse antibody" or "mouse-derived antibody" refers to an antibody in which the framework region and CDR region in the variable region are all derived from mouse immunoglobulin sequences. Furthermore, if the antibody contains a constant region, that region is also derived from a mouse immunoglobulin sequence. The mouse antibodies published here may contain amino acid residues encoded by mouse immunoglobulin sequences (e.g., mutations introduced through random or point mutations in vitro or cellular mutations in vivo), but "mouse antibody" does not include antibodies in which CDR sequences from other mammals have been inserted into the mouse framework sequence.

[0057] A "chimeric antibody" refers to an antibody created by combining genetic material from a non-human organism with genetic material from a human organism. More generally, a chimeric antibody is an antibody derived from the genetic material of one species and the genetic material of another species. In this publication, chimeric antibodies are also represented as "Xi".

[0058] A "humanized antibody" is an antibody derived from a non-human species, but whose protein sequence has been modified to increase its similarity to antibodies naturally produced by humans. In this publication, humanized antibodies are also represented as "hz".

[0059] "Isotype" refers to the antibody category (e.g., IgM or IgG1) encoded by a heavy chain constant region gene.

[0060] In this publication, the terms "antibody that recognizes an antigen" and "antibody that is specific to an antigen" are interchangeable with the term "antibody that is specifically bound to an antigen."

[0061] An antibody that "specifically binds to human ST2" means an antibody that binds to the human ST2 protein (and possibly to ST2 proteins from other non-human species), but substantially does not bind to non-ST2 proteins. Preferably, the antibody binds to human ST2 with "high affinity," i.e., a KD of 5.0 × 10⁻⁶. -8 It is less than or equal to M, and 1.0 × 10 -8 M is less than or equal to 5.0 × 10-9 is less than M and is 1.0 × 10 -9 is less than M, more preferably 5.0 × 10 -10 is less than M and is 1.0 × 10 -10 is less than M.

[0062] The term "not substantially bind to" a protein or cell means not bind to or not bind to it with high affinity. That is, the KD for binding to a protein or cell is 1.0 × 10 -6 is greater than or equal to M, preferably 1.0 × 10 -5 is greater than or equal to M and is 1.0 × 10 -4 is greater than or equal to M, more preferably 1.0 × 10 -3 is greater than or equal to M and is 1.0 × 10 -2 is greater than or equal to M.

[0063] In the case of IgG, the term "high affinity" means that the KD is 1.0 × 10 -6 is less than or equal to M and is 1.0 × 10 -7 is less than or equal to M, preferably 1.0 × 10 -8 is less than or equal to M and is 5.0 × 10 -9 is less than or equal to M, more preferably 1.0 × 10 -9 is less than or equal to M. However, in the case of other antibody isotypes, "binding with high affinity" does not mean the above meaning. For example, "binding with high affinity" of the IgM isotype means that the KD is 10 -6 is less than or equal to M, preferably 10 -7 is less than or equal to M, more preferably 10 -8 is less than or equal to M.

[0064] "Identity" means similarity between two nucleic acid sequences or between two polypeptides. This public The open sequence identity is at least 80%, 85%, 90%, or 95%, preferably at least 95%. Examples of unrestricted sequences include 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Sequence comparison and identity ratio measurements between two sequences can be performed using the default settings of the BLASTN / BLASTP algorithm on the National Center for Biotechnology Institute website.

[0065] The term "EC50 value" is also known as the half-effective concentration and refers to the antibody concentration that reaches 50% of the maximum effect after a specific exposure time.

[0066] The term "IC50 value" is also known as the half-inhibitory concentration and refers to the antibody concentration at which 50% of a given biological or biochemical function is inhibited compared to the absence of the antibody.

[0067] The terms "suppression" and "inhibition" are interchangeable and include partial and complete suppression / inhibition. In some embodiments, the ST2 antibody suppresses IL33 / ST2 binding by at least about 50%, for example, at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[0068] The term “subject” includes any person or non-human animal. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, preferably mammals, e.g., non-human primates, sheep, dogs, cats, cattle and horses.

[0069] The term "therapeutic effective dose" means an amount sufficient to prevent or improve a disease or condition, and / or to reduce the severity of the disease or condition, preferably an amount that can reduce the severity of the disease, increase the frequency and duration of asymptomatic periods, or prevent damage or incapacity caused by the disease. The therapeutic effective dose is related to the disease being treated, and those skilled in the art can easily determine the actual effective dose.

[0070] Unless otherwise specified, the use of a single number includes the plural form. Unless otherwise specified, the words "one" or "one kind" mean "at least one" or "at least one kind." Unless otherwise specified, "at least one" is equivalent to "one or more," and the use of "and / or" means "and" or "or."

[0071] Many aspects of this disclosure will be explained in more detail below.

[0072] The anti-ST2 antibody specifically binds to ST2 and inhibits the IL33 / ST2 interaction, as well as exhibiting other beneficial functional characteristics. The ST2 antibody or its antigen-binding fragment disclosed herein binds specifically to human ST2 with high affinity. The ST2 antibody or its antigen-binding fragment specifically binds to ST2 (e.g., human ST2 and monkey ST2) and inhibits IL33 / ST2 binding and its signal conduction. The ST2 antibody or its antigen-binding fragment disclosed herein is CD4 + It inhibits the secretion of IL-5 induced by IL33 / ST2 on T cells. The ST2 antibody or its antigen-binding fragment disclosed herein does not substantially bind to other IL-1R family receptors, such as IL1R1, IL1R2, IL1R3, IL1R7, IL1R8, and IL1R9. The ST2 antibody or its antigen-binding fragment disclosed herein has good physical stability (e.g., thermal stability). The ST2 antibody or its antigen-binding fragment disclosed herein has a long half-life in the body.

[0073] Preferably, the ST2 antibody disclosed herein is a monoclonal antibody. Alternatively, the antibody may be, for example, a mouse, chimeric, or humanized monoclonal antibody.

[0074] ST2 monoclonal antibody Preferably, the ST2 antibody or its antigen-binding fragment disclosed herein is an antibody having the structure and chemical properties described below. The ST2 antibody or its antigen-binding fragment includes a heavy chain CDR region and a light chain CDR region. Here, Tables 1 and 2 provide illustrative examples of heavy chain CDR sequences and light chain CDR sequences. The ST2 antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region. Here, Table 3 provides illustrative examples of heavy chain variable regions and light chain variable regions. Some antibodies have the same CDR, and some antibodies have the same VH or VL. The heavy chain constant region of the antibody may be a human IgG2 heavy chain constant region, and the light chain constant region of the antibody may be a human κ light chain constant region. These antibodies may further include a mouse IgG1 or IgG4 heavy chain constant region and / or a mouse κ light chain constant region.

[0075] The heavy-chain CDR regions in Table 1 and the light-chain CDR regions in Table 2 are defined using the Kabat numbering system. However, as is well known in this art, CDR regions are determined based on the heavy / light-chain variable region sequence by other numbering systems, such as the Chothia, IMGT, AbM, or Contact numbering systems / methods.

[0076] [Table 1]

[0077] [Table 2]

[0078] [Table 3-1]

[0079] [Table 3-2]

[0080] [Table 4-1]

[0081] [Table 4-2]

[0082] [Table 4-3]

[0083] [Table 4-4]

[0084] [Table 4-5]

[0085] [Table 4-6]

[0086] The VH and / or VL sequences (or CDR sequences) of other anti-ST2 antibodies that bind to human ST2 can be "combined, paired" with the VH and / or VL sequences (or CDR sequences) of the antibodies disclosed herein. Preferably, when combining and pairing VH and VL chains (or their CDRs), the VH sequence in a particular VH / VL pair can be replaced with a VH sequence having a similar structure. Similarly, preferably, the VL sequence in a particular VH / VL pair can be replaced with a VL sequence having a similar structure.

[0087] Therefore, in one embodiment, the antibody or antigen-binding fragment thereof disclosed herein contains, (a) Heavy chain variable region containing the amino acid sequence shown in Table 3; and (b) Includes a light chain variable region containing the amino acid sequence shown in Table 3, or a VL of another ST2 antibody that specifically binds to human ST2.

[0088] In another embodiment, the antibody or antigen-binding fragment thereof disclosed herein includes: (a) Heavy chains CDR1, CDR2 and CDR3 shown in Table 1; and (b) Includes light chain CDR1, CDR2, and CDR3 shown in Table 2, or another ST2 antibody CDR that specifically binds to human ST2;

[0089] In another embodiment, the disclosed antibody or its antigen-binding fragment includes the heavy chain CDR2 of the disclosed ST2 antibody and the CDRs of another antibody that binds to human ST2, for example, the heavy chain CDR1 and / or CDR3 and / or the light chain CDR1, CDR2 and / or CDR3 of another ST2 antibody.

[0090] Furthermore, as is well known in this field, the CDR3 domain is CDR1 and / or CDR2 domain It is independent of the nucleotide sequence, the binding specificity of antibodies against the same antigen can be determined independently, and it can be predicted that multiple antibodies with the same binding specificity can be generated based on the CDR3 sequence. For example, Klimka et al.,British J. of Cancer.83(2):252-260(2000);Beiboer et al.,J.Mol.Biol.296:833-849(2000);Rader et al.,Proc.Natl.Acad.Sci.USA95:8910-8915(1998);Barbas et al. al.,J.Am.Chem.Soc.116:2161-2162(1994);Barbas et al.,Proc.Natl.Acad.Sci.USA92:2529-2533(1995);Ditzel et al.,J. Immunol.157:739-749(1996);Berezov et al. al.,BIAjournal 8:Scientific Review 8(2001);Igarashi et al.,J.Biochem(Tokyo).117:452-7(1995);Bourgeois et al.,J. Virol.72:807-10(1998);Levi et al. al.,Proc.Natl.Acad.Sci.USA90:4374-8(1993);Polymenis and Stoller,J.Immunol.152:5218-5329(1994)and Xu and See Davis, Immunity. 13:37-45 (2000); USPat. Nos. 6,951,646; 6,914,128; 6,090,382; 6,818,216; 6,156,313; 6,827,925; 5,833,943; 5,762,905 and 5,760,185. All of these references are cited in this publication by reference.

[0091] In another embodiment, the publicly disclosed antibody or its antigen-binding fragment comprises the heavy chain CDR2 of the publicly disclosed ST2 antibody, and at least the heavy chain and / or light chain CDR3 of the publicly disclosed ST2 antibody, or the heavy chain and / or light chain CDR3 of another ST2 antibody that specifically binds to human ST2. Preferably, these antibodies and the publicly disclosed ST2 antibody (a) compete to bind to ST2; (b) maintain functional characteristics; (c) bind to the same target epitope; and / or (d) have similar binding affinity as the publicly disclosed antibody. In another embodiment, the publicly disclosed antibody or its antigen-binding fragment further comprises the light chain CDR2 of the publicly disclosed ST2 antibody, or the light chain CDR2 of another ST2 antibody that specifically binds to human ST2. The publicly disclosed antibody or its antigen-binding fragment further comprises the heavy and / or light chain CDR1 of the publicly disclosed ST2 antibody, or the heavy and / or light chain CDR1 of another ST2 antibody that specifically binds to human ST2.

[0092] conservative modification In another embodiment, the disclosed antibody or its antigen-binding fragment contains one or more conserved modified heavy chain variable regions and / or light chain variable regions CDR1, CDR2, and CDR3 sequences relative to the disclosed ST2 antibody. It should be understood in the art that some modifications of conserved sequences do not eliminate antigen binding. For example, see Brummell et al., Biochem 32:1180-8 (1993); de Wildt et al., Prot.Eng.10:835-41 (1997); Komissarov et al., J.Biol.Chem.272: 26864-26870 (1997); Hall et al., J.Immunol.149:1605-12 (1992); Kelley and O'Connell Biochem.32:6862-35 (1993); Adib-Conquy et al., Int.Immunol.10:341-6 (1998); Beers et al., Clin.Can.Res.6:2835-43 (2000).

[0093] Therefore, in one embodiment, the antibody includes a heavy chain variable region and / or a light chain variable region, the heavy chain variable region and the light chain variable region each containing CDR1, CDR2, and CDR3, respectively. (a) The CDR1 sequence in the heavy chain variable region includes the sequences shown in Table 1 and / or their conservative modifications; and / or (b) The CDR2 sequence of the heavy chain variable region includes the sequences shown in Table 1 and / or their conservative modifications; and / or (c) The CDR3 sequences in the heavy chain variable region include the sequences shown in Table 1, and / or their conservative modifications; and / or (d) The CDR1 and / or CDR2 and / or CDR3 sequences in the light chain variable region include the sequences shown in Table 2; and / or their conservative modifications; and (e) The antibody specifically binds to human ST2.

[0094] The antibodies disclosed here possess one or more functional properties, such as high affinity for human ST2 and inhibition of IL33 / ST2 binding and its signal conduction.

[0095] In some embodiments, the antibody may be a mouse antibody, a chimeric or humanized antibody, or an antigen-binding fragment thereof.

[0096] As used in this publication, the term "conservative sequence modification" refers to amino acid modifications that do not significantly affect antibody binding properties. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications are introduced into the antibodies described in this publication by standard techniques known in this field, such as point mutations and PCR-mediated mutations. Conservative amino acid substitution refers to the substitution of an amino acid residue with a similar side-chain amino acid residue. Families of similar side-chain amino acid residues are known in this field. These amino acid residue families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), electrodeless side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR region of the antibodies published here can be substituted with other amino acid residues in the same side chain family, and the resulting antibodies can be detected for retained function (i.e., the function described above) using the functional tests described here.

[0097] Engineered and modified antibodies The antibodies disclosed herein are generated by engineering modifications using antibodies having one or more VH / VL sequences of the disclosed ST2 antibodies as starting material. The antibodies can undergo genetic modifications to one or more residues within one or two variable regions (i.e., VH and / or VL) (e.g., one or more CDR regions and / or one or more framework regions). Alternatively, the antibodies can undergo engineering modifications to residues in the constant region, such as altering the effector function of the antibody.

[0098] In some embodiments, CDR transplantation can be used for genetic modification of the variable region of an antibody. Antibodies are primarily due to the interaction between amino acid residues located in six heavy and light chain complementarity-determining regions (CDRs) and the target antigen. Therefore, the amino acid sequence within each antibody CDR is more diverse than the sequence outside the CDR. Since the CDR sequence is responsible for the main antibody-antigen interaction, constructing an expression vector allows the CDR sequence of a specific native antibody to be transplanted into the skeleton sequence of a different antibody with different properties, thereby expressing a recombinant antibody that mimics the properties of a specific native antibody (Riechmann et al., Nature. 332:323-327 (1998); Jones et al., Nature. 321:522-525 (1986); Queen et al., Proc. Natl). .Acad;USA86:10029-10033(1989);USPat.Nos.5,225,539;5,530,101;5,585,089;5,693,762 and 6,180,370).

[0099] Accordingly, another embodiment of the present disclosure relates to an isolated monoclonal antibody or its antigen-binding fragment, comprising a heavy chain variable region and / or a light chain variable region. The heavy chain variable region comprises CDR1, CDR2 and CDR3 of the sequences described herein, and the light chain variable region comprises CDR1, CDR2 and CDR3 of the sequences described herein. These antibodies contain the VH and VL CDR sequences of the monoclonal antibodies described herein, but they may contain different skeleton sequences.

[0100] Such skeleton sequences can be obtained from open DNA databases or publicly available references that include germline antibody gene sequences. For example, germline DNA sequences used for human heavy chain variable region and light chain variable region genes can be obtained from the Vbase human gene sequence database (www.mrc-cpe.cam.ac.uk / vbase), as well as from Kabat et al., (1991) (same as above), and from Tomlinson et al., J.Mol.Biol.227:776-798 (1992); and Cox et al., Eur.J.Immunol.24:827-836 (1994). In another embodiment, germline DNA sequences used for human heavy chain variable region and light chain variable region genes can be obtained from the Genbank database.

[0101] The antibody protein sequence is compared with a protein sequence database using one of the methods for searching for sequence similarity of Gapped BLAST, which is known to those skilled in the art (Altschul et al., (1997), the same as described above).

[0102] The skeleton sequence of the publicly disclosed antibody is preferably similar in structure to the skeleton sequence used in the publicly disclosed antibody. The VH CDR1, CDR2, and CDR3 sequences can be transplanted into a framework region having the same sequence as the system immunoglobulin gene from which the skeleton sequence is obtained, or the CDR sequences can be transplanted into a framework region having one or more mutations compared to the system sequence. For example, in some cases, it may be beneficial to mutate residues in the framework region to maintain or enhance the antigen-binding ability of the antibody (see, e.g., USPat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0103] Another type of variable region modification involves mutating amino acid residues within the VH and / or VL CDR1, CDR2 and / or CDR3 regions to improve one or more properties of the target antibody (e.g., affinity, physicochemical properties). Mutations can be introduced by site-directed mutagenesis or PCR-mediated mutagenesis, and the effect of the mutation on antibody binding or other functional properties can be evaluated through in vitro or in vivo assays known in the art. Preferably, conservative modifications known in the art are introduced. These may be amino acid substitutions, additions, or deletions, preferably substitutions. Also, typically one, two, three, four, or five or fewer residues within each CDR are changed.

[0104] In another embodiment, this disclosure provides an isolated ST2 monoclonal antibody or its antigen-binding fragment comprising a heavy chain variable region and a light chain variable region. It comprises: (a) a VH CDR1 region comprising the sequence of this disclosure or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; (b) a VH CDR1 region comprising the sequence of this disclosure or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions. CDR2 region; (c) VH CDR3 region containing the publicly disclosed sequence or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; (d) the publicly disclosed sequence, (a) a VL CDR1 region comprising an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; (e) a VL CDR2 region comprising the publicly disclosed sequence or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; (f) a VL CDR3 region comprising the publicly disclosed sequence or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions.

[0105] The genetically modified antibodies published here include antibodies in which the VH and / or VL framework regions have been modified to improve antibody properties. Generally, such modifications to the framework regions may reduce the immunogenicity of the antibody. For example, one or more framework residues "reverse mutations" into the corresponding germline sequences. More specifically, antibodies that have undergone somatic mutations may contain framework region residues that differ from the resulting antibody germline sequence. These residues can be identified by comparing the antibody framework sequence with the germline sequence of the resulting antibody.

[0106] Another type of framework modification involves reducing certain immunogenicity by an antibody by mutating one or more residues in the framework region to remove a T cell epitope or by mutating one or more CDRs. This method is also known as “deimmunization” and is described in detail in U.S. Patent Publication 20030153043.

[0107] In addition to modifications of the framework region or CDR region, another type of modification, such as genetic engineering-based modification of the FC region of the antibodies disclosed herein, is generally used to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, FC receptor binding, and / or antigen-dependent cytotoxicity. Furthermore, the antibodies disclosed herein can also be modified to alter one or more functional properties of the antibody, such as by chemical modification (e.g., by binding to one or more chemical functional groups) or by modification to alter their glycosylation.

[0108] In one embodiment, the CH1 hinge region is modified, for example, by increasing or decreasing the number of cysteine ​​residues in the hinge region. This method is described in detail in U.S. Patent No. 5,677,425. For example, changing the number of cysteine ​​residues in the CH1 hinge region may facilitate the assembly of the light and heavy chains or improve or decrease the stability of the antibody.

[0109] In another embodiment, the Fc-hinge region of an antibody is mutated to increase or decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 region of the FC-hinge domain to reduce binding to Staphylococcus protein A (SpA) compared to the native FC-hinge domain. This method is described in detail in U.S. Patent No. 6,165,745.

[0110] In another embodiment, the glycosylation of an antibody is modified. For example, a deglycosylated antibody (i.e., an antibody lacking glycosylation) can be prepared. Such glycosylation modification can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can eliminate glycosylation sites in one or more variable region frameworks, thereby allowing glycosylation at those sites. Such deglycosylation may increase the antibody's affinity for an antigen. See, for example, U.S. Patent Nos. 5,714,350 and 6,350,861.

[0111] Furthermore, antibodies with changes in glycosylation can be prepared, such as low-fucosylated antibodies with a decrease in fucose residues, or antibodies with an increase in bisected GlcNac structures. This has been demonstrated to increase the ADCC activity of the antibody. Such glycosylation modifications can be achieved, for example, by expressing the antibody in host cells in which the glycosylation system has been altered. The glycosylation system is well known and can be used as a host cell to express the recombinant antibody published here, thereby producing antibodies with altered glycosylation. For example, since the cell lines MS704, MS705, and MS709 lack the fucosyltransferase gene FuT8 (α(1,6)-fucosyltransferase), the antibodies expressed in the MS704, MS705, and MS709 cell lines lack fucose. The Ms704, Ms705, and Ms709FUT8- / - cell lines were prepared by targeted disruption of the FuT8 gene in CHO / DG44 cells using two surrogate vectors. (See U.S. Patent 20040110704 and Ohnuki et al., Biotechnol Bioeng. 87:614-22 (2004)). As another example, EP1,176,195 describes cell lines in which FUT8 gene function is disrupted. This gene encodes fucosyltransferase, and antibodies expressed in such cell lines exhibit hypofucosylation by reducing or removing α-1,6 binding-related enzymes. EP1,176,195 describes cell lines with low or absent enzymatic activity for adding fucose to N-acetylglucosamine bound to the Fc region of the antibody, e.g., rat myeloma cell line Yb2 / 0 (ATCC CRL 1662). WO03 / 035835 describes LEC13 cells, a CHO variant cell line. This reduces the ability to add fucose to Asn(297)-related sugars, resulting in low fucosylation of antibodies expressed by host cells. (See Shields et al., J. Biol. Chem. 277:26733-26740 (2002)). Antibodies with altered glycosylation properties can also be prepared in eggs, as described in WO06 / 089231. Alternatively, antibodies with altered glycosylation properties can be prepared in plant cells such as remna.WO99 / 54342 discloses a cell line that is genetically engineered to express a glycosyltrantanase (e.g., β(1,4)-N-acetylglucosaminetransferase III (GNTIII)) that modifies glycoproteins so that antibodies expressed in the cell line enhance ADCC activity by increasing the GlcNac structure. (See Umana et al., Nat. Biotech. 17:176-180 (1999)). Alternatively, fucosidases are used to cleave fucose residues in antibodies. For example, α-L-fucosidase removes fucose residues from antibodies. (Tarentino et al., Biochem. 14:5516-23 (1975)).

[0112] Another type of modification of antibodies disclosed herein is pegylation. For example, pegylation of an antibody may increase its biological (e.g., serum) half-life. To obtain a pegylated antibody, the antibody or a fragment thereof is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups are attached to the antibody or antibody fragment. Preferably, pegylation is carried out by acylation or alkylation using an active PEG molecule (or a similar reactive water-soluble polymer). The term “polyethylene glycol” as used in this disclosure includes all forms of PEG for producing other protein derivatives, such as mono(C1-C10)alkoxy- or allyloxy polyethylene glycol or polyethylene glycol maleimide. In some embodiments, the antibody being pegylated is a deglycosylated antibody. Methods for pegylation are well known and applicable to the antibodies disclosed herein. See, for example, EP0154316 and EP0401384.

[0113] Physical properties of antibodies The antibodies published here may be characterized by their various physical properties, so the category to which they belong is determined and / or identified. For example, an antibody may contain one or more glycosylation sites in the light chain or heavy chain variable region. These glycosylation sites may increase the immunogenicity of the antibody or alter the antibody's PK value due to changes in antigen binding. Potentially brings about change (Marshall et al., Annu Rev Biochem. 41:673-702 (1972); Gala & Morrison. J Immunol. 172: 5489-94 (2004); Wallick et al., J Exp Med. 168: 1099-109 (1988); Spiro, Glycobiology. 12: 43R-56R (2002); Parekh et al., Nature 316: 452-7 (1985); Mimura et al., Mol Immunol 37: 697-706 (2000). Glycosylation is known to occur in motifs containing NXS / T sequences. In certain cases, it is preferable that the ST2 antibody does not contain glycosylation in the variable region. This can be achieved by selecting an antibody that does not contain glycosylation motifs in the variable region, or by mutating residues within the glycosylation region.

[0114] In one preferred embodiment, the antibody does not contain an asparagine isomerization site. Deamidation of asparagine occurs in NG or DG sequences, leading to the production of isoaspaginate residues, which can reduce stability.

[0115] Each antibody has a unique isoelectric point (pI), typically within the pH range of 6–9.5. The pI of IgG1 antibodies is usually within the pH range of 7–9.5, while that of IgG4 antibodies is typically within the pH range of 6–8. Antibodies with pI values ​​outside the normal range are suspected to be unstable under in vivo conditions. Therefore, ST2 antibodies with pI values ​​within the normal range are preferred. This can be achieved by selecting antibodies with pI values ​​within the normal range or by mutating surface residues.

[0116] Nucleic acid molecule encoding this publicly released antibody On the other hand, this disclosure provides nucleic acid molecules encoding heavy chain and / or light chain variable regions or CDRs of the disclosed antibodies. Nucleic acids may exist in intact cells, cell lysates, or in partially purified or substantially pure forms. By using standard techniques to purify them from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, nucleic acids are “isolated” or “substantially pure.” The nucleic acids disclosed herein may be, for example, DNA or RNA, and may or may not contain intron sequences. In one preferred embodiment, the nucleic acid is a cDNA molecule.

[0117] The nucleic acids published here can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes), the light and heavy chain cDNAs encoding the antibodies prepared from the hybridomas can be obtained using standard PCR amplification or cDNA cloning techniques. In the case of antibodies obtained from immunoglobulin gene libraries (e.g., using phage display techniques), the nucleic acids encoding such antibodies can be recovered from the gene library.

[0118] Preferably, the nucleic acid molecules in this disclosure include those encoding the VH and VL sequences or CDR of the ST2 monoclonal antibody disclosed herein. Once the DNA fragments encoding the VH and VL fragments are obtained, further operations such as converting the variable region gene to a full-length antibody chain gene, FAB fragment gene, or scFv gene can be performed using standard recombinant DNA techniques. In these operations, the DNA fragment encoding VL or VH is operably ligated to another protein, such as another DNA fragment encoding an antibody constant region or a flexible linker. As used in this disclosure, the term “operably ligated” means that the two DNA fragments are ligated such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0119] The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by manipulatively ligating the VH-encoding DNA to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are well known, and DNA fragments can obtain standard PCR amplification. The heavy chain constant region can be the IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but the IgG2 constant region is most preferred. In the case of Fab fragment heavy chain genes, the VH-encoding DNA can be manipulatively ligated to another DNA molecule encoding only the heavy chain CH1 constant region.

[0120] Isolated DNA encoding the VL region can be converted into a full-length light chain gene (and Fab light chain gene) by manipulatively ligating the VL-encoding DNA to another DNA molecule encoding the light chain constant region (CL). The sequences of human light chain constant region genes are well known, and DNA fragments can be subjected to standard PCR amplification. In preferred embodiments, the light chain constant region may be a κ or λ light chain constant region.

[0121] To prepare the scFv gene, the VH and VL sequences can be expressed as a continuous single-chain protein by manipulating the VH and VL encoding DNA fragments to another fragment encoding a flexible linker such as the amino acid sequence (Gly4-Ser)3. Here, the VL and VH regions are linked via a flexible linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc Nat.Acad.Sci.USA 85:5879-5883 (1988); McCafferty et al., Nature 348:552-554 (1990)).

[0122] Preparation of monoclonal antibodies as published here The monoclonal antibodies (mAbs) disclosed herein can be prepared using the innate somatic cell hybridization (hybridoma) technique described in Kohler and Milstein Nature 256:495 (1975). Other embodiments for preparing monoclonal antibodies include viral transformation of B lymphocytes and phage representation techniques. Chimeric or humanized antibodies are also well known in the art. See, for example, U.S. Patents 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370.

[0123] Preparation of monoclonal antibody transfectomas The antibodies disclosed herein can also be produced in host cell transfectomas using, for example, a combination of recombinant DNA technology and gene transfection methods (e.g., Morrison, S. Science 229:12021985). In one embodiment, DNA encoding a portion or full length of light and heavy chains obtained using standard molecular biology techniques is inserted into one or more expression vectors so as to be operably ligated to transcriptional and translational regulatory sequences. In this case, the term “operably ligated” refers to ligating the antibody gene to the vector such that the transcriptional and translational regulatory sequences of the vector perform a predetermined function of regulating the transcription and translation of the antibody gene.

[0124] The term "regulatory sequence" includes promoters, enhancers, and other expression regulators (e.g., polyadenylation signals) that control the transcription or translation of antibody genes. Such regulatory sequences are described in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). Preferably, regulatory sequences expressed in mammalian host cells include cytomegalovirus (CMV), Si MIA virus 40 (SV40) contains viral elements that enable high levels of protein expression in mammalian cells, such as adenovirus-derived promoters and enhancers, including adenovirus major late promoter (AdMLP). Alternatively, non-viral regulatory sequences such as ubiquitin promoters and β-globin promoters are used. Furthermore, regulatory factors consist of sequences from different origins, such as the SRα promoter system, which comprises sequences from the SV40 early promoter and sequences containing long terminal repeats from human T-cell leukemia virus type 1 (Takebe et al., Mol (Cell. Biol. 8:466-472 (1988)). Expression vectors and expression regulatory sequences are selected to be compatible with the expression host cells used.

[0125] The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or different expression vectors. In a preferred embodiment, a full-length antibody gene can be created by inserting a variable region into an expression vector encoding the heavy chain constant region and light chain constant region of the desired isotype, such that VH is operably linked to CH in the vector and VL is operably linked to CL in the vector. Alternatively, the recombinant expression vector can encode a signal peptide that promotes the secretion of the antibody chain from host cells. The antibody chain gene can be cloned into a vector such that the signal peptide is in-frame linked to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin protein).

[0126] In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors disclosed herein may contain other sequences, such as sequences that regulate vector replication in host cells (e.g., replication origins) or selectable marker genes. Selectable marker genes can be used to select host cells into which the vector has been introduced (see, for example, U.S. Patents 4,399,216; 4,634,665 and 5,179,017). For example, selectable marker genes typically confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (dhfr) gene (for methotrexate selection / amplification in DHFR host cells) and the neo gene (for G418 selection).

[0127] To express the light and heavy chains, host cells are transfected with expression vectors encoding the heavy and light chains using standard techniques. The term "transfect" includes various techniques for introducing exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, and DEAE-dextran transfection. While it is theoretically possible to express the antibodies disclosed herein in prokaryotic or eukaryotic host cells, it is preferable to express the antibodies in eukaryotic cells, and most preferably in mammalian host cells. This is because eukaryotic cells, especially mammalian cells, are more likely to fold and secrete antibodies that are immunologically active and better folded than prokaryotic cells.

[0128] Preferred mammalian host cells for expressing the recombinant antibodies disclosed herein include Chinese hamster ovary cells (CHO cells) (including dhfr-CHO cells administered with DHFR-selectable markers, e.g., as described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220 (1980). DHFR-selectable markers are described, e.g., as described in RJ Kaufman and PA Sharp. J. Mol. Biol. 159:601-621 (1982)), NSO myeloma cells, COS cells, and SP2 cells. Another appropriate expression system, particularly when using NSO myeloma cells, is GS gene expression, as disclosed in WO87 / 04462, WO89 / 01036, and EP338,841. This is a system where a recombinant expression vector encoding an antibody gene is introduced into mammalian host cells. Antibodies are then prepared by culturing the host cells for a sufficient period to allow antibody expression, or by allowing sufficient time for antibody secretion into the culture medium in which the host cells grow. The antibodies can be recovered from the culture medium using protein purification methods.

[0129] Polypeptide fusion On the other hand, this disclosure involves a polypeptide fusion comprising one or more antibodies or antigen-binding fragments thereof, wherein the antibody or antigen-binding fragment is linked to at least one other functional molecule. The other functional molecule may be a peptide, protein, or non-protein. In one embodiment, the polypeptide fusion comprises an immunoconjugate, which comprises one or more antibodies or antigen-binding fragments thereof, and a therapeutic agent such as a steroid linked to at least one of the antibodies or antigen-binding fragments thereof. In one embodiment, the polypeptide fusion comprises a multifunctional molecule, which comprises one or more antibodies or antigen-binding fragments thereof, and an immune cytokine or receptor ligand linked to at least one of the antibodies or antigen-binding fragments thereof. The immune cytokine or receptor ligand may be for IgE, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-9, IL-13, IL-13R, IL-17, IL-23, IL-33, OX40 variant (OX40L), GM-CSF, or TSLP, TSLPR / IL7R. In one embodiment, the multifunctional molecule has a third function in addition to FC receptor binding and ST2 binding. The third function may be for IgE, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-9, IL-13, IL-13R, IL-17, IL-23, IL-33, OX40 variant (OX40L), GM-CSF, or TSLP, TSLPR / IL7R. As used herein, “multifunctional molecule” includes molecules having three or more functions. In other embodiments, the polypeptide fusions described herein also include other forms. These and other forms of polypeptide fusions can be prepared by genetic engineering, chemical methods, and the like.

[0130] multispecific molecules On the other hand, this publication relates to a multispecific molecule comprising one or more antibodies or antigen-binding fragments thereof disclosed herein. Here, the antibody or antigen-binding fragment thereof disclosed herein is ligated to at least one other functional moiety having a different specificity from the antibody or antigen-binding fragment disclosed herein. The other functional moiety includes another peptide or protein (e.g., another antibody or antigen-binding fragment thereof). This then generates a multispecific molecule that binds to at least two different binding sites or targets. Thus, as used in this publication, “multispecific molecule” includes two types of specificity (i.e., a bispecific molecule), three types of specificity (i.e., a triplicate molecule), four types of specificity (i.e., a quadruplicate molecule), or more specific molecules.

[0131] In one particular embodiment, the multispecific molecules disclosed herein may have one or more other specificities in addition to anti-FC binding specificity and ST2 binding specificity. Exemplarily, the other specificities may be for IgE, IL4, IL4R, IL5, IL5R, IL6, IL9, IL13, IL13R, IL17, IL23, IL33, or TSLP, TSLPR / IL7R.

[0132] In one particular embodiment, multispecific molecules may exist in various shapes and sizes. Exemplarily, at one end of the size spectrum, a bispecific molecule retains the conventional antibody format, but the difference is that the two binding arms are not the same specificity but have different specificities, and at the other end, a bispecific molecule consisting of two single-chain antibody fragments (scFv's) linked by a peptide chain, so-called Bs(scFv) ) is a two-construct. Intermediate-sized bispecific molecules contain two different F(ab) fragments linked by a peptidyl linker. These and other formats of bispecific molecules can be prepared by genetic engineering, somatic cell hybridization, or chemical methods. See, for example, Kufer et al., cited supra; Cao and Suresh, Bioconjugate Chemistry. 9(6).635-644 (1998); and van Spriel et al., Immunology Today. 21(8):391-397 (2000).

[0133] Viral vector On the other hand, the antibodies or antigen-binding fragments disclosed herein are encoded by or supported by viral vectors. Furthermore, the antibodies or antigen-binding fragments disclosed herein can be used together with viral vectors, or viral vectors encoding or supporting the antibodies or antigen-binding fragments can be introduced into the human body.

[0134] This disclosure further provides the polypeptide fusion, multispecific molecule, viral vector, and pharmaceutical composition comprising pharmaceutically acceptable excipients, diluents, or carriers.

[0135] Pharmaceutical composition On the other hand, this publication provides pharmaceutical compositions obtained by formulating an antibody or antigen-binding fragment disclosed herein together with a pharmaceutically acceptable excipient, diluent, or carrier. The pharmaceutical composition may contain one or more pharmaceutically active ingredients, such as another antibody or drug, for example, another ST2 antibody, an anti-IgE antibody, another anti-inflammatory drug, an anti-asthmatic drug, an anti-chronic obstructive pulmonary disease drug, an anti-ulcerative colitis drug, an anti-abnormal dermatitis drug, or an anti-psoriasis drug.

[0136] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or dermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient may be encapsulated in the material to protect it from the action of acids or other natural conditions that may inactivate it. The term "parenteral administration" means a mode of administration other than enteral and topical administration, which is usually by injection, and includes, but is not limited to, injections and infusions into the venous, intramuscular, intra-arterial, subarachnoid, intracapsular, intraorbital, intracardiac, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal regions. Alternatively, the pharmaceutical compositions disclosed herein may be administered via external, such as intranasal, oral, vaginal, rectal, sublingual, or topical, or parenteral, such as dermal or mucosal administration routes.

[0137] Pharmaceutical compositions may be in the form of sterile aqueous solutions or dispersions. They may also be formulated into microemulsions, liposomes, or other ordered structures suitable for high drug concentrations.

[0138] The drug regimen is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus dose may be administered, multiple divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation. To facilitate administration and ensure uniformity of the dose, it is particularly advantageous to formulate the composition for parenteral administration in dose unit form. As used herein, dose unit form refers to a physically distinct unit appropriate as a unit dose for the subject being treated, each unit containing a predetermined amount calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier. Alternatively, the antibody may be administered as a sustained-release formulation, in which case the required frequency of administration is reduced.

[0139] When administering the composition, the dosage should be approximately 0.0001 to 100 m³ per kg of host body weight. g, or more typically in the range of 0.01 to 5 mg. For example, the dosage may be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, or 10 mg / kg body weight, or in the range of 1 to 10 mg / kg body weight. An exemplary treatment plan may require administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months.

[0140] The pharmaceutical composition may be a controlled-release formulation including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used.

[0141] In some embodiments, the antibodies disclosed herein can be formulated to ensure appropriate distribution in vivo. For example, to ensure that the antibodies disclosed herein cross the blood-brain barrier, they can be formulated into liposomes and may further include a targeting moiety to enhance selective transport to specific cells or organs.

[0142] Uses and Methods of This Publication The antibodies or antigen-binding fragments disclosed herein (encoding nucleic acid molecules, pharmaceutical compositions, polypeptide fusions, multimolecular molecules, or viral vectors) have a variety of in vitro and in vivo applications related to diagnosis. They are involved in the treatment and / or prevention of IL33 / ST2-mediated diseases and conditions. The IL33 / ST2-mediated diseases and conditions include, but are not limited to, asthma, allergic rhinitis, chronic obstructive pulmonary disease, eosinophilic bronchiolitis, eosinophilic esophagitis, atopic dermatitis, psoriasis, systemic lupus erythematosus, bullous pemphigoid, rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, pulmonary fibrosis, hepatic fibrosis, systemic sclerosis, sarcoidosis, graft-versus-host disease (GVHD), diabetic diseases, cardiovascular diseases, or combinations thereof. The antibodies or antigen-binding fragments disclosed herein can be applied to subjects to alleviate, mitigate, or treat the aforementioned diseases or conditions. During diagnosis, the amount of ST2 protein in a subject sample can be detected by contacting the antibody or its antigen-binding fragment with the sample under conditions in which the antibody and ST2 form a complex.

[0143] These and other disclosures will be described further below.

[0144] Combination therapy This disclosure provides the administration of the ST2 antibody or its antigen-binding fragment (coding nucleic acid molecule, pharmaceutical composition, polypeptide fusion, multispecific molecule, or viral vector) in combination with one or more other antibodies or pharmaceuticals, which can reduce, alleviate, or treat IL33 / ST2-mediated related diseases and conditions in a subject. The IL33 / ST2-mediated related diseases and conditions include, but are not limited to, asthma, allergic rhinitis, chronic obstructive pulmonary disease, eosinophilic bronchiolitis, eosinophilic esophagitis, atopic dermatitis, psoriasis, systemic lupus erythematosus, bullous pemphigoid, rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, pulmonary fibrosis, hepatic fibrosis, systemic sclerosis, sarcoidosis, graft-versus-host disease (GVHD), diabetic disease, cardiovascular disease, or combinations thereof. In one embodiment, this disclosure provides a method for treating asthma, chronic obstructive pulmonary disease, and atopic dermatitis in a subject. Herein, this disclosure describes the use of an ST2 antibody or its antigen-binding fragment together with one or more other antibodies. For example, TSLP antibody, TSLPR antibody, IL4 antibody, IL4R antibody, IL13 antibody, IL13R antibody, IL5 antibody, IL5R antibody and / or IgE antibody. In another embodiment, this disclosure provides a method for treating asthma, chronic obstructive pulmonary disease, and atopic dermatitis in a subject. Herein, the ST2 antibody or its antigen-binding fragment of this disclosure is used together with at least one other pharmaceutical agent. For example, an anti-asthmatic agent, an anti-chronic obstructive pulmonary disease agent, or an anti-abnormal dermatitis agent. In some embodiments, the subject is a human being. Other therapies that can be used in combination with the ST2 antibody or its antigen-binding fragment include reducing or avoiding allergen exposure, hormone therapy, and surgery.

[0145] The combinations of therapeutic agents (i.e., combinations) discussed in this publication can be administered simultaneously as a single composition on a pharmaceutically acceptable carrier, or each agent can be administered simultaneously as a separate composition on a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents can be applied sequentially.

[0146] Furthermore, when combination therapy is administered multiple times and the drugs are administered sequentially, the sequence of administration at each time point can be reversed or maintained, and sequential administration can be combined with simultaneous administration or any combination thereof.

[0147] For the purpose to be clearly understood, the invention described above is explained in detail through examples and embodiments, but according to the doctrine of this public, it is clear that an ordinary person skilled in the art can make several changes and modifications to this in general without departing from the spirit and the scope of the patent. This publication is further explained by the following examples, but is not intended to be limited thereto. A person skilled in this art will readily identify a variety of non-critical parameters that can be changed or modified to produce substantially similar results.

[0148] Unless otherwise indicated, the operations described in the current disclosure employ conventional methods in protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the scope of this art. [Examples]

[0149] Example 1: Preparation of ST2 antigen and detection protein The full-length human UniProt Interleukin-1 receptor-like 1 (ST2) isoform A (SEQ ID NO: 1) gene (hST2) was used as a template for the ST2 disclosed herein to obtain the gene sequences encoding the antigen and detection proteins disclosed herein. For mouse immunization or subsequent screening assays, these can be recombined with a mouse antibody heavy chain Fc fragment (such as mouse IgG2a) to form hST2-mFc, or with a human antibody heavy chain Fc to form hST2-hFc. cDNA encoding a recombinant protein containing an hST2 extracellular domain with a mouse antibody heavy chain Fc tag (hST2-ECD-mFc, SEQ ID NO: 2) and cDNA encoding a recombinant protein containing an hST2 extracellular domain with a human antibody heavy chain Fc tag (hST2-ECD-hFc, SEQ ID NO: 4) (SEQ ID NOs: 3 and 5, respectively) were obtained by gene synthesis and subcloned into pcDNA3.1 expression vectors (Invitrogen, V-790), respectively. Expi293 cells (Thermo, A14527) were transfected with the vector constructed above and transiently expressed. Subsequently, the recombinant hST2-ECD-mFc and hST2-ECD-hFc proteins were purified using a Protein A column (GE Healthcare).

[0150] A cDNA encoding a recombinant protein containing human IL33 with avitag (SEQ ID NO: 7) was obtained by gene synthesis and cloned into a GST-tagged expression vector. The vector constructed above was transformed into BL21 competent cells for inducible expression. The GST-IL33avitag protein was purified using a GST purification column, digested with thrombin enzyme (Sigma-Ignaphalium, T4648-1KU), and GST was removed to obtain hIL33avitag (SEQ ID NO: 6).

[0151] The cDNA encoding the human IL33 recombinant protein (SEQ ID NO: 9) was obtained by gene synthesis and cloned into a GST-tagged expression vector. The cells were transformed into BL21 competent cells for inducible expression. The GST-IL33 protein was purified using a GST purification column, digested with thrombin enzyme (Sigma-America, T4648-1KU), and GST was removed to obtain the hIL33 protein (SEQ ID NO: 8).

[0152] In the examples, ST2 antibody RG6149 refers to antibody AB2 in CN104334582 prepared in-house, and its heavy and light chain amino acid sequences are as described in SEQ ID NOs. 83 and 84 of this disclosure. ST2 antibody GSK3772847 refers to antibody STLM208 in CN104411333 prepared in-house, and its heavy and light chain amino acid sequences are as described in SEQ ID NOs. 85 and 86 of this disclosure.

[0153] Example 2: Preparation of anti-ST2 hybridoma monoclonal antibody Purified hST2-ECD-mFc recombinant protein (100 μg / mouse) was thoroughly emulsified with the same volume of complete Freund's adjuvant (Sigma, F5881-10X10ML) (primary immunization) or incomplete Freund's adjuvant (Sigma, F5506-10X10ML) (booster immunization), and BALB / C mice were subcutaneously immunized every two weeks for a total of eight weeks. Three days before fusion, adjuvant-free hST2-ECD-mFc antigen (50 μg / mouse) was intraperitoneally injected as a booster immunization. Splenocytes (1 × 10⁶) derived from immunized mice were then tested. 8 ) and SP2 / 0 myeloma cells (2 × 10 7 Hybridoma cells were obtained by fusing the cells in a PEG-mediated fusion step. After fusion, the cells were resuspended in HAT complete medium (Gibco, 21060017), dispensed at 0.1 mL / well into 96-well plates, and cultured at 37°C in a 5% CO2 incubator. Generally, approximately 10-15 days after fusion, the ST2 binding activity of the cell culture supernatant was measured by ELISA (see Example 5), and the cell culture supernatant of the positive hole was selected, and the blocking activity against IL33 / ST2 binding was measured by ELISA (see Example 6).

[0154] Wells in which ST2 is specifically bound and blocking IL33 / ST2 binding were selected and expanded to 24-well plates according to cell density. After the cell lines transferred to the 24-well plates were retested, preservation and the first subcloning were performed. During the first subcloning, cell lines that were determined to be positive were preserved, and a second subcloning was performed. During the second subcloning, cell lines that were determined to be positive were preserved and subjected to protein expression.

[0155] Example 3: Acquisition of cDNA for anti-ST2 antibody and construction of chimeric antibody structure Using a total RNA extraction kit, total RNA was isolated from hybrid tumor cells exhibiting the binding and blocking functions described above. Using this as a template, the first strand of cDNA was synthesized by SuperScript III reverse transcriptase (Thermo, 18080051) according to the instructions. Next, the variable region sequence of the antibody was amplified via PCR using degenerate mouse IgG primers.

[0156] The PCR mixture was electrophoresed in a 1% agarose / trisborate gel containing 0.5 μg / mL ethidium bromide. DNA fragments of expected size (approximately 500 bp for heavy and light chains) were excised from the gel and purified. The purified PCR products were cloned into the pMD-19T vector (Takara, 6013) and transformed into DH5α-competent Escherichia coli (Takara, 9057). Five colonies were selected from the LB culture plate and subjected to DNA sequencing. The heavy chain variable region sequences and light chain variable region sequences of the antibodies were obtained: 7D1 (SEQ ID NOs. 35, 36), 3C6 (SEQ ID NOs. 37, 38), 31C8 (SEQ ID NOs. 39, 40), 26A1 (SEQ ID NOs. 41, 42), 8F4 (SEQ ID NOs. 43, 44), and 17E2 (SEQ ID NOs. 45, 46).

[0157] Regarding the construction and expression of chimeric antibodies, the mouse VL region gene synthesis fragment is double-destroyed. The fragment was linked to the human κ chain constant region via compounding, constructing a chimeric light chain. The mouse VH region gene synthesis fragment was linked to the human IgG2 constant region via double digestion, constructing a chimeric heavy chain.

[0158] The DNA vector containing the above-mentioned chimeric light chain and the DNA vector containing the above-mentioned chimeric heavy chain are used with Expi CHO cells (50 mL system, 6 × 10⁶ cells). 6 The cells were cotransfected (1 μg / mL cells, 1 μg / mL DNA), transiently expressed, and cultured for 7 days. Next, the chimeric antibody in the cell culture supernatant was purified using a Protein A column (GE Healthcare).

[0159] Example 4: Measurement of the affinity of anti-ST2 antibody against hST2 antigen. Anti-mouse IgG antibody (for capturing human ST2 antigen, Cytiva, 29215281) or anti-his antibody (for capturing Cynomolgus Monkey ST2 antigen, Cytiva, 29234602) was bound to a CM5 biochip (Cytiva, BR-1000-12) using the method described in the product instructions. A series of anti-ST2 antibodies (32.8 nM, 16.4 nM, 8.2 nM, 4.1 nM, 2.05 nM, 1.0259 nM, 0.51297 nM) were flowed onto the chip surface, and the reaction signal was detected in real time using a Biacore instrument (Cytiva, Biacore T200), after which binding-dissociation curves were obtained. After dissociation was complete in each cycle, the biochip was regenerated with a regeneration solution for the next capture. This procedure was repeated until the affinity of each ST2 antibody was determined. The obtained data were finally analyzed using GE BIAvaluation software with a 1:1 (langmuir) binding model to measure the binding rate constant ka (kon) and dissociation rate constant kd (koff), and the dissociation constant KD was calculated using KD = kd / ka. The affinity of the anti-ST2 antibody for the human ST2 antigen (hST2-ECD-mFc), the Cynomolgus Monkey ST2 antigen (Cyno-ST2-his, Sino biologica1, 90915-C08H), and the affinity of the anti-ST2 chimeric antibody for ST2 are shown in Table 5.

[0160] [Table 5]

[0161] Example 5: Binding analysis of anti-ST2 antibody by ELISA ST2 binding screening and antibody analysis were performed by ELISA using hST2-ECD-mFc (for the measurement of chimeric and humanized antibodies), hST2-ECD-hFc protein (for the measurement of hybridoma antibodies), and Cyno-ST2-his (Sino biological, 90915-C08H) as antigens. 2 μg / mL hST2-ECD-mFc or Cyno-ST2-his antigen was coated at 100 μL / well on a high-adsorption 96-well plate (Costar, 9018) and incubated overnight at 4°C. After thoroughly washing away unadsorbed antigen, nonspecific binding sites were blocked with blocking buffer (PBS containing 2% bovine serum albumin). The plate was washed three times with washing buffer (PBS containing 0.05% (v / v) Tween 20) and then subjected to anti-S2 binding. T2 antibody (eight concentrations starting from an initial concentration of 10 nM: 3.333 nM, 1.111 nM, 0.370 nM, 0.123 nM, 0.041 nM, 0.014 nM, 0.005 nM, and 0.002 nM) was added at 100 μL / well, and the plate was incubated at room temperature for 1 hour. After washing the plate with wash buffer, horseradish peroxidase (HRP)-conjugated secondary antibody was added, and the plate was incubated for a further 60 minutes. After washing the plate with wash buffer, substrate TMB solution (Thermo, 00-4201-56) was added at 100 μL / well, and the plate was incubated at room temperature for 2 minutes. Stop solution (2N H2SO4) was added at 100 μL / well to terminate the reaction. A colorimetric signal was generated and read at 450 nm using a microplate reader (PE, Envision). The data were analyzed using GraphPad Prism5 and EC50 values ​​were calculated. The EC50 values ​​for the binding of anti-ST2 chimeric antibodies to hST2 and Cyno-ST2 are shown in Table 6 and Figure 1-2.

[0162] [Table 6]

[0163] Example 6: Blocking analysis of anti-ST2 antibody by ELISA The ability of anti-ST2 antibody to block the binding of hIL33 to hST2 was determined based on ELISA. 2 μg / mL hST2-ECD-hFc was coated onto a highly adsorbent 96-well plate at 100 μL / well and incubated overnight at 4°C. After thoroughly washing away unadsorbed antigen, nonspecific binding sites were blocked with blocking buffer (PBST containing 1% bovine serum albumin). The plate was washed three times with washing buffer (PBST, PBS containing 0.05% (v / v) Tween 20), and then 100 μL / well of anti-ST2 antibody (diluted 2-fold from an initial concentration of 66.67 nM to eight different concentrations) was added and incubated at 37°C for 1 hour. After washing the plate three times with washing buffer, 100 μL of biotin-labeled hIL33 Avitag (at a concentration of 0.1 μg / mL) was added to each well and incubated at 37°C for 1 hour. After washing the plate three times with washing buffer, 100 μL / well of avidin HRP secondary antibody (Jackson immunoresearch, 016-030-084) diluted 1:1000 was added and incubated at room temperature for 1 hour. After washing the plate with washing buffer, 100 μL / well of substrate TMB solution (Thermo, 00-4201-56) was added and the plate was incubated at room temperature for 3 minutes. To terminate the reaction, 50 μL of stop solution (2N H2SO4) was added. A colorimetric signal was generated and read at 450 nm using a microplate reader (PE, Envision). The data were analyzed using GraphPad Prism5 and IC50 values ​​were calculated. The IC50 values ​​blocking IL33 / ST2 binding of the anti-ST2 chimeric antibody are shown in Table 7 and Figure 3.

[0164] [Table 7]

[0165] Example 7: Cell-based binding analysis of anti-ST2 antibody Based on FACS methods, binding assays were performed between an HEK293T cell line overexpressing hST2 (SEQ ID NO: 1) (HEK293T-hST2-NFκB-Luciferase) and a HEK293T cell line overexpressing cynomolgus monkey ST2 (SEQ ID NO: 10) (HEK293T-Cyno-ST2-NFκB-Luciferase) to analyze the binding ability of anti-ST2 antibodies to hST2-overexpressing cell lines and cyno-ST2-overexpressing cell lines. The cell line HEK293T-hST2-NFκB-luciferasae was constructed by transfecting HEK293 cells with the pLenti6.3-hST2 plasmid and the pLenti6.3-NFκB-luciferasae plasmid using a lentiviral transfection system. The cell line HEK293T-Cyno-ST2-NFκB-Luciferase was constructed by transfecting HEK293T cells with the pLenti6.3-cynoST2 and pLenti6.3-NFκB-luciferasae plasmids.

[0166] 3 x 10 5 HEK293T-hST2-NFκB-Luciferase or HEK293T-Cyno-ST2-NFκB-Luciferase cells were added to a 96-well culture plate. Serially diluted anti-ST2 antibody (in the binding test with HEK293T-hST2-NFκB-Luciferase cells, the initial concentration of anti-ST2 antibody was 667 nM, and eight concentrations were diluted according to a 4-fold concentration gradient. In the binding experiment with HEK293T-Cyno-ST2-NFκB-Luciferase, the initial concentration of anti-ST2 antibody was 400 nM, and eight concentrations were diluted according to a 4-fold concentration gradient) was added to the cell suspension. After incubating the plate at room temperature for 60 minutes, the cells were washed three times with PBS. A 1:200 diluted PE goat-anti-human-IgG secondary antibody (Jackson) was added. Immunoresearch (109-116-170) was added at 100 μL / well, and the plates were incubated at room temperature for 30 minutes. Cells were washed three times with PBS, resuspended in 100 μL of PBS, and then the fluorescence signal was analyzed and detected using a flow cytometer (BD, Accuri C6). The binding ability of anti-ST2 antibodies to hST2 and cyno-ST2 on the cell line surface was measured from the mean fluorescence intensity (MFI) of staining. Data were analyzed using GraphPad Prism5, and EC50 values ​​were calculated. The EC50 values ​​of anti-ST2 chimeric antibody binding to hST2 and cyno-ST2 at the cellular level are shown in Table 8 and Figures 7-8.

[0167] [Table 8]

[0168] Example 8: Assay of an anti-ST2 antibody blocking the interaction between IL33 protein and an hST2 overexpressing cell line (HEK293T-hST2-NFκB-Luciferase). HEK293T cells spontaneously express high levels of IL1RAcP protein. Using a lentiviral transfection system, HEK293 cells were transfected with the pLenti6.3-hST2 plasmid and the pLenti6.3-NFκB-Luciferasae plasmid to construct a stable transfected cell line, HEK293T-hST2-NFκB-Luciferase. When IL33 binds to ST2, it recruits IL-1RAcP and activates the downstream NFκB signaling pathway, thus initiating luciferase expression. The presence of an anti-ST2 antibody in the system blocks the binding of IL33 to ST2, and consequently blocks luciferase expression. The blocking activity of the anti-ST2 antibody was evaluated by detecting changes in the fluorescence intensity of the reaction substrate.

[0169] HEK293T-hST2-NFκB-Luciferasae cells in good condition during the log phase were collected and measured in 2.5 × 10⁶ units. 5The cells were diluted to cells / mL. The cell dilution was added to a 384-well plate (Thermo, 262360) at a rate of 20 μL / well. Next, serially diluted anti-ST2 antibody (serially diluted 4-fold from an initial concentration of 667 nM) was added at a rate of 15 μL / well. The plate was incubated at 37°C for 30 minutes. After incubation, 15 μL of hIL33 protein was added to each well (final concentration 50 pM). The mixture was thoroughly mixed and incubated at 37°C for 5 hours. Next, 50 μL of ONE-Glo was added. (商標) Luciferase Assay (Promega, E6120) was added to each well, and the mixture was allowed to react in the dark for 2–5 minutes. Chemiluminescence signals were read using a microplate reader (PE, Envision). The data were analyzed using GraphPad Prism5, and IC50 values ​​were calculated. Table 9 and Figure 9 show the IC50 values ​​for blocking of IL33 / ST2 signaling by anti-ST2 chimeric antibodies at the cellular level.

[0170] [Table 9]

[0171] Example 9, Assay of an anti-ST2 antibody blocking the interaction between IL 33 protein and cells spontaneously expressing hST2 ((KU812-NFκB-Luciferasae)). KU812 cells spontaneously express ST2 and IL1RAcP proteins. Using a lentiviral transfection system, pLenti6.3-NFκB-Luciferasae was transfected into Ku812 cells to construct a stable transfected cell line, KU812-NFκB-Luciferasae. See Example 8 for experimental principles. Good condition logarithmic phase KU812-NFκB-Luciferasae cells were harvested, and 2.5 × 10⁶ cells were collected. 5The cells were diluted to cells / mL. The cell dilution was added to a 384-well plate (Thermo, 262360) at a rate of 20 μL / well. Next, serially diluted anti-ST2 antibody (serially diluted 4-fold from an initial concentration of 667 nM) was added at a rate of 15 μL / well. The plate was incubated at 37°C for 30 minutes. After incubation, 15 μL of hIL33 protein was added to each well (final concentration 200 pM). The mixture was thoroughly mixed and incubated at 37°C for 5 hours. Next, 50 μL of ONE-Glo was added. (商標) Luciferase Assay (Promega, E6120) was added to each well, and the mixture was allowed to react in the dark for 2–5 minutes. The chemiluminescence signal was read using a microplate reader (PE, Envision). The data was analyzed using GraphPad Prism5, and the IC50 value was calculated.

[0172] Example 10, IL33 and IL2 co-stimulated CD4 + Assay of an anti-ST2 antibody that blocks interaction with T cells Under co-stimulation by IL33 and IL2, IL33 stimulates CD4 + It binds to ST2 on the surface of T cells, activating downstream signaling pathways and inducing the secretion of the cytokine IL-5. If an anti-ST2 antibody is present in the system, IL-33 CD4 + It blocks T cell binding to ST2 and inhibits IL-5 secretion. IL-33 and IL-2 co-stimulation CD4 + The ability of anti-ST2 antibodies to block T cell interactions was analyzed by measuring their IL-5 content.

[0173] Using a human CD4+ T cell sorting kit (stemcell, 17952), CD4+ T cells were selected from human PBMCs. + Select T cells, 2.5 × 10 5 Individual cells were seeded into a 96-well U-bottom cell culture plate, and the volume was increased to 60 μL / well. Next, anti-ST2 antibody (3-fold dilution from the initial concentration of 53 nM), diluted to 15 μL / well, was added. The plate was incubated at 37°C for 30 minutes. After incubation, 15 μL of hIL33 protein (final concentration 4) was added. CD4 by IL33 / ST2 of anti-ST2 chimeric antibody was added to each well. 15 μL of IL2 (final concentration 10 ng / mL) (PrimeGene, GMP-101-02) was added. The mixture was thoroughly mixed and incubated at 37°C for 48 hours. After incubation, the supernatant was collected and the IL5 content of the supernatant was measured according to the instructions for the IL5 assay kit (R&D, DY205). OD450 values ​​were read using a microplate reader (PE, Envision). The data were analyzed using GraphPad Prism5 and IC50 values ​​were calculated. + Table 10 shows the IC50 values ​​for blocking IL5-induced secretion in T cells.

[0174] [Table 10]

[0175] Example 11: Humanization of anti-human ST2 hybridoma monoclonal antibody 31C8 (sequence number 39 for the heavy chain variable region sequence; sequence number 40 for the light chain variable region sequence) was selected for humanization design. An established CDR transplantation method was employed to select a human antibody framework region that could be used for humanization of a mouse antibody. A human germline antibody or its subtype with the best sequence identity (i.e., sequence similarity) to the amino acids of the mouse antibody variable region was selected. The heavy chain variable region CDR and light chain variable region CDR of the mouse antibody were inserted into the selected framework region, and residues in the framework region were mutated. Some CDRs were further mutated to improve the antibody properties, such as physicochemical properties and antidote properties. Humanized antibodies hz31C8-1.1 and hz31C8-1.2 were obtained. Here, the amino acid sequences of the heavy chain variable region and light chain variable region of hz31C8-1.1 were sequence number 71 and sequence number 79, respectively. The DNA sequences encoding the heavy chain variable region and light chain variable region of the humanized antibody hz31C8-1.1 were sequence number 72 and sequence number 80, respectively. The amino acid sequences of the heavy chain variable region and light chain variable region of the humanized antibody hz31C8-1.2 were SEQ ID NO: 73 and SEQ ID NO: 79, respectively. The DNA sequences encoding the heavy chain variable region and light chain variable region of the humanized antibody hz31C8-1.2 were SEQ ID NO: 74 and SEQ ID NO: 80, respectively.

[0176] Humanized VL region gene synthesis fragments were ligated to the human κ chain constant region by double digestion to construct a humanized light chain, and human region gene synthesis fragments were ligated to the human IgG2 constant region by double digestion to construct a humanized heavy chain. Humanized heavy and light chain DNA corresponding to each antibody was transfected into expression vectors. Protein expression was performed using the ExpiCHO expression system. Next, the humanized antibodies in the cell culture supernatant were purified via a Protein A column.

[0177] Example 12: Biological function assay of humanized anti-ST2 antibody Humanized antibody hz31C81-1.1 (heavy chain sequence number 75, light chain sequence number 75) was produced according to the methods of Examples 4, 5, 6, 7, 8, 9 and 10. Affinity, binding, inhibition, and cellular function analyses were performed on antibody 81) and hz31C8-1.2 (heavy chain sequence number 77, light chain sequence number 81). In this publication, "hz" and "xi" represent humanized antibodies and chimeric antibodies, respectively. For example, "hz31C81-1.1" and "hz31C81-1.2" represent humanized 31C81-1.1 and 31C81-1.2 antibodies, and "xi31C8" represents a chimeric 31C8 antibody.

[0178] The affinity results between the anti-ST2 humanized antibody and ST2 are shown in Table 11. The EC50 values ​​of the anti-ST2 humanized antibody bound to hST2 and Cyno-ST2 in ELISA experiments are shown in Table 12 and Figures 4-5. The IC50 values ​​of the anti-ST2 humanized antibody inhibiting IL33 / ST2 binding in ELISA are shown in Table 13 and Figure 6. The EC50 values ​​of the anti-ST2 humanized antibody bound to cellular-level hST2 are shown in Table 14 and Figure 10. The IC50 values ​​of the anti-ST2 humanized antibody inhibiting cellular-level IL33 / ST2 signaling are shown in Table 15 and Figures 11-12. The anti-ST2 humanized antibody is shown to bind to CD4 + Table 16 and Figure 13 show the IC50 values ​​that inhibit IL33 / ST2-induced IL5 secretion in T cells.

[0179] [Table 11]

[0180] [Table 12]

[0181] [Table 13]

[0182] [Table 14]

[0183] [Table 15]

[0184] [Table 16]

[0185] Example 13: Stability assay of humanized anti-ST2 antibody The thermal stability of each antibody is determined by DSC (Differential Scanning). The results were determined by calorimetry (differential scanning calorimetry). Samples were dissolved in PBS buffer (pH 7.4) and measured using MicroCal VP-Capillary DSC (Malvern Panalytical). As shown in Table 17, both the humanized anti-ST2 antibodies hz31C8-1.1 and hz31C8-1.2 of this disclosure exhibited excellent thermal stability, superior to the anti-ST2 antibodies RG6149 and GSK3772847 used as controls.

[0186] [Table 17]

[0187] The periodic stability of the antibodies was measured by SEC-HPLC under specific concentration conditions. For example, the stability was compared when the antibody concentration was controlled at approximately 1 mg / ml and stored in PBS buffer (pH 7.2) at 40°C for 2 weeks. This was performed using LC-20ADXR / DGU (Shimadzu). As shown in Table 18, both the humanized anti-ST2 antibodies hz31C8-1.1 and hz31C8-1.1 of this disclosure showed good stability.

[0188] [Table 18]

[0189] Anti-ST2 antibody was diluted to a concentration of 1.5 mg / mL in PBS buffer and treated at 72°C for 5 minutes, after which its binding activity to the antigen was measured by ELISA. The ELISA procedure was as follows: A 96-well plate was coated with 100 μL / well of hST2-ECD-mFc as the antigen at 2 μg / ml and incubated overnight at 4°C. After thoroughly washing away any unadsorbed antigen, nonspecific binding sites were blocked with blocking buffer (PBST containing 1% bovine serum albumin). The plate was washed three times with washing buffer (PBS, PBST containing 0.05% (v / v) Tween 20), and then 100 μL / well of anti-ST2 antibody (diluted 3-fold from the initial concentration to eight concentrations starting at 1.5 μg / ml) was added, and the plate was incubated at 37°C for 1 hour. After washing the plate with washing buffer, anti-human IgGFcγ secondary antibody (Jackson ImmunoResearch, 109-035-008) was added, and the plate was incubated at 37°C for 1 hour. After washing the plate with washing buffer, 100 μL / well of substrate TMB solution (Thermo, 00-4201-56) was added for color development. After incubating the plate at room temperature for 5 minutes, a stop solution (2N H2) was added to stop the reaction. SO4 was added. Using a plate reader (PE, Envision), the signal was read at 450 nm, and the data was analyzed with a GraphPad Prism5 to calculate the EC50 values. As shown in Table 19, both the humanized anti-ST2 antibodies hz31C8-1.1 and hz31C8-1.1 of this disclosure showed excellent thermal stability.

[0190] [Table 19]

[0191] Example 14: Cross-reactivity of humanized anti-ST2 antibodies using IL1R family receptors ST2 belongs to the IL1R receptor family. Cross-reactivity of anti-ST2 antibodies with IL1R family receptors was determined by ELISA. The IL1R family receptors include cIL1R1 / CD121a (Sino biological, 10126-H08H), IL1R2 / CD121b (Sino biological, 10111-H08H), IL1R3 / IL1RAP (Sino biological, 10121-H08H), IL1R7 / IL-18RAcP (Sino biological, 10176-H08H), IL1R8 / IL1RAPL1 (Sino biological, 10177-H08H), and IL1R9 / IL1RAPL2 (Sino biological, 10156-H08H). The ELISA assay was performed using the above proteins as antigens (2 μg / ml). For specific ELISA procedures, please refer to Example 13. Non-binding means that the highest concentration of antibody does not bind to the antigen. Weak binding means that the highest concentration of antibody binds weakly to the antigen, while the lower concentration of antibody does not bind. Strong binding means that the binding data fits a very good binding curve.

[0192] [Table 20]

[0193] Example 15: Pharmacokinetic evaluation of humanized anti-ST2 antibody For the experiment, 18 Balb / c mice purchased from Nanjing University's animal model were used, with 6 mice per group, and they were given 12 / 12 hours of visual adjustment and free access to drinking water. On the experimental day, each mouse was administered a 1 mg / ml sample by intravenous injection at a dose of 10 mg / kg. Orbital blood serum was collected before administration (0 minutes, or at least 1 day before administration) and at 30 minutes, 8 hours, 24 hours, 2 days (48 hours), 4 days (96 hours), 7 days, and 14 days after administration, and antibody concentrations were measured by ELISA. The ELISA procedure was as follows: 96-well plates were coated with 2 μg / mL ST2-His (Synobiology, 10105-H08H) as 100 μL / well antigen and incubated overnight at 4°C. After sufficient washing away of unadsorbed antigen, nonspecific binding sites were blocked with blocking buffer (PBST containing 1% bovine serum albumin). After washing the plate three times with washing buffer (PBS, PBST containing 0.05% (v / v) Tween 20), 100 μl of test serum was added and the plate was incubated at 37°C for 1 hour. After washing the plate with washing buffer, anti-mouse IgGFcγ secondary antibody was added and the plate was incubated at 37°C for 1 hour. After washing the plate with washing buffer, 100 μL / well of substrate TMB solution (Thermo, 00-4201-56) was added for color development. After incubating the plate at room temperature for 5 minutes, stop solution (2N H2SO4) was added to stop the reaction. The signal was read at 450 nm using a plate reader (PE, Envision), and the data was analyzed with a GraphPad Prism5. Referring to Table 21, PK analysis showed that the humanized anti-ST2 antibodies hz31C8-1.1 and hz31C8-1.1 of this disclosure had half lives of approximately 9.7 d and 7.33 d, respectively, demonstrating superiority over the control anti-ST2 antibody RG6149.

[0194] [Table 21]

[0195] Although this disclosure is described in detail above through the general description and specific embodiments, it will be apparent to those skilled in the art that modifications or improvements can be made based on this disclosure. Therefore, all such modifications and improvements made without deviating from the spirit of this disclosure are within the scope of protection of this disclosure.

Claims

1. An isolated ST2 antibody or its antigen-binding fragment, The antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 39, 71, or 73, containing heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 40 or 79, wherein CDR is defined according to the Chothia, IMGT, AbM, or Contact number system, and is an antibody or antigen-binding fragment thereof.

2. The antibody or antigen-binding fragment according to Claim 1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 39, heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 40, wherein CDR is defined according to the Chothia, IMGT, AbM, or Contact number system.

3. The antibody or antigen-binding fragment according to Claim 1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 71, heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 79, wherein CDR is defined according to the Chothia, IMGT, AbM, or Contact number system.

4. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 73, heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 79, wherein CDR is defined according to the Chothia, IMGT, AbM, or Contact number system.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof is a chimeric or humanized antibody.

6. The antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, (1) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 39 or an amino acid sequence having at least 90% identity thereto, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 40 or an amino acid sequence having at least 90% identity thereto; (2) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 71 or an amino acid sequence having at least 90% identity thereto, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 79 or an amino acid sequence having at least 90% identity thereto; or, (3) The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 73 or an amino acid sequence having at least 90% identity thereto, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 79 or an amino acid sequence having at least 90% identity thereto.

7. The antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, (1) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 39, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 40; (2) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 71, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 79; or, (3) The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 73, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

79.

8. The antibody or its antigen-binding fragment comprises a heavy chain and a light chain, (1) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 67 or an amino acid sequence having at least 90% identity thereto, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 69 or an amino acid sequence having at least 90% identity thereto; (2) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 75 or an amino acid sequence having at least 90% identity thereto, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 81 or an amino acid sequence having at least 90% identity thereto; or, (3) The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 77 or an amino acid sequence having at least 90% identity thereto, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 81 or an amino acid sequence having at least 90% identity thereto.

9. The antibody or its antigen-binding fragment comprises a heavy chain and a light chain, (1) The heavy chain contains the amino acid sequence shown in SEQ ID NO: 67, and the light chain contains the amino acid sequence shown in SEQ ID NO: 69; (2) The heavy chain contains the amino acid sequence shown in SEQ ID NO: 75, and the light chain contains the amino acid sequence shown in SEQ ID NO: 81; or, (3) The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 77 and the light chain comprises the amino acid sequence shown in SEQ ID NO:

81.

10. The antibody or antigen-binding fragment according to any one of claims 1 to 9, wherein the antibody or antigen-binding fragment is selected from a monoclonal antibody, a Fab fragment, an F(ab')2 fragment, an Fv fragment, a single-chain Fv molecule, or a combination thereof.

11. The antibody or its antigen-binding fragment specifically binds to human or monkey ST2 and inhibits the binding and signal conduction of IL33 / ST2; The antibody or its antigen-binding fragment does not substantially bind to IL1R1, IL1R2, IL1R3, IL1R7, IL1R8, and IL1R9; or, The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antibody or antigen-binding fragment thereof inhibits IL-5 secretion induced by IL-33 / ST2 in CD4+ T cells.

12. A separated nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 11.

13. An expression vector comprising the nucleic acid molecule described in Claim 12.

14. A host cell comprising the nucleic acid molecule described in claim 12 or the expression vector described in claim 13.

15. A polypeptide fusion comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 11.

16. A multispecific molecule comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 11.

17. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

18. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 for the manufacture of a pharmaceutical product for treating or relieving IL33 / ST2-mediated related diseases and conditions in a subject in need.

19. The use according to claim 18, wherein the associated diseases and conditions mediated by IL33 / ST2 include asthma, allergic rhinitis, chronic obstructive pulmonary disease, eosinophilic bronchiolitis, eosinophilic esophagitis, atopic dermatitis, psoriasis, systemic lupus erythematosus, bullous pemphigoid, rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, pulmonary fibrosis, hepatic fibrosis, systemic sclerosis, sarcoidosis, graft-versus-host disease (GVHD), diabetic disease, cardiovascular disease, or a combination thereof.

20. The use according to claim 19, wherein the antibody or its antigen-binding fragment is administered together with one or more other antibodies or pharmaceuticals.

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