ST2 antigen binding proteins

Abstract

The present invention provides ST2 antigen binding proteins, such as mice, humans, chimeric or humanized antibodies or antigen binding fragments thereof that specifically bind ST2, and also provides nucleic acid molecules encoding the antibodies and antigen binding fragments thereof, as well as expression vectors and host cells for expressing the antibodies or antigen binding fragments thereof. Further provided are methods of making and using the antibodies of the invention and antigen-binding fragments thereof, including the treatment and prevention of IL33 / ST2 mediated related diseases and disorders.

Description

ST2 antigen-binding protein

[0001] This application is a divisional application of the invention patent application filed on May 11, 2021, with Chinese application number 202180030957.9 and invention title "ST2 antigen-binding protein".

[0002] Reference to related applications: This disclosure claims priority to Chinese Patent Application No. 202010397572.3, filed on May 12, 2020, the contents of which are incorporated herein by reference in their entirety and for all purposes. Technical Field

[0003] This disclosure relates to an isolated antibody. Specifically, this disclosure provides mouse, chimeric, and humanized monoclonal antibodies or antigen-binding fragments thereof that specifically bind to ST2, as well as nucleic acids, expression vectors, and host cells for producing said antibodies or antigen-binding fragments thereof. This disclosure further provides polypeptide fusions comprising said antibodies or antigen-binding moieties thereof, multispecific molecules, viral vectors, and pharmaceutical compositions, as well as diagnostic and treatment methods using said antibodies or antigen-binding fragments thereof. Background Technology

[0004] Interleukin 33 (IL33) is a member of the IL-1 cytokine family, expressed by endothelial or epithelial cells. When cells receive stimuli such as stress, infection, and injury, IL33 is expressed as an "alarmin" in damaged cells, transmitting external antigenic stimulation signals to the Th2 pathway. The IL33 receptor consists of two proteins: IL-1 receptor-associated protein (IL-1RL1, ST2) and IL-1 receptor accessory protein (IL-1RAP). IL33 binding to ST2 requires the participation of IL-1RAP to induce signal transduction, but IL-1RAP cannot directly bind to either IL33 or ST2 (Chackerian et al., (2007) J Immunol. 179:2551-2555).

[0005] ST2 belongs to the Toll / IL1 receptor family and has three subtypes: transmembrane ST2L, soluble sST2, and mutant ST2V. Among them, ST2L mainly mediates the intracellular signaling response of IL33. ST2L is mainly expressed on the surface of Th2 cells, ILC2 cells, mast cells, and Treg cells, and can also be expressed on the surface of NK cells, NKT cells, macrophages, eosinophils, and basophils.

[0006] IL33 binds to ST2L on the cell surface, recruits IL-1RAcP, thereby activating the MyD88 / NFκB cell pathway and inducing mast cells, Th2 cells, Treg cells, and ILC2 cells to secrete pro-inflammatory factors such as IL-5, IL-6, IL-13, TNF, and INF-γ, as well as chemokines such as CCL17, CCL22, and CXCL8, thus inducing an inflammatory response. IL-33 expression is abnormally elevated in diseases of mucosal tissue inflammation, joint inflammation, and chronic skin inflammation, such as allergic rhinitis (Kamekura R et al., (2012) ClinExp Allergy. 42:218-28), rheumatoid arthritis, ankylosing spondylitis, atopic dermatitis (Savinko T et al., (2012) J Invest Dermatol. 132:1392–1400), and psoriasis; and IL-33 / ST2 signaling pathway dysregulation is associated with asthma, chronic obstructive pulmonary disease (Hacker, Lampers et al., (2009) J Clin Lab Anal. 23:372-9), bronchitis, and inflammatory bowel disease (Beltran CJ et al., (2010) Inflamm Bowel Dis.). IL33 (16:1097-107) is closely related to immune-mediated diseases such as systemic lupus erythematosus, liver fibrosis, and systemic sclerosis. Gene-level analysis has revealed several single nucleotide polymorphisms (SNPs) in the IL33 and ST2 genes, which are associated with basophil counts and are also closely related to the occurrence of atopic dermatitis and asthma-like 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 has become an important direction for the treatment of inflammatory immune-related diseases. Summary of the Invention

[0007] This disclosure provides a high-affinity, high-specificity, and high-biological-activity ST2 antibody suitable for the treatment of IL33 / ST2-mediated diseases.

[0008] This disclosure 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).

[0009] The antibodies or antigen-binding fragments disclosed herein have a variety of uses, including the detection of ST2 protein, and the treatment and prevention of IL33 / ST2-mediated diseases and conditions, including asthma, allergic rhinitis, chronic obstructive pulmonary disease, eosinophilic bronchitis, eosinophilic esophagitis, atopic dermatitis, psoriasis, systemic lupus erythematosus, pemphigoid, rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, pulmonary fibrosis, liver fibrosis, systemic sclerosis, sarcoidosis, transplant anti-host disease (GVHD), diabetes, cardiovascular disease, or combinations thereof.

[0010] On the one hand, this disclosure provides isolated antibodies or antigen-binding fragments thereof comprising heavy chain CDRs, said heavy chain CDRs comprising heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, wherein, (1) heavy chain CDR1 comprises 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 with it, heavy chain CDR2 comprises the sequence shown in SEQ ID NO: 15 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 heavy chain CDR3 comprises the sequence shown in SEQ ID NO: 15. (2) The heavy chain CDR1 comprises 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 with it; and the heavy chain CDR2 comprises 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 with it. 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, and the heavy chain CDR3 contains 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;(3) Heavy chain CDR1 comprises 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 with it; heavy chain CDR2 comprises 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%, 98%, or 99% identity with it; and heavy chain CDR3 comprises the sequence shown in SEQ ID NO:12. (3) The sequence shown in 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; (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 with it, and heavy chain CDR2 contains the sequence shown in SEQ ID NO: 13. 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, and the heavy chain CDR3 contains 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;(5) Heavy chain CDR1 comprises 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 with it; heavy chain CDR2 comprises 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%, 96%, 97%, 98%, or 99% identity with it; and heavy chain CDR3 comprises the sequence shown in SEQ ID NO: 19. 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 with it; or (6) the 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 with it, and the heavy chain CDR2 contains the sequence shown in SEQ ID NO: 11. The sequence shown in SEQ ID NO: 24 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 heavy chain CDR3 containing the sequence shown in SEQ ID NO: 24 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.

[0011] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2, wherein 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.

[0012] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2, wherein 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.

[0013] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2, wherein 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.

[0014] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2, wherein 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.

[0015] In some specific implementations, the isolated antibody or its antigen-binding fragment binds to ST2, and it is a monoclonal antibody (e.g., mouse, chimeric, or humanized antibody).

[0016] In some embodiments, the isolated antibody or its antigen-binding fragment comprises a heavy chain variable region, the amino acid sequence of which comprises the sequence shown in SEQ ID NOs: 35, 37, 39, 41, 43, 45, 71 or 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 with the sequence shown in SEQ ID NOs: 35, 37, 39, 41, 43, 45, 71 or 73.

[0017] In some specific implementations, the isolated antibody or its antigen-binding fragment binds to ST2, and it is a monoclonal antibody (e.g., mouse, chimeric, or humanized antibody).

[0018] In some specific implementations, the amino acid represented by SEQ ID NO: 71 can be encoded by the nucleic acid represented by SEQ ID NO: 72, and the amino acid represented by SEQ ID NO: 73 can be encoded by the nucleic acid represented by SEQ ID NO: 74.

[0019] In some embodiments, the isolated antibody or its antigen-binding fragment comprises a light chain CDR, the light chain CDR comprising light chain CDR1, light chain CDR2 and light chain CDR3, wherein, (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 with it, light chain CDR2 comprises 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%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, and light chain CDR3 comprises the sequence shown in SEQ ID NO: 29. (1) The sequence shown in SEQ ID NO: 26 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; (2) Light chain CDR1 contains the sequence shown in SEQ ID NO: 26 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 light chain CDR2 contains the sequence shown in SEQ ID NO: 26. 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 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;(3) Light chain CDR1 comprises 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%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; light chain CDR2 comprises 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 light chain CDR3 comprises the sequence shown in SEQ ID NO:27. The sequence shown in 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; or (4) the light chain CDR1 contains the sequence shown in SEQ ID NO: 28 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 CDR2 contains the sequence shown in SEQ ID NO: The sequence shown in SEQ ID NO: 31, 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 comprising the sequence shown in SEQ ID NO: 33, 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.

[0020] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2, wherein the amino acid sequence of SEQ ID NO: 30 is QX4SNLAS, where X4 = M or L.

[0021] In some specific implementations, the isolated antibody or its antigen-binding fragment binds to ST2, and it is a monoclonal antibody (e.g., mouse, chimeric, or humanized antibody).

[0022] In some embodiments, the isolated antibody or its antigen-binding fragment comprises a light chain variable region, the amino acid sequence of which comprises the sequence shown in SEQ ID NOs: 36, 38, 40, 42, 44, 46 or 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 with the sequence shown in SEQ ID NOs: 36, 38, 40, 42, 44, 46 or 79.

[0023] In some specific implementations, the isolated antibody or its antigen-binding fragment binds to ST2, and it is a monoclonal antibody (e.g., mouse, chimeric, or humanized antibody).

[0024] In some specific implementations, the amino acid represented by SEQ ID NO: 79 may be encoded by the nucleic acid represented by SEQ ID NO: 80.

[0025] 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, wherein, (1) heavy chain CDR1 comprises 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 with it; heavy chain CDR2 comprises the sequence shown in SEQ ID NO:15 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 heavy chain CDR3 comprises the sequence shown in SEQ ID NO:15. The light chain CDR1 comprises 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; the light chain CDR2 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 with it; and the light chain CDR2 comprises the sequence shown in SEQ ID NO:20. The sequence shown in NO:29 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:32 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;(2) Heavy chain CDR1 comprises 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 with it; heavy chain CDR2 comprises the sequence shown in SEQ ID NO: 16 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; heavy chain CDR3 comprises the sequence shown in SEQ ID NO: 16. The light chain CDR1 comprises the sequence shown in SEQ ID NO: 26 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 CDR2 comprises the sequence shown in SEQ ID NO: 26 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 CDR2 comprises the sequence shown in SEQ ID NO: 26. 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 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;(3) Heavy chain CDR1 comprises 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 with it; heavy chain CDR2 comprises 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%, 98%, or 99% identity with it; heavy chain CDR3 comprises the sequence shown in SEQ ID NO: 17. The light chain CDR1 comprises 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%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, and the light chain CDR2 comprises 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%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, and the light chain CDR2 comprises the sequence shown in SEQ ID NO: 27. 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 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 comprises 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 with it; heavy chain CDR2 comprises 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 with it; heavy chain CDR3 comprises the sequence shown in SEQ ID NO: 18. The light chain CDR1 comprises 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%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, and the light chain CDR2 comprises 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%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, and the light chain CDR2 comprises the sequence shown in SEQ ID NO: 27. 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 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;(5) Heavy chain CDR1 comprises 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 with it; heavy chain CDR2 comprises 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%, 96%, 97%, 98%, or 99% identity with it; heavy chain CDR3 comprises the sequence shown in SEQ ID NO: 19. The light chain CDR1 comprises 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%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, and the light chain CDR2 comprises 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%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, and the light chain CDR2 comprises the sequence shown in SEQ ID NO: 27. 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 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;Or (6) 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 with it; 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 with it; heavy chain CDR3 contains the sequence shown in SEQ ID NO: 18. The light chain CDR1 comprises the sequence shown in SEQ ID NO: 28 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 CDR2 comprises the sequence shown in SEQ ID NO: 28 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 CDR2 comprises the sequence shown in SEQ ID NO: 28. The sequence shown in SEQ ID NO: 31, 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 comprising the sequence shown in SEQ ID NO: 33, 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.

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

[0027] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2; wherein, the amino acid sequence of SEQ ID NO: 17 is AIDPETGDTVYX1X2KFX3G, 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, X4=M or L.

[0028] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2; wherein, the amino acid sequence of SEQ ID NO: 17 is AIDPETGDTVYX1X2KFX3G, 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, X4=M or L.

[0029] In some specific embodiments, the isolated antibody or its antigen-binding fragment binds to ST2; wherein, the amino acid sequence of SEQ ID NO: 17 is AIDPETGDTVYX1X2KFX3G, 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, X4=M or L.

[0030] In some specific implementations, the isolated antibody or its antigen-binding fragment binds to ST2, and it is a monoclonal antibody (e.g., mouse, chimeric, or humanized antibody).

[0031] In some embodiments, the isolated antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein (1) the heavy chain variable region comprises a sequence as shown in SEQ ID NO: 35 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 variable region comprises a sequence as 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; (2) the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 35 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; The sequence shown in NO: 37 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 variable region comprises 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 with it; (3) the heavy chain variable region comprises the sequence shown in SEQ ID NO: 38. The sequence shown in NO: 39 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 variable region comprising the sequence shown in SEQ ID NO: 40 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) The heavy chain variable region comprises 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 with it, and the light chain variable region comprises the sequence shown in SEQ ID NO: 42 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; (5) The heavy chain variable region comprises the sequence shown in SEQ ID NO: 42. The sequence shown in 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 with it, and the light chain variable region comprises the sequence shown in SEQ ID NO: 44 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; (6) the heavy chain variable region comprises the sequence shown in SEQ ID NO: 44. The sequence shown in SEQ ID NO: 45 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 variable region comprising 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 with it;(7) The heavy chain variable region comprises 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 with it, and the light chain variable region comprises 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%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; or (8) The heavy chain variable region comprises the sequence shown in SEQ ID NO: 79. 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 with it, and the light chain variable region comprising 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%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0032] In some specific implementations, the isolated antibody or its antigen-binding fragment binds to ST2, and it is a monoclonal antibody (e.g., mouse, chimeric, or humanized antibody).

[0033] In some embodiments, the isolated antibody or its antigen-binding fragment comprises a heavy chain whose amino acid sequence comprises the sequence shown in SEQ ID NOs: 47, 51, 55, 59, 63, 67, 75 or 77; 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 the sequence shown in SEQ ID NOs: 47, 51, 55, 59, 63, 67, 75 or 77.

[0034] In some specific implementations, the isolated antibody or its antigen-binding fragment binds to ST2, and it is a monoclonal antibody (e.g., mouse, chimeric, or humanized antibody).

[0035] In some specific implementations, the amino acids represented by SEQ ID NOs: 47, 51, 55, 59, 63, 67, 75 and 77 can be encoded by the nucleic acids represented by SEQ ID NOs: 48, 52, 56, 60, 64, 68, 76 and 78, respectively.

[0036] In some embodiments, the isolated antibody or its antigen-binding fragment comprises a light chain whose amino acid sequence comprises the sequence shown in SEQ ID NOs: 49, 53, 57, 61, 65, 69 or 81; 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 the sequence shown in SEQ ID NOs: 49, 53, 57, 61, 65, 69 or 81.

[0037] In some specific implementations, the isolated antibody or its antigen-binding fragment binds to ST2, and it is a monoclonal antibody (e.g., mouse, chimeric, or humanized antibody).

[0038] In some specific implementations, the amino acids represented by SEQ ID NOs: 49, 53, 57, 61, 65, 69 and 81 can be encoded by the nucleic acids represented by SEQ ID NOs: 50, 54, 58, 62, 66, 70 and 82, respectively.

[0039] In some embodiments, the isolated antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein (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 with it, and the light chain comprises the sequence shown in SEQ ID NO: 49 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; (2) the heavy chain comprises the sequence shown in SEQ ID NO: 49. The sequence shown in NO: 51 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 comprises the sequence shown in SEQ ID NO: 53 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; (3) the heavy chain comprises the sequence shown in SEQ ID NO: 53. The light chain comprises the sequence shown in SEQ ID NO: 55 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) The heavy chain comprises the sequence shown in SEQ ID NO: 59 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 comprises the sequence shown in SEQ ID NO: 61 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; (5) The heavy chain comprises the sequence shown in SEQ ID NO: 61. The light chain comprises the sequence shown in SEQ ID NO: 63 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 heavy chain comprises the sequence shown in SEQ ID NO: 65 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; (6) the heavy chain comprises the sequence shown in SEQ ID NO: 65. The light chain comprises the sequence shown in SEQ ID NO: 67 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 heavy chain comprises the sequence shown in SEQ ID NO: 69 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; (7) the heavy chain comprises the sequence shown in SEQ ID NO: 69. The light chain comprises the sequence shown in SEQ ID NO: 75 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 comprises the sequence shown in SEQ ID NO: 81 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;Or (8) the heavy chain comprises the sequence shown in SEQ ID NO: 77 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 comprises the sequence shown in SEQ ID NO: 81 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.

[0040] In some specific implementations, the isolated antibody or its antigen-binding fragment binds to ST2, and it is a monoclonal antibody (e.g., mouse, chimeric, or humanized antibody).

[0041] 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 comprising a heavy chain variable region and a heavy chain constant region, the light chain comprising a light chain variable region and a light chain constant region, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequence described above, the heavy chain constant region having a human IgG1, IgG2, or IgG4 constant region, preferably a human IgG2 constant region; the light chain constant region having a human κ constant region or a λ constant region, wherein the antibody or antigen-binding fragment binds to ST2.

[0042] In some embodiments, the antibody of this disclosure comprises, or is composed of, two heavy chains and two light chains, interconnected by disulfide bonds. Each heavy chain contains the aforementioned heavy chain constant region, heavy chain variable region, or CDR sequence, and each light chain contains the aforementioned light chain constant region, light chain variable region, or CDR sequence. The C-terminus of the heavy chain variable region is attached to the N-terminus of the heavy chain constant region, and the C-terminus of the light chain variable region is attached to the N-terminus of the light chain constant region. The antibody of this disclosure may be, for example, a full-length antibody of an IgG1, IgG2, or IgG4 isotype. In another embodiment, the antibody of this disclosure may be a single-chain antibody (scFv) or an antibody fragment, such as a Fab, F(ab')2 fragment, Fd fragment, Fv fragment, dAb, or a separated CDR region.

[0043] On one hand, this disclosure also provides polypeptide fusions comprising the antibody or antigen-binding fragment of this disclosure, and other functional molecules, said other functional molecules being peptides, proteins, or non-proteins, wherein the polypeptide fusion has the function of binding ST2 and one or more other functions. On another hand, this disclosure also provides multispecific molecules comprising the antibody or antigen-binding fragment of this disclosure, and at least one other functional portion different from the specificity of said antibody or antigen-binding fragment, said other functional portion being another peptide or protein, said multispecific molecule being able to bind ST2 and at least one other disease-related protein, such as IgE. On yet another hand, the ST2 antibody or antigen-binding fragment of this disclosure may also be encoded by or carried by a viral vector.

[0044] On the one hand, this disclosure also provides pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof of the present disclosure, or a multispecific molecule, polypeptide fusion, or viral vector comprising the present disclosure, and pharmaceutically acceptable excipients, diluents, or carriers.

[0045] On the one hand, this disclosure also provides isolated nucleic acid molecules encoding antibodies or antigen-binding fragments thereof, expression vectors containing said nucleic acid molecules, and host cells containing said expression vectors.

[0046] On the one hand, this disclosure also provides a method for preparing ST2 antibody or antigen-binding fragment thereof, comprising the steps of: (i) expressing the antibody or antigen-binding fragment thereof in host cells, and (ii) isolating the antibody or antigen-binding fragment thereof from host cells or cell cultures thereof.

[0047] On the other hand, this disclosure provides a method for detecting ST2 expression levels in a subject sample, the method comprising contacting the sample with the antibody or antigen-binding fragment thereof under conditions allowing the antibody or antigen-binding fragment thereof of this disclosure to bind to ST2 (or under conditions allowing a complex to form with ST2). On the other hand, this disclosure provides a method for treating or alleviating IL33 / ST2-mediated diseases and conditions in a subject in need, said IL33 / ST2-mediated diseases and conditions including but not limited to: asthma, allergic rhinitis, chronic obstructive pulmonary disease, eosinophilic bronchitis, eosinophilic esophagitis, atopic dermatitis, psoriasis, systemic lupus erythematosus, pemphigoid, rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, pulmonary fibrosis, liver fibrosis, systemic sclerosis, sarcoidosis, transplant anti-host disease (GVHD), diabetes, cardiovascular disease, or combinations thereof, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof of this disclosure, or a pharmaceutical composition thereof. In some embodiments, the method includes administering to the subject a therapeutically effective amount of a nucleic acid molecule encoding an antibody of the present disclosure or an antigen-binding fragment thereof. In some embodiments, the method includes administering to the subject a therapeutically effective amount of a polypeptide fusion of the present disclosure, a multispecific molecule, a viral vector, or a pharmaceutical composition thereof. In some embodiments, the multispecific molecule may bind ST2 and at least one other disease-associated protein, such as TSLP, IgE, IL4, IL13, or IL-5. In some embodiments, at least one other antibody may be administered together with an antibody of the present disclosure or an antigen-binding fragment thereof, such as a TSLP antibody, TSLPR antibody, IL4 antibody, IL4R antibody, or IgE antibody. In some embodiments, at least one additional drug may be administered together with an antibody of the present disclosure or an antigen-binding fragment thereof, such as an anti-asthmatic drug, an anti-chronic obstructive pulmonary disease drug, an anti-ulcerative colitis drug, an anti-atopic dermatitis drug, or an anti-psoriasis drug.

[0048] Other features and advantages of this disclosure will become clearer from the following specific description and embodiments, which should not be construed as limiting. All documents, Genbank records, patents, and published patent applications cited in this disclosure are expressly included in this disclosure by reference. Attached Figure Description

[0049] Figure 1: ELISA detection of the binding of chimeric anti-ST2 antibodies to hST2 within a certain concentration range. Figure 1 shows the binding of chimeric antibodies xi17E2, xi8F4, xi26A1, xi31C8, xi3C6, and xi7D1 to hST2, with the binding of RG6149 shown as a control.

[0050] Figure 2: ELISA detection of the binding of chimeric anti-ST2 antibodies to cyno-ST2 within a certain concentration range. 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.

[0051] Figure 3: ELISA detection of the blocking effect of chimeric anti-ST2 antibodies on IL33 / ST2 binding within a certain antibody concentration range. Figure 3 shows the blocking effect of chimeric antibodies xi26A1, xi7D1, xi17E2, xi31C8, xi3C6, and xi8F4 on IL33 / ST2 binding, with the blocking effect of RG6149 shown as a control.

[0052] Figure 4: ELISA detection of the binding of humanized anti-ST2 antibody to hST2 within a certain concentration range. Figure 4 shows the binding of humanized antibodies hz31C8-1.1 and hz31C8-1.2 to hST2, with the binding of chimeric antibodies xi31C8, RG6149, and GSK3772847 shown as controls, and the binding of IgG2 shown as a negative control.

[0053] Figure 5: ELISA detection of the binding of humanized anti-ST2 antibody to cyno-ST2 within a certain concentration range. Figure 5 shows the binding of humanized antibodies hz31C8-1.1 and hz31C8-1.2 to cyno-ST2, with the binding of chimeric antibodies xi31C8, RG6149, and GSK3772847 shown as controls, and the binding of IgG2 shown as a negative control.

[0054] Figure 6: ELISA detection of the blocking effect of humanized anti-ST2 antibodies on IL33 / ST2 binding within a certain antibody concentration range. Figure 6 shows the blocking effect of humanized antibodies hz31C8-1.1 and hz31C8-1.2 on IL33 / ST2 binding, with the blocking effects of chimeric antibodies xi31C8, RG6149, and GSK3772847 shown as controls.

[0055] Figure 7: The binding of chimeric anti-ST2 antibodies to hST2-overexpressing cells within a specific antibody concentration range was detected by FACS. Figure 7 shows the binding of chimeric antibodies xi31C8, xi26A1, xi7D1, xi3C6, xi8F4, and xi17E2 to HEK293T-hST2-NFκB-Luciferase cells overexpressing hST2. Binding of RG6149 is shown as a control, and binding of IgG4 is shown as a negative control.

[0056] Figure 8: The binding of chimeric anti-ST2 antibodies to cyno-ST2 overexpressing cells within a certain antibody concentration range was detected by FACS. Figure 8 shows the binding of chimeric antibodies xi31C8, xi26A1, xi7D1, xi3C6, xi8F4, and xi17E2 to HEK293T-Cyno-ST2-NFκB-Luciferase cells overexpressing cyno-ST2. Binding of RG6149 is shown as a control, and binding of IgG4 is shown as a negative control.

[0057] Figure 9: Cell viability assays show the blocking effect of chimeric anti-ST2 antibodies on IL33 / ST2 binding within a specific antibody concentration range. Figure 9 illustrates the blocking effect of chimeric antibodies xi31C8, xi26A1, xi7D1, and xi3C6 on the interaction between IL33 protein and HEK293T-hST2-NFκB-Luciferase cells expressing hST2. The blocking effect of RG6149 is shown as a control.

[0058] Figure 10: The binding of humanized anti-ST2 antibodies to hST2-overexpressing cells within a certain antibody concentration range was detected by FACS. Figure 10 shows the binding of humanized antibodies hz31C8-1.1 and hz31C8-1.2 to HEK293T-hST2-NFκB-Luciferase cells overexpressing hST2. The binding of chimeric antibodies xi31C8, RG6149, and GSK3772847 is shown as a control, and the binding of IgG2 is shown as a negative control.

[0059] Figure 11: Cell viability assays show the blocking effect of humanized anti-ST2 antibodies on IL33 / ST2 binding within a specific antibody concentration range. Figure 11 shows that humanized antibodies hz31C8-1.1 and hz31C8-1.2 blocked the interaction between IL33 protein and HEK293T-hST2-NFκB-Luciferase cells expressing hST2. The blocking effects of chimeric antibodies xi31C8, RG6149, and GSK3772847 are shown as controls, and the blocking effect of IgG2 is shown as a negative control.

[0060] Figure 12: Cell viability assays show the blocking effect of humanized anti-ST2 antibodies on IL33 / ST2 binding within a specific antibody concentration range. Figure 12 shows that humanized antibodies hz31C8-1.1 and hz31C8-1.2 blocked the interaction between IL33 protein and hST2-expressing KU812-NFκB-Luciferasae cells. The blocking effects of chimeric antibodies xi31C8, RG6149, and GSK3772847 are shown as controls, and the blocking effect of IgG2 is shown as a negative control.

[0061] Figure 13: Cell viability assay of humanized anti-ST2 antibody on CD4 within a certain antibody concentration range. + Blocking IL5 secretion induced by IL33 / ST2 on T cells. Figure 13 shows the effects of humanized antibodies hz31C8-1.1 and hz31C8-1.2 on CD4 cells. + Blocking IL33 / ST2-induced IL5 secretion on T cells, with the chimeric antibodies xi31C8, RG6149 and GSK3772847 serving as controls, is shown. It should be understood that the terminology used in this disclosure is for describing particular embodiments only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0063] "ST2" includes ST2 variants, homologs, orthologs, and paralogs. For example, in some embodiments, antibodies specific to human ST2 protein may cross-react with ST2 protein of another species (e.g., monkey) under certain conditions. In other embodiments, antibodies specific to human ST2 protein may be completely specific to human ST2 protein without cross-reacting with ST2 protein of other species or other types of proteins, or may cross-react with ST2 protein of some other species but not all other species.

[0064] The terms "human ST2" or "hST2" are used interchangeably in this disclosure and refer to a protein having the human ST2 amino acid sequence, such as the human ST2 amino acid sequence shown in SEQ ID NO: 1, wherein 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 used interchangeably in this disclosure and refer to a protein having the monkey ST2 amino acid sequence, such as the monkey ST2 amino acid sequence shown in SEQ ID NO: 10.

[0065] The “antibody” disclosed herein includes full-length antibodies and any antigen-binding fragments (i.e., “antigen-binding portions”) or single chains. A full-length antibody is a glycoprotein comprising 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 can also be divided into hypervariable regions, namely complementarity-determining regions (CDRs), and relatively conserved backbone regions (FRs). Each VH and VL consists of three CDRs and four FRs, from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0066] An antibody's "antigen-binding fragment" or "antibody-binding portion" refers to one or more fragments of an antibody that retain the function of specifically binding to an antigen (e.g., the ST2 protein). It has been demonstrated that the antigen-binding function of an antibody can be exercised by fragments of the full-length antibody. Examples encompassed in the term "antigen-binding portion / fragment" of an antibody include: (i) Fab fragments: composed of V... L V H(ii) a monovalent fragment consisting of the CL and CH1 domains; (iii) an F(ab')2 fragment, a bivalent fragment containing two Fab fragments connected by a disulfide bridge in the hinge region; (iv) an Fd fragment consisting of the VH and CH1 domains; (v) an Fv fragment consisting of the VL and VH domains of the antibody single arm; (v) a dAb fragment consisting of the VH domain (see Ward et al., Nature. 341:544-546(1989)); (vi) a separated complementarity-determining region (CDR); and (vii) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by different genes, VH and VL can be linked into a single protein chain via synthetic linkers using a recombination approach, where 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 encompassed in the term antigen-binding portion / fragment. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments can be functionally screened using the same methods as full-length antibodies.

[0067] "Isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., isolated antibodies that specifically bind to the ST2 protein are substantially free of antibodies that specifically bind to antigens other than the ST2 protein). However, isolated antibodies that specifically bind to the human ST2 protein may have cross-binding properties with other antigens (e.g., ST2 proteins from other species). Furthermore, isolated antibodies are substantially free of other cellular components and / or chemicals.

[0068] "Mouse antibody" or "mouse-derived antibody" refers to an antibody in which both the backbone region and the CDR region in the variable region are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, that constant region is also derived from mouse germline immunoglobulin sequences. The mouse antibodies disclosed herein may include amino acid residues not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced through in vitro random or point mutations or through in vivo somatic mutations), but "mouse antibody" does not include antibodies in which a CDR sequence derived from other mammalian germlines is inserted into the mouse backbone sequence.

[0069] A "chimeric antibody" is an antibody created by combining genetic material from a non-human source with genetic material from a human source. Or, more generally, a chimeric antibody is an antibody having genetic material from one species and genetic material from another species. In this disclosure, a chimeric antibody is also referred to as "Xi".

[0070] "Humanized antibody" refers to an antibody derived from a non-human species whose protein sequence has been modified to increase its similarity to naturally occurring human antibodies. In this disclosure, humanized antibody is also referred to as "hz".

[0071] "Isotype" refers to the antibody class (e.g., IgM or IgG1) encoded by the heavy chain constant region gene.

[0072] In this disclosure, the terms “antibody that recognizes an antigen” and “antibody that is specific to an antigen” are used interchangeably with the term “antibody that binds specifically to an antigen”.

[0073] Antibodies that "specifically bind to human ST2" refer to antibodies that bind to human ST2 protein (and possibly ST2 proteins from other non-human species) but substantially do 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 M or smaller, 1.0 × 10 -8 M or smaller, preferably 5.0 × 10 -9 M or smaller, 1.0 × 10 -9 M or smaller, more preferably 5.0 × 10 -10 M or smaller, 1.0 × 10 -10 M or smaller.

[0074] The term "substantially non-binding" to proteins or cells refers to substances that do not bind to proteins or cells, or do not bind to them with high affinity, meaning that the KD (kinetic density) for binding to proteins or cells is 1.0 × 10⁻⁶. -6 M or higher, preferably 1.0 × 10 -5 M or higher, 1.0×10 -4 M or higher, more preferably 1.0 × 10 -3 M or higher, 1.0×10 -2 M or higher.

[0075] For IgG, the term "high affinity" refers to a KD of 1.0 × 10⁻⁶. -6 M or smaller, 1.0 × 10 -7 M or smaller, preferably 1.0 × 10 -8 M or smaller, 5.0×10 -9 M or smaller, more preferably 1.0 × 10 -9 M or smaller. However, "high affinity" binding may differ for other antibody isotypes. For example, "high affinity" binding for IgM isotypes refers to a KD of 10. -6 M or smaller, preferably 10 -7 M or smaller, preferably 10 -8 M or smaller.

[0076] "Identity" refers to the similarity between two nucleic acid sequences or two polypeptides. The sequence identity of this disclosure is at least 80%, 85%, 90%, or 95%, preferably at least 95%. Non-limiting embodiments include: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Sequence comparisons and identity percentage determinations between two sequences can be performed using the default settings of the BLASTN / BLASTP algorithm on the National Center for Biotechnology Institute website.

[0077] The term "EC50," also known as the half-maximal effective concentration, refers to the antibody concentration that achieves 50% of the maximum effect after a specific exposure time.

[0078] The term "IC50," also known as the half-maximal inhibitory concentration, refers to the antibody concentration that inhibits specific biological or biochemical functions by 50% relative to the absence of antibodies.

[0079] The terms “inhibition” and “blocking” are used interchangeably and include partial and complete inhibition / blocking. In some embodiments, the ST2 antibody inhibits the binding of IL33 / ST2 by at least about 50%, for example, at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[0080] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, with a preference for mammals, such as non-human primates, sheep, dogs, cats, cattle, and horses.

[0081] The term "therapeutic effective amount" refers to an amount sufficient to prevent or improve a disease or symptom and / or reduce its severity, preferably an amount that has a preventive effect on the severity of disease symptoms, the frequency and duration of asymptomatic periods, or the damage or incapacity caused by the disease. Therapeutic effective amount is related to the disease being treated, and the actual effective amount can be readily determined by those skilled in the art.

[0082] Unless otherwise specified, the use of the singular includes the plural. Unless otherwise specified, the words “a” or “an” mean “at least one” or “at least one type”. Unless otherwise specified, the phrase “at least one” has the same meaning as “one or more”, and the use of “and / or” means “and” or “or”.

[0083] Several aspects of this disclosure are described in more detail below.

[0084] Anti-ST2 antibodies specifically bind to ST2 and block IL33 / ST2 interaction, as well as other beneficial functional characteristics. The disclosed ST2 antibody or its antigen-binding fragment binds specifically to human ST2 with high affinity. The disclosed ST2 antibody or its antigen-binding fragment specifically binds to ST2 (e.g., human ST2 and monkey ST2) and blocks IL33 / ST2 binding and its signaling. The disclosed ST2 antibody or its antigen-binding fragment is located on CD4... + Blocks IL5 secretion induced by IL33 / ST2 on T cells. The ST2 antibody or its antigen-binding fragment disclosed herein substantially does not 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 exhibits good physical stability (e.g., thermal stability). The ST2 antibody or its antigen-binding fragment disclosed herein has a relatively long in vivo half-life.

[0085] Preferably, the ST2 antibody disclosed herein is a monoclonal antibody. Furthermore, the antibody may be, for example, a mouse, chimeric, or humanized monoclonal antibody.

[0086] ST2 monoclonal antibody Preferably, the ST2 antibody or its antigen-binding fragment of this disclosure is an antibody with the structure and chemical properties described below. The ST2 antibody or its antigen-binding fragment comprises a heavy chain CDR region and a light chain CDR region, wherein exemplary heavy chain CDR sequences and light chain CDR sequences are provided in Tables 1 and 2 below; the ST2 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein exemplary heavy chain variable regions and light chain variable regions are provided in Table 3 below. 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 the human IgG2 heavy chain constant region, and the light chain constant region of the antibody may be the human κ light chain constant region. These antibodies may also comprise a mouse IgG1 or IgG4 heavy chain constant region and / or a mouse κ light chain constant region.

[0087] The heavy chain CDR regions in Table 1 and the light chain CDR regions in Table 2 are defined by the Kabat numbering system. However, as is well known in the art, CDR regions can also be determined by other numbering systems such as Chothia, IMGT, AbM, or the Contact numbering system / method based on the heavy / light chain variable region sequence.

[0088] Table 1. Amino acid sequence listing of the heavy chain CDR region

[0089] Table 2. Amino acid sequence listing of the CDR region of the light chain

[0090] Table 3. Amino acid sequence listing of the variable regions of the heavy / light chains (underlined regions indicate CDR regions in order).

[0091] Table 4. Amino acid sequence listing of heavy / light chains (underlined regions indicate constant regions)

[0092] The VH and / or VL sequences (or CDR sequences) of other anti-ST2 antibodies that bind to human ST2 can be "mixed and paired" with the VH and / or VL sequences (or CDR sequences) of the antibodies disclosed herein. Preferably, when the VH and VL chains (or their CDRs) are mixed and paired, the VH sequence in a particular VH / VL pair can be replaced by a structurally similar VH sequence. Similarly, it is preferable to replace the VL sequence in a particular VH / VL pair with a structurally similar VL sequence.

[0093] Therefore, in one embodiment, the antibody or antigen-binding fragment thereof disclosed herein comprises: (a) a heavy chain variable region containing an amino acid sequence listed in Table 3; and (b) a light chain variable region containing an amino acid sequence listed in Table 3, or a VL of another ST2 antibody, wherein the antibody specifically binds to human ST2.

[0094] In another embodiment, the antibody or antigen-binding fragment thereof disclosed herein comprises: (a) the heavy chain CDR1, CDR2 and CDR3 listed in Table 1; and (b) the light chain CDR1, CDR2 and CDR3 listed in Table 2, or CDRs of another ST2 antibody, wherein the antibody specifically binds to human ST2.

[0095] In another embodiment, the antibody or antigen-binding fragment thereof disclosed herein includes the heavy chain CDR2 of the ST2 antibody disclosed herein and other CDRs binding to human ST2 antibodies, such as the heavy chain CDR1 and / or CDR3 of another ST2 antibody, and / or the light chain CDR1, CDR2 and / or CDR3.

[0096] Furthermore, it is known in the art that the CDR3 domain, independent of the CDR1 and / or CDR2 domains, can be used to determine the binding specificity of an antibody to the same antigen, and can predict the generation of multiple antibodies with the same binding specificity based on the CDR3 sequence. See, e.g., 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. USA .95:8910-8915(1998); Barbas et al., J. Am. Chem. Soc. 116: 2161-2162 (1994); Barbas et al., Proc. Natl. Acad. Sci. USA .92: 2529-2533 (1995); Ditzel et al., J. .Immunol.157:739-749(1996); Berezov et al., BIAjournal8: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., Proc.Natl.Acad.Sci.U.S.A .90:4374-8(1993);Polymenis and Stoller,J.Immunol.152:5218-5329(1994)and Xu and Davis,Immunity. 13:37-45 (2000);U.S.Pat.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 incorporated herein by reference.

[0097] In another embodiment, the antibody or antigen-binding fragment thereof disclosed herein comprises the heavy chain CDR2 of the ST2 antibody of this disclosure and at least the heavy chain and / or light chain CDR3 of the ST2 antibody of this disclosure, or the heavy chain and / or light chain CDR3 of another ST2 antibody, wherein the antibody specifically binds to human ST2. Preferably, these antibodies (a) compete with the ST2 antibody of this disclosure for binding to ST2; (b) retain functional characteristics; (c) bind the same epitopes; and / or (d) have similar binding affinity to the ST2 antibody of this disclosure. In another embodiment, the antibody or antigen-binding fragment thereof disclosed herein may further comprise the light chain CDR2 of the ST2 antibody of this disclosure, or the light chain CDR2 of another ST2 antibody, wherein the antibody specifically binds to human ST2. In another embodiment, the antibody or antigen-binding fragment thereof disclosed herein may further comprise the heavy and / or light chain CDR1 of the ST2 antibody of this disclosure, or the heavy and / or light chain CDR1 of another ST2 antibody, wherein the antibody specifically binds to human ST2.

[0098] Conservative modification In another embodiment, the antibody or its antigen-binding fragment of this disclosure comprises CDR1, CDR2, and CDR3 sequences of one or more conserved modified heavy chain variable regions and / or light chain variable regions, similar to the ST2 antibody of this disclosure. It will be understood in the art that some conserved sequence modifications do not result in the loss of antigen binding. See, for example, Brummell et al., Biochem 32:1180-8 (1993); de Wildt et al., Prot. Eng. 10:835-41 (1997); Komissarovet al., J. Biol. Chem. 272: 26864-26870 (1997); Hall et al. 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).

[0099] Therefore, in one embodiment, the antibody comprises a heavy chain variable region and / or a light chain variable region, the heavy chain variable region and the light chain variable region comprising CDR1, CDR2 and CDR3 respectively, wherein: (a) the CDR1 sequence of the heavy chain variable region comprises the sequences listed in Table 1, and / or their conserved modifications; and / or (b) the CDR2 sequence of the heavy chain variable region comprises the sequences listed in Table 1, and / or their conserved modifications; and / or (c) the CDR3 sequence of the heavy chain variable region comprises the sequences listed in Table 1, and / or their conserved modifications; and / or (d) the CDR1 and / or CDR2 and / or CDR3 sequences of the light chain variable region comprise the sequences listed in Table 2, and / or their conserved modifications; and (e) the antibody specifically binds to human ST2.

[0100] The antibody disclosed herein has one or more of the functional properties described above, such as high affinity for human ST2 and blocking IL33 / ST2 binding and its signal transduction.

[0101] In several implementations, the antibody may be a mouse, chimeric, or humanized antibody or an antigen-binding fragment thereof.

[0102] As used in this disclosure, the term "conserved sequence modification" refers to amino acid modifications that do not significantly affect or alter antibody binding properties. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of this disclosure using standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conserved amino acid substitution refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are known in the art. These families of amino acid residues include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), nonpolar 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 antibody of this disclosure may be replaced by other amino acid residues of the same side chain family, and the resulting antibody may be tested for retention of function (i.e., the function described above) using the functional assays described in this disclosure.

[0103] Engineered and modified antibodiesThe antibodies disclosed herein can be engineered to produce modified antibodies using antibodies possessing one or more VH / VL sequences of the ST2 antibody of this disclosure as starting material. The antibodies can be genetically modified with one or more residues within one or two variable regions (i.e., VH and / or VL) (e.g., in one or more CDR regions and / or one or more backbone regions). Alternatively, the antibodies can be engineered with residues in constant regions, for example, to alter the effector function of the antibody.

[0104] In some implementations, CDR transplantation can be used to genetically modify the variable regions of antibodies. Antibodies primarily interact with target antigens through amino acid residues located in the six complementarity-determining regions (CDRs) of the heavy and light chains. Therefore, the amino acid sequences within the CDRs of individual antibodies are more diverse than those outside the CDRs. Since the CDR sequence is responsible for the main antibody-antigen interaction, recombinant antibodies that mimic the characteristics of a specific natural antibody can be expressed by constructing expression vectors in which the CDR sequence of a specific natural antibody is transplanted into the backbone sequence of different antibodies with different characteristics (Riechmann et al., Nature. 332: 323-327 (1998); Jones et al., Nature. 321: 522-525 (1986); Queen et al., Proc. Natl. Acad; USA 86: 10029-10033 (1989); US Pat. Nos. 5,225, 539; 5,530, 101; 5,585, 089; 5,693, 762 and 6,180, 370).

[0105] Therefore, another embodiment of this disclosure relates to isolated monoclonal antibodies or antigen-binding fragments thereof, comprising heavy chain variable regions and / or light chain variable regions, wherein the heavy chain variable regions comprise CDR1, CDR2, and CDR3 of the sequences described above in this disclosure, and the light chain variable regions comprise CDR1, CDR2, and CDR3 of the sequences described above in this disclosure. Although these antibodies comprise the CDR sequences of the VH and VL of the monoclonal antibodies of this disclosure, they may contain different backbone sequences.

[0106] Such backbone sequences can be obtained from publicly available DNA databases or references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy chain variable region and light chain variable region genes can be obtained from the Vbase Human Germline Sequence Database (www.mrc-cpe.cam.ac.uk / vbase) and from Kabat et al., (1991), ibid.; 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 for human heavy chain variable region and light chain variable region genes can be obtained from the Genbank database.

[0107] The antibody protein sequence was compared with a protein sequence database using one of the sequence similarity search methods known to those skilled in the art (Altschulet al., (1997), ibid.).

[0108] The backbone sequences of the antibodies disclosed herein are preferably those structurally similar to those used in the backbone sequences of the antibodies disclosed herein. The VH CDR1, CDR2, and CDR3 sequences can be transplanted into backbone regions having the same sequence as the germline immunoglobulin gene from which the backbone sequence is obtained, or the CDR sequences can be transplanted into backbone regions having one or more mutations compared to the germline sequence. For example, in some cases, mutation of residues in the backbone region is beneficial and can maintain or enhance the antigen-binding capacity of the antibody (see, for example, US Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0109] Another type of variable region modification involves mutating amino acid residues in the CDR1, CDR2, and / or CDR3 regions of VH and / or VL to improve one or more properties of the target antibody (e.g., affinity, physicochemical properties). Mutations can be introduced by deterministic mutagenesis or PCR-mediated mutagenesis, and the effect of the mutation on antibody binding or other functional properties can be assessed by in vitro or in vivo assays known in the art. Preferably, conserved modifications known in the art are introduced. This can be substitution, addition, or deletion of amino acids, preferably substitution. Furthermore, typically no more than one, two, three, four, or five residues in each CDR region are altered.

[0110] Furthermore, 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, comprising: (a) a VH CDR1 region containing the sequence of this disclosure, or having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; (b) a VH CDR2 region containing the sequence of this disclosure, or having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; (c) a VH CDR3 region containing the sequence of this disclosure, or having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; (d) a VL CDR1 region containing the sequence of this disclosure, or having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; (e) a VLCDR2 region containing the sequence of this disclosure, or having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; (f) a VL CDR2 region containing the sequence of this disclosure, or having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; and (f) a VL CDR3 ...c) a VL CDR2 region containing the sequence of this disclosure, or having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; and (d) a VL CDR3 region containing the sequence of this disclosure, or having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; and (e) a VLCDR2 The CDR3 region contains the sequence of this disclosure, or has 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions.

[0111] The genetically modified antibodies disclosed herein include those in which backbone residues of VH and / or VL are modified to improve antibody properties. Typically, such backbone modifications can reduce the immunogenicity of the antibody. For example, one or more backbone residues are “reverted” to the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutations may contain backbone residues that differ from the resulting antibody germline sequence. These residues can be identified by comparing the antibody backbone sequence with the germline sequence of the resulting antibody.

[0112] Another type of backbone modification involves mutating one or more residues in the backbone region, or even one or more CDR regions, to remove T cell epitopes, thereby reducing the immunogenicity that antibodies may cause. This method is also known as "deimmunization," and is described in more detail in U.S. Patent Publication No. 20030153043.

[0113] In addition to modifications to the backbone or CDR region, other modifications, such as genetic modification of the Fc region of the antibody disclosed herein, are typically used to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antigen-dependent cytotoxicity. Furthermore, the antibodies of this disclosure can also be chemically modified (e.g., by attaching one or more chemical functional groups), or modified to alter their glycosylation, to change one or more functional properties of the antibody.

[0114] 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 specifically described in U.S. Patent No. 5,677,425. Changing the number of cysteine ​​residues in the CH1-hinge region can, for example, promote the assembly of light and heavy chains or increase or decrease antibody stability.

[0115] In another embodiment, the Fc-hinge region of the antibody is mutated to increase or decrease the antibody's biological half-life. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 region of the Fc-hinge region, such that the antibody has weakened binding to staphylococcal protein A (SpA) relative to the native Fc-hinge domain. This method is described in more detail in U.S. Patent No. 6,165,745.

[0116] In another embodiment, the glycosylation of the 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 within the antibody sequence. For example, one or more amino acid substitutions can be performed to eliminate glycosylation sites in one or more variable region backbones, thereby eliminating glycosylation at that site. This deglycosylation can increase the antibody's affinity for the antigen. See, for example, U.S. Patent Nos. 5,714,350 and 6,350,861.

[0117] Furthermore, antibodies with altered glycosylation types can be prepared, such as hypofucosylated antibodies with reduced fucose residues or antibodies with increased bipartite GlcNac structures. It has been demonstrated that alterations in glycosylation form increase the ADCC activity of antibodies. Such glycosylation modifications can be achieved, for example, by expressing antibodies in host cells with altered glycosylation systems, which are known in the art and can be used as host cells for expressing the recombinant antibodies of this disclosure to prepare antibodies with altered glycosylation. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosylation gene FUT8 (α(1,6)-fucosylation), and therefore antibodies expressed in Ms704, Ms705, and Ms709 cell lines lack fucose. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were prepared by directionally disrupting the FUT8 gene in CHO / DG44 cells using two alternative vectors (see U.S. Patent 20040110704 and Ohnuki et al., Biotechnol Bioeng. 87:614-22 (2004)). As another example, EP1,176,195 describes a cell line with disrupted FUT8 gene function, which encodes fucosyltransferase, such that antibodies expressed in this cell line exhibit hypofucosylation by reducing or eliminating α-1,6 bond-related enzymes. EP1,176,195 also describes a cell line with low or absent enzymatic activity for adding fucose to N-acetylglucosamine bound to the Fc region of an antibody, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). WO03 / 035835 describes a CHO variant cell line, Lec13 cells, with reduced ability to add fucosylate to Asn(297)-related sugars, resulting in hypofucosylated antibodies expressed by this host cell (see Shields et al., J. Biol. Chem. 277:26733-26740 (2002)). Antibodies with altered glycosylation characteristics can also be prepared in eggs, as described in WO06 / 089231. Alternatively, antibodies with altered glycosylation characteristics can be prepared in plant cells such as Lemna. WO99 / 54342 discloses a cell line genetically modified to express a glycosyltransferase (e.g., β(1,4)-N-acetylglucosamine transferase III (GnTIII)) that causes antibodies expressed in this cell line to exhibit an increased bipartite GlcNac structure, thereby resulting in enhanced ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 17:176-180 (1999)).Alternatively, fucosidases can be used to cleave the fucose residues of the antibody, such as α-L-fucosidase to remove the fucose residues from the antibody (Tarentino et al., Biochem.14:5516-23 (1975)).

[0118] Another modification of the antibodies disclosed herein is polyethylene glycol (PEGylation). PEGylation of antibodies can, for example, increase the biological (e.g., serum) half-life of the antibody. 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 that attach one or more PEG groups to the antibody or antibody fragment. Preferably, this is carried out by an acylation or alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). The term "polyethylene glycol" as used herein includes any form of PEG used to derive other proteins, such as mono(C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In some embodiments, the antibody to be PEGylated is a deglycosylated antibody. Methods of PEGylation are known in the art and can be applied to the antibodies of this disclosure. See, for example, EP0154316 and EP0401384.

[0119] Physical properties of antibodies The antibodies disclosed herein can be characterized by a variety of physical properties to detect and / or differentiate their classification. For example, antibodies may contain one or more glycosylation sites in the variable region of the light or heavy chain. These glycosylation sites may lead to increased immunogenicity of the antibody or changes in the antibody pK value due to alterations in antigen binding (Marshall et al., Annu Rev Biochem .41:673-702 (1972); Gala and 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 some cases, it is preferable that the ST2 antibody does not contain glycosylated variable regions. This can be achieved by selecting antibodies that do not contain glycosylated motifs in the variable regions or by mutating residues within the glycosylated regions.

[0120] In a preferred embodiment, the antibody does not contain an asparagine isomer site. Deamidation of asparagine may occur in the NG or DG sequence, resulting in the production of isoaspartic residues and reduced stability.

[0121] Each antibody possesses a unique isoelectric point (pI), typically within a pH range of 6–9.5. The pI of IgG1 antibodies is generally in the pH range of 7–9.5, while that of IgG4 antibodies is typically in the pH range of 6–8. It has been speculated that antibodies with pI values ​​outside the normal range may undergo some unfolding and become 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.

[0122] Nucleic acid molecules encoding the antibodies disclosed herein On the other hand, this disclosure provides nucleic acid molecules encoding the variable regions, or CDRs, of the heavy and / or light chains of the antibodies of this disclosure. The nucleic acids may be present in intact cells, cell lysates, or in partially purified or substantially pure forms. When purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, using standard techniques, the nucleic acids are “isolated” or “substantially pure.” The nucleic acids of this disclosure may be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0123] The nucleic acids disclosed herein can be obtained using standard molecular biology techniques. For antibodies expressed in hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes), the light and heavy chain cDNA encoding the antibodies prepared from the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), the nucleic acids encoding such antibodies can be recovered from the gene library.

[0124] Preferably, the nucleic acid molecule of this disclosure comprises a nucleic acid molecule encoding the VH and VL sequences or CDR of the ST2 monoclonal antibody of this disclosure. Once the DNA fragments encoding the VH and VL fragments are obtained, they can be further manipulated using standard recombinant DNA techniques, such as converting the variable region gene into a full-length antibody chain gene, a Fab fragment gene, or an scFv gene. In these manipulations, the DNA fragment encoding VL or VH is operatively ligated to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used in this disclosure, the term "operatively ligated" means ligating two DNA fragments together such that the amino acid sequences encoded by both DNA fragments are within the reading frame.

[0125] By operatively linking the DNA encoding VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3), a separated DNA encoding the VH region can be converted into a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art, and these DNA fragments can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but the IgG2 constant region is most preferred. For Fab fragment heavy chain genes, the DNA encoding VH can be operatively linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0126] By operatively linking the DNA encoding the VL region to another DNA molecule encoding the light chain constant region (CL), the isolated DNA encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene). The sequences of human light chain constant region genes are known in the art, and these DNA fragments can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region may be a κ or λ light chain constant region.

[0127] To prepare the scFv gene, the DNA fragments encoding VH and VL are operatively linked to another fragment encoding a flexible linker, such as the amino acid sequence (Gly4-Ser)3, so that the VH and VL sequences can be expressed as a continuous single-stranded protein, wherein the VL and VH regions are linked by 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)).

[0128] This disclosure pertains to the preparation of monoclonal antibodies. The monoclonal antibodies (mAbs) disclosed herein can be prepared using the somatic cell hybridization (hybridoma) technique described in Kohler and Milstein Nature. 256:495 (1975). Other methods of preparing monoclonal antibodies include viral or oncogenic transformation of B lymphocytes and phage display 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.

[0129] Preparation of monoclonal antibodies from transfected tumorsThe antibodies disclosed herein can also be generated in host cells transfected with tumors using, for example, recombinant DNA technology combined with gene transfection methods (e.g., Morrison, S. Science 229:12021985). In one embodiment, DNA encoding a partial or full-length light and heavy chain, obtained by standard molecular biology techniques, is inserted into one or more expression vectors such that the gene is operatively linked to transcriptional and translational regulatory sequences. In this case, the term "operatively linked" refers to linking the antibody gene to a vector such that the transcriptional and translational regulatory sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene.

[0130] The term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody genes. Such regulatory sequences have been described in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), and adenoviruses, such as the adenovirus major late promoter (AdMLP). Alternatively, non-viral regulatory sequences, such as ubiquitin promoters or β-globin promoters, can be used. In addition, regulatory elements are composed of sequences from different sources, such as the SRα promoter system, which contains sequences from the SV40 early promoter and sequences from human T-cell leukemia type I long terminal repeat sequences (Takebe et al., Mol (Cell. Biol. 8:466-472 (1988)). The expression vector and expression regulatory sequences are compatible with the expression host cells used.

[0131] Antibody light chain genes and antibody heavy chain genes can be inserted into the same or different expression vectors. In a preferred embodiment, the variable region is used to construct a full-length antibody gene by inserting it into an expression vector that already encodes the heavy chain constant region and light chain constant region of the desired isotype, thereby operatively linking VH to CH in the vector and VL to CL in the vector. Alternatively, the recombinant expression vector can encode a signal peptide that promotes the secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector, such that the signal peptide is linked to the amino terminus of the antibody chain gene within the reading frame. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin).

[0132] In addition to the antibody chain gene and regulatory sequence, the recombinant expression vector of this disclosure may also carry other sequences, such as sequences regulating vector replication in host cells (e.g., origin of replication) and 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 in 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).

[0133] To express the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells using standard techniques. The term "transfection" encompasses various techniques for introducing exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-glucan transfection, etc. While expressing the antibodies of this disclosure in prokaryotic or eukaryotic host cells is theoretically feasible, expression in eukaryotic cells, and most preferably in mammalian host cells, is preferred because eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete appropriately folded and immunologically active antibodies.

[0134] Preferred mammalian host cells for expressing the recombinant antibodies of this disclosure include Chinese hamster ovary (CHO) cells (including dhfr-CHO cells administered with DHFR selective markers, as described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220 (1980), DHFR selective markers as described in RJ Kaufman and PA Sharp. J. Mol. Biol. 159:601-621 (1982)), NSO myeloma cells, COS cells, and SP2 cells. Particularly when using NSO myeloma cells, another preferred expression system is the GS gene expression system disclosed in WO87 / 04462, WO89 / 01036, and EP338,841. When the recombinant expression vector encoding the antibody gene is introduced into mammalian host cells, the antibody is prepared by culturing the host cells for a period sufficient for antibody expression in the host cells, or preferably sufficient for antibody secretion into the culture medium in which the host cells grow. Antibodies can be recovered from the culture medium using protein purification methods.

[0135] peptide fusionOn the other hand, this disclosure relates to polypeptide fusions comprising one or more antibodies or antigen-binding fragments of the disclosed herein, wherein the antibody or antigen-binding fragment of the disclosed herein is linked to at least one other functional molecule, which may be a peptide, protein, or non-protein. In one embodiment, the polypeptide fusion of the disclosed herein comprises an immunoconjugate comprising one or more antibodies or antigen-binding fragments of the disclosed herein and at least one therapeutic agent, such as a steroid, linked to the antibody or antigen-binding fragment of the disclosed herein. In one embodiment, the polypeptide fusion of the disclosed herein comprises a bifunctional molecule comprising one or more antibodies or antigen-binding fragments of the disclosed herein and at least one immunocytokine or receptor ligand linked to the antibody or antigen-binding fragment of the disclosed herein, wherein the immunocytokine or receptor ligand may be for IgE, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-9, IL13, IL13R, IL-17, IL-23, IL-33, OX40 ligand (OX40L), GM-CSF, or TSLP, TSLPR / IL7R. In one embodiment, in addition to Fc receptor binding and ST2 binding functions, the bifunctional molecule also has a third function. This third function may be targeting IgE, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-9, IL-13, IL-13R, IL-17, IL-23, IL-33, OX40 ligand (OX40L), GM-CSF, or TSLP, TSLPR / IL7R. The term "bifunctional molecule" as used herein encompasses molecules having three or more functions. In other embodiments, the peptide fusions of this disclosure also comprise other forms. These and other forms of peptide fusions can be prepared by genetic modification, chemical methods, etc.

[0136] Multispecific molecules On the other hand, this disclosure relates to multispecific molecules comprising one or more antibodies or antigen-binding fragments of the present disclosure, wherein the antibody or antigen-binding fragment of the present disclosure is linked to at least one other functional portion having a specificity different from that of the antibody or antigen-binding fragment of the present disclosure, said other functional portion comprising another peptide or protein (e.g., another antibody or antigen-binding fragment of the present disclosure) to produce a multispecific molecule that binds to at least two different binding sites or targets. Therefore, the term "multispecific molecule" as used in this disclosure encompasses molecules having two (i.e., bispecific molecules), three (i.e., trispecific molecules), four (i.e., tetraspecific molecules), or more specificities.

[0137] In one specific embodiment, the multispecific molecule of this disclosure may have one or more other specificities in addition to its anti-Fc binding specificity and ST2 binding specificity. For example, the other specificities may be against IgE, IL4, IL4R, IL5, IL5R, IL6, IL9, IL13, IL13R, IL17, IL23, IL33, or TSLP, TSLPR / IL7R.

[0138] In a specific implementation, multispecific molecules can appear in a variety of different forms and sizes. For example, at one end of the size spectrum, bispecific molecules retain the traditional antibody form, but with two binding arms, each with a different specificity, instead of two binding arms with the same specificity. At the other end, bispecific molecules consist of two single-chain antibody fragments (scFv) linked by peptide chains, referred to as the Bs(scFv)2 construct. Bispecific molecules of intermediate sizes comprise two distinct F(ab) fragments linked by peptide linkers. These and other forms 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).

[0139] Viral vector On the other hand, the antibodies or antigen-binding fragments of this disclosure may also be encoded by or carried by a viral vector. Furthermore, the antibodies or antigen-binding fragments of this disclosure may also be used in conjunction with a viral vector, or a viral vector encoding or carrying the antibodies or antigen-binding fragments of this disclosure may be introduced into the human body.

[0140] This disclosure also provides pharmaceutical compositions comprising polypeptide fusions, multispecific molecules, viral vectors, and pharmaceutically acceptable excipients, diluents, or carriers.

[0141] Pharmaceutical Composition On the other hand, this disclosure provides pharmaceutical compositions comprising an antibody of this disclosure or an antigen-binding fragment thereof, formulated together with a pharmaceutically acceptable excipient, diluent, or carrier. The pharmaceutical composition may optionally comprise one or more other 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-atopic dermatitis drug, or an anti-psoriasis drug.

[0142] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient may be encapsulated in a material to protect it from acids and other natural conditions that could inactivate it. “Parenteral administration” refers to a method other than intestinal and topical application, typically by injection, including but not limited to intravenous, intramuscular, intraarterial, intramembranous, intracystic, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, sub-bursular, subarachnoid, spinal, supradural, and intrasternal injection and bolus. Alternatively, the pharmaceutical compositions of this disclosure may be administered via non-parenteral routes, such as topical, epidermal, or mucosal administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical application.

[0143] Pharmaceutical compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated in microemulsions, liposomes, or other ordered structures suitable for high concentrations of drugs.

[0144] The dosing regimen is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single large dose can be administered, multiple fractional doses can be administered over time, or the dose can be proportionally reduced or increased depending on the severity of the treatment condition. Particularly advantageous are parenteral compositions formulated in convenient and uniformly dose-unit formats. A dose-unit format refers to physically separate units suitable for a single dose to the therapeutic subject; each unit contains a predetermined amount of active ingredient calculated to produce the desired therapeutic effect together with the pharmaceutical carrier. Alternatively, antibodies can be administered as extended-release formulations, in which case the required dosing frequency is reduced.

[0145] For the administration of the composition, the dosage can be from about 0.0001 to 100 mg / kg of host body weight, more commonly from 0.01 to 5 mg / kg. For example, the dosage can 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 1-10 mg / kg. Exemplary treatment regimens require administration twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every two months, once every three months, or once every three to six months.

[0146] The pharmaceutical composition can be a sustained-release agent, including implants, transdermal patches, and microcapsule delivery systems. Biodegradable and biocompatible polymers such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used.

[0147] In some embodiments, the antibodies of this disclosure may be formulated to ensure appropriate in vivo distribution. For example, to ensure that the therapeutic antibodies of this disclosure cross the blood-brain barrier, the antibodies may be formulated in liposomes, which may additionally contain targeting functional groups to enhance selective delivery to specific cells or organs.

[0148] Uses and methods of this disclosure The antibodies or antigen-binding fragments thereof disclosed herein (encoding nucleic acid molecules, pharmaceutical compositions, polypeptide fusions, multispecific molecules, or viral vectors) have various in vitro and in vivo applications related to the diagnosis, treatment, and / or prevention of IL33 / ST2-mediated diseases and conditions. These IL33 / ST2-mediated diseases and conditions include, but are not limited to: asthma, allergic rhinitis, chronic obstructive pulmonary disease, eosinophilic bronchitis, eosinophilic esophagitis, atopic dermatitis, psoriasis, systemic lupus erythematosus, pemphigoid, rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, pulmonary fibrosis, liver fibrosis, systemic sclerosis, sarcoidosis, transplant-versus-host disease (GVHD), diabetes, cardiovascular disease, or combinations thereof. The antibodies or antigen-binding fragments thereof disclosed herein can be administered to human subjects to reduce, alleviate, or treat the aforementioned diseases or conditions. In diagnostic procedures, the antibodies of this disclosure can be contacted with a subject's sample, provided that the antibodies or antigen-binding fragments thereof are allowed to bind to ST2, to detect the amount of ST2 protein in the subject's sample.

[0149] These and other methods disclosed herein are discussed further below.

[0150] combination therapyThis disclosure provides that the ST2 antibody or its antigen-binding fragment (encoding a nucleic acid molecule, pharmaceutical composition, polypeptide fusion, multispecific molecule, or viral vector) can be co-administered with one or more other antibodies or drugs to reduce, alleviate, or treat IL33 / ST2-mediated diseases and conditions in subjects, including but not limited to: asthma, allergic rhinitis, chronic obstructive pulmonary disease, eosinophilic bronchitis, eosinophilic esophagitis, atopic dermatitis, psoriasis, systemic lupus erythematosus, pemphigoid, rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, pulmonary fibrosis, liver fibrosis, systemic sclerosis, sarcoidosis, transplant anti-host disease (GVHD), diabetes, cardiovascular disease, or combinations thereof. In one embodiment, this disclosure provides a method for treating a subject with asthma, chronic obstructive pulmonary disease (COPD), and atopic dermatitis, wherein the disclosed ST2 antibody or its antigen-binding fragment is administered together with one or more other antibodies, such as 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 a subject with asthma, COPD, and atopic dermatitis, wherein the disclosed ST2 antibody or its antigen-binding fragment is administered together with at least one additional drug, such as an anti-asthmatic drug, an anti-COPD drug, or an anti-atopic dermatitis drug. In some embodiments, the subject is a human being. Other therapies that can be combined with the disclosed ST2 antibody or its antigen-binding fragment include: reducing or avoiding allergen exposure, hormone therapy, surgery, etc.

[0151] The combination (i.e., combination) of therapeutic agents discussed in this disclosure can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as separate compositions wherein each agent is contained in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents can be administered sequentially.

[0152] Furthermore, if multiple combination therapies are administered and the drugs are administered sequentially, the order of administration at each time point can be reversed or kept the same, and sequential administration can be combined with simultaneous administration or any combination thereof.

[0153] Although the foregoing invention has been described in detail by way of example and embodiments for clarity of understanding, it will be apparent to those skilled in the art, based on the teachings of this disclosure, that certain changes and modifications may be made to this disclosure without departing from the spirit and scope of the appended claims. This disclosure is further illustrated by the following embodiments and is not intended to be limiting. Those skilled in the art will readily identify various non-critical parameters that can be changed or modified to produce substantially similar results.

[0154] Unless otherwise indicated, the practice of this disclosure will employ conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the scope of the art.

[0155] Example 1: Preparation of ST2 antigen and detection protein. Using the full-length gene (hST2) of human UniProt Interleukin-1 receptor-like 1 (ST2) isoform A (SEQ ID NO: 1) as the template of ST2 disclosed in this invention, the gene sequence encoding the antigen and detection protein of this invention is obtained. It can be recombined with mouse antibody heavy chain Fc fragment (such as mouse IgG2a) to form hST2-mFc, or recombined with human antibody heavy chain Fc fragment to form hST2-hFc, for use in mouse immunization or subsequent screening detection. The cDNAs encoding the recombinant hST2 extracellular region protein with a mouse antibody heavy chain Fc tag (hST2-ECD-mFc, SEQ ID NO: 2) and the recombinant hST2 extracellular region protein with a human antibody heavy chain Fc tag (hST2-ECD-hFc, SEQ ID NO: 4) (SEQ ID NOs: 3 and 5, respectively) were synthesized and subcloned into the expression vector pcDNA3.1 (Invitrogen, V-790). These constructed vectors were transfected into Expi293 cells (Thermo, A14527) for transient expression. The hST2-ECD-mFc and hST2-ECD-hFc recombinant proteins were then purified using a Protein A column (GE Healthcare).

[0156] The cDNA encoding the recombinant protein of human IL33 containing Avitag (SEQ ID NO: 7) was obtained through gene synthesis and cloned into a GST-tagged expression vector. The constructed vector was transformed into BL21 competent cells for induced expression. The GST-IL33 Avitag protein was purified using a GST purification column, and GST was removed by thrombin enzyme digestion (Sigma, T4648-1KU) to obtain hIL33 Avitag (SEQ ID NO: 6).

[0157] The cDNA encoding the recombinant protein of human IL33 (SEQ ID NO: 9) was obtained through gene synthesis and cloned into a GST-tagged expression vector. The constructed vector was transformed into BL21 competent cells for induced expression. The GST-IL33 protein was purified using a GST purification column, and the GST was removed by thrombin enzyme digestion (Sigma, T4648-1KU) to obtain the hIL33 protein (SEQ ID NO: 8).

[0158] The ST2 antibody RG6149 in the examples refers to antibody Ab2 in CN104334582, which was prepared internally, and the amino acid sequences of the heavy and light chains are shown in SEQ ID NO: 83 and SEQ ID NO: 84 of this disclosure; the ST2 antibody GSK3772847 refers to antibody STLM208 in CN104411333, which was prepared internally, and the amino acid sequences of the heavy and light chains are shown in SEQ ID NO: 85 and SEQ ID NO: 86 of this disclosure.

[0159] Example 2: Preparation of Anti-ST2 Hybridoma Monoclonal Antibody. Purified hST2-ECD-mFc recombinant protein (100 μg / mouse) was thoroughly mixed and emulsified with an equal volume of complete Freund's adjuvant (Sigma, F5881-10X10ML) (for initial immunization) or incomplete Freund's adjuvant (Sigma, F5506-10X10ML) (for booster immunization). BALB / c mice were subcutaneously immunized every 2 weeks for 8 weeks. Three days before fusion, mice were boosted by intraperitoneal injection of adjuvant-free hST2-ECD-mFc antigen (50 μg / mouse). A PEG-mediated fusion step was used to fusion with spleen cells (1 × 10⁻⁶) from immunized mice. 8 ) and SP2 / 0 myeloma cells (2 × 10 7 Hybridoma cells were obtained by fusion. After fusion, the cells were resuspended in HAT complete medium (Gibco, 21060017) and dispensed into 96-well plates at 0.1 mL / well, and cultured in an incubator at 37°C and 5% CO2. Typically, around day 10-15 post-fusion, the binding activity of the cell culture supernatant to ST2 was detected using ELISA (see Example 5). Cell culture supernatants from wells showing positive results were selected, and their blocking activity against IL33 / ST2 binding was detected using ELISA (see Example 6).

[0160] Wells that specifically bind to ST2 and block IL33 / ST2 binding were selected, and cells were promptly expanded into 24-well plates according to cell density. Cell lines transferred to the 24-well plates were retested and then preserved for the first subcloning. Cells showing positive results in the first subcloning were preserved and subjected to a second subcloning. Cells showing positive results in the second subcloning were then preserved and subjected to protein expression.

[0161] Example 3: cDNA Acquisition and Chimeric Antibody Construction of Anti-ST2 Antibody. Total RNA was isolated from hybridoma cells with binding and blocking functions using a total RNA extraction kit. First-strand cDNA was synthesized using Superscript III reverse transcriptase (Thermo, 18080051) according to the manufacturer's instructions. The variable region sequence of the antibody was then amplified by PCR using degenerate mouse IgG primers.

[0162] The PCR mixture was separated by electrophoresis on a 1% agarose / Tris-borate gel containing 0.5 μg / mL ethidium bromide. DNA fragments of the expected size (approximately 500 bp for both heavy and light chains) were excised from the gel and purified. The purified PCR product was cloned into the pMD-19T vector (Takara, 6013) and transformed into DH5α competent *E. coli* cells (Takara, 9057). Five colonies were picked from LB agar plates for DNA sequencing. The heavy chain variable region and light chain variable region sequences of the antibody 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).

[0163] Construction and expression of chimeric antibodies: A chimeric light chain was constructed by ligating a mouse VL region gene synthesis fragment to the human κ chain constant region via a double enzyme digestion reaction. A chimeric heavy chain was constructed by ligating a mouse VH region gene synthesis fragment to the human IgG2 constant region via a double enzyme digestion reaction.

[0164] The DNA vector containing the above-mentioned chimeric light chain and the DNA vector containing the above-mentioned chimeric heavy chain were co-transfected into Expi CHO cells (50 mL system, 6 × 10⁶ cells / year). 6 Transient expression of the chimeric antibody (1 μg / mL cells, 1 μg / mL DNA) was performed and cultured for 7 days. The chimeric antibody was then purified from the cell culture supernatant using a Protein A column (GE Healthcare).

[0165] Example 4: Determination of the affinity of anti-ST2 antibody for hST2 antigen. Following the instructions in the product manual, anti-mouse IgG antibody (for capturing human ST2 antigen, Cytiva, 29215281) or anti-his antibody (for capturing cynomolgus monkey ST2 antigen, Cytiva, 29234602) was conjugated onto a CM5 biochip (Cytiva, BR-1000-12). A series of concentrations (32.8 nM, 16.4 nM, 8.2 nM, 4.1 nM, 2.05 nM, 1.0259 nM, 0.51297 nM) of anti-ST2 antibody were then passed through the chip surface. The reaction signal was detected in real time using a Biacore instrument (Cytiva, Biacore T200) to obtain binding and dissociation curves. After each cycle of dissociation, the biochip was regenerated using a regeneration solution for the next capture cycle, repeating the process to determine the affinity of different antibodies for ST2. Finally, the data obtained were analyzed using GEBIA evaluation software at a 1:1 (Langmuir) ratio with the model to determine the association rate constant ka (kon) and the dissociation rate constant kd (koff). The dissociation constant KD was calculated using KD = kd / ka. Affinity data of the anti-ST2 antibody against human ST2 antigen (hST2-ECD-mFc) and cynomolgus monkey ST2 antigen (Cyno-ST2-his, Sino biologica1, 90915-C08H) were measured. The affinity results of the anti-ST2 chimeric antibody with ST2 are shown in Table 5.

[0166] Table 5. Affinity of anti-ST2 chimeric antibody to ST2

[0167] Example 5: ELISA-based Binding Analysis of Anti-ST2 Antibodies. Using ELISA, hST2-ECD-mFc (for chimeric and humanized antibody detection), hST2-ECD-hFc protein (for hybridoma antibody detection), and Cyno-ST2-his (Sino Biologica 1, 90915-C08H) were used as antigens for ST2 binding screening and analysis. 2 μg / mL of hST2-ECD-mFc or Cyno-ST2-his antigen was coated at 100 μL / well in a high-adsorption 96-well plate (Costar, 9018) and incubated overnight at 4°C. After thoroughly washing away unadsorbed antigen, non-specific binding sites were blocked using blocking buffer (PBS containing 2% bovine serum albumin). After washing the plates three times with wash buffer (PBS containing 0.05% (v / v) Tween 20), add 100 μL / well of anti-ST2 antibody (initial concentration 10 nM, serially diluted 3-fold to eight concentrations, i.e., approximately 3.333 nM, 1.111 nM, 0.370 nM, 0.123 nM, 0.041 nM, 0.014 nM, 0.005 nM, 0.002 nM, etc.) and incubate at room temperature for 1 hour. After washing the plates with wash buffer, add secondary antibody conjugated with horseradish peroxidase (HRP) and incubate for another 60 minutes. After washing the plates with wash buffer, add 100 μL / well of substrate TMB solution (Thermo, 00-4201-56) and incubate the plates at room temperature for 2 minutes. Add 100 μL / well of stop solution (2N H2SO4) to stop the reaction. Colorimetric signals were generated and read at 450 nm using a microplate reader (PE, Envision). Data were analyzed using a GraphPad Prism5, and EC50 was calculated. The EC50 values ​​of the anti-ST2 chimeric antibody binding to hST2 and Cyno-ST2 are shown in Table 6 and Figures 1-2.

[0168] Table 6. EC50 values ​​of anti-ST2 chimeric antibodies binding to hST2 and Cyno-ST2 in ELISA assays.

[0169] Example 6: Blocking Assay of Anti-ST2 Antibody Based on ELISA. This study used an ELISA method to detect whether anti-ST2 antibody could block the binding of hIL33 to hST2. 2 μg / mL hST2-ECD-hFc was coated at 100 μL / well in a high-adsorption 96-well plate and incubated overnight at 4°C. After thoroughly washing away unadsorbed antigen, non-specific binding sites were blocked using blocking buffer (PBST containing 1% bovine serum albumin). The plates were washed three times with washing buffer (PBST containing 0.05% (v / v) Tween 20). Then, 100 μL / well of anti-ST2 antibody (initial concentration 66.67 nM, serially diluted 2-fold to 8 concentrations) was added, and the plates were incubated at 37°C for 1 hour. After washing the plates three times with washing buffer, 100 μL of biotin-labeled hIL33 Avitag (concentration 0.1 μg / mL) was added to each well, and the plates were incubated at 37°C for 1 hour. After washing the plates three times with wash buffer, add 100 μL / well of 1:1000 diluted secondary antibody Avidin HRP (Jacksonimmunoresearch, 016-030-084) and incubate at room temperature for 1 hour. After washing the plates again with wash buffer, add 100 μL / well of substrate TMB solution (Thermo, 00-4201-56) and incubate the plates at room temperature for 3 minutes. Stop the reaction by adding 50 μL of stop solution (2N H2SO4). A colorimetric signal was generated and read at 450 nm using a plate reader (PE, Envision). Analyze the data using a GraphPad Prism5 and calculate the IC50 value. The IC50 values ​​for the anti-ST2 chimeric antibody blocking IL33 / ST2 binding are shown in Table 7 and Figure 3.

[0170] Table 7. IC50 of anti-ST2 chimeric antibodies blocking IL33 / ST2 binding in ELISA assays

[0171] Example 7: Cell-based binding assay of anti-ST2 antibody. Based on the FACS method, the binding ability of anti-ST2 antibody to the HEK293T cell line overexpressing hST2 (SEQ ID NO: 1) (HEK293T-hST2-NFκB-Luciferase) and the HEK293T cell line overexpressing cyno-ST2 (SEQ ID NO: 10) (HEK293T-Cyno-ST2-NFκB-Luciferase) was analyzed by binding experiments. Using a lentiviral transfection system, the pLenti6.3-hST2 and pLenti6.3-NFκB-luciferasae plasmids were transfected into HEK293 cells to construct the cell line HEK293T-hST2-NFκB-Luciferase; the pLenti6.3-cynoST2 and pLenti6.3-NFκB-luciferasae plasmids were transfected into HEK293T cells to construct the cell line HEK293T-Cyno-ST2-NFκB-Luciferase.

[0172] 3×10 5 HEK293T-hST2-NFκB-Luciferase or HEK293T-Cyno-ST2-NFκB-Luciferase cells were added to 96-well culture plates. Serially diluted anti-ST2 antibody (in the HEK293T-hST2-NFκB-Luciferase cell binding assay, the initial concentration of anti-ST2 antibody was 667 nM, with 8 concentrations serially diluted 4-fold; in the HEK293T-Cyno-ST2-NFκB-Luciferase binding assay, the initial concentration of anti-ST2 antibody was 400 nM, with 8 concentrations serially diluted 4-fold) was added to the cell suspension. After incubation at room temperature for 60 minutes, the cells were washed three times with PBS, and 100 μL of PE goat-anti-human-IgG secondary antibody (Jackson Immunoresearch, 109-116-170) diluted 1:200 was added to each well, and the plates were incubated at room temperature for 30 minutes. Cells were washed three times with PBS and resuspended in 100 μL PBS. Fluorescence signals were then analyzed using a flow cytometer (BD, Accuri C6). The binding affinity of the anti-ST2 antibody to hST2 and cyno-ST2 on the cell line surface was measured by mean fluorescence intensity (MFI). Data were analyzed using a GraphPad Prism5, and EC50 values ​​were calculated. The EC50 values ​​of the anti-ST2 chimeric antibody binding to hST2 and Cyno-ST2 at the cellular level are shown in Table 8 and Figures 7-8.

[0173] Table 8. EC50 values ​​of anti-ST2 chimeric antibodies binding to hST2 and Cyno-ST2 at the cellular level.

[0174] NA: Detection line not reached. Example 8: Analysis of the interaction between anti-ST2 antibody and IL-1RAcP protein in an overexpressing hST2 cell line (HEK293T-hST2-NFκB-Luciferase). HEK293T cells naturally overexpress IL-1RAcP protein. Based on this, a lentiviral transfection system was used to transfect pLenti6.3-hST2 and pLenti6.3-NFκB-Luciferasae plasmids into HEK293 cells, constructing a stable transfected cell line HEK293T-hST2-NFκB-Luciferase. IL-33 binds to ST2, recruiting IL-1RAcP, which activates the downstream NFκB signaling pathway, thereby initiating Luciferase expression. When anti-ST2 antibody is present in the system, it blocks the binding of IL-33 to ST2, thereby blocking Luciferase expression. The blocking activity of the anti-ST2 antibody was evaluated by detecting changes in the fluorescence intensity of the reaction substrate.

[0175] HEK293T-hST2-NFκB-Luciferase cells in good exponential growth phase were diluted to 2.5 × 10⁻⁶. 5 20 μL of anti-ST2 antibody (initial concentration 667 nM, serially diluted 4-fold) was added to each well of a 384-well plate (Thermo, 262360); then 15 μL of serially diluted anti-ST2 antibody (initial concentration 667 nM, serially diluted 4-fold) was added to each well, and the plate was incubated at 37°C for 30 minutes; after incubation, 15 μL of hIL33 protein (final concentration 50 pM) was added to each well; the mixture was mixed, and the plate was incubated at 37°C for 5 hours. Then 50 μL of ONE-Glo™ Luciferase Assay (Promega, E6120) was added to each well, and the plate was incubated in the dark for 2–5 minutes. The chemiluminescence signal was read using a microplate reader (PE, Envision). Data were analyzed using a GraphPad Prism5, and the IC50 value was calculated. The IC50 values ​​of the anti-ST2 chimeric antibody blocking the IL33 / ST2 signaling at the cellular level are shown in Table 9 and Figure 9.

[0176] Table 9. IC50 values ​​of anti-ST2 chimeric antibodies in blocking IL33 / ST2 signaling at the cellular level.

[0177] "ND" indicates untested. Example 9: Analysis of the interaction between anti-ST2 antibody and naturally expressed hST2 cells (KU812-NFκB-Luciferasae). KU812 cells naturally express ST2 and IL1RAcP proteins. pLenti6.3-NFκB-Luciferasae was transfected into KU812 cells using a lentiviral transfection system to construct a stable transfected cell line KU812-NFκB-Luciferasae. The experimental principle is described in Example 8. KU812-NFκB-Luciferasae cells in good exponential growth phase were diluted to 2.5 × 10⁻⁶. 5 20 μL of anti-ST2 antibody (initial concentration 667 nM, serially diluted 4-fold) was added to each well of a 384-well plate (Thermo, 262360). Then, 15 μL of serially diluted anti-ST2 antibody (initial concentration 667 nM, serially diluted 4-fold) was added to each well, and the plate was incubated at 37°C for 30 minutes. After incubation, 15 μL of hIL33 protein (final concentration 200 pM) was added to each well; the mixture was mixed, and the plate was incubated at 37°C for 5 hours. Then, 50 μL of ONE-Glo™ Luciferase Assay (Promega, E6120) was added to each well, and the plate was incubated in the dark for 2–5 minutes. The chemiluminescence signal was read using a microplate reader (PE, Envision). Data were analyzed using a GraphPad Prism5, and IC50 values ​​were calculated.

[0178] Example 10: Anti-ST2 antibody blocks IL33 and IL2 co-stimulation of CD4 + Analysis of T cell interactions under co-stimulation of IL33 and IL2: IL33 and CD4 + ST2 binding on the surface of T cells activates downstream signaling pathways, inducing the secretion of the cytokine IL-5. When anti-ST2 antibodies are present in the system, they can block the binding of IL-33 and CD4+. + ST2 binding on T cells inhibits IL-5 secretion. IL-5 levels are measured to analyze the effect of anti-ST2 antibodies blocking the co-stimulation of CD4 by IL-33 and IL-2. + The ability of T cells to interact.

[0179] CD4+ T cells were sorted from human PBMCs using a human CD4+ T cell sorting kit (stemcell, 17952). + T cells, at 2.5 × 10 560 μL of cells / well were seeded into 96-well U-bottom cell culture plates. Then, 15 μL of diluted anti-ST2 antibody (initial concentration 53 nM, serially diluted 3-fold) was added to each well, and the plates were incubated at 37°C for 30 minutes. After incubation, 15 μL of hIL33 protein (final concentration 4 ng / mL) and 15 μL of IL2 (final concentration 10 ng / mL) (PrimeGene, GMP-101-02) were added to each well, mixed, and incubated at 37°C for 48 hours. The supernatant was collected after incubation, and the IL5 content in the supernatant was measured according to the instructions of the IL5 assay kit (R&D, DY205). OD450 values ​​were read using a microplate reader (PE, Envision). Data were analyzed using a GraphPad Prism5, and IC50 values ​​were calculated. The anti-ST2 chimeric antibody was detected on CD4... + The IC50 results for blocking IL5 secretion induced by IL33 / ST2 on T cells are shown in Table 10.

[0180] Table 10 Anti-ST2 chimeric antibodies in CD4 + IC50 on T cells that blocks IL33 / ST2-induced IL5 secretion

[0181] "ND" indicates untested. Example 11: Humanization of anti-human ST2 hybridoma monoclonal antibody. Humanization design was performed using 31C8 (heavy chain variable region sequence SEQ ID NO: 39; light chain variable region sequence SEQ ID NO: 40). Using an established CDR transplantation method, human antibody backbone regions suitable for mouse antibody humanization were selected. Human germline antibodies or their subtypes with the highest sequence identity (i.e., sequence similarity) to the variable region amino acids of the mouse antibody were screened. The heavy chain and light chain variable region CDRs of the mouse antibody were inserted into the screened backbone regions, and residues in the backbone regions were mutated. Some CDRs were further mutated to improve antibody properties, such as improving physicochemical properties or drug-likeness. Humanized antibodies hz31C8-1.1 and hz31C8-1.2 were obtained. The amino acid sequences of the heavy chain variable region and the light chain variable region of hz31C8-1.1 are SEQ ID NO: 71 and SEQ ID NO: 79, respectively; the DNA sequences encoding the heavy chain variable region and the light chain variable region of humanized antibody hz31C8-1.1 are SEQ ID NO: 72 and SEQ ID NO: 80, respectively; the amino acid sequences of the heavy chain variable region and the light chain variable region of humanized antibody hz31C8-1.2 are SEQ ID NO: 73 and SEQ ID NO: 79, respectively; the DNA sequences encoding the heavy chain variable region and the light chain variable region of humanized antibody hz31C8-1.2 are SEQ ID NO: 74 and SEQ ID NO: 80, respectively.

[0182] Humanized light chains were constructed by ligating the humanized VL region gene fragment to the human κ chain constant region via double enzyme digestion, and humanized heavy chains were constructed by ligating the humanized VH region gene fragment to the human IgG2 constant region via double enzyme digestion. The humanized heavy and light chains corresponding to each antibody were transfected into expression vectors, and protein expression was performed using the ExpiCHO expression system. The humanized antibodies in the cell culture supernatant were then purified using a Protein A column.

[0183] Example 12: Biological Function Analysis of Humanized Anti-ST2 Antibody The affinity, binding, blocking, and cellular function analyses of humanized antibodies hz31C8-1.1 (heavy chain sequence SEQ ID NO: 75, light chain sequence SEQ ID NO: 81) and hz31C8-1.2 (heavy chain sequence SEQ ID NO: 77, light chain sequence SEQ ID NO: 81) were performed according to the methods described in Examples 4, 5, 6, 7, 8, 9, and 10. In this disclosure, "hz" and "xi" represent humanized antibody and chimeric antibody, respectively. For example, "hz31C8-1.1" and "hz31C8-1.2" represent humanized 31C8-1.1 and 31C8-1.2 antibodies, and "xi31C8" represents a chimeric 31C8 antibody.

[0184] The affinity results of anti-ST2 humanized antibody for ST2 are shown in Table 11; the EC50 of anti-ST2 humanized antibody binding to hST2 and Cyno-ST2 in ELISA experiments are shown in Table 12 and Figures 4-5; the IC50 of anti-ST2 humanized antibody blocking IL33 / ST2 binding in ELISA experiments are shown in Table 13 and Figure 6; the EC50 of anti-ST2 humanized antibody binding to hST2 at the cellular level is shown in Table 14 and Figure 10; the IC50 of anti-ST2 humanized antibody blocking IL33 / ST2 signaling at the cellular level is shown in Table 15 and Figures 11-12; the affinity of anti-ST2 humanized antibody for CD4+ + The IC50 values ​​for blocking IL33 / ST2-induced IL5 secretion on T cells are shown in Table 16 and Figure 13.

[0185] Table 11 Affinity of anti-ST2 humanized antibody to ST2

[0186] Table 12 EC50 values ​​of anti-ST2 humanized antibodies binding to hST2 and Cyno-ST2 in ELISA assays

[0187] Table 13 IC50 of anti-ST2 humanized antibodies blocking IL33 / ST2 binding in ELISA assays

[0188] Table 14 EC50 of anti-ST2 humanized antibodies binding to hST2 at the cellular level

[0189] Table 15 IC50 of anti-ST2 humanized antibodies blocking IL33 / ST2 signaling at the cellular level

[0190] Table 16 Anti-ST2 humanized antibodies in CD4 + IC50 on T cells that blocks IL33 / ST2-induced IL5 secretion

[0191] Example 13: Stability Analysis of Humanized Anti-ST2 Antibodies. The thermostability of different antibodies was detected using DSC (Differential Scanning Calorimetry). Samples were dissolved in PBS buffer (pH 7.4), and the results were analyzed using a MicroCal VP-Capillary DSC (Malvern Panalytical). The results are shown in Table 17. The humanized anti-ST2 antibodies hz31C8-1.1 and hz31C8-1.2 disclosed in this paper both exhibited good thermostability, which was superior to the control anti-ST2 antibodies RG6149 and GSK3772847.

[0192] Table 17 Thermostability of Antibodies Detected by DSC

[0193] The periodic stability of the antibodies under certain concentration conditions was detected by SEC-HPLC. Exemplary conditions included controlling the antibody concentration at approximately 1 mg / mL and comparing the stability after storage at 40°C for 2 weeks in PBS buffer (pH 7.2). LC-20ADXR / DGU (Shimadzu) was used for detection. The results are shown in Table 18 below. Both the humanized anti-ST2 antibodies hz31C8-1.1 and hz31C8-1.2 disclosed in this paper exhibited good stability.

[0194] Table 18. SEC-HPLC Detection of Antibody Periodic Stability

[0195] Anti-ST2 antibody was diluted with PBS buffer to a concentration of 1.5 mg / mL. After treatment at 72°C for 5 minutes, the binding activity of the sample to the antigen was detected by ELISA. The ELISA assay procedure was as follows: 2 μg / mL hST2-ECD-mFc was used as the antigen, and 100 μL / well was coated onto a 96-well plate and incubated overnight at 4°C. After thoroughly washing away unadsorbed antigen, non-specific binding sites were blocked with blocking buffer (PBST containing 1% bovine serum albumin). The plates were washed three times with washing buffer (PBST containing 0.05% (v / v) Tween 20). 100 μL / well of anti-ST2 antibody (initial concentration 1.5 μg / mL, serially diluted 3-fold to 8 concentrations) was added, and the plates were incubated at 37°C for 1 hour. After washing the plates with washing buffer, anti-Human IgG Fcγ secondary antibody (Jackson ImmunoResearch, 109-035-008) was added, and the plates were incubated at 37°C for 1 hour. After washing the plates with washing buffer, 100 μL / well of substrate TMB solution (Thermo, 00-4201-56) was added for color development. The reaction was terminated with stop solution (2N H2SO4) after incubation at room temperature for 5 minutes. Signals were read at 450 nm using a plate reader (PE, Envision), and the data were analyzed using a GraphPad Prism5 to calculate EC50 values. The results are shown in Table 19 below. Both the humanized anti-ST2 antibodies hz31C8-1.1 and hz31C8-1.2 disclosed in this paper exhibit good thermal stability.

[0196] Table 19 ELISA Detection of Antibody Binding Activity After Heating

[0197] Example 14: Cross-reactivity of humanized anti-ST2 antibody with IL1R family receptors. ST2 belongs to the IL1R receptor family. The cross-reactivity of anti-ST2 antibody with IL1R family receptors was detected by ELISA. The IL1R family receptors include IL1R1 / 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 above-mentioned protein was used as the antigen (2 μg / mL) for ELISA detection. The specific ELISA detection procedure is described in Example 13. No binding means that the antibody does not bind to the antigen even at its highest concentration; weak binding means that the antibody binds weakly to the antigen at its highest concentration but not at low concentrations; strong binding means that the binding data can fit a very good binding curve.

[0198] Table 20 ELISA method for detecting cross-reactivity between humanized ST2 antibody and IL1R family receptors

[0199] (-: not bound; +: weakly bound; ++++: strongly bound) Example 15: Pharmacokinetic Evaluation of Humanized Anti-ST2 Antibody. Eighteen Balb / c mice (n=6 per group) were used in the experiment. They were conditioned for 12 / 12 hours, allowed free access to food and water, and purchased from the Institute of Model Animals, Nanjing University. On the day of the experiment, mice were intravenously injected with the drug at a dose of 10 mg / kg, with a sample concentration of 1 mg / mL. Blood samples were collected from the orbital sinus at 0 min (or one day or more in advance) and 30 min, 8 h, 24 h, 2 d (48 h), 4 d (96 h), 7 d, and 14 d after administration. Serum was collected, and the antibody concentration in the serum was detected by ELISA. The detection procedure was as follows: 2 μg / mL ST2-His (sinobiological, 10105-H08H) was used as the antigen, and 100 μL / well was coated into a 96-well plate and incubated overnight at 4°C. After thoroughly washing away unadsorbed antigen, non-specific binding sites were blocked using blocking buffer (PBST containing 1% bovine serum albumin). The plates were washed three times with wash buffer (PBST containing 0.05% (v / v) Tween 20), and 100 μL of the test serum was added. The plates were incubated at 37°C for 1 h. After washing the plates with wash buffer, anti-Mouse IgG Fcγ secondary antibody was added, and the plates were incubated at 37°C for 1 h. After washing the plates with wash buffer, 100 μL / well of substrate TMB solution (Thermo, 00-4201-56) was added for color development. The reaction was terminated with stop solution (2N H2SO4) after incubation at room temperature for 5 minutes. The signal was read at 450 nm using a plate reader (PE, Envision), and the data were analyzed using a GraphPad Prism5. Referring to Table 21, the PK analysis results show that the humanized anti-ST2 antibodies hz31C8-1.1 and hz31C8-1.2 disclosed herein have half-lives of approximately 9.7 days and 7.33 days in mice, respectively, which are superior to the control group anti-ST2 antibody RG6149.

[0200] Table 21. T1 / 2 of humanized ST2 antibody in mice

[0201] Although this disclosure has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, such modifications or improvements made without departing from the spirit of this disclosure are all within the scope of protection claimed by this disclosure.

Claims

1. An isolated ST2 antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising: (i) heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, wherein, (1) Heavy chain CDR1 contains the sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% identity with it; heavy chain CDR2 contains the sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 80% identity with it; and heavy chain CDR3 contains the sequence shown in SEQ ID NO: 20 or an amino acid sequence having at least 80% identity with it; (2) Heavy chain CDR1 contains the sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% identity with it; heavy chain CDR2 contains the sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 80% identity with it; and heavy chain CDR3 contains the sequence shown in SEQ ID NO: 21 or an amino acid sequence having at least 80% identity with it; (3) Heavy chain CDR1 contains the sequence shown in SEQ ID NO: 12 or an amino acid sequence having at least 80% identity with it; heavy chain CDR2 contains the sequence shown in SEQ ID NO: 17 or an amino acid sequence having at least 80% identity with it; and heavy chain CDR3 contains the sequence shown in SEQ ID NO: 10 or an amino acid sequence having at least 80% identity with it. (3) The sequence shown in SEQ ID NO: 22 or an amino acid sequence having at least 80% 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% identity with it, heavy chain CDR2 contains the sequence shown in SEQ ID NO: 18 or an amino acid sequence having at least 80% identity with it, and heavy chain CDR3 contains the sequence shown in SEQ ID NO: 23 or an amino acid sequence having at least 80% identity with it; (5) Heavy chain CDR1 contains the sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 80% identity with it, heavy chain CDR2 contains the sequence shown in SEQ ID NO: 19 or an amino acid sequence having at least 80% identity with it, and heavy chain CDR3 contains the sequence shown in SEQ ID NO: 22 or an amino acid sequence having at least 80% identity with it; or (6) Heavy chain CDR1 contains the sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% identity with it, heavy chain CDR2 contains the sequence shown in SEQ ID NO: 22 or an amino acid sequence having at least 80% identity with it, and heavy chain CDR2 contains the sequence shown in SEQ ID NO: 22 or an amino acid sequence having at least 80% identity with it. The sequence shown in SEQ ID NO: 24 or an amino acid sequence having at least 80% identity with it, and the heavy chain CDR3 contains the sequence shown in SEQ ID NO: 24 or an amino acid sequence having at least 80% identity with it;(ii) Light chain CDR1, light chain CDR2, and light chain CDR3, wherein, (1) light chain CDR1 comprises the sequence shown in SEQ ID NO: 25 or an amino acid sequence having at least 80% identity with it, light chain CDR2 comprises the sequence shown in SEQ ID NO: 29 or an amino acid sequence having at least 80% identity with it, and light chain CDR3 comprises the sequence shown in SEQ ID NO: 32 or an amino acid sequence having at least 80% identity with it; (2) light chain CDR1 comprises the sequence shown in SEQ ID NO: 26 or an amino acid sequence having at least 80% identity with it, light chain CDR2 comprises the sequence shown in SEQ ID NO: 30 or an amino acid sequence having at least 80% identity with it, and light chain CDR3 comprises the sequence shown in SEQ ID NO: 34 or an amino acid sequence having at least 80% identity with it; (3) light chain CDR1 comprises the sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 80% identity with it, light chain CDR2 comprises the sequence shown in SEQ ID NO: 29 or an amino acid sequence having at least 80% identity with it, and light chain CDR3 comprises the sequence shown in SEQ ID NO: 29 or an amino acid sequence having at least 80% identity with it. The sequence shown in SEQ ID NO: 30 or an amino acid sequence having at least 80% identity with it and the light chain CDR3 contain the sequence shown in SEQ ID NO: 34 or an amino acid sequence having at least 80% identity with it; or (4) the light chain CDR1 contains the sequence shown in SEQ ID NO: 28 or an amino acid sequence having at least 80% identity with it, the light chain CDR2 contains the sequence shown in SEQ ID NO: 31 or an amino acid sequence having at least 80% identity with it, and the light chain CDR3 contains the sequence shown in SEQ ID NO: 33 or an amino acid sequence having at least 80% identity with it; or (iii) the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 as shown in (i), and the light chain CDR1, light chain CDR2 and light chain CDR3 as shown in (ii); wherein the amino acid sequence of SEQ ID NO: 17 is AIDPETGDTVYX1X2KFX3G, X1=N or A, X2=Q, E or K, X3=K or Q; SEQ ID The amino acid sequence of NO:30 is QX4SNLAS, where X4 = M or L.

2. The antibody or its antigen-binding fragment according to claim 1, wherein, The amino acid sequence of SEQ ID NO: 17 is AIDPETGDTVYX1X2KFX3G, where (i) X1=N or A, X2=Q, X3=K or Q; (ii) X1=N or A, X2=E, X3=K or Q; or (iii) X1=N or A, X2=K, X3=K or Q.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, wherein, (1) Heavy chain CDR1 contains the sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% identity with it; heavy chain CDR2 contains the sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 80% identity with it; heavy chain CDR3 contains the sequence shown in SEQ ID NO: 20 or an amino acid sequence having at least 80% identity with it; light chain CDR1 contains the sequence shown in SEQ ID NO: 25 or an amino acid sequence having at least 80% identity with it; light chain CDR2 contains the sequence shown in SEQ ID NO: 29 or an amino acid sequence having at least 80% identity with it; and light chain CDR3 contains the sequence shown in SEQ ID NO: 32 or an amino acid sequence having at least 80% identity with it; (2) Heavy chain CDR1 contains the sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% identity with it; heavy chain CDR2 contains the sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 80% identity with it; and heavy chain CDR3 contains the sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% identity with it; and light ...2 contains the sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% identity with it; and light chain CDR3 contains the sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% identity with it; and light chain CDR3 contains the sequence shown in SEQ ID NO: 11 or an amino (2) The sequence shown in SEQ ID NO: 21 or an amino acid sequence having at least 80% identity with it, light chain CDR1 contains the sequence shown in SEQ ID NO: 26 or an amino acid sequence having at least 80% identity with it, light chain CDR2 contains the sequence shown in SEQ ID NO: 30 or an amino acid sequence having at least 80% identity with it, and light chain CDR3 contains the sequence shown in SEQ ID NO: 34 or an amino acid sequence having at least 80% identity with it; (3) heavy chain CDR1 contains the sequence shown in SEQ ID NO: 12 or an amino acid sequence having at least 80% identity with it, heavy chain CDR2 contains the sequence shown in SEQ ID NO: 17 or an amino acid sequence having at least 80% identity with it, heavy chain CDR3 contains the sequence shown in SEQ ID NO: 22 or an amino acid sequence having at least 80% identity with it, light chain CDR1 contains the sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 80% identity with it, light chain CDR2 contains the sequence shown in SEQ ID NO: 24 or an amino acid sequence having at least 80% identity with it, and light chain CDR3 contains the sequence shown in SEQ ID NO: 22 or an amino acid sequence having at least 80% identity with it, light chain CDR1 contains the sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 80% identity with it, and light chain CDR2 contains the sequence shown in SEQ ID NO: 24 or an amino acid sequence having at least 80% identity with it. The sequence shown in SEQ ID NO: 30 or an amino acid sequence having at least 80% identity with it and the light chain CDR3 contain the sequence shown in SEQ ID NO: 34 or an amino acid sequence having at least 80% 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% identity with it; heavy chain CDR2 contains the sequence shown in SEQ ID NO: 18 or an amino acid sequence having at least 80% identity with it; heavy chain CDR3 contains the sequence shown in SEQ ID NO: 23 or an amino acid sequence having at least 80% identity with it; light chain CDR1 contains the sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 80% identity with it; light chain CDR2 contains the sequence shown in SEQ ID NO: 30 or an amino acid sequence having at least 80% identity with it; and light chain CDR3 contains the sequence shown in SEQ ID NO: 34 or an amino acid sequence having at least 80% identity with it; (5) Heavy chain CDR1 contains the sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 80% identity with it; heavy chain CDR2 contains the sequence shown in SEQ ID NO: 19 or an amino acid sequence having at least 80% identity with it; and heavy chain CDR3 contains the sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 80% identity with it; and light chain CDR3 contains the sequence shown in SEQ ID NO: 19 or an amino acid sequence having at least 80% identity with it. The sequence shown in SEQ ID NO: 22 or an amino acid sequence having at least 80% identity with it, light chain CDR1 contains the sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 80% identity with it, light chain CDR2 contains the sequence shown in SEQ ID NO: 30 or an amino acid sequence having at least 80% identity with it, and light chain CDR3 contains the sequence shown in SEQ ID NO: 34 or an amino acid sequence having at least 80% identity with it; or (6) heavy chain CDR1 contains the sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% identity with it, heavy chain CDR2 contains the sequence shown in SEQ ID NO: 18 or an amino acid sequence having at least 80% identity with it, heavy chain CDR3 contains the sequence shown in SEQ ID NO: 24 or an amino acid sequence having at least 80% identity with it, light chain CDR1 contains the sequence shown in SEQ ID NO: 28 or an amino acid sequence having at least 80% identity with it, light chain CDR2 contains the sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% identity with it, and light chain CDR2 contains the sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% identity with it. The sequence shown in SEQ ID NO: 31 or an amino acid sequence having at least 80% identity with it and the light chain CDR3 contain the sequence shown in SEQ ID NO: 33 or an amino acid sequence having at least 80% identity with it; wherein, the amino acid sequence of SEQ ID NO: 17 is AIDPETGDTVYX1X2KFX3G, wherein, (i) X1=N or A, X2=Q, X3=K or Q; (ii) X1=N or A, X2=E, X3=K or Q; or (iii) X1=N or A, X2=K, X3=K or Q;Furthermore, the amino acid sequence of SEQ ID NO: 30 is QX4SNLAS, where X4 = M or L.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof is chimeric or humanized.

5. An isolated ST2 antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising: (i) a heavy chain variable region comprising a sequence as shown in SEQ ID NOs: 35, 37, 39, 41, 43, 45, 71 or 73, or an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NOs: 35, 37, 39, 41, 43, 45, 71 or 73; (ii) a light chain variable region comprising a sequence as shown in SEQ ID NOs: 36, 38, 40, 42, 44, 46 or 79, or an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NOs: 36, 38, 40, 42, 44, 46 or 79; or (iii) a heavy chain variable region as shown in (i) and a light chain variable region as shown in (ii).

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein, (1) The heavy chain variable region comprises the sequence shown in SEQ ID NO: 35 or an amino acid sequence having at least 80% identity with it, and the light chain variable region comprises the sequence shown in SEQ ID NO: 36 or an amino acid sequence having at least 80% identity with it; (2) The heavy chain variable region comprises the sequence shown in SEQ ID NO: 37 or an amino acid sequence having at least 80% identity with it, and the light chain variable region comprises the sequence shown in SEQ ID NO: 38 or an amino acid sequence having at least 80% identity with it; (3) The heavy chain variable region comprises the sequence shown in SEQ ID NO: 39 or an amino acid sequence having at least 80% identity with it, and the light chain variable region comprises the sequence shown in SEQ ID NO: 40 or an amino acid sequence having at least 80% identity with it; (4) The heavy chain variable region comprises the sequence shown in SEQ ID NO: 41 or an amino acid sequence having at least 80% identity with it, and the light chain variable region comprises the sequence shown in SEQ ID NO: 42 or an amino acid sequence having at least 80% identity with it; (5) The heavy chain variable region comprises the sequence shown in SEQ ID NO: 36 or an amino acid sequence having at least 80% identity with it. (6) The heavy chain variable region contains the sequence shown in SEQ ID NO: 43 or an amino acid sequence having at least 80% identity with it, and the light chain variable region contains the sequence shown in SEQ ID NO: 44 or an amino acid sequence having at least 80% identity with it; (7) The heavy chain variable region contains the sequence shown in SEQ ID NO: 71 or an amino acid sequence having at least 80% identity with it, and the light chain variable region contains the sequence shown in SEQ ID NO: 79 or an amino acid sequence having at least 80% identity with it; or (8) The heavy chain variable region contains the sequence shown in SEQ ID NO: 73 or an amino acid sequence having at least 80% identity with it, and the light chain variable region contains the sequence shown in SEQ ID NO: 79 or an amino acid sequence having at least 80% identity with it.

7. An isolated ST2 antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising: (i) a heavy chain comprising a sequence as shown in SEQ ID NOs: 47, 51, 55, 59, 63, 67, 75 or 77, or an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NOs: 47, 51, 55, 59, 63, 67, 75 or 77; (ii) a light chain comprising a sequence as shown in SEQ ID NOs: 49, 53, 57, 61, 65, 69 or 81, or an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NOs: 49, 53, 57, 61, 65, 69 or 81; or (iii) a heavy chain as shown in (i) and a light chain as shown in (ii).

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein the antibody or antigen-binding fragment comprises a heavy chain and a light chain, wherein, (1) The heavy chain comprises the sequence shown in SEQ ID NO: 47 or an amino acid sequence having at least 80% identity with it, and the light chain comprises the sequence shown in SEQ ID NO: 49 or an amino acid sequence having at least 80% identity with it; (2) The heavy chain comprises the sequence shown in SEQ ID NO: 51 or an amino acid sequence having at least 80% identity with it, and the light chain comprises the sequence shown in SEQ ID NO: 53 or an amino acid sequence having at least 80% identity with it; (3) The heavy chain comprises the sequence shown in SEQ ID NO: 55 or an amino acid sequence having at least 80% identity with it, and the light chain comprises the sequence shown in SEQ ID NO: 57 or an amino acid sequence having at least 80% identity with it; (4) The heavy chain comprises the sequence shown in SEQ ID NO: 59 or an amino acid sequence having at least 80% identity with it, and the light chain comprises the sequence shown in SEQ ID NO: 61 or an amino acid sequence having at least 80% identity with it; (5) The heavy chain comprises the sequence shown in SEQ ID NO: 49 or an amino acid sequence having at least 80% identity with it. (6) The heavy chain contains the sequence shown in SEQ ID NO: 63 or an amino acid sequence having at least 80% identity with it, and the light chain contains the sequence shown in SEQ ID NO: 65 or an amino acid sequence having at least 80% identity with it; (7) The heavy chain contains the sequence shown in SEQ ID NO: 75 or an amino acid sequence having at least 80% identity with it, and the light chain contains the sequence shown in SEQ ID NO: 81 or an amino acid sequence having at least 80% identity with it; or (8) The heavy chain contains the sequence shown in SEQ ID NO: 77 or an amino acid sequence having at least 80% identity with it, and the light chain contains the sequence shown in SEQ ID NO: 81 or an amino acid sequence having at least 80% identity with it.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1-8, wherein the antibody or antigen-binding fragment thereof is selected from monoclonal antibodies, Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, dAb, isolated CDR regions, single-chain Fv molecules, or combinations thereof.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1-9, wherein the antibody or antigen-binding fragment thereof specifically binds to ST2 in humans or monkeys and blocks the binding of IL33 / ST2 and its signal transduction.

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