Bifunctional fusion proteins and their uses

Abstract

The present invention provides a bifunctional fusion protein that targets both TGFβ ligand and AREG and simultaneously blocks TGFβ and AREG signaling. The present invention further provides a nucleic acid molecule encoding the bifunctional fusion protein, an expression vector for producing the bifunctional fusion protein, a host cell for producing the bifunctional fusion protein, and a method for preparing and / or characterizing the bifunctional fusion protein.

Description

[Background technology]

[0001] Transforming growth factor-β (TGFβ) is a pleiotropic cytokine that plays important roles under physiological and pathological conditions. TGFβ isomers, β1, β2, and β3, are approximately 25 kDa homodimeric polypeptides. These ligands transduce signals through cell surface receptors (including transforming growth factor-β receptors I, II, and III (TRI, TRII, and TRIII)) and intracellular SMAD effector proteins, such as SMAD2 and 3. Signal transduction influences cellular processes, including cell survival, proliferation, differentiation, cell motility, and extracellular matrix (ECM) production.

[0002] TGF-β is thought to be a central regulator of fibrogenesis. Studies have shown that TGF-β1 induces fibrosis in multiple organs by activating myofibroblasts, causing excessive production of ECM components, and inhibiting ECM degradation. Blocking TGF-β signaling can prevent and inhibit abnormal remodeling and scar formation in many organs, including the lung, liver, and kidney. TGF-β has also been shown to play an important role in the immune system and is considered one of the most effective immunosuppressants in both the innate and adaptive immune responses. Furthermore, TGF-β has been reported to exhibit tumor-promoting activity in certain cancers by acting directly on tumor cells and / or the tumor environment.

[0003] Anti-TGFβ therapies are being developed for fibrosis, certain cancers, and other diseases. TGFβ inhibitors include antisense oligonucleotides, small molecules that inhibit receptor kinase activity, monoclonal antibodies against TGFβ ligands or receptors, and bifunctional proteins engineered to trap TGFβ. Specifically, TGFβ traps involve engineered modifications of TGFβ receptors through artificial dimerization of the extracellular domains of these receptors.

[0004] Amphiregulin (AREG) is a low-affinity ligand of the epidermal growth factor (EGF) family. The AREG protein is synthesized from a 252-amino acid transmembrane precursor and undergoes proteolytic cleavage within the extracellular domain by plasma membrane proteases, primarily tumor necrosis factor-α-invertase (TACE). Mature soluble AREG then directly binds to the epidermal growth factor receptor (EGFR) to activate downstream signaling. This triggers major intracellular signaling cascades, including MAPK / ERK signaling, which regulate cell survival, proliferation, and motility.

[0005] In pulmonary fibrosis models and patients with idiopathic pulmonary fibrosis (IPF), AREG is specifically upregulated in type II pneumocytes (AT2s). Some reports demonstrate that AREG is both necessary and sufficient for the development of pulmonary fibrosis. Specifically, in a progressive pulmonary fibrosis model, reducing AREG expression significantly attenuates the development of pulmonary fibrosis. Overexpression of AREG in mouse AT2s induces lung remodeling and fibrotic changes. Furthermore, AREG expression levels are upregulated in liver and kidney fibrosis, and AREG has been reported to be necessary for the development of liver, kidney, and skin fibrosis. Therefore, as a profibrotic factor, AREG is an attractive target point not only for pulmonary fibrosis but also for fibrosis in other organs.

[0006] AREG-EGFR signaling also plays a role in the immune system and tumorigenesis. AREG is expressed in various immune cells under inflammatory conditions. The presence of AREG in various types of immune cells and the activation patterns of these immune cells suggest that immune-derived AREG is involved in type 2 immune-mediated (Th2) resistance and tolerance mechanisms. Furthermore, AREG is upregulated in various cancers. Functional studies demonstrate that AREG functions as a proto-oncogene in certain cancers. These findings suggest that targeting AREG activity may represent a novel approach for the treatment of chronic inflammation-related diseases and cancer.

[0007] However, to date, no combination of two separate drugs with anti-TGFβ and anti-AREG activity, or a therapy that delivers a single bifunctional protein that simultaneously has the ability to inhibit TGFβ and AREG activity, has been proposed or tested for the treatment of the above-mentioned diseases (including diseases associated with fibrosis, cancer, and chronic inflammation). Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention provides a bifunctional fusion protein that simultaneously targets a TGFβ ligand and AREG and simultaneously blocks TGFβ and AREG signaling. The bifunctional fusion protein is an ideal candidate for treating fibrotic diseases, including but not limited to renal fibrosis, liver fibrosis, and lung fibrosis, particularly IPF, cancer, and diseases associated with chronic inflammation. The present application also provides nucleic acid molecules encoding the bifunctional fusion protein, expression vectors for producing the bifunctional fusion protein, host cells for producing the bifunctional fusion protein, and methods for preparing and / or characterizing the bifunctional fusion protein. The present invention also provides uses of the bifunctional fusion protein for treating, preventing, and / or diagnosing diseases, such as fibrotic diseases, including but not limited to renal fibrosis, liver fibrosis, and lung fibrosis, particularly IPF, cancer, and diseases associated with chronic inflammation. [Means for solving the problem]

[0009] In a first aspect, the present invention provides a bifunctional fusion protein, which comprises at least two domains, wherein the domains are capable of binding to AREG or a fragment thereof and / or capable of binding to a TGFβ ligand or a fragment thereof.

[0010] In some embodiments, the bifunctional fusion protein comprises a first domain and a second domain, wherein the first domain is capable of binding to AREG or a fragment thereof, and the second domain is capable of chelating to a TGFβ ligand or a fragment thereof.

[0011] In some embodiments, the first domain is an antibody or antigen-binding fragment thereof that binds to AREG or a fragment thereof, and the second domain is at least a portion of the extracellular domain of TGFβ receptor II (TGFβRII, TRII) or a mutant thereof.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof is an anti-AREG antibody or fragment thereof that can bind to both human AREG (hAREG) and mouse AREG (mAREG).

[0013] In some embodiments, the anti-AREG antibody or fragment thereof according to the present invention is a human anti-AREG antibody, a murine anti-AREG antibody, a chimeric anti-AREG antibody, or a humanized anti-AREG antibody.

[0014] In some embodiments, the anti-AREG antibody or fragment thereof according to the present invention is capable of binding to a soluble form of AREG. Preferably, the anti-AREG antibody is capable of binding to the EGF-like domain of a soluble form of AREG.

[0015] In some embodiments, the anti-AREG antibody or fragment thereof according to the present invention is a single-chain antibody, a disulfide-linked Fv, a DART, a diabody, or a fragment containing a VL or VH domain. The fragment may be an IgG, Fab, Fab', F(ab')2, Fv, or scFv. The fragment also includes any synthetic or genetically engineered protein containing an immunoglobulin variable region that functions like an antibody by binding to a specific antigen to form a complex. Regardless of structure, an antibody fragment binds to the same antigen recognized by the intact antibody.

[0016] In some embodiments, an anti-AREG antibody or fragment thereof according to the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein:

[0017] HCDR1, HCDR2, and HCDR3 are as follows: (1) HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, and HCDR3 shown in SEQ ID NO:3; (2) HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, and HCDR3 shown in SEQ ID NO:4; (3) HCDR1 shown in SEQ ID NO:5, HCDR2 shown in SEQ ID NO:2, and HCDR3 shown in SEQ ID NO:6; (4) HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, and HCDR3 shown in SEQ ID NO:9; (5) HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:10, and HCDR3 shown in SEQ ID NO:9; (6) HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, and HCDR3 shown in SEQ ID NO:9. (7) HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, HCDR3 shown in SEQ ID NO:12, (8) HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:13, HCDR3 shown in SEQ ID NO:14, (9) HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:13, HCDR3 shown in SEQ ID NO:140, (10) HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:15, HCDR3 shown in SEQ ID NO:16, (11) HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:18, HCDR3 shown in SEQ ID NO:19, (12) HCDR1 shown in SEQ ID NO:17, SEQ ID HCDR2 as set forth in SEQ ID NO: 18, HCDR3 as set forth in SEQ ID NO: 20, and (13) HCDR1, HCDR2, HCDR3 as set forth in (1) to (12), wherein at least one of them is selected from the group consisting of one, two, three, four, or five amino acid additions, deletions, conservative amino acid substitutions, or a combination thereof; andLCDR1, LCDR2, and LCDR3 are (1) LCDR1 shown in SEQ ID NO:21, LCDR2 shown in SEQ ID NO:22, and LCDR3 shown in SEQ ID NO:23; (2) LCDR1 shown in SEQ ID NO:21, LCDR2 shown in SEQ ID NO:22, and LCDR3 shown in SEQ ID NO:24; (3) LCDR1 shown in SEQ ID NO:25, LCDR2 shown in SEQ ID NO:26, and LCDR3 shown in SEQ ID NO:27; (4) LCDR1 shown in SEQ ID NO:28, LCDR2 shown in SEQ ID NO:29, and LCDR3 shown in SEQ ID NO:30; (5) LCDR1 shown in SEQ ID NO:31, LCDR2 shown in SEQ ID NO:32, and LCDR3 shown in SEQ ID NO:30; (6) LCDR1 shown in SEQ ID NO:33, LCDR2 shown in SEQ ID NO:34, and LCDR3 shown in SEQ ID NO:35. LCDR2 shown in SEQ ID NO:34, LCDR3 shown in SEQ ID NO:30, (7) LCDR1 shown in SEQ ID NO:35, LCDR2 shown in SEQ ID NO:34, LCDR3 shown in SEQ ID NO:30, (8) LCDR1 shown in SEQ ID NO:36, LCDR2 shown in SEQ ID NO:37, LCDR3 shown in SEQ ID NO:38, (9) LCDR1 shown in SEQ ID NO:39, LCDR2 shown in SEQ ID NO:40, LCDR3 shown in SEQ ID NO:38, (10) LCDR1 shown in SEQ ID NO:41, LCDR2 shown in SEQ ID NO:42, LCDR3 shown in SEQ ID NO:38, (11) LCDR1 shown in SEQ ID NO:43, LCDR2 shown in SEQ ID NO:44, LCDR3 shown in SEQ ID NO:38, (12) LCDR LCDR1 shown in SEQ ID NO:39, LCDR2 shown in SEQ ID NO:40, LCDR3 shown in SEQ ID NO:38, (13) LCDR1 shown in SEQ ID NO:45, LCDR2 shown in SEQ ID NO:42, LCDR3 shown in SEQ ID NO:46, (14) LCDR1 shown in SEQ ID NO:47, SEQ IDLCDR2 shown in SEQ ID NO:44, LCDR3 shown in SEQ ID NO:46, (15) LCDR1 shown in SEQ ID NO:48, LCDR2 shown in SEQ ID NO:37, LCDR3 shown in SEQ ID NO:49, (16) LCDR1 shown in SEQ ID NO:50, LCDR2 shown in SEQ ID NO:40, LCDR3 shown in SEQ ID NO:51, (17) LCDR1 shown in SEQ ID NO:50, LCDR2 shown in SEQ ID NO:40, LCDR3 shown in SEQ ID NO:52, (18) LCDR1 shown in SEQ ID NO:50, LCDR2 shown in SEQ ID NO:40, LCDR3 shown in SEQ ID NO:53, (19) LCDR1 shown in SEQ ID NO:54, LCDR2 shown in SEQ ID NO:42, LCDR3 shown in SEQ ID NO:55, (20) SEQ ID LCDR1 shown in SEQ ID NO: 56, LCDR2 shown in SEQ ID NO: 44, LCDR3 shown in SEQ ID NO: 55, and LCDR1, LCDR2, LCDR3 as shown in (21)(1) to (20), at least one of which is selected from the group consisting of one, two, three, four, or five amino acid additions, deletions, conservative amino acid substitutions, or combinations thereof.

[0018] In one embodiment, an anti-AREG antibody or fragment thereof according to the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein:

[0019] HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are: (1) HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:3, LCDR1 shown in SEQ ID NO:21, LCDR2 shown in SEQ ID NO:22 and LCDR3 shown in SEQ ID NO:23; (2) HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:4, LCDR1 shown in SEQ ID NO:21, LCDR2 shown in SEQ ID NO:22 and LCDR3 shown in SEQ ID NO:24; (3) HCDR1 shown in SEQ ID NO:5, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:6, LCDR1 shown in SEQ ID NO:25, HCDR LCDR2 shown in SEQ ID NO:26, LCDR3 shown in SEQ ID NO:27, (4) HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, HCDR3 shown in SEQ ID NO:9, LCDR1 shown in SEQ ID NO:28, LCDR2 shown in SEQ ID NO:29, LCDR3 shown in SEQ ID NO:30, (5) HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:10, HCDR3 shown in SEQ ID NO:9, LCDR1 shown in SEQ ID NO:31, LCDR2 shown in SEQ ID NO:32, LCDR3 shown in SEQ ID NO:30, (6) HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, HCDR3 shown in SEQ ID NO:11, LCDR1 shown in SEQ ID NO:33, SEQ ID LCDR2 shown in SEQ ID NO:34, LCDR3 shown in SEQ ID NO:30, (7) HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:35, LCDR2 shown in SEQ ID NO:34, SEQ IDLCDR3 shown in SEQ ID NO:30, (8) HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:13, HCDR3 shown in SEQ ID NO:14, LCDR1 shown in SEQ ID NO:36, LCDR2 shown in SEQ ID NO:37, LCDR3 shown in SEQ ID NO:38, (9) HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:13, HCDR3 shown in SEQ ID NO:140, LCDR1 shown in SEQ ID NO:39, LCDR2 shown in SEQ ID NO:40, LCDR3 shown in SEQ ID NO:38, (10) HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:13, HCDR3 shown in SEQ ID NO:140, LCDR1 shown in SEQ ID NO:41, LCDR2 shown in SEQ ID NO:42, SEQ ID LCDR3 shown in SEQ ID NO: 38, (11) HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 13, HCDR3 shown in SEQ ID NO: 140, LCDR1 shown in SEQ ID NO: 43, LCDR2 shown in SEQ ID NO: 44, LCDR3 shown in SEQ ID NO: 38, (12) HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 15, HCDR3 shown in SEQ ID NO: 16, LCDR1 shown in SEQ ID NO: 39, LCDR2 shown in SEQ ID NO: 40, LCDR3 shown in SEQ ID NO: 38, (13) HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 15, HCDR3 shown in SEQ ID NO: 16, LCDR1 shown in SEQ ID NO: 45, LCDR2 shown in SEQ ID NO: 42, SEQ ID (14) HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 15, HCDR3 shown in SEQ ID NO: 16, LCDR1 shown in SEQ ID NO: 47, LCDR2 shown in SEQ ID NO: 44, SEQ IDLCDR3 shown in SEQ ID NO: 46, (15) HCDR1 shown in SEQ ID NO: 17, HCDR2 shown in SEQ ID NO: 18, HCDR3 shown in SEQ ID NO: 19, LCDR1 shown in SEQ ID NO: 48, LCDR2 shown in SEQ ID NO: 37, LCDR3 shown in SEQ ID NO: 49, (16) HCDR1 shown in SEQ ID NO: 17, HCDR2 shown in SEQ ID NO: 18, HCDR3 shown in SEQ ID NO: 20, LCDR1 shown in SEQ ID NO: 50, LCDR2 shown in SEQ ID NO: 40, LCDR3 shown in SEQ ID NO: 51, (17) HCDR1 shown in SEQ ID NO: 17, HCDR2 shown in SEQ ID NO: 18, HCDR3 shown in SEQ ID NO: 20, LCDR1 shown in SEQ ID NO: 50, LCDR2 shown in SEQ ID NO: 40, SEQ ID LCDR3 shown in SEQ ID NO: 52, (18) HCDR1 shown in SEQ ID NO: 17, HCDR2 shown in SEQ ID NO: 18, HCDR3 shown in SEQ ID NO: 20, LCDR1 shown in SEQ ID NO: 50, LCDR2 shown in SEQ ID NO: 40, LCDR3 shown in SEQ ID NO: 53, (19) HCDR1 shown in SEQ ID NO: 17, HCDR2 shown in SEQ ID NO: 18, HCDR3 shown in SEQ ID NO: 20, LCDR1 shown in SEQ ID NO: 54, LCDR2 shown in SEQ ID NO: 42, LCDR3 shown in SEQ ID NO: 55, (20) HCDR1 shown in SEQ ID NO: 17, HCDR2 shown in SEQ ID NO: 18, HCDR3 shown in SEQ ID NO: 20, LCDR1 shown in SEQ ID NO: 56, LCDR2 shown in SEQ ID NO: 44, SEQ IDThe LCDR3 shown in NO:55, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 shown in (21)(1) to (20), at least one of which is selected from the group consisting of those containing one, two, three, four, or five amino acid additions, deletions, conservative amino acid substitutions, or combinations thereof.

[0020] In some embodiments, an anti-AREG antibody or fragment thereof according to the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence selected from SEQ ID NOs: 57 to 69, or an amino acid sequence that has at least 95% sequence identity with any one of SEQ ID NOs: 57 to 69 and retains epitope-binding activity; and Here, the light chain variable region has an amino acid sequence selected from SEQ ID NOs: 70 to 89, or an amino acid sequence that has at least 95% sequence identity with any one of SEQ ID NOs: 70 to 89 and retains epitope binding activity.

[0021] In some embodiments, the anti-AREG antibody or fragment thereof according to the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are selected from the group consisting of (1) SEQ ID NO: 57 and SEQ ID NO: 70, (2) SEQ ID NO: 58 and SEQ ID NO: 71, (3) SEQ ID NO: 59 and SEQ ID NO: 72, (4) SEQ ID NO: 60 and SEQ ID NO: 73, (5) SEQ ID NO: 61 and SEQ ID NO: 74, (6) SEQ ID NO: 62 and SEQ ID NO: 75, (7) SEQ ID NO: 63 and SEQ ID NO: 76, (8) SEQ ID NO: 64 and SEQ ID NO: 77, (9) SEQ ID NO: 65 and SEQ ID NO: 78, (10) SEQ ID NO: 66 and SEQ ID NO: 79, and (11) SEQ ID NO: 66 and SEQ ID NO: 79. NO:80, (12) SEQ ID NO:66 and SEQ ID NO:81, (13) SEQ ID NO:67 and SEQ ID NO:79, (14) SEQ ID NO:67 and SEQ ID NO:82, (15) SEQ ID NO:67 and SEQ ID NO:83, (16) SEQ ID NO:68 and SEQ ID NO:84, (17) SEQ ID NO:69 and SEQ ID NO:85, (18) SEQ ID NO:69 and SEQ ID NO:86, (19) SEQ ID NO:69 and SEQ ID NO:87, (20) SEQ ID NO:69 and SEQ ID NO:88, (21) SEQ ID NO:69 and SEQ ID No. 89 and (22) each having an amino acid sequence selected from two amino acid sequences that have at least 95% sequence identity with any one of (1) to (21) and retain epitope binding activity.

[0022] In some embodiments, anti-AREG antibodies or fragments thereof according to the present invention are of the IgG, IgM, IgA, IgE, or IgD isotype or a mutant thereof. In some embodiments, anti-AREG antibodies or fragments thereof according to the present invention are of the IgG1, IgG2, IgG3, or IgG4 isotype or a mutant thereof.

[0023] In some embodiments, an antibody of the invention is a human monoclonal antibody (mAb), a murine mAb, a humanized mAb, or a chimeric mAb.

[0024] Preferably, the humanized monoclonal antibodies (mAbs) of the present invention are comprised of constant regions derived from human constant regions.

[0025] Preferably, the humanized monoclonal antibodies (mAbs) of the invention have a human light chain constant region derived from a kappa or lambda light chain constant region.

[0026] Preferably, the humanized monoclonal antibodies (mAbs) of the invention have a human heavy chain constant region derived from a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.

[0027] In some embodiments, the second domain is the extracellular domain of TRII or a variant thereof.

[0028] In some embodiments, the variant of the TRII extracellular domain is a variant comprising a point mutation and / or a deletion.

[0029] In some embodiments, the extracellular domain of TRII has the amino acid sequence set forth in SEQ ID NO:90, amino acids numbered 1 to 136 from the N-terminus to the C-terminus, or an amino acid sequence having at least 85% sequence identity to SEQ ID NO:90.

[0030] In some embodiments, the point mutations include one or more point mutations selected from K7, T16, D17, R34, R66, K67, K103 and K104, based on the numbering from the N-terminus to the C-terminus of SEQ ID NO:90.

[0031] In some embodiments, the point mutations include one or more point mutations selected from K7Q, T16S, D17N, R34S, R34H, R66S, K67S, K103S and K104S, based on the numbering from the N-terminus to the C-terminus of SEQ ID NO:90.

[0032] In some embodiments, the point mutations include T16S and D17N.

[0033] In some embodiments, the point mutations include K7Q and D17N.

[0034] In some embodiments, the point mutation comprises K7Q.

[0035] In some embodiments, the point mutation comprises R34S.

[0036] In some embodiments, the point mutation comprises R34H.

[0037] In some embodiments, the point mutations include R66S and K67S.

[0038] In some embodiments, the point mutations include K103S and K104S.

[0039] In some embodiments, the point mutations include K7Q and R34S.

[0040] In some embodiments, the point mutations include K7Q, R66S, and K67S.

[0041] In some embodiments, the point mutations include K7Q, K103S, and K104S.

[0042] In some embodiments, the point mutations include K7Q, R34S, R66S, and K67S.

[0043] In some embodiments, the point mutations include K7Q, R34S, K103S, and K104S.

[0044] In some embodiments, the point mutations include K7Q, R66S, K67S, K103S, K104S.

[0045] In some embodiments, the point mutations include K7Q, R34S, R66S, K67S, K103S, and K104S.

[0046] In some embodiments, variants of the TRII extracellular domain having one or two mutations selected from K7Q, R34S, R66S, K67S, K103S and K104S exhibit reduced splicing of the bifunctional fusion protein.

[0047] In some embodiments, variants of the TRII extracellular domain having a mutation selected from R34S, R66S, K67S, K103S and K104S exhibit reduced splicing.

[0048] In some embodiments, a variant of the TRII extracellular domain having the mutation K7Q exhibits a significant reduction in splicing.

[0049] In some embodiments, the variants of the TRII extracellular domain are variants having N-terminal deletions, preferably 4 amino acids, 7 amino acids, 9 amino acids, 13 amino acids, 17 amino acids and 21 amino acids, based on the amino acid numbering from the N-terminus to the C-terminus of SEQ ID NO:90.

[0050] In some embodiments, the mutant TRII extracellular domain is a mutant containing point mutations T16S and D17N and having an N-terminal 7 amino acid deletion.

[0051] In some embodiments, the second domain is the extracellular domain of TRII or a variant thereof, having an amino acid sequence set forth in any one of SEQ ID NOs:90-107, or an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs:90-107.

[0052] In some embodiments, the C-terminus of the first domain is linked to the N-terminus of the second domain via a linker, or vice versa.

[0053] The linker may be a small molecule, a PEG polymer or a linker peptide. Preferably, the linker is a linker peptide.

[0054] In some embodiments, the first and second domains are linked by a short linker peptide of 2 to about 30 amino acids, preferably 6 to 26 amino acids. The linker can be rich in glycine to increase flexibility, or can contain serine, threonine, glutamic acid, alanine, or lysine to increase solubility. The C-terminus of the heavy or light chain of the anti-AREG antibody can be linked to the N-terminus of the extracellular domain of TRII or a mutant thereof, or vice versa. The linker peptide can be (G4S) n , (G4S) n G, S (G4S) n G, SG (EAAAK) n SG, S(GEGES)nG, (EAAAK) nwhere n is an integer of 1 to 5. In some embodiments, the linker can comprise an amino acid sequence selected from SEQ ID NOs: 108 to 117. In some embodiments, the C-terminus of the heavy chain or light chain of an anti-AREG antibody, preferably the C-terminus of the heavy chain, is linked directly to the N-terminus of the extracellular domain of TRII or a mutant thereof or via a linker peptide, or vice versa.

[0055] In some embodiments, the N-terminus of the heavy or light chain of an anti-AREG antibody, preferably the N-terminus of the heavy chain, is linked directly to the C-terminus of the extracellular domain of TRII or a variant thereof, or vice versa, via a linker peptide.

[0056] Such bifunctional fusion proteins still retain the specificity of the original immunoglobulin despite the introduction of the linker.

[0057] In some embodiments, the bifunctional fusion protein according to the present invention comprises a heavy or light chain, preferably a heavy chain, of an anti-AREG antibody linked directly or via a linker to the extracellular domain of TRII or a mutant thereof.

[0058] In some embodiments, a bifunctional fusion protein according to the present invention comprises the heavy or light chain of an anti-AREG antibody, preferably the heavy chain, the N-terminus of which is linked directly or via a linker to the C-terminus of the extracellular domain of TRII or a mutant thereof.

[0059] In some embodiments, a bifunctional fusion protein according to the present invention comprises the heavy or light chain of an anti-AREG antibody, preferably the heavy chain, the C-terminus of which is linked directly or via a linker to the N-terminus of the extracellular domain of TRII or a mutant thereof.

[0060] In some embodiments, a bifunctional fusion protein according to the present invention comprises the heavy chain of an anti-AREG antibody, the C-terminus of which is linked via a linker to the N-terminus of the extracellular domain of TRII. In some embodiments, a bifunctional fusion protein according to the present invention comprises the amino acid sequence set forth in any one of SEQ ID NOs: 118 to 139, or an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 118 to 139.

[0061] Preferably, the bifunctional fusion protein according to the present invention further comprises the light chain of an anti-AREG antibody.

[0062] The bifunctional fusion protein according to the present invention is in the form of a heterotetramer.

[0063] In some embodiments, the Fc region of an anti-AREG antibody can comprise a hinge portion, a CH3 portion, and a CH2 portion.

[0064] In some embodiments, the Fc region can further comprise a domain that promotes heterodimerization, preferably heterodimerization of two heavy chains.

[0065] In some embodiments, the constant region contains various modifications to increase half-life, improve stability, increase or decrease ADCC and / or CDC.

[0066] The bifunctional fusion proteins of the present invention simultaneously possess anti-TGFβ and anti-AREG activity, have the ability to simultaneously inhibit TGFβ and AREG, and can simultaneously block pathways related to TGFβ and AREG, and can more effectively alleviate and treat diseases, including those associated with fibrosis, cancer, and chronic inflammation. In particular, the bifunctional fusion proteins of the present invention comprise at least a portion of the extracellular domain of TRII capable of binding to a TGFβ ligand and an antibody or antigen-binding fragment that binds to and neutralizes AREG. The bifunctional proteins can simultaneously block TGFβ and AREG signaling. Therefore, the bifunctional fusion proteins of the present invention can be used to treat fibrotic diseases (including, but not limited to, kidney fibrosis, liver fibrosis, and pulmonary fibrosis, particularly IPF), cancer, and diseases associated with chronic inflammation.

[0067] In a second aspect, the present invention provides an isolated nucleic acid encoding the bifunctional fusion protein of the first aspect.

[0068] In a third aspect, the present invention provides an expression vector comprising the isolated nucleic acid of the second aspect.

[0069] In a fourth aspect, the present invention provides a host cell comprising the isolated nucleic acid of the second aspect or the expression vector of the third aspect.

[0070] The host cell may be any conventional host cell in the art, as long as the expression vector of the third aspect is capable of stably expressing the carried nucleic acid into the bifunctional fusion protein of the first aspect. Preferably, the host cell is a prokaryotic cell and / or a eukaryotic cell, with the prokaryotic cell preferably being an Escherichia coli (E. coli) cell such as TG1 or BL21, and the eukaryotic cell preferably being an HEK293 cell, a CHO cell, or a derived cell line. The host cell of the present invention can be obtained by transfecting the expression vector of the third aspect. The transfection method may be any conventional transfection method in the art, preferably chemical transfection, heat shock, or electroporation.

[0071] In a fifth aspect, the present invention provides a method for preparing a bifunctional fusion protein according to the first aspect.

[0072] In some embodiments, the method comprises culturing a host cell of the fourth aspect.

[0073] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the bifunctional fusion protein of the first aspect and a pharmaceutically acceptable vector.

[0074] In some embodiments, the pharmaceutical composition comprises other ingredients, such as other small molecule drugs or antibodies or polypeptides, as active ingredients.

[0075] The pharmaceutical composition may be administered parenterally, by injection, or orally. The pharmaceutical composition may be in a form suitable for administration, such as a solid, semi-solid, or liquid form, such as an aqueous solution, non-aqueous solution or suspension, powder, tablet, capsule, granule, injection, or infusion. The pharmaceutical composition may be administered intravenously, subcutaneously, intraperitoneally, intramuscularly, by inhalation, intranasally, tracheal instillation, or intrapleural instillation. The pharmaceutical composition may be administered in the form of an aerosol or spray, such as intranasally, intrathecally, intramedullary, or intracerebroventricularly, or may be administered transdermally, topically, enterally, vaginally, sublingually, or rectally.

[0076] In some embodiments, the bifunctional fusion protein and one or more other active ingredients are administered simultaneously or sequentially.

[0077] In a seventh aspect, the present invention provides the use of the bifunctional fusion protein of the first aspect, the isolated nucleic acid of the second aspect, and the pharmaceutical composition of the sixth aspect in the prevention, treatment, and / or diagnosis of fibrotic diseases, cancer, and diseases associated with chronic inflammation in a subject, including, but not limited to, kidney fibrosis, liver fibrosis, and pulmonary fibrosis, particularly IPF.

[0078] In a ninth aspect, the present invention provides a method for preventing, treating and / or diagnosing fibrotic diseases, cancer, and diseases associated with chronic inflammation in a subject, the method comprising administering to the subject a therapeutically effective amount of the bifunctional fusion protein of the first aspect. Fibrotic diseases include, but are not limited to, kidney fibrosis, liver fibrosis, and pulmonary fibrosis, in particular IPF. [Effects of the Invention]

[0079] The bifunctional fusion protein of the present invention has the following technical effects.

[0080] 1. As an antagonist of TGFβ and AREG,

[0081] 2. As a single bifunctional protein, it has the ability to inhibit TGFβ and AREG, effectively blocking the two driving factors of fibrosis development and causing a synergistic effect to treat tissue fibrosis, especially pulmonary fibrosis;

[0082] 3. As a single bifunctional protein, it simultaneously blocks TGFβ and AREG signaling,

[0083] 4. As a single bifunctional protein, it binds to both TGFβ and AREG specifically;

[0084] 5. Has the ability to inhibit AREG-induced pEGFR,

[0085] 6. It has the ability to bind to TGFβ ligands, thereby inhibiting downstream activation of the TGFβ signaling pathway;

[0086] 7. A bifunctional fusion protein comprising a TRII extracellular domain mutant having one or two mutations selected from K7Q, R34S, R66S, K67S, K103S and K104S, exhibiting reduced splicing of the bifunctional fusion protein;

[0087] 8. A bifunctional fusion protein comprising a TRII extracellular domain mutant having a mutation selected from R34S, R66S, K67S, K103S and K104S exhibits reduced splicing;

[0088] 9. A bifunctional fusion protein containing a TRII extracellular domain mutant with the mutation K7Q showed a significant reduction in splicing.

[0089] 10. Bifunctional fusion proteins comprising TRII extracellular domain mutants, herein, mutants with N-terminal deletions, exhibiting better stability and less splicing, having 4 amino acids, 7 amino acids, 9 amino acids, 13 amino acids, 17 amino acids, or 21 amino acids N-terminal deletions, based on the amino acid numbering from the N-terminus to the C-terminus of SEQ ID NO:90.

[0090] Definition:

[0091] Transforming growth factor-β (TGFβ) is a pleiotropic cytokine that plays important roles under physiological and pathological conditions. TGFβ isomers, namely β1, β2, and β3, are approximately 25 kDa homodimeric polypeptides. These ligands transduce signals through cell surface receptors (including transforming growth factor-β receptors I, II, and III (TRI, TRII, and TRIII)) and intracellular SMAD effector proteins, such as SMAD2 and 3. Signal transduction influences a range of cellular processes, including cell survival, proliferation, differentiation, cell motility, and extracellular matrix (ECM) production.

[0092] Amphiregulin (AREG) is a low-affinity ligand of the epidermal growth factor (EGF) family. The AREG protein is synthesized from a 252-amino acid transmembrane precursor, which is proteolytically cleaved within its extracellular domain by plasma membrane proteases, primarily tumor necrosis factor-α-invertase (TACE). Mature soluble AREG then directly binds to the epidermal growth factor receptor (EGFR) to activate downstream signaling. This triggers major intracellular signaling cascades, including MAPK / ERK signaling, that regulate cell survival, proliferation, and motility.

[0093] As used herein, the articles "a" and "an" refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.

[0094] Unless the context clearly dictates otherwise, the term "or" is used herein to mean, and can be used interchangeably with, the term "and / or."

[0095] "About" and "approximately" generally refer to an acceptable level of error for a measured quantity given the nature or precision of the measurement. Exemplary error levels are within 20%, typically less than 10%, and more typically less than 5% of a given value or range of values.

[0096] The products and methods disclosed herein include polypeptides and polynucleotides having a specified sequence or a sequence identical or similar thereto, for example, a sequence having at least about 85% or 95% sequence identity (identity) to the specified sequence. In the context of amino acid sequences, the term "85% or 95% sequence identity (identity)" is used herein to refer to a first amino acid sequence containing a sufficient or minimum number of amino acid residues i) identical to aligned amino acid residues in a second amino acid sequence, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence, such that the first and second amino acid sequences have a common domain and / or a common functional activity. For example, an amino acid sequence containing a common domain that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence (e.g., a sequence provided herein).

[0097] In the context of nucleic acids, the term "85% or 95% sequence identity (homology)" is used herein to refer to a first nucleic acid sequence that contains a sufficient or maximum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode polypeptides having a common functional activity, or encode a common structural polypeptide domain or common functional polypeptide activity. For example, a nucleotide sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a reference sequence (e.g., a sequence provided herein).

[0098] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., for optimal alignment, gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences can be ignored for comparison purposes). In a preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, for example, at least 40%, 50%, 60%, for example, at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position.

[0099] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length.

[0100] The terms "nucleic acid," "nucleic acid sequence," "nucleotide sequence" or "polynucleotide sequence" and "polynucleotide" are used interchangeably.

[0101] As used herein, the term "antibody" refers to a protein comprising at least one immunoglobulin variable domain sequence, such as an immunoglobulin chain or a fragment thereof. The term "antibody" is meant to include, for example, monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region). In one embodiment, an antibody comprises a full-length antibody or a full-length immunoglobulin chain. In one embodiment, an antibody comprises an antigen-binding or functional fragment of a full-length antibody, or a full-length immunoglobulin chain. As used herein, it is understood by those skilled in the art that an antibody "binds" to an antigen. In one embodiment, the dissociation constant (KD) for an antibody binding to an antigen is about 1 x 10 -5 M or less, 1 x 10 -6 M or less, or 1 x 10 -7 M or less.

[0102] For example, an antibody can comprise a heavy (H) chain variable domain sequence (abbreviated herein as VH) and a light (L) chain variable domain sequence (abbreviated herein as VL). In one embodiment, an antibody consists of a heavy chain and a light chain, or a heavy chain and a light chain. In another example, an antibody comprises two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequences, thereby forming two antigen-binding sites, such as Fab, Fab', F(ab')2, Fc, Fd, Fd', Fv, single-chain antibodies (e.g., scFv), single variable domain antibodies, diabodies (DAbs) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which can be produced by modifying whole antibodies or by de novo antibody synthesis using recombinant DNA technology. These functional antibody fragments retain the ability to selectively bind to their respective antigens or receptors. Antibodies and antibody fragments can be derived from any class of antibody, including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and any subclass of antibody (e.g., IgG1, IgG2, IgG3, and IgG4). Antibody preparations can be monoclonal or polyclonal. The antibody can be human, humanized, CDR-grafted, or in vitro-produced. The antibody can have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. The antibody can have a light chain selected from, for example, kappa or lambda. As used herein, the term "immunoglobulin" (Ig) is used interchangeably with the term "antibody."

[0103] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Antibody fragments bind to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes synthetic or genetically engineered proteins that function like antibodies by binding to a specific antigen to form a complex.

[0104] Examples of antigen-binding fragments of antibodies include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH regions; (ii) an F(ab')2 fragment, a bivalent fragment containing two Fab fragments connected by a disulfide bond at the hinge region; (iii) an Fd fragment consisting of the VH and CH regions; (iv) an Fv fragment consisting of the VL and VH regions of a single antibody arm; (v) a diabody (dAb) fragment consisting of the VH region; (vi) a camel or camelized variable region; (vii) a single-chain Fv (scFv); and (viii) a single-part antibody. These antibody fragments can be obtained using any suitable method, including conventional techniques known to those skilled in the art, and these fragments can be screened for utility in the same manner as intact antibodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that functions like an antibody by binding to a specific antigen to form a complex.

[0105] "Single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin.

[0106] In some embodiments, the regions are linked via a peptide with a short linker of 2 to about 30 amino acids, preferably 6 to 26 amino acids. The linker can be rich in glycine to provide flexibility, or can contain serine, threonine, glutamic acid, alanine, or lysine, and can link the N-terminus of the VH of an anti-AREG antibody or fragment thereof to the C-terminus of the extracellular domain of TRII or a variant thereof, or vice versa.

[0107] Light and heavy chains are divided into "constant" and "variable" regions. The light chain variable domain (VL) and heavy chain variable domain (VH) partially determine antigen recognition and specificity. Conversely, the light chain constant domain (CL) and heavy chain constant domain (CH1, CH2, or CH3) confer important biological properties such as secretion, placental transport, Fc receptor binding, and complement fixation. The N-terminal portions are the variable region domains, and the C-terminal portions are the constant region domains; the CH3 and CL portions actually comprise the carboxy termini of the heavy and light chains, respectively.

[0108] The variable region enables an antibody to selectively recognize and specifically bind to an epitope on an antigen. The VL and VH portions of an antibody, or a subset of complementarity-determining regions (CDRs), combine to form the variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs from each of the VH and VL chains (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3).

[0109] As used herein, the terms "complementarity-determining region" and "CDR" refer to amino acid sequences within an antibody variable region that confer antigen specificity and binding affinity. In some embodiments, each heavy chain variable region has three CDRs (HCDR1, HCDR2, and HCDR3), and each light chain variable region has three CDRs (LCDR1, LCDR2, and LCDR3).

[0110] The precise boundaries of a given CDR amino acid sequence are determined using the well-known protocol described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme).

[0111] Each VH and VL typically comprises three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0112] "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject in need of diagnosis, prognosis, or treatment, particularly a mammalian subject. Mammals include humans, farm animals, farm animals, zoo animals, sport animals, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, dairy cows, etc.

[0113] As used herein, the phrase "patient in need of treatment" or "subject in need of treatment" includes a subject, e.g., a mammalian subject, who would benefit from the administration of an antibody or composition of the disclosure, wherein said antibody or composition is used, e.g., for detection, diagnostic procedures and / or treatment.

[0114] As used herein, the term "epitope" refers to a portion of an antigen (e.g., human AREG) that specifically interacts with an antibody. Such a portion, also referred to herein as an epitopic determinant, typically comprises or is a portion of an amino acid side chain or a sugar side chain. Epitopes can be defined by methods known in the art or disclosed herein, such as crystallography or hydrogen-deuterium exchange. At least one or several portions of an antibody that specifically interact with an epitopic determinant are typically located in a CDR. Epitopes typically have specific three-dimensional structural characteristics. Epitopes typically have specific charge characteristics. Some epitopes are linear epitopes, while others are conformational epitopes.

[0115] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibodies having a single molecular component. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Monoclonal antibodies can be prepared by hybridoma technology or methods that do not use hybridoma technology (e.g., library selection, screening, or recombinant methods).

[0116] The antibody can be a polyclonal or monoclonal antibody. In other embodiments, the antibody can be produced recombinantly by yeast display, phage display, or combinatorial methods.

[0117] In one embodiment, the antibody is a fully human antibody (e.g., an antibody produced by yeast display, an antibody produced by phage display, or an antibody prepared in a mouse genetically engineered to produce antibodies to human immunoglobulin sequences), or a non-human antibody, such as a murine (mouse or rat), goat, primate (e.g., monkey), or camel antibody. Methods for producing rodent antibodies are known in the art.

[0118] Transgenic mice carrying human immunoglobulin genes (instead of the mouse system) can be used to produce human monoclonal antibodies. Splenocytes obtained from these transgenic mice immunized with an antigen of interest or its fragment are used to generate hybridomas secreting human mAbs with specific affinity for the epitope.

[0119] The antibody may be an antibody whose variable region or a portion thereof (e.g., CDR) is produced in a non-human organism (e.g., a rat or a mouse). Chimeric antibodies, CDR-grafted antibodies, and humanized antibodies are within the scope of the present invention. Antibodies produced in a non-human organism, such as a rat or a mouse, and then modified, for example, in the variable framework or constant region to reduce antigenicity in humans are all within the scope of the present invention.

[0120] The scope of the present invention further includes humanized antibodies in which specific amino acids have been substituted, deleted, or added. Standards for selecting amino acids from donors are described in US 5,585,089, e.g., US 5,585,089, columns 12-16, the contents of which are incorporated herein by reference. Padlan et al. described other techniques for antibody humanization in EP 519596A1, published December 23, 1992.

[0121] In other embodiments, the antibody has a heavy chain constant region selected from, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, and particularly, the heavy chain constant region selected from, e.g., IgG1, IgG2, IgG3, and IgG4 (e.g., human).

[0122] It is understood that the molecules of the present invention can have additional conservative or non-essential amino acid substitutions that do not substantially affect their function.

[0123] As shown in Table 1, a "conservative amino acid substitution" refers to a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0124] [Table 1] [Brief explanation of the drawings]

[0125] [Figure 1] 1 shows the structure of an exemplary anti-AREG / TRII bifunctional fusion protein.

[0126] [Figure 2] 1 shows the characterization of splicing material in anti-AREG / TRII mutants with different linkers.

[0127] [Figure 3] Cleavage hotspots (bold-italic alphabet) in anti-AREG / TRII are indicated.

[0128] [Figure 4] 1 shows that the K7 mutation (014) in anti-AREG / TRII reduces splicing.

[0129] [Figure 5]1 shows that the N-terminal deletion of TRII in the anti-AREG / TRII mutant reduces heavy chain splicing.

[0130] [Figure 6] 1 shows that N-terminal deletions of TRII (013, 017, 028, 029, 030) in anti-AREG / TRII reduce heavy chain splicing.

[0131] [Figure 7] The purity of anti-AREG / TRII variants 013, 029, and 030 is shown.

[0132] [Figure 8] 1 shows the inhibitory effect of mutant 010 on pEGFR.

[0133] [Figure 9] Inhibition of mutants 010, 013 and 030 on the SRE reporter gene is shown.

[0134] [Figure 10] Mutants 010, 013 and 030 are shown to block nuclear localization of pSMAD2.

[0135] [Figure 11] 1 shows that mutant 013 inhibits pSMAD2 in immunoblot assays.

[0136] [Figure 12] 1 shows the inhibitory effects of mutants 010, 013, and 030 on TGFβ signaling as measured by an SBE luciferase reporter gene.

[0137] [Figure 13] 1 shows that the bifunctional anti-AREG / TRII fusion protein simultaneously targets AREG and TGFβ.

[0138] [Figure 14]Pharmacokinetic characteristics of variants 010, 013 and 030 are shown.

[0139] [Figure 15] 1 shows the efficacy of anti-AREG / TRII fusion protein in a Cdc42 AT2 null fibrosis model. DETAILED DESCRIPTION OF THE INVENTION

[0140] The descriptions of specific embodiments and examples are provided by way of illustration and not limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield substantially similar results.

[0141] Table 2 below shows the schematic structures of different anti-AREG / TRII variants.

[0142] [Table 2] TIFF0007804295000003.tif104156 [Example]

[0143] Example 1. Production of anti-AREG / TRII bifunctional fusion protein

[0144] Anti-AREG / TRII is a bifunctional protein consisting of an anti-AREG antibody and the extracellular domain of transforming growth factor β receptor II (TGFβRII, TRII). The light chain variable region of the molecule is identical to that of an anti-AREG antibody (SEQ ID Nos.: 70-89). The heavy chain of the molecule is a fusion protein containing the heavy chain of an anti-AREG antibody (SEQ ID Nos.: 57-69) fused to the N-terminus of soluble TRII (SEQ ID Nos.: 90-107) via a flexible linker (SEQ ID Nos.: 108, 109, 113, and 116-117). At the fusion junction, the C-terminal lysine residue of the antibody heavy chain is mutated to alanine to reduce proteolytic cleavage.

[0145] The following exemplary procedure is used to construct the plasmid.

[0146] The fragment was amplified by polymerase chain reaction (PCR) (TOYOBO, KOD-201). After electrophoresis, the PCR product was separated on a 1.5% agarose gel and recovered using a DNA purification kit (Magen, D2111-03). The fragment and vector were digested with restriction enzymes and ligated using T4 DNA ligase (New England Biolabs, M0202L). The ligated construct was transformed into E. coli Top10 strain (CWBIO, CW0807) for positive clone selection. The cloned plasmid was used for protein expression in a eukaryotic expression system.

[0147] The following exemplary procedure is used to produce the protein.

[0148] Two expression vectors carrying the heavy and light chains are transfected into FreeStyle™ 293-F cells (Invitrogen, R79007) at a 1:1 ratio. The day before transfection, the 293-F cells are subcultured and expanded, and grown overnight. On the day of transfection, the cells are harvested by centrifugation and then resuspended in fresh FreeStyle™ 293 Expression Medium (Gibco, 12336-018) at a final density of 1.2 x 10 6 Resuspend the cells at 1 μg / mL. Transiently co-transfect the plasmids at the indicated molar ratio with polyethyleneimine (Polysciences, 23966) at a final concentration of 1 μg / mL. Five to six days after transfection, harvest the cell culture supernatant.

[0149] Example 2. Characterization of anti-AREG / TRII bifunctional fusion proteins with different linkers

[0150] Exemplary anti-AREG / TRII bifunctional fusion proteins (anti-AREG / TRII) 001, 005, 008, and 009 comprise a light chain (SEQ ID Nos. 70-89) and a heavy chain fused to TRII via different linkers (SEQ ID Nos. 118-121). The structures of the bifunctional fusion proteins are as shown in FIG. 1.

[0151] The following exemplary procedure is used to evaluate splicing of fusion proteins. Fusion proteins are collected after transfection and then purified by one-step Protein A chromatography. All samples are adjusted to a concentration of 0.5 mg / mL and then incubated at 37°C for stability testing. Samples are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions.

[0152] Figure 2 shows that after SDS-PAGE, two bands of approximately 25 kDa and 70 kDa were detected in all samples. These bands represent the light and heavy chains of the bifunctional protein. After incubation at 37°C for 2 days, an additional band of approximately 50-60 kDa was detected, indicating the presence of various splicing products in the variants after additional incubation.

[0153] Example 3. Determination of cleavage site of anti-AREG / TRII bifunctional fusion protein

[0154] Anti-AREG / TRII with different linkers exhibit varying degrees of splicing, which are represented by different molecular weights on SDS-PAGE. Therefore, further studies will be performed to determine the site of splicing.

[0155] 0.5 mg / mL of 001 or 009 was incubated at 37°C. After 2 days of incubation, the sample was loaded onto a gel. The spliced ​​fragments were separated and collected. The fragments were analyzed by mass spectrometry (MS) (LTQ Orbitrap, ThermoFisher Scientific). The analysis results, as shown in Figure 3, indicate that Lys7 / Ser8, Arg34 / Phe35, Arg66 / Lys67, and Lys103 / Lys104 are hotspots for TGFβ trap splicing.

[0156] Predict potential cleavage sites at https: / / web.expasy.org / peptide_cutter / . The results of these analyses indicate that Lys7 / Ser8, Arg34 / Phe35, Arg66 / Lys67, and Lys103 / Lys104 are also potential cleavage sites.

[0157] Example 4. Study of anti-AREG / TRII mutants that reduce splicing

[0158] To develop TRII-containing molecules that significantly reduce splicing, we designed various traps containing TRII-targeting mutants (SEQ ID NOs: 90-107).

[0159] An exemplary anti-AREG / TRII antibody comprises a light chain (SEQ ID NO: 83) and a heavy chain (SEQ ID NOs: 122-139) fused to a TRII variant. 010 is an anti-AREG / TRII variant with a wild-type TRII extracellular domain. 014, 015, 016, 017, and 018 are anti-AREG / TRII variants, in which the TRII has one or two mutations at the above sites. The purified sample is incubated at 37°C for 3 days. The spliced ​​material is evaluated by the method described in Example 3.

[0160] Figure 4 shows that the K7 mutation (014) significantly reduces heavy chain splicing. Compared to 010, the R34 (016), R66 / K67 (017), and K103 / K104 (018) mutations also reduce splicing.

[0161] Considering that K7 is the most important residue mediating splicing, a new mutant, 026, was designed by deleting seven residues at the N-terminus of TRII. Combinations of the K7 mutation with R34, R66 / K67, and K103 / K104 mutations (019, 020, 021, 022, 024, 025, and 026) were also designed and subjected to splicing evaluation. As shown in Figure 5, among all mutants, 026 showed the least splicing after 6 days of incubation at 37°C.

[0162] Compared with point mutations or combinations of point mutations, deletion mutant 026 exhibits better stability. Therefore, additional deletion mutants were designed, including a four-residue deletion (027), a nine-residue deletion (028), a 13-residue deletion (029), a 17-residue deletion (030), and a 21-residue deletion (013). As shown in Figure 6, after 5 days of incubation at 37°C, 029, 030, and 013 show less splicing products than 010 on SDS-PAGE.

[0163] The purity of anti-AREG / TRII variants is evaluated using size exclusion chromatography (SEC). Figure 7 shows the exemplary maps of 013, 029 and 030. The purity after purification is more than 98%. The level of high molecular weight (HMW) and low molecular weight (LMW) is less than 2%.

[0164] Example 5. Detection of binding of anti-AREG / TRII mutants to AREG and TGFβ by SPR

[0165] Surface plasmon resonance (SPR) is used to characterize the binding kinetics of anti-AREG / TRII mutants to AREG and TGFβs.

[0166] The following exemplary procedure is used to measure the equilibrium dissociation constant.

[0167] SPR measurements are performed using a Biacore T200 instrument (GE Life Sciences). Samples are captured on the surface of a Protein A / G CM5 biosensor chip. Binding of human TGFβ3 (R&D, 243-B3-010) to various mutants is detected. Serially diluted TGFβ protein is injected onto the surface that binds to the anti-AREG / TRII mutants, followed by a dissociation phase. A one-to-one Langmuir binding model (BIA evaluation software, GE Life Sciences) is used to calculate the association rate (ka) and dissociation rate (kd). KD is calculated as the ratio of kd to ka.

[0168] As shown in Table 3, 010, 013 and 030 bind to TGFβ1 / 2 / 3 and AREG with similar kinetics.

[0169] [Table 3]

[0170] Example 6. Measurement of the inhibitory effect of anti-AREG / TRII mutants on EGFR by detecting phosphorylated EGFR

[0171] AREG binds to epidermal growth factor receptor (EGFR) and activates the receptor, which can be measured by phosphorylated EGFR (pEGFR). Exemplary anti-AREG / TRII variant 010 and its ability to inhibit AREG-induced pEGFR are detected.

[0172] The following exemplary procedure is used: 2 x 10 A431 cells 5Cells were seeded into 6-well plates at 1000 cells / well and cultured overnight at 37°C. The following day, cells were starved for 2 hours using serum-free medium and then treated with 10 nM recombinant human AREG (Peprotech, 96-100-55B-50) and various concentrations of anti-AREG / TRII mutants for 1 hour. Treated cells were lysed and samples were subjected to Western blotting. Primary antibodies used for the assay included anti-pEGFR (Abcam, ab40815), anti-EGFR (Cell Signaling Technology, 2232), and anti-GAPDH (Nakasugi Jinqiao, TA-08).

[0173] 8 shows that mutant 010 inhibits AREG-induced pEGFR with an IC50 of 5.56 nM. The data demonstrate that mutant 010 can effectively block AREG-induced EGFR activation.

[0174] Example 7. Characterization of anti-AREG / TRII mutants by luciferase reporter gene via MAPK / ERK signaling

[0175] The inhibitory effect of anti-AREG / TRII mutants on AREG-EGFR downstream signaling is detected using a serum response regimen and a (SRE) luciferase reporter gene, which is typically used to evaluate EGFR-MAPK / ERK signaling.

[0176] The following exemplary procedure is used: An SRE luciferase reporter gene is constructed and HEK293T cells are transfected in a 96-well plate using a Lipo3000 transfection kit (Invitrogen, L3000-015). Each well is co-transfected with a TK-Renilla plasmid as an internal control. Six hours after transfection, the cells are serum-starved and then treated with recombinant human AREG (Peprotech, 96-100-55B-50) and anti-AREG / TRII mutants for six hours. The processed luciferase signal is detected using a Dual-Glo® Luciferase Measurement System (Promega, E2940).

[0177] 9 shows that anti-AREG / TRII mutants, including 010, 013, and 030, inhibit AREG-induced SRE reporter gene expression with an IC99 of approximately 25 nM. The data indicate that anti-AREG / TRII mutants can effectively inhibit AREG-induced EGFR-MAPK / ERK signaling.

[0178] Example 8. Evaluation of the inhibitory effect of anti-AREG / TRII mutants on TGFβ signaling by pSMAD2 nuclear localization

[0179] The extracellular domain of transforming growth factor beta receptor II in the anti-AREG / TRII fusion protein is designed to chelate with TGFβ ligands, thereby inhibiting downstream activation of the TGFβ signaling pathway. Nuclear localization of phosphorylated SMAD2 (pSMAD2) is frequently used to assess activation of the pathway. pSMAD2 immunofluorescence staining is used to detect pSMAD2 nuclear localization, i.e., the effect of anti-AREG / TRII mutants on TGFβ signal activation.

[0180] The following exemplary procedure was used to detect nuclear localization of pSMAD2. A549 cells were seeded into 96-well microwell plates at a density of 5,000 cells per well and cultured overnight at 37°C using DMEM containing 10% FBS. The following day, cells were serum-starved and then treated for 6 hours with 0.078 nM TGFβ1 (R&D, 240-B-101) and 0.39 nM and 1.95 nM anti-AREG / TRII mutants, respectively. Cells were rinsed with PBS, fixed, and stained overnight at 4°C using anti-pSMAD2 antibody (Cell Signaling Technology, 18338). The following day, samples were treated with a secondary antibody (Jackson Immuno Research, 711-064-152) using the Elite ABC kit (VECTOR, PK-6100) for 30 minutes, followed by staining with tyramide fluorescein (Pekin-Elmer, FP1013). Fluorescent images are acquired using a Perkin-Elmer Opera LX 20x air lens.

[0181] As shown in Figure 10, 1.95 nM of anti-AREG / TRII variants 010, 013, and 030 effectively blocked pSMAD2 nuclear localization induced by 1 ng / ml of TGFβ1, indicating that anti-AREG / TRII variants 010, 013, and 030 bind to and chelate TGFβ2 and inhibit downstream TGFβ signaling.

[0182] Example 9. Measurement and quantification of the inhibitory effect of anti-AREG / TRII mutants on TGFβ signaling by pSMAD2 immunoblotting

[0183] The expression level of pSMAD2 protein shown by Western blotting (WB) is also used to quantitatively measure the inhibitory effect of anti-AREG / TRII mutants on TGFβ1.

[0184] The following exemplary procedure was used: 2 x 10 A549 cells were cultured. 5Cells were seeded into 6-well plates at 100 cells / well and cultured overnight at 37°C. The next day, cells were starved for 2 hours using serum-free medium and then treated with recombinant human TGFβ1 (R&D, 240-B-101) and different concentrations of anti-AREG / TRII for 2 hours. The treated cells were lysed and samples were analyzed by Western blotting. Primary antibodies included anti-pSMAD2 (Cell Signaling Technology, 18338), anti-SMAD2 / 3 (Cell Signaling Technology, 8685), and anti-GAPDH (Nakasugi Jinqiao, TA-08).

[0185] Figure 11 shows that when 1 ng / mL TGFβ1 was added to the culture, variant 030 inhibited pSMAD2 with an IC50 of 0.034 nM. These data demonstrate that the anti-AREG / TRII TRII-ECD moiety can effectively capture ligands such as TGFβ1 and inhibit the activation of downstream signaling.

[0186] Example 10. Quantification and measurement of the inhibitory effect of anti-AREG / TRII mutants on TGFβ signaling using an SBE reporter gene

[0187] The inhibitory effect of exemplary anti-AREG / TRII variants 010, 013, and 030 on TGFβ downstream signaling is further quantified and measured using a SMAD binding entity (SBE) luciferase reporter gene, which is frequently used to assess the activity of TGFβ signaling.

[0188] The following exemplary procedure is used: An SBE luciferase reporter gene is constructed and transfected into HEK293T cells in a 96-well plate using a Lipo 3000 transfection kit (Invitrogen, L3000-015). Each well is co-transfected with a TK Renilla plasmid as an internal control. Six hours after transfection, the cells are serum-starved and then treated with human TGFβ1 (R&D, 240-B-101) and anti-AREG / TRII for six hours. The processed luciferase signal is then detected using the Dual-Glo® Luciferase Measurement System (Promega, E2940).

[0189] As shown in Figure 12, anti-AREG / TRII variants 010, 013, and 030 all blocked TGFβ1 (0.015 nM) induction of the SBE reporter gene with IC50 values ​​of 0.045 nM, 0.085 nM, and 0.06 nM, respectively.

[0190] Example 11. Anti-AREG / TRII mutant that simultaneously targets AREG and TGFβ

[0191] The anti-AREG / TRII bifunctional fusion protein refers to the simultaneous targeting of AREG and TGFβ ligands through a single molecule. To test this, a CHO-hAREG cell line was constructed that overexpressed the AREG-EGF-like domain, which primarily binds to the cell membrane. When anti-AREG / TRII was added to CHO-hAREG cells, the bifunctional fusion protein bound to the membrane AREG-EGF-like domain via the anti-AREG portion. The membrane-bound bifunctional molecule can also block the activation of TGFβ signaling by capturing free TGFβ ligands.

[0192] The following exemplary procedure is used: CHO and CHO-hAREG cells are harvested and seeded in DMEM supplemented with 10% FBS in a 96-well microwell plate at a density of 8,000 cells per well. The well plate is incubated overnight in a 37°C CO2 incubator. The next day, the cells are starved in serum-free DMEM for 4 hours and then treated with DMEM containing 10 nM mutant for 2 hours. After washing away the free mutant, 0.078 nM TGFβ1 is added and incubated with the cells for 1 hour in a CO2 incubator. After treatment, the cells are washed twice with PBS, fixed with 4% PFA, and then stained with anti-pSMAD2 antibody (Cell Signaling technology, 18338). After staining, fluorescent images are acquired using a 20x air lens on a Pekin-Elmer high-content cell analysis system.

[0193] FIG. 13 shows that mutants 010, 013, and 030 effectively blocked TGFβ1-induced signaling and simultaneously bound to AREG, demonstrating that the bifunctional fusion molecule can simultaneously block AREG and TGFβ signaling.

[0194] Example 12. Pharmacokinetics of anti-AREG / TRII mutants in mice

[0195] C57 / B16 mice were administered a single dose of anti-AREG / TRII variants 010, 013, and 030 at a dose of 15 mg / kg. Blood samples were collected pre-dose and 3, 8, 24, 48, 72, 120, 168, 336, and 504 hours post-dose. Serum samples were separated using standard protocols and then stored at -60°C or below until analysis.

[0196] The analysis program is as follows:

[0197] 1) Coating: Add 100 µL of 3 µg / mL streptavidin (Sigma, S4762) to each well, then seal the well plate and incubate overnight at 2–8 °C.

[0198] 2) Blocking: Discard the contents of the wells, wash each well three times with 300 μL of wash buffer, and allow the well plate to dry. Add 100 μL of biotinylated AREG-hFc (1 μg / mL in dilution buffer) to each well. Seal the well plate and incubate at 30°C for approximately 1 hour.

[0199] 3) Adding samples: Discard the contents of the wells, wash each well three times with 300 μL of wash buffer, and allow the well plate to dry. Dilute the serum samples and standard curve samples with dilution buffer. Next, add 100 μL of each treatment sample to each well of the well plate. Seal the well plate and incubate at 30°C for approximately 1 hour.

[0200] 4) Addition of anti-TRII antibody working solution: Discard the contents of the wells. Wash each well six times with 300 μL of wash buffer and allow the well plate to dry. Add 100 μL of 5 μg / mL anti-TRII antibody working solution to each well of the well plate and incubate at 30 °C for approximately 1 hour.

[0201] 5) Addition of antibody detection working solution: Discard the contents of the wells. Wash each well six times with 300 μL of wash buffer and allow the well plate to dry. Add 100 μL of antibody detection working solution to each well of the well plate and incubate at 30°C for approximately 0.5 hours.

[0202] 6) Color development: Discard the contents of the wells. Wash each well six times with 300 μL of wash buffer and allow the well plate to dry. Add 100 μL of substrate solution to each well. Incubate the well plate at room temperature in the dark for 10 minutes.

[0203] 7) Read the plate: Add 50 μL of stop solution to each well and read the well plate within 30 minutes using a plate reader at a wavelength of 450 nm, with the reference wavelength set at 630 nm.

[0204] 8) Result output: Draw a calibration standard curve with the calibration concentration on the X-axis and the corresponding absorbance (OD) values ​​(OD450nm-OD630nm, without subtracting the blank) on the Y-axis, and calculate the sample concentrations.

[0205] As shown in Figure 14, after intraperitoneal injection of 010, 013, and 030 at a single dose of 15 mg / kg, these variants exhibit similar Cmax (200-250 μg / ml) and half-lives of approximately 4-10 days.

[0206] Example 13. In vivo efficacy of anti-AREG / TRII fusion protein

[0207] Using a surrogate molecule, we demonstrate the in vivo efficacy of anti-AREG / TRII fusion proteins in an animal model of progressive pulmonary fibrosis. In this animal model, deletion of Cdc42 (encoding CDC42, a homolog of cell division control protein 42) in type II pneumocytes (AT2s) leads to impaired alveolar regeneration and progressive pulmonary fibrosis in mice after pneumonectomy (PNX)-induced lung injury. Fibrosis in this model (hereinafter referred to as the Cdc42 AT2 null model) is characterized by progressive scar formation from the periphery to the center, recapitulating the disease progression seen in IPF patients. Cdc42 AT2-deficient mice suffer from progressive fibrosis, and their body weight is significantly reduced and their survival rate is significantly reduced. The anti-AREG / TRII fusion protein comprises a light chain (SEQ ID NO: 141) and a heavy chain (SEQ ID NO: 142) fused to TRII via a linker, wherein anti-AREG specifically binds to AREG in an animal model.

[0208] The detailed generation of the mouse fibrosis model has been described previously (WO2020237587A1). _flox / floxMice were crossed with Spc-CreER-rtTA mice and injected with tamoxifen to specifically delete Cdc42 in AT2. These transgenic mice then underwent partial pneumonectomy (PNX) to remove the left lung lobe, thereby increasing mechanical tension and inducing fibrosis. Starting on day 14 after PNX, Cdc42 AT2 null mice were administered the anti-AREG / TRII surrogate molecule at a dose of 15 mg / kg every 5 days until day 60 after PNX. Body weight was measured every 5 days. Treatment with the surrogate molecule consistently demonstrated efficacy, as manifested by a significant improvement in survival (P = 0.0083, Figure 15A), a significant reduction in weight loss (P < 0.0001, Figure 15B), and a significant reduction in fibrotic foci and area as measured by fibrosis score (P < 0.0001, Figure 15C). Thus, anti-AREG / TRII mechanisms are sufficient to reduce the progression of fibrosis.

[0209] Discussion The inventors demonstrate that the constructed bifunctional anti-AREG / TRII fusion protein 1) effectively blocks AREG-EGFR signaling in pEGFR immunoblot assays and SRE reporter gene measurements, and 2) effectively inhibits TGFβ signaling, including preventing pSMAD2 nuclear localization as detected by immunostaining, suppressing SMAD2 phosphorylation as detected by immunoblotting, and inhibiting TGFβ1-induced SBE reporter gene induction. Furthermore, the inventors discovered that the anti-AREG / TRII mutant can simultaneously target AREG and TGFβ signaling. These results collectively demonstrate that the anti-AREG / TRII bifunctional fusion protein can block AREG and TGFβ signaling and can be used as a therapeutic molecule for fibrosis, chronic inflammation, and cancer. The present invention is as follows. [1] A bifunctional fusion protein, The bifunctional fusion protein, characterized in that it comprises at least two domains, wherein the two domains are capable of binding to AREG or a fragment thereof, and / or a TGFβ ligand or a fragment thereof. [2] A polypeptide comprising a first domain and a second domain, wherein the first domain is capable of binding to AREG or a fragment thereof, and the second domain is capable of binding to a TGFβ ligand or a fragment thereof. The bifunctional fusion protein according to [1] above. [3] The first domain is an antibody or an antigen-binding fragment thereof that binds to AREG or a fragment thereof, and the second domain comprises a portion of the extracellular domain of TGFβ receptor II (TRII) or a mutant thereof. The bifunctional fusion protein according to [2] above. [4] The antibody or antigen-binding fragment thereof is an anti-AREG antibody or fragment thereof capable of binding to AREG, preferably both human AREG and mouse AREG, or the anti-AREG antibody or fragment thereof is capable of binding to human AREG and has weak or no cross-reactivity with mouse AREG. The bifunctional fusion protein according to [3] above. [5] The anti-AREG antibody or fragment thereof is a human anti-AREG antibody, a murine anti-AREG antibody, a chimeric anti-AREG antibody, or a humanized anti-AREG antibody, and is preferably a human monoclonal antibody (mAb), a murine mAb, a chimeric mAb, or a humanized mAb. The bifunctional fusion protein according to [3] above. [6] The anti-AREG antibody or a fragment thereof is capable of binding to a soluble form of AREG, preferably to the epidermal growth factor (EGF)-like domain of a soluble form of AREG, and more preferably to the C-terminus within the EGF-like domain of a soluble form of AREG. The bifunctional fusion protein according to [3] above. [7] The anti-AREG antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein: HCDR1, HCDR2 and HCDR3 are (1) HCDR1:SYAMS(SEQ ID NO:1), HCDR2:AISGSGGSTYYADSVKG(SEQ ID NO:2), HCDR3:PTSRYSYGYDY(SEQ ID NO:3), (2) HCDR1:SYAMS(SEQ ID NO:1), HCDR2:AISGSGGSTYYADSVKG(SEQ ID NO:2), HCDR3:PTSRYSYSYNN(SEQ ID NO:4), (3) HCDR1:SHAMS(SEQ ID NO:5), HCDR2:AISGSGGSTYYADSVKG(SEQ ID NO:2), HCDR3:VDTKFDP(SEQ ID NO:6), (4) HCDR1:SYPMS(SEQ ID NO:7), HCDR2:TISTGGTYTYYPDSVKG(SEQ ID NO:8), HCDR3:QGPIYYGNYYYAMDY(SEQ ID NO:9), (5) HCDR1:SYPMS(SEQ ID NO:7), HCDR2:TISTGGRYTYYPDSVKG(SEQ ID NO:10), HCDR3:QGPIYYGNYYYAMDY(SEQ ID NO:9), (6) HCDR1:SYPMS(SEQ ID NO:7), HCDR2:TISTGGTYTYYPDSVKG(SEQ ID NO:8), HCDR3:QGPILRKNYYYGMDV(SEQ ID NO:11), (7) HCDR1:SYPMS(SEQ ID NO:7), HCDR2:TISTGGTYTYYPDSVKG(SEQ ID NO:8), HCDR3:QGPIYYGNYYYGMDV(SEQ ID NO:12), (8) HCDR1:SYAMS(SEQ ID NO:1), HCDR2:TISTGGSHTYYPDSVKG(SEQ ID NO:13), HCDR3:HGYLLYDGYYEWYFDV(SEQ ID NO:14), (9) HCDR1:SYAMS(SEQ ID NO:1), HCDR2:TISTGGSHTYYPDSVKG(SEQ ID NO:13), HCDR3:HGYLLYDGYYEWYFDY(SEQ ID NO:140), (10) HCDR1:SYAMS(SEQ ID NO:1), HCDR2:TISTGGSHTYYPESVKG(SEQ ID NO:15), HCDR3:HGYLLYEGYYEWYFDY(SEQ ID NO:16), (11) HCDR1:GYPMS(SEQ ID NO:17), HCDR2:TISTGARHTYYPDSVKG(SEQ ID NO:18), HCDR3:HEGLRRGKYHCIMDY(SEQ ID NO:19), (12) HCDR1: GYPMS (SEQ ID NO: 17), HCDR2: TISTGARHTYYPDSVKG (SEQ ID NO: 18), HCDR3: HEGLRRGKYHSIMDY (SEQ ID NO: 20), and (13) HCDR1, HCDR2, and HCDR3 as set forth in (1) to (12), with the proviso that at least one of them is selected from the group consisting of one, two, three, four, or five amino acid additions, deletions, conservative amino acid substitutions, or combinations thereof; and LCDR1, LCDR2 and LCDR3 are (1) LCDR1: TGNSNNVGDQGAV (SEQ ID NO: 21), LCDR2: RNNNRPS (SEQ ID NO: 22), LCDR3: STWDSGLNSVV (SEQ ID NO: 23), (2) LCDR1: TGNSNNVGDQGAV (SEQ ID NO: 21), LCDR2: RNNNRPS (SEQ ID NO: 22), LCDR3: STWDKNNKSVV (SEQ ID NO: 24), (3) LCDR1:SGSSSNIGSNTVN(SEQ ID NO:25), LCDR2:SNNQRPS(SEQ ID NO:26), LCDR3:EVWDDSLNGPV(SEQ ID NO:27), (4)LCDR1:RSSQSLVHSDGNTYLH(SEQ ID NO:28), LCDR2:KVSNRFS(SEQ ID NO:29),LCDR3:SQSTHVPYT(SEQ ID NO:30), (5)LCDR1:RSSQSLVDGEDGTYLN(SEQ ID NO:31)、LCDR2:KVSERFD(SEQ ID NO:32)、LCDR3:SQSTHVPYT(SEQ ID NO:30)、 (6)LCDR1:RSSQSLVDGQDGTYLH(SEQ ID NO:33)、LCDR2:KVSNRFD(SEQ ID NO:34)、LCDR3:SQSTHVPYT(SEQ ID NO:30)、 (7)LCDR1:RSSQSLVNQEGETYLH(SEQ ID NO:35)、LCDR2:KVSNRFD(SEQ ID NO:34)、LCDR3:SQSTHVPYT(SEQ ID NO:30); (8)LCDR1:KASQSVDYDGHSFLN(SEQ ID NO:36)、LCDR2:AASNLES(SEQ ID NO:37)、LCDR3:QQSTEDPPYT(SEQ ID NO:38)、 (9)LCDR1:RASESVDYDGHSFIN(SEQ ID NO:39)、LCDR2:AASNKDT(SEQ ID NO:40)、LCDR3:QQSTEDPPYT(SEQ ID NO:38)、 (10)LCDR1:RASQSVDYDGHSFLN(SEQ ID NO:41)、LCDR2:AASNLQS(SEQ ID NO:42)、LCDR3:QQSTEDPPYT(SEQ ID NO:38)、 (11)LCDR1:KSSQSVDYDGHSFLN(SEQ ID NO:43)、LCDR2:AASNRES(SEQ ID NO:44)、LCDR3:QQSTEDPPYT(SEQ ID NO:38)、 (12)LCDR1:RASESVDYDGHSFIN(SEQ ID NO:39)、LCDR2:AASNKDT(SEQ ID NO:40)、LCDR3:QQSTEDPPYT(SEQ ID NO:38)、 (13)LCDR1:RASQSVDYEGHSFLN(SEQ ID NO:45)、LCDR2:AASNLQS(SEQ ID NO:42)、LCDR3:QQSTENPPYT(SEQ ID NO:46)、 (14)LCDR1:KSSQSVDYEGHSFLN(SEQ ID NO:47)、LCDR2:AASNRES(SEQ ID NO:44)、LCDR3:QQSTENPPYT(SEQ ID NO:46)、 (15)LCDR1:KASQSIDYDGDSFLN(SEQ ID NO:48), LCDR2:AASNLES(SEQ ID NO:37),LCDR3:HQCNEDPYM(SEQ ID NO:49), (16)LCDR1:RASESVDYDGDSFIN(SEQ ID NO:50),LCDR2:AASNKDT(SEQ ID NO:40),LCDR3:HQSNEDPYM(SEQ ID NO:51), (17)LCDR1:RASESVDYDGDSFIN(SEQ ID NO:50),LCDR2:AASNKDT(SEQ ID NO:40),LCDR3:HQSNEDPYL(SEQ ID NO:52), (18)LCDR1:RASESVDYDGDSFIN(SEQ ID NO:50),LCDR2:AASNKDT(SEQ ID NO:40),LCDR3:HQSNEDPYV(SEQ ID NO:53), (19)LCDR1:RASQSIDYDGDSFLN(SEQ ID NO:54), LCDR2:AASNLQS(SEQ ID NO:42),LCDR3:QQSNEDPYV(SEQ ID NO:55), (20) LCDR1: KSSQSIDYDGDSFLN (SEQ ID NO: 56), LCDR2: AASNRES (SEQ ID NO: 44), LCDR3: QQSNEDPYV (SEQ ID NO: 55), and (21) LCDR1, LCDR2, and LCDR3 as set forth in (1) to (20), wherein at least one of them is selected from the group consisting of one, two, three, four, or five amino acid additions, deletions, conservative amino acid substitutions, or combinations thereof. The bifunctional fusion protein according to [3] above. [8] The anti-AREG antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are (1)HCDR1:SYAMS(SEQ ID NO:1)、HCDR2:AISGSGGSTYYADSVKG(SEQ ID NO:2)、HCDR3:PTSRYSYGYDY(SEQ ID NO:3)、LCDR1:TGNSNNVGDQGAV(SEQ ID NO:21)、LCDR2:RNNNRPS(SEQ ID NO:22)、LCDR3:STWDSGLNSVV(SEQ ID NO:23)、 (2)HCDR1:SYAMS(SEQ ID NO:1)、HCDR2:AISGSGGSTYYADSVKG(SEQ ID NO:2)、HCDR3:PTSRYSYSYNN(SEQ ID NO:4)、LCDR1:TGNSNNVGDQGAV(SEQ ID NO:21)、LCDR2:RNNNRPS(SEQ ID NO:22)、LCDR3:STWDKNNKSVV(SEQ ID NO:24)、 (3)HCDR1:SHAMS(SEQ ID NO:5)、HCDR2:AISGSGGSTYYADSVKG(SEQ ID NO:2)、HCDR3:VDTKFDP(SEQ ID NO:6)、LCDR1:SGSSSNIGSNTVN(SEQ ID NO:25)、LCDR2:SNNQRPS(SEQ ID NO:26)、LCDR3:EVWDDSLNGPV(SEQ ID NO:27)、 (4)HCDR1:SYPMS(SEQ ID NO:7)、HCDR2:TISTGGTYTYYPDSVKG(SEQ ID NO:8)、HCDR3:QGPIYYGNYYYAMDY(SEQ ID NO:9)、LCDR1:RSSQSLVHSDGNTYLH(SEQ ID NO:28)、LCDR2:KVSNRFS(SEQ ID NO:29)、LCDR3:SQSTHVPYT(SEQ ID NO:30)、 (5)HCDR1:SYPMS(SEQ ID NO:7)、HCDR2:TISTGGRYTYYPDSVKG(SEQ ID NO:10)、HCDR3:QGPIYYGNYYYAMDY(SEQ ID NO:9)、LCDR1:RSSQSLVDGEDGTYLN(SEQ ID NO:31)、LCDR2:KVSERFD(SEQ ID NO:32)、LCDR3:SQSTHVPYT(SEQ ID NO:30)、 (6)HCDR1:SYPMS(SEQ ID NO:7)、HCDR2:TISTGGTYTYYPDSVKG(SEQ ID NO:8)、HCDR3:QGPILRKNYYYGMDV(SEQ ID NO:11)、LCDR1:RSSQSLVDGQDGTYLH(SEQ ID NO:33)、LCDR2:KVSNRFD(SEQ ID NO:34)、LCDR3:SQSTHVPYT(SEQ ID NO:30)、 (7)HCDR1:SYPMS(SEQ ID NO:7)、HCDR2:TISTGGTYTYYPDSVKG(SEQ ID NO:8)、HCDR3:QGPIYYGNYYYGMDV(SEQ ID NO:12)、LCDR1:RSSQSLVNQEGETYLH(SEQ ID NO:35)、LCDR2:KVSNRFD(SEQ ID NO:34)、LCDR3:SQSTHVPYT (SEQ ID NO:30)、 (8)HCDR1:SYAMS(SEQ ID NO:1)、HCDR2:TISTGGSHTYYPDSVKG(SEQ ID NO:13)、HCDR3:HGYLLYDGYYEWYFDV(SEQ ID NO:14)、LCDR1:KASQSVDYDGHSFLN(SEQ ID NO:36)、LCDR2:AASNLES(SEQ ID NO:37)、LCDR3:QQSTEDPPYT(SEQ ID NO:38)、 (9)HCDR1:SYAMS(SEQ ID NO:1)、HCDR2:TISTGGSHTYYPDSVKG(SEQ ID NO:13)、HCDR3:HGYLLYDGYYEWYFDY(SEQ ID NO:140)、LCDR1:RASESVDYDGHSFIN(SEQ ID NO:39)、LCDR2:AASNKDT(SEQ ID NO:40)、LCDR3:QQSTEDPPYT(SEQ ID NO:38)、 (10)HCDR1:SYAMS(SEQ ID NO:1)、HCDR2:TISTGGSHTYYPDSVKG(SEQ ID NO:13)、HCDR3:HGYLLYDGYYEWYFDY(SEQ ID NO:140)、LCDR1:RASQSVDYDGHSFLN(SEQ ID NO:41)、LCDR2:AASNLQS(SEQ ID NO:42)、LCDR3:QQSTEDPPYT(SEQ ID NO:38)、 (11)HCDR1:SYAMS(SEQ ID NO:1)、HCDR2:TISTGGSHTYYPDSVKG(SEQ ID NO:13)、HCDR3:HGYLLYDGYYEWYFDY(SEQ ID NO:140)、LCDR1:KSSQSVDYDGHSFLN(SEQ ID NO:43)、LCDR2:AASNRES(SEQ ID NO:44)、LCDR3:QQSTEDPPYT(SEQ ID NO:38)、 (12)HCDR1:SYAMS(SEQ ID NO:1)、HCDR2:TISTGGSHTYYPESVKG(SEQ ID NO:15)、HCDR3:HGYLLYEGYYEWYFDY(SEQ ID NO:16)、LCDR1:RASESVDYDGHSFIN(SEQ ID NO:39)、LCDR2:AASNKDT(SEQ ID NO:40)、LCDR3:QQSTEDPPYT(SEQ ID NO:38)、 (13)HCDR1:SYAMS(SEQ ID NO:1)、HCDR2:TISTGGSHTYYPESVKG(SEQ ID NO:15)、HCDR3:HGYLLYEGYYEWYFDY(SEQ ID NO:16)、LCDR1:RASQSVDYEGHSFLN(SEQ ID NO:45)、LCDR2:AASNLQS(SEQ ID NO:42)、LCDR3:QQSTENPPYT(SEQ ID NO:46)、 (14)HCDR1:SYAMS(SEQ ID NO:1)、HCDR2:TISTGGSHTYYPESVKG(SEQ ID NO:15)、HCDR3:HGYLLYEGYYEWYFDY(SEQ ID NO:16)、LCDR1:KSSQSVDYEGHSFLN(SEQ ID NO:47)、LCDR2:AASNRES(SEQ ID NO:44)、LCDR3:QQSTENPPYT(SEQ ID NO:46)、 (15)HCDR1:GYPMS(SEQ ID NO:17)、HCDR2:TISTGARHTYYPDSVKG(SEQ ID NO:18)、HCDR3:HEGLRRGKYHCIMDY(SEQ ID NO:19)、LCDR1:KASQSIDYDGDSFLN(SEQ ID NO:48)、LCDR2:AASNLES(SEQ ID NO:37)、LCDR3:HQCNEDPYM(SEQ ID NO:49)、 (16)HCDR1:GYPMS(SEQ ID NO:17), HCDR2:TISTGARHTYYPDSVKG(SEQ ID NO:18), HCDR3:HEGLRRGKYHSIMDY(SEQ ID NO:20), LCDR1:RASESVDYDGDSFIN(SEQ ID NO:50), LCDR2:AASNKDT(SEQ ID NO:40) LCDR3:HQSNEDPYM(SEQ ID NO:51) (17)HCDR1:GYPMS(SEQ ID NO:17), HCDR2:TISTGARHTYYPDSVKG(SEQ ID NO:18), HCDR3:HEGLRRGKYHSIMDY(SEQ ID NO:20), LCDR1:RASESVDYDGDSFIN(SEQ ID NO:50), LCDR2:AASNKDT(SEQ ID NO:40) LCDR3:HQSNEDPYL(SEQ ID NO:52) (18)HCDR1:GYPMS(SEQ ID NO:17), HCDR2:TISTGARHTYYPDSVKG(SEQ ID NO:18), HCDR3:HEGLRRGKYHSIMDY(SEQ ID NO:20), LCDR1:RASESVDYDGDSFIN(SEQ ID NO:50), LCDR2:AASNKDT(SEQ ID NO:40) LCDR3:HQSNEDPYV(SEQ ID NO:53) (19)HCDR1:GYPMS(SEQ ID NO:17), HCDR2:TISTGARHTYYPDSVKG(SEQ ID NO:18), HCDR3:HEGLRRGKYHSIMDY(SEQ ID NO:20), LCDR1:RASQSIDYDGDSFLN(SEQ ID NO:54), LCDR2:AASNLQS(SEQ). ID NO:42) LCDR3:QQSNEDPYV(SEQ ID NO:55) (20)HCDR1:GYPMS(SEQ ID NO:17), HCDR2:TISTGARHTYYPDSVKG(SEQ ID NO:18), HCDR3:HEGLRRGKYHSIMDY(SEQ ID NO:20), LCDR1:KSSQSIDYDGDSFLN(SEQ ID NO:56), LCDR2:AASNRES(SEQ ID NO:56). NO:44) LCDR3:QQSNEDPYV(SEQ ID NO:55). (21) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as shown in (1) to (20), with the proviso that: at least one of which is selected from the group consisting of one, two, three, four or five amino acid additions, deletions, conservative amino acid substitutions or combinations thereof. The bifunctional fusion protein according to [3] above. [9] The anti-AREG antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region has an amino acid sequence selected from SEQ ID NOs: 57 to 69, or an amino acid sequence that has at least 95% sequence identity with any one of SEQ ID NOs: 57 to 69 and retains epitope binding activity; wherein the light chain variable region has an amino acid sequence selected from SEQ ID NOs: 70 to 89, or an amino acid sequence that has at least 95% sequence identity with any one of SEQ ID NOs: 70 to 89 and retains epitope binding activity. The bifunctional fusion protein according to [3] above.

[10] The anti-AREG antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region are (1) SEQ ID NO: 57 and SEQ ID NO: 70, (2) SEQ ID NO: 58 and SEQ ID NO: 71; (3) SEQ ID NO: 59 and SEQ ID NO: 72; (4) SEQ ID NO: 60 and SEQ ID NO: 73; (5) SEQ ID NO: 61 and SEQ ID NO: 74, (6) SEQ ID NO: 62 and SEQ ID NO: 75; (7) SEQ ID NO: 63 and SEQ ID NO: 76, (8) SEQ ID NO: 64 and SEQ ID NO: 77, (9) SEQ ID NO: 65 and SEQ ID NO: 78, (10) SEQ ID NO: 66 and SEQ ID NO: 79, (11) SEQ ID NO: 66 and SEQ ID NO: 80, (12) SEQ ID NO: 66 and SEQ ID NO: 81, (13) SEQ ID NO: 67 and SEQ ID NO: 79, (14) SEQ ID NO: 67 and SEQ ID NO: 82, (15) SEQ ID NO: 67 and SEQ ID NO: 83, (16) SEQ ID NO: 68 and SEQ ID NO: 84, (17) SEQ ID NO: 69 and SEQ ID NO: 85, (18) SEQ ID NO: 69 and SEQ ID NO: 86, (19) SEQ ID NO: 69 and SEQ ID NO: 87, (20) SEQ ID NO: 69 and SEQ ID NO: 88, (21) SEQ ID NO: 69 and SEQ ID NO: 89, and (22) A method for identifying a target polypeptide, comprising: selecting from the group consisting of two amino acid sequences each having at least 95% sequence identity with any one of (1) to (21) and retaining epitope binding activity; The bifunctional fusion protein according to [3] above.

[11] The anti-AREG antibody or fragment thereof is an IgG, IgM, IgA, IgE, or IgD isotype or a mutant thereof, preferably an IgG1, IgG2, IgG3, or IgG4 isotype or a mutant thereof. The bifunctional fusion protein according to [3] above.

[12] The second domain comprises the extracellular domain of TRII or a mutant thereof. The bifunctional fusion protein according to [3] above.

[13] The mutant is characterized in that it contains point mutations and / or deletions. The bifunctional fusion protein according to

[12] above.

[14] The extracellular domain of the TRII has an amino acid sequence set forth in SEQ ID NO: 90 or an amino acid sequence having at least 85% identity with SEQ ID NO: 90. The bifunctional fusion protein according to

[12] above.

[15] The point mutations include one or more point mutations at positions selected from K7, T16, D17, R34, R66, K67, K103, and K104, where the position numbering is based on the N-terminus to the C-terminus of SEQ ID NO: 90. The bifunctional fusion protein according to

[13] above.

[16] The point mutations include one or more site mutations selected from K7Q, T16S, D17N, R34S, R34H, R66S, K67S, K103S, and K104S, and the position numbering is based on the N-terminus to C-terminus of SEQ ID NO: 90. The bifunctional fusion protein according to

[13] above.

[17] The point mutations are selected from T16S and D17N, K7Q and D17N, K7Q, R34S, R34H, R66S and K67S, K103S and K104S, K7Q and R34S, K7Q, R66S and K67S, K7Q, K103S and K104S, K7Q, R34S, R66S and K67S, K7Q, R34S, K103S and K104S, K7Q, R66S, K67S, K103S and K104S, R34S, R66S, K67S, K103S and K104S, K7Q, R34S, R66S, K67S, and K103S and K104S. The bifunctional fusion protein according to

[13] above.

[18] The variants have an N-terminal deletion of 4 to 21 amino acids, preferably 4, 7, 9, 13, 17, and 21 amino acids, and the position numbering is based on the N-terminus to C-terminus of SEQ ID NO: 90. The bifunctional fusion protein according to

[13] above.

[19] The mutant is characterized by containing point mutations T16S and D17N and an N-terminal deletion of seven amino acids. The bifunctional fusion protein according to

[13] above.

[20] The second domain comprises the extracellular domain of TRII or a variant thereof, and has any one of the amino acid sequences shown in SEQ ID NOs: 90 to 107, or an amino acid sequence having at least 85% identity with any one of SEQ ID NOs: 90 to 107. The bifunctional fusion protein according to

[12] above.

[21] The C-terminus of the first domain is fused to the N-terminus of the second domain via a linker, preferably a linker peptide, and vice versa. The bifunctional fusion protein according to [3] above.

[22] The C-terminus of the heavy or light chain of the anti-AREG antibody is fused directly or via a linker to the N-terminus of the extracellular domain of TRII or a variant thereof, or the N-terminus of the heavy or light chain of the anti-AREG antibody is linked directly or via a linker to the C-terminus of the extracellular domain of TRII or a variant thereof. The bifunctional fusion protein according to [3] above.

[23] The bifunctional fusion protein comprises a heavy chain of an anti-AREG antibody, which is linked directly or via a linker to the extracellular domain of TRII or a mutant thereof. The bifunctional fusion protein according to [3] above.

[24] The bifunctional fusion protein comprises a heavy chain of an anti-AREG antibody, the N-terminus of which is linked, directly or via a linker, to the C-terminus of the extracellular domain of TRII or a mutant thereof, or the C-terminus of the heavy chain of the anti-AREG antibody is linked, directly or via a linker, to the N-terminus of the extracellular domain of TRII or a mutant thereof. The bifunctional fusion protein according to [3] above.

[25] The linker has the formula (G 4 S) n 、(G 4 S) n G, S(G 4 S) n G, SG (EAAAK) n SG, S(GEGES)nG or (EAAAK) n wherein n is an integer of 1 to 5, and preferably the linker peptide has an amino acid sequence shown in any one of SEQ ID NOs: 108 to 117. The bifunctional fusion protein according to any one of

[21] to

[24] above.

[26] The bifunctional fusion protein comprises a heavy chain of an anti-AREG antibody, the C-terminus of which is linked to the N-terminus of the extracellular domain of the TRII via a linker, and has an amino acid sequence of any one of SEQ ID NOs: 118 to 139, or an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 118 to 139. The bifunctional fusion protein according to [3] above.

[27] The method further comprising the light chain of an anti-AREG antibody. The bifunctional fusion protein according to [3] above.

[28] Characterized by a heterotetrameric structure The bifunctional fusion protein according to [2] above.

[29] An isolated nucleic acid molecule comprising: The isolated nucleic acid molecule, characterized in that it encodes the bifunctional fusion protein according to any one of [1] to

[28] above.

[30] An expression vector, An expression vector characterized by comprising the isolated nucleic acid molecule described in

[29] above.

[31] A host cell, A host cell characterized by comprising the isolated nucleic acid molecule according to

[29] above or the expression vector according to

[30] above.

[32] A method for preparing the bifunctional fusion protein according to any one of [1] to

[28] above, comprising: A method for preparing the bifunctional fusion protein according to any one of [1] to

[28] above, comprising culturing the host cell according to

[31] above.

[33] A pharmaceutical composition, comprising the bifunctional fusion protein according to any one of [1] to

[28] above and a pharmaceutically acceptable carrier.

[34] Use of the bifunctional fusion protein according to any one of [1] to

[28] above, the isolated nucleic acid molecule according to

[29] above, or the pharmaceutical composition according to

[33] above for preventing, treating, and / or diagnosing fibrotic diseases, cancer, and diseases associated with chronic inflammation in a subject, Preferably, the fibrotic disease includes, but is not limited to, renal fibrosis, liver fibrosis, and pulmonary fibrosis, in particular idiopathic pulmonary fibrosis (IPF).

[35] A method for preventing, treating, and / or diagnosing fibrotic diseases, cancer, and diseases associated with chronic inflammation in a subject, comprising: A method for preventing, treating and / or diagnosing fibrotic diseases, cancer, and diseases associated with chronic inflammation in a subject, comprising administering to the subject a therapeutically effective amount of the bifunctional fusion protein according to any one of the above [1] to

[28] or the pharmaceutical composition according to the above

[33] , wherein the fibrotic diseases include, but are not limited to, renal fibrosis, hepatic fibrosis, and pulmonary fibrosis.

Claims

1. A bifunctional fusion protein comprising a first domain and a second domain, the first domain is capable of binding to AREG or a fragment thereof and is an anti-AREG antibody or antigen-binding fragment thereof that binds to AREG or a fragment thereof; the second domain is capable of binding to a TGFβ ligand or a fragment thereof and comprises a portion of the extracellular domain of TGFβ receptor II (TRII) or a variant thereof; the anti-AREG antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2 and LCDR3; wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are as follows: (1) HCDR1: SYAMS (SEQ ID NO: 1), HCDR2: AISGSGGSTYYADSVKG (SEQ ID NO: 2), HCDR3: PTSRYSYGYDY (SEQ ID NO: 3), LCDR1: TGNSNNVGDQGAV (SEQ ID NO: 21), LCDR2: RNNNRPS (SEQ ID NO: 22), LCDR3: STWDSGLNSVV (SEQ ID NO: 23), (2) HCDR1: SYAMS (SEQ ID NO: 1), HCDR2: AISGSGGSTYYADSVKG (SEQ ID NO: 2), HCDR3: PTSRYSYSYNN (SEQ ID NO: 4), LCDR1: TGNSNNVGDQGAV (SEQ ID NO: 21), LCDR2: RNNNRPS (SEQ ID NO: 22), LCDR3: STWDKNNKSVV (SEQ ID NO: 24), (3) HCDR1: SHAMS (SEQ ID NO: 5), HCDR2: AISGSGGSTYYADSVKG (SEQ ID NO: 2), HCDR3: VDTKFDP (SEQ ID NO: 6), LCDR1: SGSSSNIGSNTVN (SEQ ID NO: 25), LCDR2: SNNQRPS (SEQ ID NO: 26), LCDR3: EVWDDSLNGPV (SEQ ID NO: 27), (4)HCDR1:SYPMS(SEQ ID NO:7)、HCDR2:TISTGGTYTYYPDSVKG(SEQ ID NO:8)、HCDR3:QGPIYYGNYYYAMDY(SEQ ID NO:9)、LCDR1:RSSQSLVHSDGNTYLH(SEQ ID NO:28)、LCDR2:KVSNRFS(SEQ ID NO:29)、LCDR3:SQSTHVPYT(SEQ ID NO:30)、 (5)HCDR1:SYPMS(SEQ ID NO:7)、HCDR2:TISTGGRYTYYPDSVKG(SEQ ID NO:10)、HCDR3:QGPIYYGNYYYAMDY(SEQ ID NO:9)、LCDR1:RSSQSLVDGEDGTYLN(SEQ ID NO:31)、LCDR2:KVSERFD(SEQ ID NO:32)、LCDR3:SQSTHVPYT(SEQ ID NO:30)、 (6)HCDR1:SYPMS(SEQ ID NO:7)、HCDR2:TISTGGTYTYYPDSVKG(SEQ ID NO:8)、HCDR3:QGPILRKNYYYGMDV(SEQ ID NO:11)、LCDR1:RSSQSLVDGQDGTYLH(SEQ ID NO:33)、LCDR2:KVSNRFD(SEQ ID NO:34)、LCDR3:SQSTHVPYT(SEQ ID NO:30)、 (7)HCDR1:SYPMS(SEQ ID NO:7)、HCDR2:TISTGGTYTYYPDSVKG(SEQ ID NO:8)、HCDR3:QGPIYYGNYYYGMDV(SEQ ID NO:12)、LCDR1:RSSQSLVNQEGETYLH(SEQ ID NO:35)、LCDR2:KVSNRFD(SEQ ID NO:34)、LCDR3:SQSTHVPYT(SEQ ID NO:30)、 (8)HCDR1:SYAMS(SEQ ID NO:1)、HCDR2:TISTGGSHTYYPDSVKG(SEQ ID NO:13)、HCDR3:HGYLLYDGYYEWYFDV(SEQ ID NO:14)、LCDR1:KASQSVDYDGHSFLN(SEQ ID NO:36)、LCDR2:AASNLES(SEQ ID NO:37)、LCDR3:QQSTEDPPYT(SEQ ID NO:38)、 (9)HCDR1:SYAMS(SEQ ID NO:1)、HCDR2:TISTGGSHTYYPDSVKG(SEQ ID NO:13)、HCDR3:HGYLLYDGYYEWYFDY(SEQ ID NO:140)、LCDR1:RASESVDYDGHSFIN(SEQ ID NO:39)、LCDR2:AASNKDT(SEQ ID NO:40)、LCDR3:QQSTEDPPYT(SEQ ID NO:38)、 (10)HCDR1:SYAMS(SEQ ID NO:1)、HCDR2:TISTGGSHTYYPDSVKG(SEQ ID NO:13)、HCDR3:HGYLLYDGYYEWYFDY(SEQ ID NO:140)、LCDR1:RASQSVDYDGHSFLN(SEQ ID NO:41)、LCDR2:AASNLQS(SEQ ID NO:42)、LCDR3:QQSTEDPPYT(SEQ ID NO:38)、 (11)HCDR1:SYAMS(SEQ ID NO:1)、HCDR2:TISTGGSHTYYPDSVKG(SEQ ID NO:13)、HCDR3:HGYLLYDGYYEWYFDY(SEQ ID NO:140)、LCDR1:KSSQSVDYDGHSFLN(SEQ ID NO:43)、LCDR2:AASNRES(SEQ ID NO:44)、LCDR3:QQSTEDPPYT(SEQ ID NO:38)、 (12)HCDR1:SYAMS(SEQ ID NO:1)、HCDR2:TISTGGSHTYYPESVKG(SEQ ID NO:15)、HCDR3:HGYLLYEGYYEWYFDY(SEQ ID NO:16)、LCDR1:RASESVDYDGHSFIN(SEQ ID NO:39)、LCDR2:AASNKDT(SEQ ID NO:40)、LCDR3:QQSTEDPPYT(SEQ ID NO:38)、 (13)HCDR1:SYAMS(SEQ ID NO:1)、HCDR2:TISTGGSHTYYPESVKG(SEQ ID NO:15)、HCDR3:HGYLLYEGYYEWYFDY(SEQ ID NO:16)、LCDR1:RASQSVDYEGHSFLN(SEQ ID NO:45)、LCDR2:AASNLQS(SEQ ID NO:42)、LCDR3:QQSTENPPYT(SEQ ID NO:46)、 (14)HCDR1:SYAMS(SEQ ID NO:1)、HCDR2:TISTGGSHTYYPESVKG(SEQ ID NO:15)、HCDR3:HGYLLYEGYYEWYFDY(SEQ ID NO:16)、LCDR1:KSSQSVDYEGHSFLN(SEQ ID NO:47)、LCDR2:AASNRES(SEQ ID NO:44)、LCDR3:QQSTENPPYT(SEQ ID NO:46)、 (15)HCDR1:GYPMS(SEQ ID NO:17)、HCDR2:TISTGARHTYYPDSVKG(SEQ ID NO:18)、HCDR3:HEGLRRGKYHCIMDY(SEQ ID NO:19)、LCDR1:KASQSIDYDGDSFLN(SEQ ID NO:48)、LCDR2:AASNLES(SEQ ID NO:37)、LCDR3:HQCNEDPYM(SEQ ID NO:49)、 (16)HCDR1:GYPMS(SEQ ID NO:17)、HCDR2:TISTGARHTYYPDSVKG(SEQ ID NO:18)、HCDR3:HEGLRRGKYHSIMDY(SEQ ID NO:20)、LCDR1:RASESVDYDGDSFIN(SEQ ID NO:50)、LCDR2:AASNKDT(SEQ ID NO:40)、LCDR3:HQSNEDPYM(SEQ ID NO:51)、 (17)HCDR1:GYPMS(SEQ ID NO:17)、HCDR2:TISTGARHTYYPDSVKG(SEQ ID NO:18)、HCDR3:HEGLRRGKYHSIMDY(SEQ ID NO:20)、LCDR1:RASESVDYDGDSFIN(SEQ ID NO:50)、LCDR2:AASNKDT(SEQ ID NO:40)、LCDR3:HQSNEDPYL(SEQ ID NO:52)、 (18)HCDR1:GYPMS(SEQ ID NO:17)、HCDR2:TISTGARHTYYPDSVKG(SEQ ID NO:18)、HCDR3:HEGLRRGKYHSIMDY(SEQ ID NO:20)、LCDR1:RASESVDYDGDSFIN(SEQ ID NO:50)、LCDR2:AASNKDT(SEQ ID NO:40)、LCDR3:HQSNEDPYV(SEQ ID NO:53)、 (19) HCDR1: GYPMS (SEQ ID NO: 17), HCDR2: TISTGARHTYYPDSVKG (SEQ ID NO: 18), HCDR3: HEGLRRGKYHSIMDY (SEQ ID NO: 20), LCDR1: RASQSIDYDGDSFLN (SEQ ID NO: 54), LCDR2: AASNLQS (SEQ ID NO: 42), LCDR3: QQSNEDPYV (SEQ ID NO: 55), and (20) HCDR1: GYPMS (SEQ ID NO: 17), HCDR2: TISTGARHTYYPDSVKG (SEQ ID NO: 18), HCDR3: HEGLRRGKYHSIMDY (SEQ ID NO: 20), LCDR1: KSSQSIDYDGDSFLN (SEQ ID NO: 56), LCDR2: AASNRES (SEQ ID NO: 44), LCDR3: QQSNEDPYV (SEQ ID NO: 55) selected from the group consisting of The bifunctional fusion protein, wherein the second domain comprises the extracellular domain of TRII or a variant thereof and has an amino acid sequence set forth in any one of SEQ ID NOs: 90 to 107.

2. The antibody or antigen-binding fragment thereof is capable of binding to both human AREG and mouse AREG, or the anti-AREG antibody or fragment thereof is capable of binding to human AREG and has weak or no cross-reactivity to mouse AREG, and / or The anti-AREG antibody or fragment thereof is a human anti-AREG antibody, a murine anti-AREG antibody, a chimeric anti-AREG antibody, or a humanized anti-AREG antibody; The bifunctional fusion protein of claim 1. Claim 3: The AREG antibody or fragment thereof is characterized in that it is a human monoclonal antibody (mAb), a murine mAb, a chimeric mAb, or a humanized mAb; and / or The anti-AREG antibody or fragment thereof is characterized in that it is capable of binding to a soluble form of AREG; The bifunctional fusion protein of claim 1.

4. The AREG antibody or fragment thereof is characterized in that it is capable of binding to the epidermal growth factor (EGF)-like domain of a soluble form of AREG, and / or The anti-AREG antibody or fragment thereof is an isotype of IgG, IgM, IgA, IgE, or IgD, or a variant thereof; The bifunctional fusion protein of claim 1.

5. The bifunctional fusion protein described in claim 1, characterized in that the AREG antibody or fragment thereof is of an isotype of IgG1, IgG2, IgG3, or IgG4 or a mutant thereof.

6. the anti-AREG antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 57 to 69, and an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NOs: 57 to 69 and retaining epitope binding activity; The bifunctional fusion protein of claim 1, wherein the light chain variable region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 70 to 89, or an amino acid sequence that has at least 95% sequence identity with any one of SEQ ID NOs: 70 to 89 and retains epitope binding activity.

7. The anti-AREG antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region are as follows: (1) SEQ ID NO: 57 and SEQ ID NO: 70; (2) SEQ ID NO: 58 and SEQ ID NO: 71; (3) SEQ ID NO: 59 and SEQ ID NO: 72; (4) SEQ ID NO: 60 and SEQ ID NO: 73; (5) SEQ ID NO: 61 and SEQ ID NO: 74; (6) SEQ ID NO: 62 and SEQ ID NO: 75; (7) SEQ ID NO: 63 and SEQ ID NO: 76; (8) SEQ ID NO: 64 and SEQ ID NO: 77; (9) SEQ ID NO: 65 and SEQ ID NO: 78; (10) SEQ ID NO: 66 and SEQ ID NO: 79; (11) SEQ ID NO: 66 and SEQ ID NO: 80; (12) SEQ ID NO: 66 and SEQ ID NO: 81; (13) SEQ ID NO: 67 and SEQ ID NO: 79; (14) SEQ ID NO: 67 and SEQ ID NO: 82; (15) SEQ ID NO: 67 and SEQ ID NO: 83; (16) SEQ ID NO: 68 and SEQ ID NO: 84; (17) SEQ ID NO: 69 and SEQ ID NO: 85; (18) SEQ ID NO: 69 and SEQ ID NO: 86; (19) SEQ ID NO: 69 and SEQ ID NO: 87; (20) SEQ ID NO: 69 and SEQ ID NO: 88; (21) SEQ ID NO: 69 and SEQ ID NO: 89, and (22) Two amino acid sequences each having at least 95% sequence identity with any one of (1) to (21) and retaining epitope binding activity. The bifunctional fusion protein according to claim 1, characterized in that it is selected from the group consisting of:

8. The bifunctional fusion protein of claim 1, characterized in that the C-terminus of the heavy or light chain of the anti-AREG antibody is fused directly or via a linker to the N-terminus of the extracellular domain of TRII or a mutant thereof, or the N-terminus of the heavy or light chain of the anti-AREG antibody is linked directly or via a linker to the C-terminus of the extracellular domain of TRII or a mutant thereof.

9. The bifunctional fusion protein of claim 1, characterized in that the bifunctional fusion protein comprises a heavy chain of an anti-AREG antibody, the N-terminus of which is linked, directly or via a linker, to the C-terminus of the extracellular domain of TRII or a mutant thereof, or comprises a heavy chain of an anti-AREG antibody, the C-terminus of which is linked, directly or via a linker, to the N-terminus of the extracellular domain of TRII or a mutant thereof.

10. The bifunctional fusion protein of claim 1, wherein the bifunctional fusion protein comprises a light chain of an anti-AREG antibody and / or is in a heterotetrameric form.

11. The linker has the formula (G ４ S) ｎ , (G ４ S) ｎ G, S (G ４ S) ｎ G, SG (EAAAK) ｎ SG, S(GEGES)nG or (EAAAK) ｎ 10. The bifunctional fusion protein of claim 9, comprising a linker peptide represented by the formula:

12. The bifunctional fusion protein of claim 11, wherein the linker peptide has an amino acid sequence shown in any one of SEQ ID NOs: 108 to 117.

13. The bifunctional fusion protein according to claim 1, characterized in that it comprises a heavy chain of an anti-AREG antibody, the C-terminus of which is linked to the N-terminus of the extracellular domain of the TRII via a linker, and has an amino acid sequence shown in any one of SEQ ID NOs: 118 to 139 and 142, or an amino acid sequence having at least 85% identity with any one of SEQ ID NOs: 118 to 139 and 142.

14. An isolated nucleic acid molecule, characterized in that it encodes the bifunctional fusion protein described in claim 1.

15. An expression vector, characterized in that it contains the isolated nucleic acid molecule described in claim 14.

16. A host cell characterized by containing the isolated nucleic acid molecule described in claim 14.

17. A host cell characterized by containing the expression vector described in claim 15.

18. A pharmaceutical composition comprising the bifunctional fusion protein of claim 1 and a pharmaceutically acceptable carrier.

19. A pharmaceutical composition comprising the bifunctional fusion protein of claim 1 for use in the prevention, treatment and / or diagnosis of fibrotic diseases, cancer and diseases associated with chronic inflammation in a subject.

20. A pharmaceutical composition comprising the isolated nucleic acid molecule of claim 14 for use in the prevention, treatment and / or diagnosis of fibrotic diseases, cancer and diseases associated with chronic inflammation in a subject.

21. The pharmaceutical composition described in claim 19 or 20, characterized in that the fibrotic disease includes renal fibrosis, hepatic fibrosis, and pulmonary fibrosis.

22. The pharmaceutical composition described in claim 19 or 20, characterized in that the fibrotic disease includes idiopathic pulmonary fibrosis (IPF).

Images