St2 antigen binding proteins

Abstract

Provided are ST2 antigen binding proteins, such as mouse, human, chimeric or humanized antibodies or antigen binding fragments thereof that specifically bind to ST2, nucleic acid molecules encoding the above 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 and antigen binding fragments thereof, including treating and preventing IL33 / ST2-mediated related diseases and disorders.

Description

[0001] REFERENCE TO RELATED APPLICATIONS

[0002] The present disclosure claims priority to Chinese Patent Application No. 202010397572.3, filed May 12, 2020, the contents of which are incorporated by reference in their entirety and for all purposes. TECHNICAL FIELD

[0003] The present disclosure relates to an isolated antibody, in particular, the present 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, host cells for producing the antibodies or antigen-binding fragments thereof. The present disclosure further provides polypeptide fusions, multi-specific molecules, viral vectors and pharmaceutical compositions comprising the antibodies or antigen-binding portions thereof, and diagnostic and therapeutic methods using the antibodies or antigen-binding fragments thereof. BACKGROUND

[0004] Interleukin 33 (IL33) is a member of the IL-1 cytokine family, expressed by tissue endothelial or epithelial cells, and acts as an “alarmin” when cells receive stimuli such as stress, infection and injury, and expresses in damaged cells to transmit antigenic stimulation signals from the outside to the Th2 pathway. The receptor of IL33 is composed of two proteins: IL-1 receptor associated protein (IL-1RL1, ST2) and IL-1 receptor accessory protein (IL-1RAP). The binding of IL33 to ST2 can induce signal transduction with the participation of IL-1RAP, but IL-1RAP cannot directly bind to IL33 or ST2 (Chackerian et al., (2007) J Immunol. 179:2551-2555).

[0005] ST2 belongs to the Toll / IL1 receptor family, and there are three subtypes: transmembrane type ST2L, soluble type sST2 and mutant type ST2V, of which ST2L mainly mediates intracellular signal response of IL33. ST2L is mainly expressed on the surface of Th2 cells, ILC2 cells, mast cells, 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, activates MyD88 / NFκB cell pathway, induces mast cells, Th2 cells, Treg cells and ILC2 cells to secrete IL-5, IL-6, IL-13, TNF, INF-γ and other pro-inflammatory factors and CCL17, CCL22, CXCL8 and other chemotactic factors, and induces inflammatory response. IL-33 is abnormally elevated in the expression in mucosal tissue inflammation, joint inflammation and chronic skin inflammation diseases, such as allergic rhinitis (Kamekura R et al., (2012) Clin Exp Allergy. 42:218-28), rheumatoid arthritis, ankylosing spondylitis, atopic dermatitis (Savinko T et al., (2012) J Invest Dermatol. 132: 1392-1400), psoriasis; and IL33 / ST2 signaling pathway disorder is closely related to asthma, chronic obstructive pulmonary disease (Hacker, Lambers et al., (2009) J Clin Lab Anal. 23:372-9, bronchitis, inflammatory bowel disease (Beltran CJ et al., (2010) Inflamm Bowel Dis. 16: 1097-107), systemic lupus erythematosus, liver fibrosis, systemic sclerosis and other immune-mediated diseases. Genetic analysis found that there are some single nucleotide polymorphism (SNP) sites in IL33 and ST2 genes, which are related to the number of basophils, and are also closely related to the occurrence of atopic dermatitis and asthma 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, inhibiting the IL33 / ST2 signaling pathway and the induced inflammatory response, become an important direction for the treatment of inflammation and immune-related diseases. SUMMARY

[0007] The present disclosure provides a ST2 antibody with high affinity, high specificity and high biological activity, which is suitable for treating IL33 / ST2 mediated related diseases.

[0008] The present disclosure provides an isolated antibody, for example, a mouse, human, chimeric or humanized monoclonal antibody or an antigen-binding fragment thereof that binds to ST2 (e.g., human ST2 and monkey ST2).

[0009] The antibodies or antigen-binding fragments thereof of the present disclosure have a variety of uses, including detecting ST2 protein, and treating and preventing IL33 / ST2-mediated related diseases and conditions, including asthma, allergic rhinitis, chronic obstructive pulmonary disease, eosinophilic bronchitis, eosinophilic esophagitis, atopic dermatitis, psoriasis, systemic lupus erythematosus, dermatitis herpetiformis, rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, pulmonary fibrosis, liver fibrosis, systemic sclerosis, sarcoidosis, graft versus host disease (GVHD), diabetes mellitus disease, cardiovascular disease, or a combination thereof.

[0010] In one aspect, the present disclosure provides an isolated antibody or antigen binding fragment thereof, comprising heavy chain CDRs comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, wherein (1) the heavy chain CDR1 comprises a sequence as set forth in SEQ ID NO: 11 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the heavy chain CDR2 comprises a sequence as set forth in SEQ ID NO: 15 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the heavy chain CDR3 comprises a sequence as set forth in SEQ ID NO: 20 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; or (2) the heavy chain CDR1 comprises a sequence as set forth in SEQ ID NO: 11 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the heavy chain CDR2 comprises a sequence as set forth in SEQ ID NO: 16 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the heavy chain CDR3 comprises a sequence as set forth in SEQ ID NO: 21 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.(3) heavy chain CDR1 comprises a sequence as set forth in SEQ ID NO: 12 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, heavy chain CDR2 comprises a sequence as set forth in SEQ ID NO: 17 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and heavy chain CDR3 comprises a sequence as set forth in SEQ ID NO: 22 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; (4) heavy chain CDR1 comprises a sequence as set forth in SEQ ID NO: 13 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, heavy chain CDR2 comprises a sequence as set forth in SEQ ID NO: 18 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and heavy chain CDR3 comprises a sequence as set forth in SEQ ID NO: 23 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;(5) the heavy chain CDR1 comprises a sequence as set forth in SEQ ID NO: 14 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the heavy chain CDR2 comprises a sequence as set forth in SEQ ID NO: 19 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto and the heavy chain CDR3 comprises a sequence as set forth in SEQ ID NO: 22 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; or (6) the heavy chain CDR1 comprises a sequence as set forth in SEQ ID NO: 11 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the heavy chain CDR2 comprises a sequence as set forth in SEQ ID NO: 18 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto and the heavy chain CDR3 comprises a sequence as set forth in SEQ ID NO: 24 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

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

[0012] In some embodiments, the isolated antibody or antigen-binding fragment thereof binds to ST2, wherein the amino acid sequence of SEQ ID NO: 17 is AIDPETGDTVYXiX2KFX3G, wherein Xi = N or A, X2= Q, X3= K or Q; Xi = N or A, X2= E, X3= K or Q; or Xi = N or A, X2= K, X3= K or Q.

[0013] In some embodiments, the isolated antibody or antigen-binding fragment thereof binds to ST2, wherein the amino acid sequence of SEQ ID NO: 17 is AIDPETGDTVYXiX2KFX3G, wherein Xi = N or A, X2= Q, X3= K or Q; Xi = N or A, X2= E, X3= K or Q; or Xi = N or A, X2= K, X3= K or Q.

[0014] In some embodiments, the isolated antibody or antigen-binding fragment thereof binds to ST2, wherein the amino acid sequence of SEQ ID NO: 17 is AIDPETGDTVYXiX2KFX3G, wherein Xi = N or A, X2= Q, X3= K or Q; Xi = N or A, X2= E, X3= K or Q; or Xi = N or A, X2= K, X3= K or Q.

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

[0016] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence comprising a sequence as set forth in SEQ ID NOs: 35, 37, 39, 41, 43, 45, 71, or 73; or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence as set forth in SEQ ID NOs: 35, 37, 39, 41, 43, 45, 71, or 73.

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

[0018] In some particular embodiments, the amino acid set forth in SEQ ID NO: 71 can be encoded by the nucleic acid set forth in SEQ ID NO: 72, and the amino acid set forth in SEQ ID NO: 73 can be encoded by the nucleic acid set forth in SEQ ID NO: 74.

[0019] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, wherein (1) the heavy chain CDR1 comprises a sequence as set forth in SEQ ID NO: 1 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the heavy chain CDR2 comprises a sequence as set forth in SEQ ID NO: 5 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the heavy chain CDR3 comprises a sequence as set forth in SEQ ID NO: 9 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; (2) the heavy chain CDR1 comprises a sequence as set forth in SEQ ID NO: 2 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the heavy chain CDR2 comprises a sequence as set forth in SEQ ID NO: 6 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the heavy chain CDR3 comprises a sequence as set forth in SEQ ID NO: 10 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; (3) the heavy chain CDR1 comprises a sequence as set forth in SEQ ID NO: 3 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the heavy chain CDR2 comprises a sequence as set forth in SEQ ID NO: 7 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the heavy chain CDR3 comprises a sequence as set forth in SEQ ID NO: 11 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; or (4) the heavy chain CDR1 comprises a sequence as set forth in SEQ ID NO: 4 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the heavy chain CDR2 comprises a sequence as set forth in SEQ ID NO: 8 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the heavy chain CDR3 comprises a sequence as set forth in SEQ ID NO: 12 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.(3) the light chain CDR1 comprises a sequence as set forth in SEQ ID NO: 27 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the light chain CDR2 comprises a sequence as set forth in SEQ ID NO: 30 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto and the light chain CDR3 comprises a sequence as set forth in SEQ ID NO: 34 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; or (4) the light chain CDR1 comprises a sequence as set forth in SEQ ID NO: 28 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the light chain CDR2 comprises a sequence as set forth in SEQ ID NO: 31 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto and the light chain CDR3 comprises a sequence as set forth in SEQ ID NO: 33 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

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

[0021] In some specific embodiments, the isolated antibody or antigen binding fragment thereof binds to ST2 and is a monoclonal antibody (e.g., a 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 embodiments, 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 antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein (1) the heavy chain CDR1 comprises a sequence as set forth in SEQ ID NO: 11 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the heavy chain CDR2 comprises a sequence as set forth in SEQ ID NO: 15 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the heavy chain CDR3 comprises a sequence as set forth in SEQ ID NO: 20 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the light chain CDR1 comprises a sequence as set forth in SEQ ID NO: 25 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the light chain CDR2 comprises a sequence as set forth in SEQ ID NO: 29 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain CDR3 comprises a sequence as set forth in SEQ ID NO: 32 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;(2) the heavy chain CDR1 comprises a sequence as set forth in SEQ ID NO: 11 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the heavy chain CDR2 comprises a sequence as set forth in SEQ ID NO: 16 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the heavy chain CDR3 comprises a sequence as set forth in SEQ ID NO: 21 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the light chain CDR1 comprises a sequence as set forth in SEQ ID NO: 26 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the light chain CDR2 comprises a sequence as set forth in SEQ ID NO: 30 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain CDR3 comprises a sequence as set forth in SEQ ID NO: 34 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;(3) the heavy chain CDR1 comprises a sequence as set forth in SEQ ID NO: 12 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the heavy chain CDR2 comprises a sequence as set forth in SEQ ID NO: 17 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the heavy chain CDR3 comprises a sequence as set forth in SEQ ID NO: 22 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the light chain CDR1 comprises a sequence as set forth in SEQ ID NO: 27 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the light chain CDR2 comprises a sequence as set forth in SEQ ID NO: 30 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain CDR3 comprises a sequence as set forth in SEQ ID NO: 34 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;(4) the heavy chain CDR1 comprises a sequence as set forth in SEQ ID NO: 13 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the heavy chain CDR2 comprises a sequence as set forth in SEQ ID NO: 18 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the heavy chain CDR3 comprises a sequence as set forth in SEQ ID NO: 23 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the light chain CDR1 comprises a sequence as set forth in SEQ ID NO: 27 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the light chain CDR2 comprises a sequence as set forth in SEQ ID NO: 30 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain CDR3 comprises a sequence as set forth in SEQ ID NO: 34 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;(5) the heavy chain CDR1 comprises a sequence as set forth in SEQ ID NO: 14 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the heavy chain CDR2 comprises a sequence as set forth in SEQ ID NO: 19 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the heavy chain CDR3 comprises a sequence as set forth in SEQ ID NO: 22 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the light chain CDR1 comprises a sequence as set forth in SEQ ID NO: 27 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the light chain CDR2 comprises a sequence as set forth in SEQ ID NO: 30 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain CDR3 comprises a sequence as set forth in SEQ ID NO: 34 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;(6) the heavy chain CDR1 comprises a sequence as set forth in SEQ ID NO: 11 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the heavy chain CDR2 comprises a sequence as set forth in SEQ ID NO: 18 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the heavy chain CDR3 comprises a sequence as set forth in SEQ ID NO: 24 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the light chain CDR1 comprises a sequence as set forth in SEQ ID NO: 28 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, the light chain CDR2 comprises a sequence as set forth in SEQ ID NO: 31 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain CDR3 comprises a sequence as set forth in SEQ ID NO: 33 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0026] In some specific embodiments, the isolated antibody or antigen binding fragment thereof 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 embodiments, the isolated antibody or antigen-binding fragment thereof binds to ST2; wherein the amino acid sequence of SEQ ID NO: 17 is AIDPETGDTVYXiX2KFX3G, 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 embodiments, the isolated antibody or antigen-binding fragment thereof binds to ST2; wherein the amino acid sequence of SEQ ID NO: 17 is AIDPETGDTVYXiX2KFX3G, 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 embodiments, the isolated antibody or antigen-binding fragment thereof binds to ST2; wherein the amino acid sequence of SEQ ID NO: 17 is AIDPETGDTVYXiX2KFX3G, X1= N, X2= Q, X3= K or Q; 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 embodiments, the isolated antibody or antigen-binding fragment thereof binds to ST2 and is a monoclonal antibody (e.g., a mouse, chimeric or humanized antibody).

[0031] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein (1) the heavy chain variable region comprises a sequence as set forth in SEQ ID NO: 35 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence as set forth in SEQ ID NO: 36 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; (2) the heavy chain variable region comprises a sequence as set forth in SEQ ID NO: 37 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence as set forth in SEQ ID NO: 38 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; (3) the heavy chain variable region comprises a sequence as set forth in SEQ ID NO: 39 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence as set forth in SEQ ID NO: 40 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.(4) the heavy chain variable region comprises a sequence set forth in SEQ ID NO: 41 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence set forth in SEQ ID NO: 42 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; (5) the heavy chain variable region comprises a sequence set forth in SEQ ID NO: 43 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence set forth in SEQ ID NO: 44 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; (6) the heavy chain variable region comprises a sequence set forth in SEQ ID NO: 45 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence set forth in SEQ ID NO: 46 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; (7) the heavy chain variable region comprises a sequence set forth in SEQ ID NO: 71 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence set forth in SEQ ID NO: 79 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;or (8) the heavy chain variable region comprises a sequence as set forth in SEQ ID NO: 73 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence as set forth in SEQ ID NO: 79 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

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

[0033] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence comprising a sequence as set forth in SEQ ID NOs: 47, 51, 55, 59, 63, 67, 75, or 77; or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence as set forth in SEQ ID NOs: 47, 51, 55, 59, 63, 67, 75, or 77.

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

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

[0036] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain whose amino acid sequence comprises a sequence as set forth in SEQ ID NOs: 48, 52, 56, 60, 64, 68, or 80; or an amino acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity compared to a sequence as set forth in SEQ ID NOs: 48, 52, 56, 60, 64, 68, or 80.

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

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

[0039] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein (1) the heavy chain comprises a sequence as set forth in SEQ ID NO: 47 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain comprises a sequence as set forth in SEQ ID NO: 49 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; (2) the heavy chain comprises a sequence as set forth in SEQ ID NO: 51 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain comprises a sequence as set forth in SEQ ID NO: 53 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; (3) the heavy chain comprises a sequence as set forth in SEQ ID NO: 55 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain comprises a sequence as set forth in SEQ ID NO: 57 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.(4) the heavy chain comprises a sequence set forth in SEQ ID NO: 59 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain comprises a sequence set forth in SEQ ID NO: 61 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; (5) the heavy chain comprises a sequence set forth in SEQ ID NO: 63 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain comprises a sequence set forth in SEQ ID NO: 65 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; (6) the heavy chain comprises a sequence set forth in SEQ ID NO: 67 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain comprises a sequence set forth in SEQ ID NO: 69 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; (7) the heavy chain comprises a sequence set forth in SEQ ID NO: 75 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain comprises a sequence set forth in SEQ ID NO: 81 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;or (8) the heavy chain comprises a sequence set forth in SEQ ID NO: 77 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain comprises a sequence set forth in SEQ ID NO: 81 or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

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

[0041] In some embodiments, the isolated antibody (e.g., a mouse, chimeric or humanized antibody) or antigen binding fragment thereof comprises a heavy chain comprising a heavy chain variable region and a heavy chain constant region, and a 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 above-described amino acid sequences, the heavy chain constant region has a human IgGl, IgG2 or IgG4 constant region, preferably a human IgG2 constant region; and the light chain constant region has a human kappa constant region or lambda constant region, wherein the antibody or antigen binding fragment thereof binds to ST2.

[0042] In some embodiments, the antibody of the present disclosure comprises or consists of two heavy chains and two light chains, which are linked to each other by disulfide bonds, wherein each heavy chain comprises the above-described heavy chain constant region, heavy chain variable region or CDR sequences, and each light chain comprises the above-described light chain constant region, light chain variable region or CDR sequences, wherein the C-terminus of the heavy chain variable region is linked to the N-terminus of the heavy chain constant region, and the C-terminus of the light chain variable region is linked to the N-terminus of the light chain constant region. The antibody of the present disclosure can be, for example, a full-length antibody of IgGl, IgG2 or IgG4 isotype. In another embodiment, the antibody of the present disclosure can be a single chain antibody (scFv), or an antibody fragment, such as a Fab, F(ab')2 fragment, Fd fragment, Fv fragment, dAb or isolated CDR region.

[0043] In one aspect, the present disclosure also provides polypeptide fusions comprising an antibody or antigen-binding fragment thereof of the present disclosure and other functional molecules, which can be peptides or proteins or non-proteins, the polypeptide fusions having the function of binding to ST2 and one or more other functions. In one aspect, the present disclosure also provides multispecific molecules comprising an antibody or antigen-binding fragment thereof of the present disclosure and at least one other functional moiety different from the specificity of the antibody or antigen-binding fragment, which can be another peptide or protein, the multispecific molecules can bind to ST2 and at least one other disease-associated protein, such as IgE. In another aspect, the ST2 antibodies or antigen-binding fragments thereof of the present disclosure can also be encoded by or carried by viral vectors.

[0044] In one aspect, the present disclosure also provides pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof of the present disclosure, or comprising a multispecific molecule, a polypeptide fusion, or a viral vector of the present disclosure, and a pharmaceutically acceptable excipient, diluent, or carrier.

[0045] In one aspect, the present disclosure also provides isolated nucleic acid molecules encoding an antibody or antigen-binding fragment thereof of the present disclosure, expression vectors comprising the nucleic acid molecules, and host cells comprising the expression vectors.

[0046] In one aspect, the present disclosure also provides a method of making an ST2 antibody or antigen-binding fragment thereof, comprising the steps of: (i) expressing the antibody or antigen-binding fragment thereof in a host cell, and (ii) isolating the antibody or antigen-binding fragment thereof from the host cell or its cell culture.

[0047] In another aspect, the present disclosure provides a method of detecting the amount of ST2 expression in a sample of a subject, the method comprising the step of contacting the sample with an antibody or antigen-binding fragment thereof of the present disclosure under conditions that allow the antibody or antigen-binding fragment thereof to bind to (or form a complex with) ST2.

[0048] In another aspect, the present disclosure provides a method of treating or slowing an IL33 / ST2-mediated related disease or condition, including but not limited to: asthma, allergic rhinitis, chronic obstructive pulmonary disease, eosinophilic bronchitis, eosinophilic esophagitis, atopic dermatitis, psoriasis, systemic lupus erythematosus, discoid lupus, rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, pulmonary fibrosis, liver fibrosis, systemic sclerosis, sarcoidosis, graft versus host disease (GVHD), diabetes disease, cardiovascular disease, or a combination thereof, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present disclosure, or a pharmaceutical composition thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of the present disclosure. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a polypeptide fusion, a multispecific molecule, a viral vector, or a pharmaceutical composition thereof of the present disclosure. In some embodiments, the multispecific molecule can bind to ST2 and at least another disease related protein, such as TSLP, IgE, IL4, IL13 or IL-5. In some embodiments, at least one other antibody can be administered with an antibody or antigen-binding fragment thereof of the present disclosure, such as a TSLP antibody, a TSLPR antibody, an IL4 antibody, an IL4R antibody, an IgE antibody. In some embodiments, at least one additional drug can be administered with an antibody or antigen-binding fragment thereof of the present disclosure, such as an anti-asthma drug, an anti-chronic obstructive pulmonary disease drug, an anti-ulcerative colitis drug, an anti-atopic dermatitis drug, or an anti-psoriasis drug.

[0049] Other features and advantages of the present disclosure will be better understood based on the following detailed description and examples, which should not be construed as limiting. The contents of all literature, Genbank records, patents, and published patent applications cited throughout this disclosure are expressly incorporated herein by reference. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 Binding of chimeric anti-ST2 antibodies to hST2 was measured by ELISA over a range of concentrations. Figure 1 Binding of chimeric antibodies xi17E2, xi8F4, xi26A1, xi31C8, xi3C6, xi7D1 to hST2 was shown, with binding of RG6149 shown as a control.

[0051] Figure 2 Binding of chimeric anti-ST2 antibodies to cyno-ST2 was measured by ELISA over a range of concentrations. Figure 2Binding of chimeric antibodies xi17E2, xi8F4, xi26A1, xi31C8, xi3C6, xi7D1 to cyno-ST2 is shown, binding of RG6149 is shown as a control.

[0052] Figure 3 Blocking of IL33 / ST2 binding by chimeric anti-ST2 antibodies at a range of antibody concentrations is detected by ELISA. Figure 3 Blocking of IL33 / ST2 binding by chimeric antibodies xi26A1, xi7D1, xi17E2, xi31C8, xi3C6, xi8F4 is shown, blocking by RG6149 is shown as a control.

[0053] Figure 4 Binding of humanized anti-ST2 antibodies to hST2 at a range of concentrations is detected by ELISA. Figure 4 Binding of humanized antibodies hz31C8-1.1 and hz31C8-1.2 to hST2 is shown, binding of chimeric antibody xi31C8, RG6149 and GSK3772847 is shown as a control, binding of IgG2 is shown as a negative control.

[0054] Figure 5 Binding of humanized anti-ST2 antibodies to cyno-ST2 at a range of concentrations is detected by ELISA. Figure 5 Binding of humanized antibodies hz31C8-1.1 and hz31C8-1.2 to cyno-ST2 is shown, binding of chimeric antibody xi31C8, RG6149 and GSK3772847 is shown as a control, binding of IgG2 is shown as a negative control.

[0055] Figure 6 Blocking of IL33 / ST2 binding by humanized anti-ST2 antibodies at a range of antibody concentrations is detected by ELISA. Figure 6 Blocking of IL33 / ST2 binding by humanized antibodies hz31C8-1.1 and hz31C8-1.2 is shown, blocking by chimeric antibody xi31C8, RG6149 and GSK3772847 is shown as a control.

[0056] Figure 7 Binding of chimeric anti-ST2 antibodies to hST2 overexpressing cells at a range of antibody concentrations is detected by FACS. Figure 7Binding of chimeric antibodies xi31C8, xi26A1, xi7D1, xi3C6, xi8F4, xi17E2 to HEK293T-hST2-NFkB-Luciferase cells overexpressing hST2 is shown, binding of RG6149 is shown as control, binding of IgG4 is shown as negative control.

[0057] Figure 8 Binding of chimeric anti-ST2 antibodies to cyno-ST2 overexpressing cells is shown by FACS detection over a range of antibody concentrations. Figure 8 Binding of chimeric antibodies xi31C8, xi26A1, xi7D1, xi3C6, xi8F4, xi17E2 to HEK293T-Cyno-ST2-NFkB-Luciferase cells overexpressing cyno-ST2 is shown, binding of RG6149 is shown as control, binding of IgG4 is shown as negative control.

[0058] Figure 9 Blocking of IL33 / ST2 binding by chimeric anti-ST2 antibodies is shown by cell activity detection over a range of antibody concentrations. Figure 9 Blocking of the interaction of IL33 protein to HEK293T-hST2-NFkB-Luciferase cells expressing hST2 by chimeric antibodies xi31C8, xi26A1, xi7D1, xi3C6 is shown, blocking by RG6149 is shown as control.

[0059] Figure 10 Binding of humanized anti-ST2 antibodies to hST2 overexpressing cells is shown by FACS detection over a range of antibody concentrations. Figure 10 Binding of humanized antibodies hz31C8-1.1 and hz31C8-1.2 to HEK293T-hST2-NFkB-Luciferase cells overexpressing hST2 is shown, binding of chimeric antibody xi31C8, RG6149 and GSK3772847 is shown as control, binding of IgG2 is shown as negative control.

[0060] Figure 11 Blocking of IL33 / ST2 binding by humanized anti-ST2 antibodies is shown by cell activity detection over a range of antibody concentrations. Figure 11 Blocking of the interaction of IL33 protein to HEK293T-hST2-NFkB-Luciferase cells expressing hST2 by humanized antibodies hz31C8-1.1 and hz31C8-1.2 is shown, blocking by chimeric antibody xi31C8, RG6149 and GSK3772847 is shown as control, blocking by IgG2 is shown as negative control.

[0061] Figure 12 The blocking effect of humanized anti-ST2 antibody on IL33 / ST2 binding was detected by cell viability assay within a certain antibody concentration range. Figure 12 The humanized antibodies hz31C8-1.1 and hz31C8-1.2 are shown to block the interaction of IL33 protein with hST2-expressing KU812-NFκB-Luciferasae cells. The blocking of chimeric antibodies xi31C8, RG6149 and GSK3772847 is shown as a control, and the blocking of IgG2 is shown as a negative control.

[0062] Figure 13 Cell viability assays were performed to detect the activity of humanized anti-ST2 antibodies on CD4 cells within a specific antibody concentration range. + T cells block the secretion of IL5 induced by IL33 / ST2. Figure 13 The humanized antibodies hz31C8-1.1 and hz31C8-1.2 are shown to function in CD4. + Blocking IL33 / ST2-induced IL5 secretion on T cells, with the chimeric antibodies xi31C8, RG6149 and GSK3772847 serving as controls, is shown. Invention Details

[0064] It should be understood that the terminology used in this disclosure is for the purpose of 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.

[0065] "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.

[0066] The terms“human ST2” or“hST2” and the like are used interchangeably in the present disclosure to refer to a protein having the amino acid sequence of human ST2, e.g., the human ST2 amino acid sequence set forth in SEQ ID NO: 1, which consists of several domains: amino acids 1-18 correspond to a leader sequence, amino acids 19-331 correspond to an extracellular domain, amino acids 332-350 correspond to a transmembrane domain, and amino acids 351-556 correspond to an intracellular domain. The terms“monkey ST2” or“cyno-ST2” and the like are used interchangeably in the present disclosure to refer to a protein having the amino acid sequence of monkey ST2, e.g., the monkey ST2 amino acid sequence set forth in SEQ ID NO: 10.

[0067] An“antibody” of the present disclosure includes a full-length antibody and any antigen binding fragment (i.e.,“antigen binding portion”) or single chain thereof. A full-length antibody is a glycoprotein comprising two heavy (H) chains and two light (L) chains connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, i.e., CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VHand VLregions can be further subdivided into regions of hypervariability, namely, complementarity determining regions (CDR), and regions that are more conserved, termed framework regions (FR). Each VHand VLis composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0068] An“antigen binding fragment” or“antibody binding portion” of an antibody refers to one or more fragments of an antibody that retain specific binding to an antigen (e.g., a ST2 protein). It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antibody“antigen binding portions / fragments” encompassed within the term include: L 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 recombination methods, 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 covered under the term antigen-binding part / 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.

[0069] "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.

[0070] "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 of this disclosure 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.

[0071] 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".

[0072] A "humanized antibody" refers to an antibody which is from a non-human species but whose protein sequence has been modified to increase its similarity to a human naturally-occurring antibody. In the present disclosure, a humanized antibody is also denoted as "hz".

[0073] An "isotype" refers to the antibody class (e.g., IgM or IgGl) that is encoded by heavy chain constant region genes.

[0074] "Antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably in the present disclosure with the term "antibody that specifically binds to an antigen".

[0075] An antibody that "specifically binds to human ST2" refers to an antibody that binds to human ST2 protein (and possibly ST2 protein of other non-human species) but does not substantially bind non-ST2 proteins. Preferably, the antibody binds to human ST2 with "high affinity", i.e., with a KDof 5.0 x 10 -8 M or less, 1.0 x 10 -8 M or less, preferably 5.0 x 10 -9 M or less, 1.0 x 10 -9 M or less, more preferably 5.0 x 10 -10 M or less, 1.0 x 10 -10 M or less.

[0076] The term "does not substantially bind" to a protein or cell means either does not bind to the protein or cell, or does not bind to it with high affinity, i.e., with a KDof 1.0 x 10 -6 M or higher, preferably 1.0 x 10 -5 M or higher, 1.0 x 10 -4 M or higher, more preferably 1.0 x 10 -3 M or higher, 1.0 x 10 -2 M or higher.

[0077] The term "high affinity" with respect to IgG refers to a KDof 1.0 x 10 -6 M or less, 1.0 x 10 -7 M or less, preferably 1.0 x 10 -8 M or less, 5.0 x 10 -9 M or less, more preferably 1.0 x 10 -9 M or less. However, "high affinity" binding can differ for other antibody isotypes. For example, "high affinity" binding for an IgM isotype refers to a KDof 10 -6 M or less, preferably 10 -7 M or less, more preferably 10 -8 M or less.

[0078] "Identity" refers to the similarity between two nucleic acid sequences or between two polypeptides. The sequence identity of the present disclosure is at least 80%, 85%, 90%, or 95%, preferably at least 95%. Non-limiting examples 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 determination of percent identity between two sequences can be performed by the default settings of the BLASTN / BLASTP algorithm on the National Center For Biotechnology Institute website.

[0079] The term "EC50", also known as half maximal effective concentration, refers to the concentration of antibody that results in 50% of maximal effect after a specified exposure time.

[0080] The term "IC50", also known as half maximal inhibitory concentration, refers to the concentration of antibody that results in 50% inhibition of a specific biological or biochemical function relative to the absence of the antibody.

[0081] The terms "inhibit" or "block" are used interchangeably and include partial and complete inhibition / blockade. In some embodiments, the ST2 antibody inhibits binding of IL33 / ST2 by at least about 50%, e.g., at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[0082] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, and preferably mammals, e.g., non-human primates, sheep, dogs, cats, cows and horses.

[0083] The term "therapeutically effective amount" refers to an amount that is sufficient to prevent or ameliorate a disease or condition and / or reduce the severity of a disease or condition, preferably an amount that results in a decrease in the severity of symptoms of the disease, an increase in the frequency and duration of symptom-free periods, or a prophylactic ability to cause damage or disability resulting from the disease. The therapeutically effective amount is related to the disease being treated, where one of skill in the art can readily determine the actual effective amount.

[0084] The use of the singular includes the plural, unless specifically stated otherwise. The word "a" or "an" means "at least one" or "one or more" unless specifically stated otherwise. The use of the phrase "at least one" is equivalent to the use of the phrase "one or more" and the use of "and / or" means "and" or "or".

[0085] Various aspects of the present disclosure are described in greater detail below.

[0086] Anti-ST2 antibodies specifically bind to ST2, block IL33 / ST2 interaction, and other beneficial functional features

[0087] The ST2 antibodies or antigen-binding fragments thereof of the present disclosure specifically bind human ST2 with high affinity. The ST2 antibodies or antigen-binding fragments thereof of the present disclosure specifically bind ST2 (e.g., human ST2 and monkey ST2) and block IL33 / ST2 binding and signaling thereof. The ST2 antibodies or antigen-binding fragments thereof of the present disclosure block IL5 secretion induced by IL33 / ST2 on CD4 + T cells. The ST2 antibodies or antigen-binding fragments thereof of the present disclosure do not substantially bind other IL-1 R family receptors, e.g., IL1R1, IL1R2, IL1R3, IL1R7, IL1R8, and IL1R9. The ST2 antibodies or antigen-binding fragments thereof of the present disclosure have good physical stability (e.g., thermal stability). The ST2 antibodies or antigen-binding fragments thereof of the present disclosure have a long in vivo half-life.

[0088] Preferably, the ST2 antibodies of the present disclosure are monoclonal antibodies. Further, the antibodies can be, for example, mouse, chimeric, or humanized monoclonal antibodies.

[0089] ST2 monoclonal antibodies

[0090] Preferably, the ST2 antibodies or antigen-binding fragments thereof of the present disclosure are antibodies having the structural and chemical properties described below. The ST2 antibodies or antigen-binding fragments thereof comprise heavy chain CDR regions and light chain CDR regions, wherein exemplary heavy chain CDR sequences and light chain CDR sequences are provided in Tables 1 and 2 below; the ST2 antibodies or antigen-binding fragments thereof comprise heavy chain variable regions and light chain variable regions, wherein exemplary heavy chain variable regions and light chain variable regions are provided in Table 3 below. Some antibodies have the same CDRs, some antibodies have the same VH or VL. The heavy chain constant region of the antibodies can be a human IgG2 heavy chain constant region, and the light chain constant region of the antibodies can be a human kappa light chain constant region. These antibodies can also comprise a mouse IgG1 or IgG4 heavy chain constant region and / or a mouse kappa light chain constant region.

[0091] 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, the CDR regions can also be determined based on the heavy / light chain variable region sequences by other numbering systems, e.g., Chothia, IMGT, AbM, or Contact numbering systems / methods, as is well known in the art.

[0092] Table of amino acid sequences of heavy chain CDR regions in Table 1

[0093]

[0094] Table of amino acid sequences of light chain CDR regions in Table 2

[0095]

[0096] Table 3 Amino acid sequences of heavy / light chain variable regions (underlining indicates CDR regions in order)

[0097]

[0098]

[0099] Table 4 Amino acid sequences of heavy chain / light chain (underlining indicates constant region)

[0100]

[0101]

[0102]

[0103] The VH and / or VL sequences (or CDR sequences) of other anti-ST2 antibodies that bind to human ST2 can be "mixed and matched" with the VH and / or VL sequences (or CDR sequences) of the antibodies of the present disclosure. Preferably, when VH and VL chains (or CDRs thereof) are mixed and matched, the VH sequence in a particular VH / VL pair can be replaced by a structurally similar VH sequence. Likewise, the VL sequence in a particular VH / VL pair is preferably replaced with a structurally similar VL sequence.

[0104] Thus, in one embodiment, an antibody or antigen-binding fragment thereof of the present disclosure comprises:

[0105] (a) a heavy chain variable region comprising an amino acid sequence listed in Table 3; and

[0106] (b) a light chain variable region comprising an amino acid sequence listed in Table 3, or a VL of another ST2 antibody, wherein the antibody specifically binds human ST2.

[0107] In another embodiment, an antibody or antigen-binding fragment thereof of the present disclosure comprises:

[0108] (a) heavy chain CDR1, CDR2 and CDR3 listed in Table 1; and

[0109] (b) light chain CDR1, CDR2 and CDR3 listed in Table 2, or CDRs of another ST2 antibody, wherein the antibody specifically binds human ST2.

[0110] In another embodiment, an antibody or antigen-binding fragment thereof of the present disclosure comprises a heavy chain CDR2 of a ST2 antibody of the present disclosure and other CDRs of an antibody that binds human ST2, e.g., a heavy chain CDR1 and / or CDR3, and / or a light chain CDR1, CDR2 and / or CDR3 of another ST2 antibody.

[0111] Furthermore, it is well known in the art that the CDR3 domain, independent of the CDR1 and / or CDR2 domains, alone determines the binding specificity of an antibody to a cognate antigen, and that multiple antibodies having the same binding specificity can be predicted to be generated 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. U.S.A. 95:8910-8915 (1998); Barbas et al., J. Am. Chem. Soc. 116:2161-2162 (1994); Barbas et al., Proc. Natl. Acad. Sci. U.S.A. 92:2529-2533 (1995); Ditzel et al., J. Immunol. 157:739-749 (1996); Berezov et al., BIAjournal 8: Scientific Review 8 (2001); Igarashi et al., J. Biochem (Tokyo). 117:452-7 (1995); Bourgeois et al., J. Virol. 72:807-10 (1998); Levi et al., 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. Each of these references is incorporated by reference in its entirety into the present disclosure.

[0112] In another embodiment, the antibodies or antigen-binding fragments thereof of the present disclosure comprise the heavy chain CDR2 of a ST2 antibody of the present disclosure and at least the heavy and / or light chain CDR3 of a ST2 antibody of the present disclosure, or of another ST2 antibody, wherein the antibody specifically binds human ST2. Preferably, these antibodies (a) compete for binding to ST2; (b) retain a functional characteristic; (c) bind the same epitope; and / or (d) have a similar binding affinity to a ST2 antibody of the present disclosure. In another embodiment, the antibodies or antigen-binding fragments thereof of the present disclosure can further comprise the light chain CDR2 of a ST2 antibody of the present disclosure, or of another ST2 antibody, wherein the antibody specifically binds human ST2. In another embodiment, the antibodies or antigen-binding fragments thereof of the present disclosure can further comprise the heavy and / or light chain CDR1 of a ST2 antibody of the present disclosure, or of another ST2 antibody, wherein the antibody specifically binds human ST2.

[0113] Conservative modifications

[0114] In another embodiment, the antibodies or antigen-binding fragments thereof of the present disclosure comprise the CDR1, CDR2 and CDR3 sequences of the heavy and / or light chain variable region of a ST2 antibody of the present disclosure with one or more conservative modifications. It is understood in the art that some conservative sequence modifications do not remove antigen-binding ability. See, e.g., Brummell et al., Biochem 32: 1180-8 (1993); de Wildt et al., Prot. Eng. 10:835-41 (1997); Komissarov et al., J. Biol. Chem. 272:26864-26870 (1997); Hall et al., J. Immunol. 149: 1605-12 (1992); Kelley and O’Connell Biochem. 32:6862-35 (1993); Adib-Conquy et al., Int. Immunol. 10:341-6 (1998); Beers et al., Clin. Can. Res. 6:2835-43 (2000).

[0115] Thus, in one embodiment, the antibody comprises a heavy chain variable region and / or a light chain variable region comprising a CDR1, CDR2 and CDR3, respectively, wherein:

[0116] (a) the CDR1 sequence of the heavy chain variable region comprises a sequence listed in Table 1, and / or conservative modifications thereof; and / or

[0117] (b) the CDR2 sequence of the heavy chain variable region comprises a sequence as listed in Table 1, and / or conservative modifications thereof; and / or

[0118] (c) the CDR3 sequence of the heavy chain variable region comprises a sequence as listed in Table 1, and / or conservative modifications thereof; and / or

[0119] (d) the CDR1 and / or CDR2 and / or CDR3 sequence of the light chain variable region comprises a sequence as listed in Table 2; and / or conservative modifications thereof; and

[0120] (e) the antibody specifically binds to human ST2.

[0121] The antibodies of the present disclosure have one or more of the following functional properties described above, such as high affinity for human ST2, and blocking of IL33 / ST2 binding and signaling thereof.

[0122] In various embodiments, the antibody can be a mouse, chimeric or humanized antibody or antigen-binding fragment thereof.

[0123] The term "conservative sequence modifications" as used herein denotes amendments that do not significantly affect or alter the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those that do not significantly affect or alter the binding properties of the antibody. Families of amino acid residues with similar side chains are known in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in a CDR region of an antibody of the present disclosure can be substituted with other amino acid residues within the same side chain family, and the resulting antibody can be tested for retention of function (i.e., the functions described above) using the functional assays described herein.

[0124] Engineered and modified antibodies

[0125] Antibodies of the present disclosure can be engineered to produce modified antibodies using antibodies having one or more VH / VL sequences of the ST2 antibodies of the present disclosure as starting material. Antibodies can be genetically modified with respect to one or more residues within one or both variable regions (i.e., VH and / or VL), for example, within one or more CDR regions and / or one or more framework regions. Additionally, or alternatively, antibodies can be engineered with respect to residues in the constant region, for example, to alter the effector function of the antibody.

[0126] In certain embodiments, CDR grafting can be used to genetically modify the variable regions of an antibody. Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementarity determining regions (CDRs). Thus, the amino acid sequence within each of the antibody CDRs is more diverse than the sequence outside of the CDRs. Because the CDR sequences are responsible for the primary antibody-antigen interactions, a recombinant antibody that mimics the properties of a particular native antibody can be expressed by constructing an expression vector in which the CDR sequences of the particular native antibody are grafted to the framework sequences of a different antibody of different properties (Riechmann et al., Nature. 332:323-327 (1998); Jones et al., Nature. 321 :522-525 (1986); Queen et al., Proc. Natl. Acad. Sci. U.S.A. 86: 10029-10033 (1989); U.S. Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762; and 6,180,370).

[0127] Accordingly, another embodiment of the present disclosure relates to an isolated monoclonal antibody or antigen binding fragment thereof, comprising a heavy chain variable region comprising a CDR1, CDR2, and CDR3 of the above sequences of the present disclosure, and / or a light chain variable region comprising a CDR1, CDR2, and CDR3 of the above sequences of the present disclosure. Although these antibodies comprise the CDR sequences of the VH and VL of the monoclonal antibodies of the present disclosure, they can contain different framework sequences.

[0128] Such framework sequences can be obtained from public DNA databases or public 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 in the Vbase human germline sequence database (www.mrc-cpe.cam.ac.uk / vbase) as well as in Kabat et al., (1991), supra; 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 in the Genbank database.

[0129] Antibody protein sequences are compared to protein sequence databases using one of the methods of sequence similarity searching known to those of skill in the art using Gapped BLAST (Altschul et al., (1997), supra).

[0130] The framework sequences of the antibodies of the disclosure are preferably those that are structurally similar to the framework sequences used by the antibodies of the disclosure. The VH CDR1, CDR2, and CDR3 sequences can be grafted into a framework region having the same sequence as the germline immunoglobulin gene from which the framework sequence is derived, or the CDR sequences can be grafted into a framework region having one or more mutations compared to the germline sequence. For example, in some cases it is beneficial to mutate residues in the framework regions that can maintain or enhance the antigen binding ability of the antibody (see, e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370).

[0131] Another type of variable region modification is the mutation of amino acid residues within the CDR1, CDR2, and / or CDR3 regions of the VH and / or VL to improve one or more properties of the antibody of interest (e.g., affinity, physicochemical properties). Mutations can be introduced by site-directed mutagenesis or PCR-mediated mutagenesis, and the effect of the mutation on the binding or other functional properties of the antibody can be assessed by in vitro or in vivo assays known in the art. Preferably, conservative modifications known in the art are introduced. The modifications can be substitutions, additions, or deletions of amino acids, preferably substitutions. Furthermore, typically no more than one, two, three, four, or five residues within each CDR region are altered.

[0132] In another embodiment, the present disclosure provides an isolated ST2 monoclonal antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, comprising: (a) a VH CDR1 region comprising a sequence of the present disclosure, or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions, or additions; (b) a VH CDR2 region comprising a sequence of the present disclosure, or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions, or additions; (c) a VH CDR3 region comprising a sequence of the present disclosure, or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions, or additions; (d) a VL CDR1 region comprising a sequence of the present disclosure, or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions, or additions; (e) a VL CDR2 region comprising a sequence of the present disclosure, or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions, or additions; and (f) a VL CDR3 region comprising a sequence of the present disclosure, or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions, or additions.

[0133] The genetically engineered antibodies of the present disclosure include those in which modifications are made to the framework residues of the VH and / or VL to improve antibody properties. Typically, such framework modifications result in antibodies with improved

[0134] Another type of framework modification involves the mutation of one or more residues within the framework region, or even one or more CDR regions, to remove T cell epitopes, thereby reducing the immunogenicity of the antibody. This approach, which is also referred to as "deimmunization," is described in greater detail in U.S. Patent Publication No. 20030153043.

[0135] In addition to modifications in the framework regions or CDR regions, additional modifications can be made to the Fc region of an antibody of the present disclosure, typically to alter one or more functional properties of an antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity, for example, by genetic engineering. Also, antibodies of the present disclosure can be chemically modified (e.g., to link one or more chemical moieties), or modified to alter their glycosylation, to alter one or more functional properties of an antibody.

[0136] In one embodiment, the CHI-hinge region is modified, e.g., to increase or decrease the number of cysteine residues in the hinge region. This approach is described in detail in U.S. Patent No. 5,677,425. Altering the number of cysteine residues in the CHI-hinge region, e.g., can facilitate assembly of light and heavy chains or increase or decrease the stability of the antibody.

[0137] In another embodiment, the Fc-hinge region of the antibody is mutated to increase or decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 region of the Fc-hinge region such that the antibody has attenuated Staphylococcus protein A (SpA) binding relative to a native Fc-hinge domain. This approach is described in greater detail in U.S. Patent No. 6,165,745.

[0138] In another embodiment, the glycosylation of the antibody is modified. For example, an aglycosylated antibody can be generated (i.e., the antibody lacks glycosylation). Such glycosylation modifications can be accomplished by, e.g., altering one or more glycosylation sites within an antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for antigen. See, e.g., U.S. Patent Nos. 5,714,350 and 6,350,861.

[0139] In addition, antibodies with altered types of glycosylation, e.g., hypofucosylated antibodies with reduced amounts of fucosyl residues, or antibodies with increased bisecting GlcNac structures can be made. Alterations in glycosylation have been demonstrated to increase the ADCC activity of antibodies. Such glycosylation modifications can be accomplished by, e.g., expressing the antibody in a host cell with altered glycosylation system, known in the art and which can be used as host cells to express the recombinant antibodies of the present disclosure to make antibodies with altered glycosylation. For example, cell lines Ms704, Ms705 and Ms709 lack the fucosyltransferase gene FUT8 (alpha (1,6)-fucosyltransferase), and thus antibodies expressed in the Ms704, Ms705 and Ms709 cell lines lack fucoses. The Ms704, Ms705 and Ms709 FUT8- / - cell lines were made by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see U.S. Patent 20040110704 and Ohnuki et al., Biotechnol Bioeng. 87:614-22 (2004)). As another example, EP 1,176,195 describes a cell line with a disrupted FUT8 gene, which encodes a fucosyltransferase, such that antibodies expressed in this cell line exhibit hypofucosylation by reducing or eliminating the alpha-1,6 linkage-related enzyme. EP 1,176,195 also describes a cell line with lower or deficient enzyme activity that adds fucose to N-acetylglucosamine bound in the Fc region of an antibody, e.g., the rat myeloma line YB2 / 0 (ATCC CRL 1662). WO 03 / 035835 describes a CHO variant cell line, Lec13 cells, with reduced ability to add fucose to Asn(297)-linked sugars, such that antibodies expressed in this host cell are hypofucosylated (see Shields et al., J. Biol. Chem. 277:26733-26740 (2002)). Antibodies with altered glycosylation characteristics can also be made in chicken eggs, as described in WO 06 / 089231. Alternatively, antibodies with altered glycosylation characteristics can be made in plant cells, such as Lemna. WO 99 / 54342 discloses a cell line genetically engineered to express a glycosyltransferase that modifies glycoproteins (e.g., beta (1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the cell line exhibit increased bisecting GlcNac structures, resulting in enhanced ADCC activity of the antibody (see also Umana et al., Nat. Biotech. 17:176-180 (1999)).Alternatively, fucose residues are cleaved from the antibody using a fucosidase, such as a- L-fucosidase to remove fucose residues from the antibody (Tarentino et al., Biochem. 14:5516-23 (1975)).

[0140] Another modification of the antibodies of the present disclosure is pegylation. PEGylation of an antibody, for example, can increase the biological (e.g., serum) half-life of the antibody. To obtain a PEGylated antibody, an antibody, or fragment thereof, is reacted with polyethylene glycol (PEG), e.g., a reactive ester or aldehyde derivative of PEG, under conditions that allow one or more PEG groups to become attached to the antibody or antibody fragment. Preferably, the reaction is carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similarly reactive water-soluble polymer). The term "polyethylene glycol" as used herein includes any of the forms of PEG that have been used to derivatize other proteins, e.g., mono-(Ci-Cio)alkoxy- or aryloxy- polyethylene glycol or polyethylene glycol-maleimide. In certain 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 the present disclosure. See, e.g., EP 0154316 and EP 0401384.

[0141] Physical properties of antibodies

[0142] Antibodies of the present disclosure can be characterized by their various physical properties to detect and / or distinguish their classification. For example, an antibody can comprise one or more glycosylation sites in the light chain or heavy chain variable region. These glycosylation sites can result in an increase in immunogenicity of the antibody, or a change in pK of the antibody due to a change 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 a motif containing an N-X-S / T sequence. In certain cases, it is preferred that the ST2 antibody does not comprise variable region glycosylation. Antibodies can be selected that do not comprise a glycosylation motif in the variable region or residues within the glycosylation region can be mutated to achieve this.

[0143] In a preferred embodiment, the antibody does not comprise an asparagine isomerization site. Deamidation of asparagine can occur in N-G or D-G sequences and results in the production of isoaspartic acid residues, which decrease stability.

[0144] Each antibody has a unique isoelectric point (pi) that is typically within the pH range of 6-9.5. IgGl antibodies typically have a pi within the pH range of 7-9.5, while IgG4 antibodies typically have a pi within the pH range of 6-8. It is hypothesized that antibodies with pi values outside the normal range can undergo some unfolding and instability under in vivo conditions. Thus, 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 mutating surface residues.

[0145] Nucleic acid molecules encoding antibodies of the disclosure

[0146] In another aspect, the present disclosure provides nucleic acid molecules encoding the heavy and / or light chain variable regions or CDRs of the antibodies of the present disclosure. The nucleic acids can be present in whole cells, cell lysates, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques. The nucleic acids of the present disclosure can be, for example, DNA or RNA, and can or can not include intronic sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0147] Nucleic acids of the present disclosure can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes), light and heavy chain cDNAs encoding the antibodies produced by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), nucleic acid encoding such antibodies can be recovered from the gene library.

[0148] Preferably, the nucleic acid molecules of the present disclosure include nucleic acid molecules encoding the VH and VL sequences or CDRs of the ST2 monoclonal antibodies of the present disclosure. Once a DNA fragment encoding a VH and VL segment has been obtained, it can be further manipulated by standard recombinant DNA techniques, e.g., to convert the variable region genes to full-length antibody chain genes, Fab fragment genes or scFv genes. In these manipulations, a DNA fragment encoding a VL or VH is operatively linked to another DNA segment encoding another protein, such as an antibody constant region or a flexible linker. The term "operatively linked" as used in the present disclosure means that the two DNA fragments are connected in such a way that both DNA fragments retain their original translational reading frame.

[0149] The isolated DNA encoding the VL region can be converted to a full-length light chain gene by operatively linking the DNA encoding the VL region to another DNA molecule encoding a light chain constant region (CL). The sequence of human light chain constant region genes is known in the art, and DNA fragments encoding these regions can be obtained by standard PCR amplification of a cDNA or genomic library. The light chain constant region can be a kappa (κ) or lambda (λ) constant region, but is most preferably a kappa constant region.

[0150] The isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the DNA encoding the VL region to another DNA molecule encoding a light chain constant region (CL). The sequence of human light chain constant region genes is known in the art, and DNA fragments encoding these regions can be obtained by standard PCR amplification of a cDNA or genomic library. In preferred embodiments, the light chain constant region can be a kappa (K) or lambda (λ) light chain constant region.

[0151] To create a scFv gene, the DNA fragments encoding the VH and VL are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, in which the VL and VH regions are linked by the flexible linker (see, e.g., 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)).

[0152] Production of monoclonal antibodies of the disclosure

[0153] Monoclonal antibodies (mAbs) of the present disclosure can be prepared using the hybridoma (Kohler and Milstein, Nature. 256:495 (1975)) technique. Other embodiments for 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, e.g., U.S. Patents 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762; and 6,180,370.

[0154] Transfectomas for producing monoclonal antibodies

[0155] Antibodies of the present disclosure can also be produced in host cell transfectomas using, for example, recombinant DNA techniques in conjunction with gene transfection methods (e.g., Morrison, S. Science 229: 1202 1985). In one embodiment, DNA encoding partial or full-length light and heavy chains is obtained by standard molecular biology techniques and inserted into one or more expression vectors such that the genes are operably linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" means that an antibody gene is ligated into a vector in such a way that transcriptional and translational regulatory sequences within the vector are able to exert their function on the antibody gene.

[0156] The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control transcription or translation of the antibody gene. Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Preferably, the regulatory sequences for mammalian host cell expression include viral elements that control high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, and / or a promoter and / or enhancer from a retrovirus, e.g., Myeloblastosis Virus (MLV), and / or the promoter and / or enhancer from glycoprotein hormone genes, e.g., the beta- lactamase promoter. Alternatively, non-viral regulatory elements can be used, such as the ubiquitin promoter or the beta-globin promoter. In addition, the regulatory elements are composed of sequences from different sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and sequences from the long terminal repeat of human T-cell leukemia virus type I (Takebe et al., Mol. Cell. Biol. 8:466-472 (1988)). The expression vector and the expression control sequences are compatible with the expression host cell used.

[0157] 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 constructed into a full-length antibody gene by inserting into an expression vector that already encodes heavy chain constant regions and light chain constant regions of the desired isotype, such that the VHand the CHin the vector are operably linked, and the VLand the CLin the vector are operably linked. Alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain(s) from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., from a non-immunoglobulin protein).

[0158] In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of the present disclosure can carry sequences for amplification of the vectors in host cells (e.g., origins of replication) and selectable marker genes. Selectable marker genes can be used to select host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216; 4,634,665; and 5,179,017). For example, a selectable marker gene can confer resistance to drugs, such as G418, hygromycin, or methotrexate, on host cells into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0159] To express the light and heavy chains, the expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The term "transfection" includes a variety of techniques for introducing foreign DNA into a prokaryotic or eukaryotic host cell, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although expression of the antibodies of the present disclosure in prokaryotic or eukaryotic host cells is theoretically possible, it is preferred that the antibodies be expressed in eukaryotic cells, most preferably mammalian host cells, because eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody.

[0160] Preferred mammalian host cells for expressing the recombinant antibodies of the present disclosure include Chinese hamster ovary (CHO cells) (including dhfr- CHO cells that are adapted to grow in medium containing methotrexate, as described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220 (1980), the DHFR selectable marker as described in RJ Kaufman and PA Sharp. J. Mol. Biol. 159:601-621 (1982)), NSO myeloma cells, COS cells, and SP2 cells. Another preferred expression system, particularly when NSO myeloma cells are used, is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody, or preferably, for secretion of the antibody into the medium in which the host cells are grown. Antibodies can be recovered from the culture medium using protein purification procedures.

[0161] Polypeptide fusions

[0162] In another aspect, the present disclosure relates to polypeptide fusions comprising one or more antibodies or antigen-binding fragments thereof of the present disclosure, wherein the antibodies or antigen-binding fragments thereof of the present disclosure are linked to at least one other functional molecule, which can be a peptide or protein or non-protein. In one embodiment, the polypeptide fusions of the present disclosure include immunoconjugates comprising one or more antibodies or antigen-binding fragments thereof of the present disclosure and at least one therapeutic agent, e.g., a steroid, linked to the antibodies or antigen-binding fragments thereof of the present disclosure. In one embodiment, the polypeptide fusions of the present disclosure include bifunctional molecules comprising one or more antibodies or antigen-binding fragments thereof of the present disclosure and at least one immunocytokine or receptor ligand, which can be directed against 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, linked to the antibodies or antigen-binding fragments thereof of the present disclosure. In one embodiment, the bifunctional molecules have a third function in addition to the Fc receptor binding function and the ST2 binding function. The third function can be directed against 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. As used herein, "bifunctional molecules" encompass molecules having three or more functions. In other embodiments, the polypeptide fusions of the present disclosure further comprise other formats. These and other formats of polypeptide fusions can be prepared by genetic engineering, chemical methods, etc.

[0163] Multispecific molecules

[0164] In another aspect, the present disclosure relates to multispecific molecules comprising one or more antibodies or antigen-binding fragments thereof of the present disclosure, wherein the antibodies or antigen-binding fragments thereof of the present disclosure are linked to at least one other functional moiety that is specific to a different antibody or antigen-binding fragment than the antibodies or antigen-binding fragments of the present disclosure, including another peptide or protein (e.g., another antibody or antigen-binding fragment thereof) to generate a multispecific molecule that binds to at least two different binding sites or targets. Thus, as used herein, "multispecific molecules" encompass molecules having two (i.e., bispecific molecules), three (i.e., trispecific molecules), four (i.e., tetraspecific molecules) or more specificities.

[0165] In one specific embodiment, the multispecific molecules of the present disclosure can have one or more additional specificities in addition to the anti-Fc binding specificity and the ST2 binding specificity. Illustratively, the additional specificity can be for IgE, IL4, IL4R, IL5, IL5R, IL6, IL9, IL13, IL13R, IL17, IL23, IL33, or TSLP, TSLPR / IL7R.

[0166] In one specific embodiment, the multispecific molecules can occur in a variety of different formats and sizes. Illustratively, for a bispecific molecule, at one end of the size spectrum, the bispecific molecule retains the traditional antibody format, except that it has two binding arms and each arm has a different specificity, rather than having two binding arms with the same specificity. At the other end, the bispecific molecule is composed of two single chain antibody fragments (scFv) connected by a peptide chain, referred to as a Bs(scFv)2 construct. Bispecific molecules of intermediate size include two different F(ab) fragments connected by a peptide-based linker. These and other formats of bispecific molecules can be prepared by genetic engineering, somatic hybridization, or chemically. See, e.g., 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).

[0167] Viral vectors

[0168] In another aspect, the antibodies or antigen-binding fragments thereof of the present disclosure can also be encoded by or carried by a viral vector. In addition, the antibodies or antigen-binding fragments thereof of the present disclosure can also be used with a viral vector, or a viral vector encoding or carrying the antibodies or antigen-binding fragments thereof of the present disclosure can be introduced into a human.

[0169] The present disclosure also provides pharmaceutical compositions comprising the polypeptide fusions, multispecific molecules, viral vectors of the present disclosure, and a pharmaceutically acceptable excipient, diluent, or carrier.

[0170] Pharmaceutical compositions

[0171] In another aspect, the present disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the present disclosure, formulated together with a pharmaceutically acceptable excipient, diluent, or carrier. The pharmaceutical composition can optionally comprise one or more other pharmaceutically effective ingredients, such as another antibody or drug, e.g., another ST2 antibody, an anti-IgE antibody, another anti-inflammatory drug, an anti-asthma drug, an anti-chronic obstructive pulmonary disease drug, an anti- ulcerative colitis drug, an anti-atopic dermatitis drug, or an anti-psoriasis drug.

[0172] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or topical (e.g., by injection or infusion) administration. Depending on the route of administration, the active ingredient can be coated with a material to protect it from the action of acids and other natural conditions that can inactivate it. "Parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinally, epidural, and intrasternal injection and instillation. Alternatively, the pharmaceutical composition of the present disclosure can be administered by a non-parenteral route, e.g., topically, epidermally, or mucosally, e.g., intranasally, orally, vaginally, rectally, sublingually, or topically.

[0173] The pharmaceutical compositions can be in the form of a sterile solution or dispersion. They can also be formulated in a microemulsion, liposome, or other ordered structure that is suitable for high drug concentration.

[0174] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Particularly advantageous is the formulation of parenteral compositions in dosage unit form for easy practice by entities not trained in the art of pharmacy. Dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with a pharmaceutical carrier. Alternatively, the antibody can be administered in a sustained release formulation, in which case the required frequency of administration can be reduced.

[0175] For administration of the composition, the dose can be about 0.0001 to 100 mg / kg, more usually 0.01 to 5 mg / kg, of the host body weight. For example, the dose can be 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg or 10 mg / kg body weight or within the range of 1-10 mg / kg. An exemplary treatment regimen entails administration twice per week, once per 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.

[0176] The pharmaceutical compositions can be sustained release agents, including implants, transdermal patches, and microencapsule delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, poly acid anhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.

[0177] In certain embodiments, the antibodies of the present disclosure can be formulated to ensure proper in vivo distribution. For example, to ensure that the therapeutic antibodies of the present disclosure cross the blood-brain barrier, the antibodies can be formulated in a liposome, which can additionally comprise a targeting functional group to enhance selective delivery to a particular cell or organ.

[0178] Uses and methods of the disclosure

[0179] The antibodies or antigen-binding fragments thereof (encoding nucleic acid molecules, pharmaceutical compositions, polypeptide fusions, multispecific molecules, or viral vectors) of the present disclosure have a variety of in vitro and in vivo applications related to the diagnosis, treatment, and / or prevention of IL33 / ST2-mediated related diseases and disorders. The IL33 / ST2-mediated related diseases and disorders include, but are not limited to, asthma, allergic rhinitis, chronic obstructive pulmonary disease, eosinophilic bronchitis, eosinophilic esophagitis, atopic dermatitis, psoriasis, systemic lupus erythematosus, discoid lupus, rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, pulmonary fibrosis, liver fibrosis, systemic sclerosis, sarcoidosis, graft versus host disease (GVHD), diabetes mellitus, cardiovascular disease, or a combination thereof. The antibodies or antigen-binding fragments thereof of the present disclosure can be administered to a human subject to reduce, ameliorate, or treat the disease or disorder. In the diagnostic process, the antibodies of the present disclosure can be contacted with a sample of the subject under conditions that allow the antibodies or antigen-binding fragments thereof of the present disclosure to bind to ST2 to detect the amount of ST2 protein in the sample of the subject.

[0180] These and other methods of the present disclosure are further discussed below.

[0181] Combination therapies

[0182] The present disclosure provides co-administration of the ST2 antibodies or antigen binding fragments thereof (encoding nucleic acid molecules, pharmaceutical compositions, polypeptide fusions, multispecific molecules, or viral vectors) of the present disclosure with one or more other antibodies or drugs that reduce, ameliorate, or treat IL33 / ST2-mediated related diseases and conditions in a subject, including but not limited to: asthma, allergic rhinitis, chronic obstructive pulmonary disease, eosinophilic bronchitis, eosinophilic esophagitis, atopic dermatitis, psoriasis, systemic lupus erythematosus, discoid lupus, rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, pulmonary fibrosis, liver fibrosis, systemic sclerosis, sarcoidosis, graft versus host disease (GVHD), diabetes mellitus, cardiovascular disease, or combinations thereof. In one embodiment, the present disclosure provides a method of treating asthma, chronic obstructive pulmonary disease, atopic dermatitis in a subject, wherein the ST2 antibodies or antigen binding fragments thereof of the present disclosure are administered with one or more other antibodies, such as TSLP antibodies, TSLPR antibodies, IL4 antibodies, IL4R antibodies, IL13 antibodies, IL13R antibodies, IL5 antibodies, IL5R antibodies, and / or IgE antibodies. In another embodiment, the present disclosure provides a method of treating asthma, chronic obstructive pulmonary disease, atopic dermatitis in a subject, wherein the ST2 antibodies or antigen binding fragments thereof of the present disclosure are administered with at least one additional drug, such as an anti-asthma drug, an anti-chronic obstructive pulmonary disease drug, an anti-atopic dermatitis drug. In certain embodiments, the subject is a human. Other therapies that can be combined with the ST2 antibodies or antigen binding fragments thereof of the present disclosure also include: allergen avoidance, hormone therapy, surgery, etc.

[0183] The combinations of therapeutic agents discussed in the present disclosure (i.e., combinations) can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as separate compositions, where each agent is in a pharmaceutically acceptable carrier. In another embodiment, the combinations of therapeutic agents can be administered sequentially.

[0184] Furthermore, if multiple combination therapy administrations are performed, and the agents are administered sequentially, the order of sequential administration at each time point can be reversed or remain the same, and sequential administration can be combined with simultaneous administration or any combination thereof.

[0185] While the foregoing application has been described in some detail for purposes of clarity and the specific embodiments, it will be apparent to those skilled in the art having the benefit of the teachings of the present disclosure that certain changes and modifications are

[0186] Unless otherwise indicated, the practice of the present disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Examples

[0187] Example 1, Preparation of ST2 antigen and detection protein

[0188] The full-length gene of human UniProt Interleukin-1 receptor-like 1 (ST2) isoform A (hST2) was used as a template for the ST2 of the present disclosure to obtain the gene sequence encoding the antigen and detection protein of the present disclosure, which can be recombined with the Fc fragment of the heavy chain of mouse antibody (such as mouse IgG2a) to form hST2-mFc, or recombined with the Fc fragment of the heavy chain of human antibody to form hST2-hFc, for immunization of mice or later screening and detection. The cDNA (SEQ ID NOs: 3 and 5, respectively) encoding the extracellular region recombinant protein of hST2 containing the Fc tag of the heavy chain of mouse antibody (hST2-ECD-mFc, SEQ ID NO: 2) and the extracellular region recombinant protein of hST2 containing the Fc tag of the heavy chain of human antibody (hST2-ECD-hFc, SEQ ID NO: 4) were obtained by gene synthesis, and were subcloned into the expression vector pcDNA3.1 (Invitrogen, V-790), respectively. The above constructed vectors were transfected into Expi293 cells (Thermo, A14527) for transient expression. The hST2-ECD-mFc and hST2-ECD-hFc recombinant proteins were purified by Protein A column (GE healthcare).

[0189] The cDNA (SEQ ID NO: 7) encoding the recombinant protein of human IL33 containing Avitag was obtained by gene synthesis and was cloned into the expression vector with GST tag. The above constructed vector was transformed into BL21 competent cells for induced expression. The GST-IL33 Avitag protein was purified by GST purification column, and the GST was removed after enzyme digestion of Thrombin enzyme (Sigma, T4648-1KU) to obtain hIL33 Avitag (SEQ ID NO: 6).

[0190] The cDNA (SEQ ID NO: 9) encoding the recombinant protein of human IL33 was obtained by gene synthesis, and was cloned into a GST-labeled expression vector. The above constructed vector was transformed into BL21 competent cells for induction expression. The GST-IL33 protein was purified by a GST purification column, and the hIL33 protein (SEQ ID NO: 8) was obtained by removing GST after enzymatic cleavage of Thrombin enzyme (Sigma, T4648-1KU).

[0191] The ST2 antibody RG6149 in the examples refers to the antibody Ab2 in CN104334582, which is internally prepared, and the amino acid sequences of the heavy chain and the light chain are shown as SEQ ID NO: 83 and SEQ ID NO: 84 of the present disclosure; the ST2 antibody GSK3772847 refers to the antibody STLM208 in CN104411333, which is internally prepared, and the amino acid sequences of the heavy chain and the light chain are shown as SEQ ID NO: 85 and SEQ ID NO: 86 of the present disclosure.

[0192] Example 2, Preparation of Anti-ST2 Hybridoma Monoclonal Antibodies

[0193] The purified hST2-ECD-mFc recombinant protein (100 μg per mouse) was mixed and emulsified with an equal volume of complete Freund's adjuvant (Sigma, F5881-10X10ML) (first immunization) or incomplete Freund's adjuvant (Sigma, F5506-10X10ML) (boosting immunization), and BALB / c mice were subcutaneously immunized every 2 weeks for 8 weeks. Three days before fusion, the immunized mice were boosted by intraperitoneal injection of hST2-ECD-mFc antigen without adjuvant (50 μg per mouse). The spleen cells (1×10 8 ) from the immunized mice were fused with SP2 / 0 myeloma cells (2×10 7 ) by a PEG-mediated fusion procedure to obtain hybridoma cells. After fusion, the cells were resuspended with HAT complete medium (Gibco, 21060017), and were distributed into 96-well plates at 0.1 mL per well, and were cultured in a 37°C, 5% CO2 incubator. Usually about 10-15 days after fusion, the binding activity of the cell culture supernatant to ST2 was detected by an ELISA method (see Example 5), and the cell culture supernatant of the positive well was selected, and the blocking activity of IL33 / ST2 binding was detected by an ELISA method (see Example 6).

[0194] The wells that can specifically bind to ST2 and can block the binding of IL33 / ST2 were selected, and expanded to 24-well plates according to the cell density. The cell strains moved into the 24-well plates were preserved and subcloned for the first time after retesting. The positive strains of the first subcloning were preserved and subcloned for the second time. The positive strains of the second subcloning were preserved and expressed proteins.

[0195] Example 3, cDNA acquisition and chimeric antibody construction of anti-ST2 antibody

[0196] Total RNA was isolated from the hybridoma cells with binding and blocking functions described above using a total RNA extraction kit as a template, and superscript III reverse transcriptase (Thermo, 18080051) was used to synthesize the first strand cDNA according to the instructions. Then the variable region sequence of the antibody was amplified by PCR reaction using degenerate mouse IgG primers.

[0197] The PCR mixture was electrophoretically separated in a 1% agarose / Tris-borate gel containing 0.5 μg / mL ethidium bromide. The DNA fragments with the expected size (about 500 bp for heavy and light chains) were cut from the gel and purified. The purified PCR product was cloned into the pMD-19T vector (Takara, 6013) and transformed into DH5a competent E. coli cells (Takara, 9057). Five colonies were picked from the LB solid culture plate for DNA sequencing. The heavy chain variable region sequence and the light chain variable region sequence 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), 17E2 (SEQ ID NOs: 45, 46).

[0198] Construction and expression of chimeric antibodies: The gene synthesis fragment of mouse VL region was connected to the human kappa chain constant region by double enzyme digestion reaction to construct a chimeric light chain. The gene synthesis fragment of mouse VH region was connected to the human IgG2 constant region by double enzyme digestion reaction to construct a chimeric heavy chain.

[0199] 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 x 10 6 / mL cells, 1 μg / mL DNA) for transient expression, and cultured for 7 days. Then the chimeric antibody in the cell culture supernatant was purified by Protein A column (GE healthcare).

[0200] Example 4, Affinity determination of anti-ST2 antibodies to hST2 antigen

[0201] Anti-mouse IgG antibody (for capturing human ST2 antigen, Cytiva, 29215281) or anti-his antibody (for capturing cynomolgus monkey ST2 antigen, Cytiva, 29234602) was coupled to a CM5 biosensor chip (Cytiva, BR-1000-12) according to the method described in the product manual, then 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 antibodies were flowed over the chip surface, and 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 was completed, the biosensor chip was regenerated using a regeneration solution, followed by the next capture, and the cycle was repeated to complete the determination of the affinity of different antibodies to ST2. Finally, the GEBIA evaluation software was used to analyze the data obtained using a 1:1 (Langmuir) binding model to determine the association rate constant ka (kon) and the dissociation rate constant kd (koff), and the dissociation constant KD was calculated by KD = kd / ka. The affinity data of anti-ST2 antibodies to human ST2 antigen (hST2-ECD-mFc), cynomolgus monkey ST2 antigen (Cyno-ST2-his, Sino Biological, 90915-C08H) were determined, and the affinity results of anti-ST2 chimeric antibodies to ST2 are shown in Table 5.

[0202] Table 5 Affinity of anti-ST2 chimeric antibodies to ST2

[0203]

[0204] Example 5, ELISA-based binding analysis of anti-ST2 antibodies

[0205] ST2 binding screening and analysis of antibodies were performed by ELISA method using hST2-ECD-mFc (for chimeric and humanized antibody detection), hST2-ECD-hFc protein (for hybridoma antibody detection) and Cyno-ST2-his (Sino Biological, 90915-C08H) as antigens, respectively. 2 pg / mL of hST2-ECD-mFc or Cyno-ST2-his antigens were coated in 100 uL / well in high-adsorption 96-well plates (Costar, 9018) at 4°C overnight. After washing away un-adsorbed antigens, non-specific binding sites were blocked with 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), 100 uL / well of anti-ST2 antibodies (initial concentration of 10 nM, diluted at 3-fold concentration gradient to 8 concentrations, i.e. diluted to about 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.) were added and incubated at room temperature for 1 hour. After washing the plates with wash buffer, secondary antibodies conjugated with horseradish peroxidase (HRP) were added and incubated for another 60 minutes. After washing the plates with wash buffer, 100 uL / well of substrate TMB solution (Thermo, 00-4201-56) was added and the plates were incubated at room temperature for 2 minutes. 100 uL / well of stop solution (2N H2SO4) was added to stop the reaction. Colorimetric signal was generated and read at 450 nm using a microplate reader (PE, Envision). Data were analyzed using GraphPad Prism 5 and EC50 was calculated. The EC50 of anti-ST2 chimeric antibodies binding to hST2 and Cyno-ST2 are shown in Table 6 and Figures 1-2

[0206] Table 6. EC50 of anti-ST2 chimeric antibodies binding to hST2 and Cyno-ST2 in ELISA experiments

[0207]

[0208] Example 6, ELISA-based blocking analysis of anti-ST2 antibodies

[0209] ​ELISA method, to determine whether the anti-ST2 antibodies are capable of blocking the binding of hIL33 to hST2. 2 pg / mL of hST2-ECD-hFc was coated in 100 pL / well in high-adsorption 96-well plates, 4°C overnight. After washing away the un-adsorbed antigen, non-specific binding sites were blocked with blocking buffer (PBST containing 1% bovine serum albumin). After washing the plates three times with wash buffer (PBS containing 0.05% (v / v) Tween 20, PBST), 100 pL / well of anti-ST2 antibodies (initial concentration of 66.67 nM, diluted in 2-fold concentration gradient for 8 concentrations) were added, incubated at 37°C for 1 hour, and the plates were washed three times with wash buffer. 100 pL of biotin-labeled hIL33 Avitag (concentration of 0.1 pg / mL) was added per well and incubated at 37°C for 1 hour. After washing the plates three times with wash buffer, secondary antibody Avidin HRP (Jackson immunoresearch, 016-030-084) was added at 1 : 1000 dilution, 100 pL / well, and incubated at room temperature for 1 hour. After washing the plates with wash buffer, 100 pL / well of substrate TMB solution (Thermo, 00-4201-56) was added, and the plates were incubated at room temperature for 3 minutes, and the reaction was stopped by adding 50 pL of stop solution (2N H2SO4). Colorimetric signal was generated and the signal was read at 450 nm using a plate reader (PE, Envision). Data were analyzed using GraphPad Prism 5, and IC50 values were calculated. The IC50 of anti-ST2 chimeric antibodies blocking IL33 / ST2 binding is shown in Table 7 and Figure 3

[0210] Table 7. IC50 of anti-ST2 chimeric antibodies blocking IL33 / ST2 binding in ELISA experiment

[0211]

[0212]

[0213] Example 7. Anti-ST2 antibody based cell binding assay

[0214] ​Based on the FACS method, the binding ability of anti-ST2 antibody to HEK293T cell lines overexpressing hST2 (HEK293T-hST2-NFκB-Luciferase) and HEK293T cell lines 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.

[0215] 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 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 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 Prism 5, 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. Figures 7-8 .

[0216] Table 8 EC50 of anti-ST2 chimeric antibodies binding to hST2 and Cyno-ST2 at cellular level

[0217]

[0218] NA: no assayable line

[0219] Example 8, Analysis of anti-ST2 antibodies blocking IL33 protein interacting with overexpressing hST2 cell line (HEK293T-hST2-NFκB-Luciferase)

[0220] HEK293T cells naturally express high level of IL1RAcP protein, on this basis, using lentivirus transfection system, pLenti6.3-hST2 plasmid and pLenti6.3-NFκB-Luciferasae plasmid were transfected into HEK293 cells to construct stable transfected cell line HEK293T-hST2-NFκB-Luciferase. IL33 binds to ST2, recruits IL-1RAcP, which can activate the downstream NFκB signaling pathway, thereby initiating the expression of Luciferase. When there is anti-ST2 antibody in the system, it can block the binding of IL33 to ST2, thereby blocking the expression of Luciferase, and the blocking activity of anti-ST2 antibody is evaluated by detecting the change in fluorescence intensity of the reaction substrate.

[0221] Take good HEK293T-hST2-NFκB-Luciferase cells in exponential growth phase, dilute to 2.5x10 5 6 cells / mL, add 20 μL per well to a 384-well plate (Thermo, 262360); then add 15 μL of gradient-diluted anti-ST2 antibody (initial concentration of 667 nM, diluted according to 4-fold concentration gradient) per well, incubate at 37°C for 30 minutes; after incubation, add 15 μL of hIL33 protein (final concentration of 50 pM) per well; mix well, incubate at 37°C for 5 hours. Then add 50 μL of ONE-Glo TM Luciferase Assay (Promega, E6120) per well, avoid light reaction for 2-5 minutes. Chemiluminescence signal was read by microplate reader (PE, Envision). Data were analyzed using GraphPad Prism5, and IC50 value was calculated. The IC50 of anti-ST2 chimeric antibodies blocking IL33 / ST2 signaling at cellular level is shown in Table 9 and Figure 9 .

[0222] Table 9 IC50 of anti-ST2 chimeric antibodies blocking IL33 / ST2 signaling at cellular level

[0223]

[0224] “ND” represents unmeasured.

[0225] Example 9: Analysis of the interaction between anti-ST2 antibody and naturally expressed hST2 cells (KU812-NFκB-Luciferasae) blocking IL33 protein interaction.

[0226] 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 cells. 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. Finally, 50 μL of ONE-Glo was added to each well. TM Luciferase Assay (Promega, E6120) was prepared and reacted 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.

[0227] Example 10: Anti-ST2 antibody blocks IL33 and IL2 co-stimulation of CD4 + Analysis of T cell interactions

[0228] Under conditions of co-stimulation with 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.

[0229] CD4+ T cells were sorted from human PBMCs using a human CD4+ T cell sorting kit (stemcell, 17952). + T cells, at 2.5 × 105 Cells were seeded at 60 μL / well in 96-well U-bottom cell culture plates, and then 15 μL of diluted anti-ST2 antibody (initial concentration of 53 nM, diluted according to a 3-fold concentration gradient) was added to each well, and incubated at 37°C for 30 min. After incubation, 15 μL of hIL33 protein (final concentration of 4 ng / mL) and 15 μL of IL2 (final concentration of 10 ng / mL) (PrimeGene, GMP-101-02) were added to each well, mixed, and incubated at 37°C for 48 h. After incubation, the supernatant was collected, and the IL5 content in the supernatant was detected according to the instructions of the IL5 detection kit (R&D, DY205). The OD450 value was read by an enzyme-labeled instrument (PE, Envision). The data was analyzed by GraphPad Prism5, and the IC50 value was calculated. The IC50 of the anti-ST2 chimeric antibody in blocking the IL33 / ST2-induced IL5 secretion on CD4 + The IC50 of the anti-ST2 chimeric antibody in blocking the IL33 / ST2-induced IL5 secretion on CD4

[0230] The IC50 of the anti-ST2 chimeric antibody in blocking the IL33 / ST2-induced IL5 secretion on CD4 + The IC50 of the anti-ST2 chimeric antibody in blocking the IL33 / ST2-induced IL5 secretion on CD4

[0231] Antibody IC50 (nM) RG6149 0.320 xi7D1 0.120 xi3C6 ND xi31C8 0.090 xi26A1 ND xi8F4 ND xi17E2 0.080

[0232] “ND” represents not detected

[0233] Example 11, Humanization of Anti-Human ST2 Hybridoma Monoclonal Antibody

[0234] The 31C8 (heavy chain variable region sequence SEQ ID NO: 39; light chain variable region sequence SEQ ID NO: 40) was selected for humanization design. Using the established CDR grafting method, human antibody framework regions that can be used for humanization of mouse antibodies were selected. The human germline antibody or its subtype with the highest sequence identity (i.e. sequence similarity) to the amino acid of the variable region of the mouse antibody was screened, the CDR of the heavy chain variable region and the light chain variable region of the mouse antibody was inserted into the screened framework region, and the residues in the framework region were mutated. Some of the CDRs were mutated on this basis to improve the properties of the antibody, such as improving the physicochemical properties or drugability. Humanized antibodies hz31C8-1.1 and hz31C8-1.2 were obtained, wherein 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 the 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 the 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 the humanized antibody hz31C8-1.2 are SEQ ID NO: 74 and SEQ ID NO: 80, respectively.

[0235] The humanized VL region gene synthesis fragment was ligated to the human kappa chain constant region by double enzyme digestion reaction to construct the humanized light chain, and the humanized VH region gene synthesis fragment was ligated to the human IgG2 constant region by double enzyme digestion reaction to construct the humanized heavy chain. The DNA corresponding to the humanized heavy chain and light chain of each antibody was transfected into an expression vector, protein expression was performed using the ExpiCHO expression system, and then the humanized antibody in the cell culture supernatant was purified using a Protein A column.

[0236] Example 12, Analysis of the biological function of humanized anti-ST2 antibodies

[0237] 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 subjected to affinity, binding, blocking and cell function analysis according to the methods of Example 4, Example 5, Example 6, Example 7, Example 8, Example 9 and Example 10. In the present disclosure, "hz" and "xi" refer to humanized and chimeric antibodies, respectively, e.g. "hz31C8-1.1" and "hz31C8-1.2" represent humanized 31C8-1.1 and 31C8-1.2 antibodies, "xi31C8" represents chimeric 31C8 antibody.

[0238] Affinity results of anti-ST2 humanized antibodies to ST2 are shown in Table 11; EC50 of anti-ST2 humanized antibodies to bind hST2 and Cyno-ST2 in ELISA experiments are shown in Table 12 and Figures 4-5 ; IC50 of anti-ST2 humanized antibodies to block IL33 / ST2 binding in ELISA experiments are shown in Table 13 and Figure 6 ; EC50 of anti-ST2 humanized antibodies to bind hST2 at the cellular level are shown in Table 14 and Figure 10 ; IC50 of anti-ST2 humanized antibodies to block IL33 / ST2 signaling at the cellular level are shown in Table 15 and Figures 11-12 ; IC50 of anti-ST2 humanized antibodies to block IL33 / ST2 induced IL5 secretion on CD4 + T cells are shown in Table 16 and Figure 13 .

[0239] Table 11 Affinity of anti-ST2 humanized antibodies to ST2

[0240]

[0241]

[0242] Table 12 EC50 of anti-ST2 humanized antibodies to bind hST2 and Cyno-ST2 in ELISA experiments

[0243]

[0244] Table 13 IC50 of anti-ST2 humanized antibodies to block IL33 / ST2 binding in ELISA experiments

[0245] Antibody IC50 (nM) GSK3772847 2.533 RG6149 2.151 xi31C8 1.933 hz31C8-1.1 1.893 hz31C8-1.2 2.241

[0246] Table 14 EC50 of anti-ST2 humanized antibodies to bind hST2 at the cellular level

[0247]

[0248] Table 15 IC50 of anti-ST2 humanized antibodies blocking IL33 / ST2 signaling at the cellular level

[0249]

[0250]

[0251] Table 16 IC50 of anti-ST2 humanized antibodies blocking IL33 / ST2 induced IL5 secretion at CD4 + T cells

[0252] Antibody IC50 (nM) GSK3772847 0.794 RG6149 1.819 xi31C8 0.440 hz31C8-1.1 0.462 hz31C8-1.2 0.366

[0253] Example 13, Stability analysis of humanized anti-ST2 antibodies

[0254] The thermal stability of different antibodies was detected by DSC (Differential scanning calorimetry), and the samples were dissolved in PBS buffer (pH 7.4) and detected by MicroCal VP-Capillary DSC (Malvern Panalytical). The results are shown in Table 17, and the humanized anti-ST2 antibodies hz31C8-1.1 and hz31C8-1.2 of the present disclosure both showed good thermal stability, and were superior to the control anti-ST2 antibodies RG6149 and GSK3772847.

[0255] Table 17 Thermal stability of antibodies detected by DSC

[0256] Antibody Tm onset (°C) Tm1 (°C) Tm2 (°C) Tm3 (°C) hz31C8-1.1 63.12 80.12 / / hz31C8-1.2 63.84 77.92 / / RG6149 57.47 64.51 76.94 / GSK3772847 57.24 64.70 71.32 76.29

[0257] The periodic stability of antibodies under certain concentration conditions was detected by the SEC-HPLC method. Exemplary conditions include controlling the antibody concentration at about 1 mg / mL, and comparing the stability of, for example, 40°C storage for 2 weeks in PBS buffer (pH 7.2). LC-20ADXR / DGU (Shimadzu) was used for detection. The results are shown in Table 18, and the humanized anti-ST2 antibodies hz31C8-1.1 and hz31C8-1.2 of the present disclosure both showed good stability.

[0258] Table 18 Periodic stability of antibodies detected by SEC-HPLC

[0259] Antibody Purity (main area %) Aggregate content (area %) Fragment content (area %) xi31C8 96.41 3.59 0 hz31C8-1.1 94.30 5.70 0 hz31C8-1.2 99.67 0.33 0 RG6149 93.21 6.79 0 GSK3772847 79.29 10.11 10.60

[0260] Anti-ST2 antibodies were diluted to a concentration of 1.5 mg / mL in PBS buffer and treated at 72 °C for 5 minutes. The binding activity of the samples to antigen was then measured by ELISA. The ELISA procedure was as follows: 96-well plates were coated with 100 μL / well of 2 μg / mL hST2-ECD-mFc antigen at 4 °C overnight. After washing away unabsorbed antigen, non-specific binding sites were blocked with blocking buffer (PBST containing 1% bovine serum albumin). After washing the plates three times with wash buffer (PBS containing 0.05% (v / v) Tween 20, PBST), 100 μL / well of anti-ST2 antibody (initial concentration of 1.5 μg / mL, diluted at 3-fold concentration gradient to 8 concentrations) was added and incubated at 37 °C for 1 hour. After washing the plates with wash buffer, anti-Human IgG Fc gamma secondary antibody (Jackson ImmunoResearch, 109-035-008) was added and incubated at 37 °C for 1 hour. After washing the plates with wash buffer, 100 μL / well of substrate TMB solution (Thermo, 00-4201-56) was added for color development. After incubation at room temperature for 5 minutes, the reaction was stopped with stop solution (2N H2SO4). The signal was read at 450 nm using a plate reader (PE, Envision) and the data was analyzed using GraphPad Prism 5 to calculate the EC50 value. The results are shown in Table 19 below. Humanized anti-ST2 antibodies hz31C8-1.1 and hz31C8-1.2 of the present disclosure both exhibited good thermal stability.

[0261] Table 19. ELISA detection of binding activity of antibodies after heating

[0262]

[0263] Example 14. Cross-reactivity of humanized anti-ST2 antibodies with IL1R family receptors

[0264] ST2 belongs to the IL1R family of receptors. The cross-reactivity of anti-ST2 antibodies with IL1R family receptors, including 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), IL1R9 / IL1RAPL2 (Sino biological, 10156-H08H), was determined using ELISA. ELISA was performed using the above proteins as antigens (2 μg / mL). The specific ELISA procedure is described in Example 13. No binding means that the antibody still has no binding with the antigen at the highest concentration; weak binding means that the antibody has weak binding with the antigen at the highest concentration, and has no binding at low concentrations; strong binding means that the binding data can fit a good binding curve.

[0265] Table 20. ELISA method for detecting the cross-reactivity of humanized ST2 antibodies with IL1R family receptors

[0266]

[0267] (-: no binding; +: weak binding; +++++: strong binding)

[0268] Example 15. Pharmacokinetic evaluation of humanized anti-ST2 antibodies

[0269] Experimental Balb / c mice 18, 6 in each group, 12 / 12 hours observation and adjustment, free access to food and water, purchased from Nanjing University Model Animal Center. On the day of the experiment, the mice were intravenously injected with drugs, with a dose of 10 mg / kg and a sample concentration of 1 mg / mL. Blood was collected from the orbit before administration (0 min, which can be advanced for more than one day), 30 min, 8 h, 24 h, 2 d (48 h), 4 d (96 h), 7 d, 14 d after administration, and the serum was collected. Then the antibody concentration in the serum was detected by ELISA method. The detection process is as follows: 2 μg / mL of ST2-His (sinobiological, 10105-H08H) is used as antigen, coated in 96-well plates at 100 μL / well, and incubated at 4°C overnight. After washing the unabsorbed antigen thoroughly, non-specific binding sites are blocked with blocking buffer (containing 1% bovine serum albumin in PBST). After washing the plate three times with washing buffer (PBS with 0.05% (v / v) Tween 20, PBST), 100 μL of the serum to be tested is added, and incubated at 37°C for 1 h. After washing the plate with washing buffer, anti-Mouse IgG Fcγ secondary antibody is added, and incubated at 37°C for 1 h. After washing the plate with washing buffer, 100 μL / well of substrate TMB solution (Thermo, 00-4201-56) is added for color development, and incubated at room temperature for 5 minutes. Then the reaction is terminated with termination solution (2N H2SO4). The signal is read at 450 nm using a plate reader (PE, Envision), and the data is analyzed using GraphPad Prism5. Referring to Table 21, the PK analysis results show that the half-life of the humanized anti-ST2 antibodies hz31C8-1.1 and hz31C8-1.2 of the present disclosure in mice is about 9.7 days and 7.33 days, which is better than the control anti-ST2 antibody RG6149.

[0270] Table 21 T1 / 2 of humanized ST2 antibodies in mice

[0271] Antibody T1 / 2 (mean ± SD, d) RG6149 4.61±2.40 hz31C8-1.1 9.70±3.11 hz31C8-1.2 7.33±4.37

[0272] Although the present disclosure has been described in some detail by way of general instructions and specific embodiments, certain modifications or improvements can be made to the present disclosure, which will be apparent to those skilled in the art. Therefore, these modifications or improvements made on the basis of the present disclosure without departing from the spirit of the present disclosure shall fall within the scope of protection of the present disclosure.

Claims

1. An isolated ST2 antibody or antigen-binding fragment thereof, comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein, (1) the heavy chain CDR1 is set forth in SEQ ID NO: 12, the heavy chain CDR2 is set forth in SEQ ID NO: 17, the heavy chain CDR3 is set forth in SEQ ID NO: 22, the light chain CDR1 is set forth in SEQ ID NO: 27, the light chain CDR2 is set forth in SEQ ID NO: 30, and the light chain CDR3 is set forth in SEQ ID NO: 34, wherein the amino acid sequence of SEQ ID NO: 17 is AIDPETGDTVYXiX2KFX3G, the amino acid sequence of SEQ ID NO: 30 is QX4SNLAS, wherein Xi is N, X2 is Q, X3 is K, X4 is M; (2) the heavy chain CDR1 is set forth in SEQ ID NO: 12, the heavy chain CDR2 is set forth in SEQ ID NO: 17, the heavy chain CDR3 is set forth in SEQ ID NO: 22, the light chain CDR1 is set forth in SEQ ID NO: 27, the light chain CDR2 is set forth in SEQ ID NO: 30, and the light chain CDR3 is set forth in SEQ ID NO: 34, wherein the amino acid sequence of SEQ ID NO: 17 is AIDPETGDTVYXiX2KFX3G, the amino acid sequence of SEQ ID NO: 30 is QX4SNLAS, wherein Xi is A, X2 is E, X3 is Q, X4 is L; or (3) the heavy chain CDR1 is set forth in SEQ ID NO: 12, the heavy chain CDR2 is set forth in SEQ ID NO: 17, the heavy chain CDR3 is set forth in SEQ ID NO: 22, the light chain CDR1 is set forth in SEQ ID NO: 27, the light chain CDR2 is set forth in SEQ ID NO: 30, and the light chain CDR3 is set forth in SEQ ID NO: 34, wherein the amino acid sequence of SEQ ID NO: 17 is AIDPETGDTVYXiX2KFX3G, the amino acid sequence of SEQ ID NO: 30 is QX4SNLAS, wherein Xi is A, X2 is K, X3 is K, X4 is L.

2. The antibody or antigen-binding fragment thereof of claim 1, which is chimeric or humanized.

3. The antibody or antigen-binding fragment thereof of claim 1, which comprises: (i) a heavy chain variable region comprising a sequence set forth in SEQ ID NOs: 39, 71, or 73, or an amino acid sequence that is at least 80% identical to a sequence set forth in SEQ ID NOs: 39, 71, or 73; (ii) a light chain variable region comprising a sequence as set forth in SEQ ID NOs: 40 or 79, or an amino acid sequence that is at least 80% identical to a sequence as set forth in SEQ ID NOs: 40 or 79; or (iii) a heavy chain variable region as set forth in (i) and a light chain variable region as set forth in (ii).

4. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region and a light chain variable region, wherein, (1) the heavy chain variable region comprises a sequence as set forth in SEQ ID NO: 39 or an amino acid sequence that is at least 80% identical thereto, and the light chain variable region comprises a sequence as set forth in SEQ ID NO: 40 or an amino acid sequence that is at least 80% identical thereto; (2) the heavy chain variable region comprises a sequence as set forth in SEQ ID NO: 71 or an amino acid sequence that is at least 80% identical thereto, and the light chain variable region comprises a sequence as set forth in SEQ ID NO: 79 or an amino acid sequence that is at least 80% identical thereto; or (3) the heavy chain variable region comprises a sequence as set forth in SEQ ID NO: 73 or an amino acid sequence that is at least 80% identical thereto, and the light chain variable region comprises a sequence as set forth in SEQ ID NO: 79 or an amino acid sequence that is at least 80% identical thereto.

5. The antibody or antigen-binding fragment thereof of any one of claims 1-4, comprising: (i) a heavy chain comprising a sequence as set forth in SEQ ID NOs: 67, 75, or 77, or an amino acid sequence that is at least 80% identical to a sequence as set forth in SEQ ID NOs: 67, 75, or 77; (ii) a light chain comprising a sequence as set forth in SEQ ID NOs: 69 or 81, or an amino acid sequence that is at least 80% identical to a sequence as set forth in SEQ ID NOs: 69 or 81; or (iii) a heavy chain as set forth in (i) and a light chain as set forth in (ii).

6. The antibody or antigen-binding fragment thereof of any one of claims 1-4, comprising a heavy chain and a light chain, wherein, (1) the heavy chain comprises a sequence as set forth in SEQ ID NO: 67 or an amino acid sequence that is at least 80% identical thereto, and the light chain comprises a sequence as set forth in SEQ ID NO: 69 or an amino acid sequence that is at least 80% identical thereto; (2) the heavy chain comprises a sequence as set forth in SEQ ID NO: 75 or an amino acid sequence that is at least 80% identical thereto, and the light chain comprises a sequence as set forth in SEQ ID NO: 81 or an amino acid sequence that is at least 80% identical thereto; or (3) the heavy chain comprises a sequence as set forth in SEQ ID NO: 73 or an amino acid sequence that is at least 80% identical thereto, and the light chain comprises a sequence as set forth in SEQ ID NO: 79 or an amino acid sequence that is at least 80% identical thereto. (3) the heavy chain comprises a sequence set forth in SEQ ID NO: 77 or an amino acid sequence that is at least 80% identical thereto, and the light chain comprises a sequence set forth in SEQ ID NO: 81 or an amino acid sequence that is at least 80% identical thereto.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the antibody or antigen-binding fragment thereof is selected from a monoclonal antibody, a Fab fragment, a F(ab')2 fragment, or a Fv fragment.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the antibody or antigen-binding fragment thereof is selected from a single chain Fv molecule.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the antibody or antigen-binding fragment thereof specifically binds to human or monkey ST2 and blocks the binding of IL33 / ST2 and its signaling.

10. The antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein the antibody or antigen-binding fragment thereof blocks IL5 secretion induced by IL33 / ST2 on CD4 + IL5 secretion induced by IL33 / ST2 on T cells.

11. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-10.

12. An expression vector comprising the nucleic acid molecule of claim 11.

13. A host cell comprising the nucleic acid molecule of claim 11 or the expression vector of claim 12.

14. A polypeptide comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-10.

15. A viral vector comprising a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-10 or the nucleic acid molecule of claim 11.

16. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-10, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

17. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-10 or the pharmaceutical composition of claim 16 in the manufacture of a medicament for treating IL33 / ST2-mediated related diseases and disorders, which are asthma and chronic obstructive pulmonary disease.

18. The use according to claim 17, wherein, the antibody or antigen-binding fragment thereof is co-administered with one or more other antibodies or other drugs.

19. The use according to claim 18, wherein, the other antibody is selected from a TSLP antibody, a TSLPR antibody, an IL4 antibody, an IL4R antibody, an IL13 antibody, an IL13R antibody, an IL5 antibody, an IL5R antibody, or an IgE antibody.

20. The use of claim 18, wherein, the other drug is selected from an anti-asthma drug, an anti-chronic obstructive pulmonary disease drug, or an anti-atopic dermatitis drug.

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