Antibodies against areg and uses thereof

Abstract

Anti-AREG antibodies or immunoreactive fragments thereof are provided for use in the treatment, diagnosis or prevention of fibrotic diseases including, but not limited to, renal fibrosis, liver fibrosis, lung fibrosis, in particular IPF. Also provided are polynucleotides or nucleic acid molecules encoding the antibodies, expression vectors, host cells, and methods for making the antibodies. The anti-AREG antibodies specifically bind to AREG through binding residues located in the EGF-like domain and block the function of AREG.

Description

Background Technology

[0001] Fibrosis, caused by injury leading to thickening of connective tissue and scar formation, is characterized by excessive proliferation of fibroblasts and accumulation of extracellular matrix (ECM) components. This condition is common in organs such as the lungs, liver, and kidneys, causing disruption of tissue structure and resulting in severe organ dysfunction.

[0002] Pulmonary fibrosis (PF) is a lung disease that occurs when healthy lung tissue is replaced by an excessive amount of extracellular matrix. In PF, the alveolar structure is disrupted, leading to decreased lung compliance, impaired gas exchange, and ultimately respiratory failure and death. A common feature of pulmonary fibrosis is the excessive proliferation of fibroblasts around the air sacs (alveoli) of the lungs (Barkauskas and Noble, 2014). The most common type of pulmonary fibrosis is idiopathic pulmonary fibrosis (IPF). IPF is an interstitial lung disease of unknown cause with severe and progressive loss of lung function. It is most common in older adults aged 50 to 70. IPF is a fatal disease with a median survival of only 2–4 years from diagnosis (Steele and Schwartz, 2013) and can ultimately lead to respiratory failure. The pathogenesis of pulmonary fibrosis remains a mystery, and clinical treatment is very limited. Currently, only two commercially available FDA-approved drugs, nintedanib and pirfenidone, are used to treat IPF. However, both drugs can only improve the rate of decline in forced vital capacity within one year, but neither can significantly improve the patient's survival rate.

[0003] The following lists the existing technologies for anti-AREG antibodies:

[0004] U.S. Patent Application No. 10 / 774,076 relates to AREG antibodies and their use in the treatment of cancer and psoriasis. The claimed antibody is humanized PAR34;

[0005] PCT application number PCT / GB2009 / 050389 relates to antibodies that cross-react with both AREG and HBEGF. The antibodies are intended for use in methods of treating cancer and diseases related to angiogenesis. The claimed antibody is 2F7; and

[0006] U.S. Patent Application No. 15 / 271,515 relates to AREG antibodies and their use in cancer treatment. The claimed antibodies are AR30, AR37, and AR558. AR558, in particular, exhibited the best antitumor activity in a xenograft mouse tumor model. All three antibodies are murine antibodies, not humanized antibodies. Summary of the Invention

[0007] In the prior art, there are no definitive reports on key drug targets for pulmonary fibrosis, particularly idiopathic pulmonary fibrosis (IPF), and especially AREG signaling in lung AT2 cells. The inventors of this invention have established a unique link between AREG signaling in lung AT2 cells and the development of pulmonary fibrosis, particularly IPF, and have discovered that AREG signaling in lung AT2 cells can serve as a key drug target for pulmonary fibrosis, particularly IPF. Specifically, AREG was not detected in AT2 cells from normal control lungs, but it was detected in AT2 cells from all IPF samples.

[0008] Furthermore, the inventors of this invention constructed an animal model of IPF in which the Cdc42 gene was knocked out in AT2 cells. AREG was not detectable in AT2 cells of control lungs, but it was detectable in AT2 cells of Cdc42 AT2 knockout lungs. This is the first animal model that can highly mimic the pathogenesis and progression of IPF. Using this animal model, we identified AREG as a key therapeutic target for pulmonary fibrosis.

[0009] Based on the above knowledge, the inventors of this invention prepared, screened and obtained antibodies against AREG for the treatment of renal fibrosis, liver fibrosis, pulmonary fibrosis, and especially IPF.

[0010] This invention provides anti-AREG antibodies or immunoreactive fragments thereof for the treatment, diagnosis, or prevention of fibrotic diseases, including but not limited to renal fibrosis, liver fibrosis, pulmonary fibrosis, and particularly IPF. It also provides polynucleotide or nucleic acid molecules encoding said antibodies, expression vectors, host cells, and methods for preparing said antibodies. Pharmaceutical compositions comprising said antibody molecules are also provided. The anti-AREG antibodies of this invention specifically bind to AREG and block AREG function through binding residues located in an EGF-like domain. The anti-AREG antibodies disclosed herein can be used for the treatment, prevention, and / or diagnosis of fibrotic diseases, including but not limited to renal fibrosis, liver fibrosis, pulmonary fibrosis, and particularly IPF.

[0011] On one hand, the present invention provides an isolated anti-AREG antibody or a fragment thereof, which has the ability to inhibit fibrosis. Preferably, the fibrosis is renal fibrosis, liver fibrosis, pulmonary fibrosis, and especially IPF.

[0012] In some embodiments, the anti-AREG antibody or a fragment thereof according to the present invention is capable of binding to AREG.

[0013] In some embodiments, the anti-AREG antibody or a fragment thereof according to the invention binds to both human AREG (hAREG) and mouse AREG (mAREG).

[0014] In some embodiments, the anti-AREG antibody or fragment thereof according to the invention binds only to human AREG (hAREG) and not to mouse AREG (mAREG).

[0015] In some embodiments, the anti-AREG antibody or fragment thereof according to the invention is a human anti-AREG antibody, a mouse anti-AREG antibody, a humanized anti-AREG antibody, or a chimeric anti-AREG antibody.

[0016] In some embodiments, the anti-AREG antibody or fragment thereof according to the invention binds to AREG with high affinity, having a dissociation constant (KD) of less than about 10 nM, for example less than 1 nM, 0.1 nM or 0.01 nM, for example at 1 × 10⁻⁶. -8 -1×10 -11 Within the range, preferably within 1×10 -9 -1×10 -11 Within the range.

[0017] In some embodiments, the anti-AREG antibody or a fragment thereof according to the invention is capable of binding to soluble form of AREG. Preferably, the anti-AREG antibody is capable of binding to the EGF-like domain of soluble form of AREG.

[0018] In some embodiments, the anti-AREG antibody or fragment thereof according to the present invention is capable of binding to residues 101-184 of human pro-AREG. The amino acid sequence of human pro-AREG is shown in SEQ ID NO:135.

[0019] In some embodiments, the anti-AREG antibody is capable of binding to the C-terminus of the EGF-like domain of soluble AREG.

[0020] In some embodiments, the anti-AREG antibody or fragment thereof according to the present invention is capable of binding to residues 171 to 184 of human pro-AREG.

[0021] In some embodiments, the anti-AREG antibody or fragment thereof according to the present invention is capable of binding to residues 94 to 177 of mouse pro-AREG.

[0022] In some embodiments, the anti-AREG antibody or fragment thereof according to the present invention is capable of binding to the EGF-like domain of residues 135 to 177 of murine pro-AREG.

[0023] In some embodiments, the anti-AREG antibody or fragment thereof according to the invention is capable of binding to, for example, at least one, two, three, four or five amino acids within residues 101 to 184 of human pro-AREG as represented by any of SEQ ID NO: 123 to 132, preferably within residues 142 to 184 of human pro-AREG as represented by any of SEQ ID NO: 123 to 132.

[0024] In some embodiments, the anti-AREG antibody or fragment thereof according to the invention is capable of interacting with Glu149 and / or His164 of human pro-AREG.

[0025] In some embodiments, the anti-AREG antibody or fragment thereof according to the invention is capable of binding to, for example, at least one, two, three, four or five amino acids within residues 94-177 of mouse pro-AREG, preferably residues 137-177 of mouse pro-AREG.

[0026] In some embodiments, the anti-AREG antibody or a fragment thereof according to the invention is an antibody fragment that binds to AREG in a soluble form.

[0027] In some embodiments, the anti-AREG antibody or a fragment thereof according to the invention is a Fab fragment or an F(ab)2 fragment.

[0028] In some embodiments, the anti-AREG antibody or fragment thereof according to the present invention comprises: a heavy chain variable region containing heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region containing light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein:

[0029] HCDR1, HCDR2 and HCDR3 are selected from: (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:11; (7) HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8 and HCDR3 shown in SEQ ID NO:11; (8) HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, and HCDR3 shown in SEQ ID NO:12; (9) HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:13, and HCDR3 shown in SEQ ID NO:14; (10) HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:18, and HCDR3 shown in SEQ ID NO:19; (11) HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:18, and HCDR3 shown in SEQ ID NO:20; (12) HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:13, and HCDR3 shown in SEQ ID NO:14; (13) HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, and HCDR3 shown in SEQ ID NO:12; (14) HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:15, and HCDR3 shown in SEQ ID NO:16; (15) HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:18, and HCDR3 shown in SEQ ID NO:20; (16) HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:13, and HCDR3 shown in SEQ ID NO:14; (17) HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:18, and HCDR3 shown in SEQ ID NO:19; (18) HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:18, and HCDR3 shown in SEQ ID NO:20; (19) HC HCDR3 as shown in NO:136; and (13) HCDR1, HCDR2, HCDR3 as shown in (1) to (12), but at least one of them includes the addition, deletion, conserved amino acid substitution, or combination thereof of 1, 2, 3, 4, or 5 amino acids; and

[0030] LCDR1, LCDR2, and LCDR3 are selected from: (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:25, LCDR2 shown in SEQ ID NO:26, and LCDR3 shown in SEQ ID NO:27; (7) LCDR1 shown in SEQ ID NO:30, LCDR2 shown in SEQ ID NO:34, and LCDR3 shown in SEQ ID NO:30; (8) LCDR1 shown in SEQ ID NO:36, LCDR2 shown in SEQ ID NO:37, and LCDR3 shown in SEQ ID NO:38; (9) LCDR1 shown in SEQ ID NO:39, LCDR2 shown in SEQ ID NO:40, and LCDR3 shown in SEQ ID NO:38; (10) LCDR1 shown in SEQ ID NO:41, LCDR2 shown in SEQ ID NO:42, and LCDR3 shown in SEQ ID NO:38; (11) LCDR1 shown in SEQ ID NO:43, LCDR2 shown in SEQ ID NO:44, and LCDR3 shown in SEQ ID NO:38; (12) LCDR1 shown in SEQ ID NO:39, LCDR2 shown in SEQ ID NO:40, and LCDR3 shown in SEQ ID NO:38. (13) LCDR1 shown in SEQ ID NO:45, LCDR2 shown in SEQ ID NO:42, and LCDR3 shown in SEQ ID NO:46; (14) LCDR1 shown in SEQ ID NO:47, LCDR2 shown in SEQ ID NO:44, and LCDR3 shown in SEQ ID NO:46;(15) LCDR1 shown in SEQ ID NO:48, LCDR2 shown in SEQ ID NO:37, and LCDR3 shown in SEQ ID NO:49; (16) LCDR1 shown in SEQ ID NO:50, LCDR2 shown in SEQ ID NO:40, and LCDR3 shown in SEQ ID NO:51; (17) LCDR1 shown in SEQ ID NO:50, LCDR2 shown in SEQ ID NO:40, and LCDR3 shown in SEQ ID NO:52; (18) LCDR1 shown in SEQ ID NO:50, LCDR2 shown in SEQ ID NO:40, and LCDR3 shown in SEQ ID NO:53; (19) LCDR1 shown in SEQ ID NO:54, LCDR2 shown in SEQ ID NO:42, and LCDR3 shown in SEQ ID NO:55; (20) LCDR1 shown in SEQ ID NO:56, LCDR2 shown in SEQ ID NO:44, and LCDR3 shown in SEQ ID NO:49. LCDR3 as shown in NO:55; and (21) LCDR1, LCDR2, LCDR3 as shown in (1) to (20), but at least one of them includes the addition, deletion, conserved amino acid substitution, or combination thereof of one, two, three, four, or five amino acids.

[0031] In one embodiment, the anti-AREG antibody or fragment thereof according to the present invention comprises: a heavy chain variable region containing heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region containing light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein:

[0032] HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from: (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, LCDR2 shown in SEQ ID NO:26, and LCDR3 shown in SEQ ID NO:25. (4) LCDR1 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, and LCDR3 shown in SEQ ID NO:30; (5) LCDR1 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, and LCDR3 shown in SEQ ID NO:30; (6) LCDR1 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, LCDR2 shown in SEQ ID NO:34, and LCDR3 shown in SEQ ID NO:30; (7) LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:7, LCDR3 shown in SEQ ID NO:28, LCDR2 shown in SEQ ID NO:29, and LCDR3 shown in SEQ ID NO:30. HCDR2 shown in NO:8, HCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:35, LCDR2 shown in SEQ ID NO:34, and LCDR3 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, and 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:136, LCDR1 shown in SEQ ID NO:39, LCDR2 shown in SEQ ID NO:40, and 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:136, LCDR1 shown in SEQ ID NO:41, LCDR2 shown in SEQ ID NO:42, and LCDR3 shown in SEQ ID NO:38; (11) HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:1, HCDR3 shown in SEQ ID NO:14, LCDR1 shown in SEQ ID NO:36, LCDR2 shown in SEQ ID NO:47, and LCDR3 shown in SEQ ID NO:38. (12) HCDR1 shown in SEQ ID NO:13, 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, and LCDR3 shown in SEQ ID NO:38; (13) HCDR1 shown in SEQ ID NO:13, 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, and LCDR3 shown in SEQ ID NO:38; (14) HCDR1 shown in SEQ ID NO:13, 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, and LCDR3 shown in SEQ ID NO:46; (15) HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:45, LCDR2 shown in SEQ ID NO:42, and LCDR3 shown in SEQ ID NO:46; HCDR3 shown in NO:16, LCDR1 shown in SEQ ID NO:47, LCDR2 shown in SEQ ID NO:44, and LCDR3 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, and 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, and 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, and 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, and LCDR3 shown in SEQ ID NO:52; (19) HCDR2 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, and LCDR3 shown in SEQ ID NO:53; (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:54, LCDR2 shown in SEQ ID NO:42, and LCDR3 shown in SEQ ID NO:55; (21) 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, and LCDR3 shown in SEQ ID NO:55. LCDR3 as shown in NO:55; and (21) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 as shown in (1) to (20), but at least one of them includes the addition, deletion, conserved amino acid substitution, or combination thereof of one, two, three, four, or five amino acids.

[0033] Preferably, the anti-AREG antibody or fragment thereof according to the present invention comprises HCDR1, HCDR2 and HCDR3 selected from the following:

[0034] (1) HCDR1 shown in SEQ ID NO:5, HCDR2 shown in SEQ ID NO:2, and HCDR3 shown in SEQ ID NO:6;

[0035] (2) HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, and HCDR3 shown in SEQ ID NO:4; and

[0036] (3) HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:10, and HCDR3 shown in SEQ ID NO:9; and

[0037] Selected from the following LCDR1, LCDR2 and LCDR3:

[0038] (1) LCDR1 shown in SEQ ID NO:25, LCDR2 shown in SEQ ID NO:26, and LCDR3 shown in SEQ ID NO:27;

[0039] (2) LCDR1 shown in SEQ ID NO:21, LCDR2 shown in SEQ ID NO:22, LCDR3 shown in SEQ ID NO:24; and

[0040] (3) LCDR1 shown in SEQ ID NO:31, LCDR2 shown in SEQ ID NO:32, and LCDR3 shown in SEQ ID NO:30.

[0041] Preferably, the anti-AREG antibody or fragment thereof according to the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 selected from the following:

[0042] (1) 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, LCDR2 shown in SEQ ID NO:26, and LCDR3 shown in SEQ ID NO:27;

[0043] (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; and

[0044] (3) 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, and LCDR3 shown in SEQ ID NO:30.

[0045] 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 has an amino acid sequence selected from SEQ ID NO:57-69 and an amino acid sequence having at least 95% sequence identity with any of SEQ ID NO:57-69 and retaining epitope binding activity.

[0046] The light chain variable region has an amino acid sequence selected from SEQ ID NO:70-89 and an amino acid sequence that has at least 95% sequence identity with any of SEQ ID NO:70-89 and retains epitope binding activity.

[0047] 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 have an amino acid sequence selected from the following: (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:79; 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 that have at least 95% sequence identity with any one of (1) to (21) and retain epitope binding activity.

[0048] Preferably, 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 has an amino acid sequence selected from SEQ ID NO:59, SEQ ID NO:58 and SEQ ID NO:62, and

[0049] The light chain variable region has an amino acid sequence selected from SEQ ID NO:72, SEQ ID NO:71 and SEQ ID NO:75.

[0050] Preferably, 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 have an amino acid sequence selected from the following: (1) SEQ ID NO:59 and SEQ ID NO:72; (2) SEQ ID NO:58 and SEQ ID NO:71; and (3) SEQ ID NO:62 and SEQ ID NO:75.

[0051] In some embodiments, the anti-AREG antibody or fragment thereof according to the invention is an isotype of IgG, IgM, IgA, IgE, or IgD. In some embodiments, the anti-AREG antibody or fragment thereof according to the invention is an isotype of IgG1, IgG2, IgG3, or IgG4.

[0052] In some embodiments, the antibody of the present invention is a human monoclonal antibody (mAb), a mouse mAb, a humanized mAb, or a chimeric mAb.

[0053] Preferably, the human monoclonal antibody (mAb) of the present invention has a heavy chain region comprising at least two of the three CDRs shown in SEQ ID NO:1-3, and / or a light chain region comprising at least two of the three CDRs shown in SEQ ID NO:21-23.

[0054] Preferably, the human monoclonal antibody (mAb) of the present invention has a heavy chain region comprising at least two of the three CDRs shown in SEQ ID NO: 1, 2 and 4, and / or a light chain region comprising at least two of the three CDRs shown in SEQ ID NO: 21, 22 and 24.

[0055] Preferably, the human monoclonal antibody (mAb) of the present invention has a heavy chain region comprising at least two of the three CDRs shown in SEQ ID NO: 5, 2 and 6, and / or a light chain region comprising at least two of the three CDRs shown in SEQ ID NO: 25-27.

[0056] Preferably, the murine monoclonal antibody (mAb) of the present invention has a heavy chain region comprising at least two of the three CDRs shown in SEQ ID NO:7-9, and / or a light chain region comprising at least two of the three CDRs shown in SEQ ID NO:28-30.

[0057] Preferably, the murine monoclonal antibody (mAb) of the present invention has a heavy chain region comprising at least two of the three CDRs shown in SEQ ID NO:1, 13 and 14, and / or a light chain region comprising at least two of the three CDRs shown in SEQ ID NO:36-38.

[0058] Preferably, the murine monoclonal antibody (mAb) of the present invention has a heavy chain region comprising at least two of the three CDRs shown in SEQ ID NO:17-19, and / or a light chain region comprising at least two of the three CDRs shown in SEQ ID NO:48, 37 and 49.

[0059] Preferably, the humanized monoclonal antibody (mAb) of the present invention has a heavy chain region comprising at least two of the three CDRs shown in SEQ ID NO:7-9, and / or a light chain region comprising at least two of the three CDRs shown in SEQ ID NO:28-30.

[0060] Preferably, the humanized monoclonal antibody (mAb) of the present invention has a heavy chain region comprising at least two of the three CDRs shown in SEQ ID NO: 7, 10 and 9, and / or a light chain region comprising at least two of the three CDRs shown in SEQ ID NO: 31, 32 and 30.

[0061] Preferably, the humanized monoclonal antibody (mAb) of the present invention has a heavy chain region comprising at least two of the three CDRs shown in SEQ ID NO:7, 8 and 11, and / or a light chain region comprising at least two of the three CDRs shown in SEQ ID NO:33, 34 and 30.

[0062] Preferably, the humanized monoclonal antibody (mAb) of the present invention has a heavy chain region comprising at least two of the three CDRs shown in SEQ ID NO:7, 8 and 12, and / or a light chain region comprising at least two of the three CDRs shown in SEQ ID NO:35, 34 and 30.

[0063] Preferably, the humanized monoclonal antibody (mAb) of the present invention has a heavy chain region comprising at least two of the three CDRs shown in SEQ ID NO: 1, 13 and 136, and / or a light chain region comprising at least two of the three CDRs shown in SEQ ID NO: 39, 40 and 38.

[0064] Preferably, the humanized monoclonal antibody (mAb) of the present invention has a heavy chain region comprising at least two of the three CDRs shown in SEQ ID NO: 1, 13 and 136, and / or a light chain region comprising at least two of the three CDRs shown in SEQ ID NO: 41, 42 and 38.

[0065] Preferably, the humanized monoclonal antibody (mAb) of the present invention has a heavy chain region comprising at least two of the three CDRs shown in SEQ ID NO: 1, 13 and 136, and / or a light chain region comprising at least two of the three CDRs shown in SEQ ID NO: 43, 44 and 38.

[0066] Preferably, the humanized monoclonal antibody (mAb) of the present invention has a heavy chain region comprising at least two of the three CDRs shown in SEQ ID NO: 1, 15 and 16, and / or a light chain region comprising at least two of the three CDRs shown in SEQ ID NO: 39, 40 and 38.

[0067] Preferably, the humanized monoclonal antibody (mAb) of the present invention has a heavy chain region comprising at least two of the three CDRs shown in SEQ ID NO: 1, 15 and 16, and / or a light chain region comprising at least two of the three CDRs shown in SEQ ID NO: 45, 42 and 46.

[0068] Preferably, the humanized monoclonal antibody (mAb) of the present invention has a heavy chain region comprising at least two of the three CDRs shown in SEQ ID NO: 1, 15 and 16, and / or a light chain region comprising at least two of the three CDRs shown in SEQ ID NO: 47, 44 and 46.

[0069] Preferably, the humanized monoclonal antibody (mAb) of the present invention has a heavy chain region comprising at least two of the three CDRs shown in SEQ ID NO: 17, 18 and 20, and / or a light chain region comprising at least two of the three CDRs shown in SEQ ID NO: 50, 40 and 51.

[0070] Preferably, the humanized monoclonal antibody (mAb) of the present invention has a heavy chain region comprising at least two of the three CDRs shown in SEQ ID NO: 17, 18 and 20, and / or a light chain region comprising at least two of the three CDRs shown in SEQ ID NO: 50, 40 and 52.

[0071] Preferably, the humanized monoclonal antibody (mAb) of the present invention has a heavy chain region comprising at least two of the three CDRs shown in SEQ ID NO: 17, 18 and 20, and / or a light chain region comprising at least two of the three CDRs shown in SEQ ID NO: 50, 40 and 53.

[0072] Preferably, the humanized monoclonal antibody (mAb) of the present invention has a heavy chain region comprising at least two of the three CDRs shown in SEQ ID NO: 17, 18 and 20, and / or a light chain region comprising at least two of the three CDRs shown in SEQ ID NO: 54, 42 and 55.

[0073] Preferably, the humanized monoclonal antibody (mAb) of the present invention has a heavy chain region comprising at least two of the three CDRs shown in SEQ ID NO: 17, 18 and 20, and / or a light chain region comprising at least two of the three CDRs shown in SEQ ID NO: 56, 44 and 55.

[0074] Preferably, the humanized monoclonal antibody (mAb) of the present invention comprises a constant region derived from the human constant region.

[0075] Preferably, the humanized monoclonal antibody (mAb) of the present invention has a human light chain constant region derived from the κ light chain constant region.

[0076] Preferably, the humanized monoclonal antibody (mAb) of the present invention has a human heavy chain constant region derived from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4.

[0077] In some embodiments, the anti-AREG antibody or fragment thereof according to the present invention can block the binding of AREG to EGFR.

[0078] In some embodiments, the anti-AREG antibody or fragment thereof according to the present invention is capable of inhibiting EGFR phosphorylation.

[0079] On the other hand, the present invention provides an isolated polynucleotide or nucleic acid that encodes an anti-AREG antibody or a fragment thereof according to the present invention.

[0080] In some embodiments, the polynucleotides according to the invention may encode the complete heavy chain variable region or the complete light chain variable region or both on the same polynucleotide molecule or on separate polynucleotide molecules. Alternatively, the polynucleotides according to the invention may encode a portion of the heavy chain variable region or the light chain variable region or both on the same polynucleotide molecule or on separate polynucleotide molecules.

[0081] In some embodiments, the polynucleotide according to the invention comprises: a DNA sequence encoding the heavy chain variable region represented by any one of the sequences SEQ ID NO: 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 112, 115 and 117, and / or a DNA sequence encoding the light chain variable region represented by any one of the sequences SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 110, 111, 113, 114, 116, 118, 119, 120, 121 and 122.

[0082] On the other hand, the present invention provides an isolated cell or vector containing one or more polynucleotides encoding an anti-AREG antibody or a fragment thereof according to the present invention.

[0083] In some embodiments, the cells are hybridoma cells that produce anti-AREG antibodies or fragments thereof according to the present invention.

[0084] On the other hand, the present invention provides a composition comprising an anti-AREG antibody or a fragment thereof according to the present invention and a pharmaceutically acceptable carrier.

[0085] On the other hand, the present invention provides the use of the anti-AREG antibody or a fragment thereof according to the present invention in the preparation of a medicament for treating a disorder in a subject, wherein AREG is overexpressed, upregulated or activated.

[0086] The subjects may be mammalian subjects requiring diagnosis, prognosis, or treatment. Mammal subjects include humans, domesticated animals, farm animals, and zoo, sporting, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and dairy cows.

[0087] The impairment is a fibrotic disease, including but not limited to renal fibrosis, liver fibrosis, pulmonary fibrosis, and especially IPF.

[0088] On the other hand, the present invention provides a method for treating a disorder in a subject, wherein AREG is overexpressed, upregulated, or activated, the method comprising administering to the patient an anti-AREG antibody or a fragment thereof according to the present invention. The disorder is a fibrotic disease, including but not limited to renal fibrosis, liver fibrosis, pulmonary fibrosis, and particularly IPF.

[0089] The subjects may be mammalian subjects requiring diagnosis, prognosis, or treatment. Mammal subjects include humans, domesticated animals, farm animals, and zoo, sporting, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and dairy cows.

[0090] On the other hand, the present invention provides a method for determining the presence of AREG protein, the method comprising exposing cells suspected of containing AREG protein to an anti-AREG antibody or a fragment thereof according to the present invention, and determining the binding of the anti-AREG antibody or the fragment thereof to the cells.

[0091] The method can be used to diagnose disorders in subjects in which AREG is overexpressed, upregulated, or activated. The disorder is a fibrotic disease, including but not limited to renal fibrosis, liver fibrosis, pulmonary fibrosis, and particularly IPF.

[0092] The subjects may be mammalian subjects requiring diagnosis, prognosis, or treatment. Mammal subjects include humans, domesticated animals, farm animals, and zoo, sporting, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and dairy cows.

[0093] On the other hand, the present invention provides an isolated AREG protein having an amino acid sequence shown in any of SEQ ID NO:123-132 or an amino acid sequence having at least 85% identity with any of SEQ ID NO:123-132.

[0094] The isolated AREG protein can be used as an epitope to generate anti-AREG antibodies or fragments thereof according to the present invention.

[0095] The isolated AREG protein according to the present invention can be used to identify anti-AREG antibodies or fragments thereof that have no cross-reactivity or weak cross-reactivity with mouse AREG.

[0096] Two amino acids (E149 and H164, based on hAREG numbering) were identified as key epitope residues for the binding of the anti-AREG antibody or fragment thereof according to the invention to hAREG, but not to mAREG. Amino acids K149 and N164 (based on hAREG numbering) in mAREG are residues that cause the anti-AREG antibody or fragment thereof according to the invention to lack cross-reactivity with mAREG, and residue 164N is the most critical.

[0097] Preferably, the isolated AREG protein has amino acid Glu149 (using hAREG number) and / or His164 (using hAREG number).

[0098] On the other hand, the present invention provides the use of the isolated AREG protein according to the present invention for identifying anti-AREG antibodies or fragments thereof that bind to hAREG and have no cross-reactivity or weak cross-reactivity with mAREG.

[0099] definition:

[0100] When used herein, the terms “AREG” and “Areg” refer to “amphiregulin” or the gene encoding amphiregulin and are used interchangeably. “AREG(Areg)” is a member of the epidermal growth factor (EGF) family and a low-affinity ligand of the EGF receptor (EGFR). Unless otherwise stated in the specification, “AREG(Areg)” refers to human AREG(Areg). The binding of EGFR to AREG activates the major intracellular signaling cascades that control cell survival, proliferation, and motility. AREG protein is synthesized from a 252-amino acid transmembrane precursor (pro-AREG) (SEQ ID NO:135), which is cleaved in its extracellular domain by cell membrane proteases, primarily TACE / ADAM17, thereby releasing two soluble forms of AREG protein. The larger form corresponds to residues 101–184 of pro-AREG (SVRVEQVVKPPQNKTESENTSDKPKRKKKGGK NGKNRRNRKKKNPCNAEFQNFCIHGECKYIEHLEAVTCKCQQEYFGERCGEK), and the shorter form corresponds to residues 107–184 of pro-AREG (78 residues in length). AREG protein contains a heparin-binding domain (corresponding to residues 101-143 of pro-AREG, SVRVEQVVKPPQNKTESENTSDKPKRKKKGGKNGKNRRNRK) and an EGF-like domain (corresponding to residues 144-184 of pro-AREG, KKNPCNAEFQNFCIHGECKYIEH LEAVTCKCQQEYFGERCGEK). Pro-AREG activates EGFR on adjacent cells in a proclinical mode, while the soluble form of AREG activates EGFR in an autoclinical or paraclinical mode.

[0101] When used herein, a designation without a specific number (“a” and “a type of”) refers to one or more (e.g., at least one) designations.

[0102] Unless the context clearly indicates otherwise, the term “or” is used herein to mean the term “and / or” and may be used interchangeably with it.

[0103] "About" and "approximately" generally mean the acceptable degree of error in a quantity measured given the nature or precision of the measurement. An exemplary degree of error is within 20 percent (%) of a given value or range of values, typically within 10 percent, and more often within 5 percent.

[0104] When used herein to refer to cells, polynucleotides such as DNA or RNA, proteins, or polypeptides, the term "isolated" means material removed from its original or native environment (e.g., the natural environment if naturally occurring). For example, naturally occurring polynucleotides or polypeptides present in living organisms are not isolated, but the same polynucleotides or polypeptides separated from some or all of the coexisting material in a natural system through human intervention are isolated. Such polynucleotides may be part of a carrier and / or such polynucleotides or polypeptides may be part of a composition and are still isolated, therefore such carriers or compositions are not part of the environment in which they exist in nature. Isolated polynucleotides refer to molecules that are separately separated from other DNA or RNA present in the natural source of the macromolecule. Isolated polypeptides are intended to encompass both purified and recombinant polypeptides.

[0105] The products and methods disclosed herein comprise polypeptides and polynucleotides having a specified sequence or a sequence identical or similar to said specified sequence, such as having at least about 85% or 95% sequence identity (identical). In the case of amino acid sequences, the term "85% or 95% sequence identity (identical)" is used herein to refer to a first amino acid sequence containing a sufficient or minimum number of aligned amino acid residues i) identical or ii) conservedly substituted amino acid residues in a second amino acid sequence, such that the first and second amino acid sequences can have a common structural domain and / or a common functional activity. For example, an amino acid sequence containing a common structural domain has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a reference sequence, such as the sequence provided herein.

[0106] In the context of nucleotide sequences, the term "85% or 95% sequence identity" is used herein to refer to a first nucleic acid sequence containing a sufficient or minimum number of nucleotides identical to the aligned nucleotides in a second nucleic acid sequence, such that the first and second nucleotide sequences encode a polypeptide with common functional activity, or encode a common structural polypeptide domain or common functional polypeptide activity. For example, the nucleotide sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a reference sequence, such as those provided herein.

[0107] To determine the percentage identity of two amino acid or nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the aligned reference sequence is at least 30% of the length of the reference sequence, for example, at least 40%, 50%, 60%, or at least 70%, 80%, 90%, or 100%.

[0108] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably in this document and refer to a polymer of amino acids of any length.

[0109] The terms “nucleic acid,” “nucleic acid sequence,” “nucleotide sequence,” or “polynucleotide sequence” and “polynucleotide” are used interchangeably.

[0110] When used herein, the term "antibody or antibody molecule" refers to a protein containing at least one immunoglobulin variable domain sequence, such as an immunoglobulin chain or fragment thereof. The term "antibody molecule" includes, for example, monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region). In one embodiment, the antibody molecule comprises a full-length antibody or a full-length immunoglobulin chain. In one embodiment, the antibody molecule comprises an antigen-binding or functional fragment of a full-length antibody or a full-length immunoglobulin chain. When used herein, the antibody molecule is "bound" to an antigen, a binding as understood by those skilled in the art. In one embodiment, the antibody binds to an antigen with a dissociation constant (KD) of about 1 × 10⁻⁶. -5 M or lower, 1×10 -6 M or lower, or 1×10 -7 M or lower, 1×10 -8 M or lower, 1×10 -9 M or lower, 1×10 -10 M or lower, 1×10 -11 M or lower.

[0111] For example, an antibody molecule may include 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, the antibody molecule comprises, or is composed of, a heavy chain and a light chain. In another embodiment, the antibody molecule 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, bivalent antibodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which can be generated by modifying intact antibodies or antibodies synthesized de novo using recombinant DNA technology. These functional antibody fragments retain the ability to bind specifically to their respective antigens or receptors. Antibodies and antibody fragments can be derived from any class of antibodies, including but not limited to IgG, IgA, IgM, IgD, and IgE, and can be derived from any antibody subclass (e.g., IgG1, IgG2, IgG3, and IgG4). Antibody molecules can be prepared as monoclonal or polyclonal. Antibody molecules can also be human, humanized, CDR-transplanted, or in vitro generated antibodies. The antibodies may have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. The antibodies may also have a light chain selected from, for example, κ or λ. The term "immunoglobulin" (Ig) is used interchangeably with the term "antibody" herein.

[0112] When used herein, the term "antibody fragment" or "antigen-binding fragment" refers to a part of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. An antibody fragment binds to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, specimeric antibodies, and dimeric antibodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that functions similarly to an antibody by forming a complex with a specific antigen.

[0113] Examples of antigen-binding fragments of antibody molecules include: (i) Fab fragments, monovalent fragments consisting of VL, VH, CK, and CH domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bonds in the hinge region; (iii) Fd fragments, consisting of VH and CH domains; (iv) Fv fragments, consisting of the VL and VH domains of a single arm of the antibody; (v) bisomatic antibody (dAb) fragments consisting of a VH domain; (vi) camel- or camel-derived variable domains; (vii) single-chain Fv (scFv); and (viii) single-domain antibodies. These antibody fragments can be obtained using any suitable method, including conventional techniques known to those skilled in the art, and said fragments can be screened for use in the same manner as intact antibodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that functions similarly to an antibody by binding to a specific antigen to form a complex.

[0114] "Single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin. In some cases, the regions are linked together using short linker peptides of 10 to approximately 25 amino acids. The linker may be glycine-rich for flexibility and serine or threonine-rich for solubility, and the N-terminus of the VH may be linked to the C-terminus of the VL, or vice versa. Despite the removal of the constant region and the introduction of the linker, this protein retains the specificity of the original immunoglobulin. ScFv molecules are known in the art.

[0115] The light and heavy chains are divided into "constant" and "variable" regions. The variable domains of both the light chain (VL) and heavy chain (VH) determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CK) and heavy chain (CH1, CH2, or CH3) provide important biological properties such as secretion, transplacental migration, Fc receptor binding, and complement binding. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CK domains actually contain the carboxyl termini of the heavy and light chains, respectively.

[0116] Variable regions allow antibodies to selectively recognize and specifically bind to epitopes on antigens. A subset of the antibody's VL and VH domains, or complementarity-determining regions (CDRs), are combined to form variable regions that define a three-dimensional antigen-binding site. This quaternary antibody structure forms an antigen-binding site at the end of each Y arm. More specifically, the antigen-binding site is defined by three CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) on each VH and VK chain.

[0117] When used herein, the terms “complementarity-determining region” and “CDR” refer to the amino acid sequence within the antibody variable region that confers antigen specificity and binding affinity. In some embodiments, three CDRs (HCDR1, HCDR2, and HCDR3) are present in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, and LCDR3) are present in each light chain variable region.

[0118] The precise amino acid sequence boundaries of a particular CDR can be determined using any well-known scheme, including the scheme described in Kabat et al. (1991), Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme).

[0119] Each VH and VL typically includes three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0120] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject requiring diagnosis, prognosis, or treatment, especially a mammalian subject. Mammal subjects include humans, domesticated animals, farm animals, and zoo, sporting, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, dairy cows, etc.

[0121] When used herein, phrases such as “to a patient in need of treatment” or “subject in need of treatment” include subjects who will benefit from the administration of antibodies or compositions of this disclosure for purposes such as detection, diagnostic procedures and / or treatment, such as mammalian subjects.

[0122] When used herein, the term "epitaph" refers to a component of an antigen (e.g., human AREG (hAREG)) that specifically interacts with an antibody molecule. Such components, also referred to herein as epitope determinants, typically comprise, or are part of, elements such as amino acid or sugar side chains. Epitope determinants can be defined by methods known in the art or disclosed herein, such as by crystallography or mutation. At least one or more components of an antibody molecule that specifically interact with an epitope determinant are typically located on a CDR. Epitopes typically have specific three-dimensional structural features. Epitopes typically have specific charge features. Some epitopes are linear epitopes, while others are conformational epitopes.

[0123] When used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to an antibody preparation consisting of a single molecule. Monoclonal antibody compositions exhibit single binding specificity and affinity for a specific epitope. Monoclonal antibodies can be prepared using hybridoma technology or by methods that do not use hybridoma technology (e.g., library selection and screening or recombinant methods).

[0124] The antibody molecule can be a polyclonal or monoclonal antibody. In other embodiments, the antibody can be generated recombinantly, for example by yeast display, phage display, or by a combination method.

[0125] 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 mice that have been genetically engineered to produce antibodies from human immunoglobulin sequences), or a non-human antibody such as rodent (mouse or rat), goat, primate (e.g., monkey) or camel antibody. Methods for producing rodent antibodies are known in the art.

[0126] Human monoclonal antibodies can be produced using transgenic mice carrying human immunoglobulin genes instead of the mouse system. Using spleen cells from these transgenic mice immunized with antigens of interest, hybridomas secreting epitopes with specific affinities for human proteins are generated.

[0127] Antibodies can be antibodies in which a variable region or a portion thereof (e.g., a CDR) is produced in a non-human organism (e.g., a rat or mouse). Chimeric, CDR-transplanted, and humanized antibodies are within the scope of this invention. Antibodies produced in a non-human organism, such as a rat or mouse, and then modified, for example, in a variable framework or constant region to reduce antigenicity in humans, are also within the scope of this invention.

[0128] Humanized or CDR-transplanted antibodies have at least one or two, but typically all three, receptor CDRs (of the heavy or light chain of immunoglobulins) replaced by donor CDRs. The antibody may be replaced with at least a portion of a non-human CDR, or only some CDRs may be replaced with non-human CDRs. Only the number of CDRs required for binding of the humanized antibody to the AREG needs to be replaced. In some embodiments, the donor is a murine antibody, such as a rat or mouse antibody, and the receptor is a human or human common framework. Typically, the immunoglobulin providing the CDR is referred to as the "donor," and the immunoglobulin providing the framework is referred to as the "receptor." In one embodiment, the donor immunoglobulin is non-human (e.g., murine). The receptor framework is a naturally occurring (e.g., human) framework or a common framework, or a sequence having about 85% or higher, such as 90%, 95%, 99%, or higher, identity with it.

[0129] Antibodies can be humanized using methods known in the art. Humanized or CDR-transplanted antibodies can be generated by CDR transplantation or CDR replacement, in which one, two, or all of the CDRs of the immunoglobulin chain can be replaced.

[0130] The scope of this invention also includes humanized antibodies in which specific amino acids have been replaced, deleted, or added. A standard description of the selection of amino acids from donors is found in US 5,585,089, for example in columns 12-16 of US 5,585,089, the contents of which are incorporated herein by reference. Other techniques for humanizing antibodies are described in Padlan et al., EP 519596 A1, published December 23, 1992.

[0131] In other embodiments, the antibody molecule has a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, particularly a heavy chain constant region selected from, for example, the (e.g., human) heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4.

[0132] Methods for altering the constant region of an antibody are known in the art. Functionally modified antibodies, such as those with altered affinity for effector ligands like FcRs on cells or the C1 component of complement, can be produced by replacing at least one amino acid residue in the constant region of the antibody with a different residue (see, for example, EP 388,151 A1, U.S. Patent No. 5,624,821, and U.S. Patent No. 5,648,260, the contents of which are incorporated herein by reference). Amino acid mutations that stabilize antibody structures, such as S228P (Eu number) in human IgG4, are also contemplated.

[0133] It should be understood that the molecules of the present invention may be replaced with additional conserved or non-essential amino acid substitutions that do not significantly affect their function.

[0134] "Conservative" amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative amino acid substitutions are as follows:

[0135]

[0136] Attached Figure Description

[0137] Figure 1 The binding of E1H3L4 and P7 with hAREG, mAREG and hAREG-C18 is shown.

[0138] Figure 2 The inhibitory activity of anti-AREG mAb on EGFR phosphorylation in epidermal-like cancer cells expressing hEGFR was demonstrated.

[0139] Figure 3 Five hAREG-EGFd variants are shown, which are produced by changing each amino acid at five different sites of hAREG-EGFd to the corresponding amino acid of mAREG-EGFd.

[0140] Figure 4 The protocol for generating mouse lines in which the Cdc42 gene in AT2 cells is specifically deleted is shown. Mice with a 2-fold specific deletion of the exon of the Cdc42 gene in AT2 cells are named Cdc42AT2 knockout mice.

[0141] Figure 5 The study showed that loss of Cdc42 in AT2 cells led to progressive pulmonary fibrosis in PNX-treated mice.

[0142] Figure 6 The study demonstrated that the anti-AREG antibody (P7) was effective in treating pulmonary fibrosis in a mouse model of IPF-like pulmonary fibrosis.

[0143] Figure 7 This study demonstrated that treatment with the anti-AREG antibody (E1H3L4) accelerated recovery in a mouse model of bleomycin-induced pulmonary fibrosis.

[0144] Figure 8 The study demonstrated that the anti-AREG antibody (E1H3L4) was effective in treating pulmonary fibrosis in a mouse model of IPF-like pulmonary fibrosis.

[0145] Figure 9 The study demonstrated that the anti-AREG antibody hu9C12v4 significantly prolonged the life expectancy of fibrotic mice in an IPF-like pulmonary fibrosis mouse model. Detailed Implementation

[0146] The description of specific implementations and embodiments is provided in an illustrative rather than limiting manner. Those skilled in the art will readily recognize the various non-critical parameters that can be changed or modified to produce substantially similar results.

[0147] Example

[0148] Example 1. Generation of human mAbs targeting AREG from a phage library

[0149] 1. Preparation of soluble AREG proteins or peptides for library selection and screening.

[0150] DNA sequences encoding three forms of the AREG protein (listed below) were cloned into a prokaryotic expression vector (pETDuet) and expressed as fusion proteins with N-terminal tags (His6, thioredoxin (TRX), HRV3C protease cleavage site, and Avi tag). The proteins were expressed in *E. coli* (TransB) by IPTG induction, purified from the supernatant of cell lysates using Ni-NTA beads, cleaved with HRV3C protease, and then biotinylated with BirA enzyme.

[0151] The three AREG proteins are:

[0152] • hAREG: Human AREG containing residues 101–184 of the human pro-AREG and bearing an N-terminal AVI tag (GLNDIFEAQKIEWHE). The amino acid sequence of residues 101–184 of hAREG is: SVRVEQVVKPPQNKTESENTSDKPKRKKKGGKNGKNRRNRKKKNPCNAEFQNFCIHGECKYIEHLEAVTCKCQQEYFGERCGEK (SEQ ID NO: 129)

[0153] • mAREG: Mouse AREG containing residues 94–177 of the mouse pro-AREG and bearing an N-terminal AVI tag. The amino acid sequence is: GLNDIFEAQKIEWHEGGGGSGGSVRVEQVIKPKKNKTEGEKSTEKPKRKKKGGKNGKGRRNKKKKNPCTAKFQNFCIHGECRYIENLEVVTCNCHQDYFGERCGEK (SEQ ID NO: 130)

[0154] • mARG-EGFd: The EGF-like domain of mouse AREG, containing residues 135–177 of mouse Pro-AREG and bearing an N-terminal AVI tag. The amino acid sequence of residues 135–177 of mAreg is: KKNPCTAKFQNFCIHGECRYIENLEVVT CNCHQDYFGERCGEK (SEQ ID NO: 131).

[0155] In addition, a biotinylated peptide C18 was synthesized (Scilight-peptide, Beijing, China). C18 contains 14 amino acids (residues 171-184) at the C-terminus of human AREG and includes a linker (residue GSSG) at the N-terminus. The sequence of C18 is GSSGKCQQEYFGERCGEK (SEQ ID NO: 132).

[0156] 2. Selection of antibodies and further characterization from phage display antibody libraries

[0157] Phage display antibody library

[0158] A human non-immune scFv (single-chain variable fragment) antibody library was constructed from peripheral blood mononuclear cells (PBMCs) of 93 healthy donors. The library has a total density of 1.1 × 10⁻⁶ cells. 10 The size of each member (Li et al., 2017).

[0159] Selection and screening of phage antibody libraries

[0160] Phage particles expressing scFv on their surfaces (phage-ScFv) were prepared from the library and used to screen for scFv antibodies against target antigens including biotinylated AREG proteins and peptides. The antigens were captured on streptavidin-conjugated magnetic M-280 plates. (Life Technologies), and then with 5×10 prepared from the library. 12 Phage particles were incubated. Two rounds of screening were performed for each soluble AREG protein or peptide (antigen, Ab). To obtain cross-reactive human monoclonal antibodies recognizing both hAREG and mARG, hAREG and mARG were used in the first and second rounds of selection, respectively. After the second round of selection, approximately 400 phage antibody clones were screened using ELISA for cross-binding activity against both hAREG and mARG, and clones with cross-binding activity or high binding affinity to hAREG were selected for sequencing analysis to identify clones with different antibody sequences, including variable regions of the heavy chain (VH) and light chain (VL). Subsequently, some phage-antibodies were converted into human IgG1 (hIgG1) or mouse IgG2a forms, and their binding to hAREG and mARG was analyzed using enzyme-linked immunosorbent assay (ELISA) or Biacore.

[0161] 3. Preparation of full-length antibodies

[0162] The VH and VL coding sequences of scFv were subcloned into antibody heavy chain (HC) expression vectors (plasmids) and light chain (LC) expression vectors (plasmids), respectively. To prepare the full-length antibody, 293F cells were transiently co-transfected with the two expression plasmids (HC+LC plasmids) at a 1:1 ratio. Six days after transfection, the cell culture supernatant was harvested, and the antibody was purified by protein A affinity chromatography.

[0163] 4. ELISA assay

[0164] Phosphate-buffered saline (PBS) containing streptavidin (Sigma, 4 μg / mL) was added to each well of a U-bottom 96-well plate (Nunc, MaxiSorp). TM The plate was coated overnight at 4°C or for 1 hour at 37°C. Approximately 0.5 μg / mL of AREG protein or peptide was then captured on the plate at 100 μL per well by incubation at 30°C for 1 hour. For phage-scFv-based ELISA, phage-scFv was serially diluted in PBS containing 2% skim milk and added to each well at 100 μL per well. Specific binding of phage-scFv was detected by adding HRP-conjugated mouse anti-M13 antibody (GE Healthcare) and incubating at 30°C for 30 min. Between each incubation step, the ELISA plate was washed 6 times with PBST solution (PBS containing 0.05% Tween 20) at 300 μL per well. After incubation with the HRP-conjugated antibody, an ELISA signal was generated by incubation with TMB substrate (Sigma) at 30°C for 5–10 min, and the reaction was terminated with 50 μL of 2M H2SO4 per well. The absorbance was read at 450 nm using a microplate reader (Bio-Rad), and the calibration wavelength was set at 630 nm. For IgG-based ELISA, the method is essentially the same as described above for phage-scFv, except that the bound antibody is detected using an HRP-conjugated mouse anti-Fc diantibody (Thermo Fisher Scientific).

[0165] 5. E1L2 antibody engineered to improve affinity and solubility.

[0166] To enhance the affinity of E1L2 antibodies, VH-CDR3 and VL-CDR3 of E1L2 were engineered. For VH-CDR3, a phage display library with random mutations relative to E1L2's HCDR3 was constructed. The selection and screening of antibody sub-libraries were similar to the methods described above for screening antibody libraries targeting AREG. To obtain high-affinity antibodies, competitive elution using full-length E1L2 monoclonal antibodies was employed. Subsequently, single clones were picked and subjected to phage rescue to generate phage-scFv in the bacterial culture supernatant for screening for binding to hAREG. Only antibodies with higher binding affinity than E1L2 were retained. For VL-CDR3, specific amino acid mutations based on structure modeling were engineered. To improve solubility, the engineered E1L2 VL CDR was transplanted into the human IGLV1-44*01 line.

[0167] 6. SPR measurement of affinity for human monoclonal antibodies

[0168] To assess the affinity of human monoclonal antibodies, SPR assays were performed using a Biacore T200 instrument. Monoclonal antibodies were captured on the surface of an anti-human Fc CM5 biosensor chip, and the binding of the EGF domain (hAREG-EGFd-mFc or mAREG-EGFd-mFc) of the hAREG (amino acids 142–184) or mAREG (amino acids 135–177) fused to the mFc tag to the monoclonal antibody was examined. Serially diluted fusion proteins were injected onto the antibody-bound surface, followed by a dissociation phase. The binding rate (Ka) and dissociation rate (Kd) were calculated using a 1:1 Langmuir binding model (BIA analysis software, GE Life Sciences). The equilibrium dissociation constant (KD) was calculated as the ratio of kd / ka.

[0169] 7. Results

[0170] Human monoclonal antibodies E1L2 and P7 against AREG were generated from a phage library.

[0171] Using the phage antibody library selection and ELISA screening described above, we identified C1, E1L2, P5, P6, P7, and P10 anti-AREG human monoclonal antibodies. Specifically, we identified the C1 antibody by using biotinylated hAREG and mAREG proteins expressed in *E. coli* in the first and second rounds of library selection, respectively. We identified the E1L2 antibody by using biotinylated hAREG and biotinylated mAREG-EGFd (expressed in *E. coli*) in the first and second rounds, respectively. Using the hAREG-derived C18 peptide as the target for both rounds of library selection, we identified P5, P6, P7, and P10; E1L2 was also screened from this library selection. Among these antibodies, E1L2 and P7 antibodies were selected for further identification based on their binding specificity and affinity for both hAREG and mAREG.

[0172] Production of E1H3L4 antibody derived from E1L2, which has higher affinity and solubility.

[0173] The binding affinity of E1L2 was further improved through VH-CDR3 and VL-CDR3 engineering. The solubility of the engineered E1L2 was enhanced by transplanting its VL-CDR into the human IGBL1-44*01 line, resulting in the antibody E1H3L4. Compared to E1L2, E1H3L4 exhibits three amino acid changes in VH-CDR3 and four amino acid changes in VL-CDR3. Specifically, amino acid changes are observed in amino acids 100-100 of the VH-CDR3 in E1H3L4. c The amino acids at position (Kabat system) are SYNN, while in the E1L2 antibody they are GYDY; in the VL-CDR3 of E1H3L4, positions 93-95... a The amino acids at the position (Kabat system) are KNNK, while in the E1L2 antibody they are SGLN. The CDRs of E1L2, E1H3L4, and P7 are listed in Table 1. The nucleotide and amino acid sequences of VH and VL of E1L2, E1H3L4, and P7 are listed in Table 2.

[0174] Table 1

[0175]

[0176]

[0177] The difference between E1L2 and E1H3L4 is indicated by an underline.

[0178] Use the Kabat system to define CDRs.

[0179] Table 2

[0180] E1L2 SEQ ID NO:90 SEQ ID NO:57 SEQ ID NO:91 SEQ ID NO:70 E1H3L4 SEQ ID NO:92 SEQ ID NO:58 SEQ ID NO:93 SEQ ID NO:71 P7 SEQ ID NO:94 SEQ ID NO:59 SEQ ID NO:95 SEQ ID NO:72

[0181] 8. Further identification of E1H3L4 and P7 monoclonal antibodies

[0182] Comparing the binding of E1H3L4 and P7 to mAEG, E1H3L4 showed a slightly stronger binding to mAEG compared to P7. As expected, both antibodies bound to the C-terminal peptide (C18, amino acids 171–184) within the EGF domain, because the C18 peptide was the target protein used in library selection. Figure 1 ).

[0183] Example 2. Generation of monoclonal antibodies against AREG using a mouse hybridoma method and humanization of the mouse monoclonal antibodies.

[0184] 1. Preparation of antigens for immunizing mice or for SPR analysis

[0185] The human AREG (hAREG) EGF-like domain, fused with the Fc fragment of human IgG1 or mouse IgG2a, was expressed as a fusion protein in 293F cells and named hAREG-EGFd-hFc and hAREG-EGFd-mFc, respectively. 72 hours post-transfection, the cell culture supernatant was harvested and used for purification of the Fc-fusion AREG protein by protein A affinity chromatography.

[0186] 2. Production of monoclonal antibodies against the hAREG EGF domain

[0187] Six-week-old Balb / c mice (from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were immunized subcutaneously with 100 μl of a 1:1 antigen / adjuvant emulsion containing 50 μg of hAREG-EGFd-mFc. For primary immunization, complete Freund's adjuvant (Sigma) was used. For booster immunization, incomplete Freund's adjuvant (Sigma) was used. Booster immunizations were performed every two weeks. One week after the third booster immunization, the binding of mouse serum to biotin-hAREG was assessed by ELISA. Mice with high titers of anti-hAREG antibodies were boosted intraperitoneally with 50 μg of adjuvant-free hAREG-EGFd-mFc. Three days after the booster, spleen cells were isolated and fused with SP2 / 0 cells according to standard hybridoma fusion methods.

[0188] The binding activity of hybridoma clone supernatant to biotinylated hAREG was examined by ELISA. Clones with high binding activity were selected and amplified for subsequent subcloning, and the supernatant of the subclones was analyzed by ELISA and SPR. The SPR analysis was performed using a Biacore T200 instrument (GE Life Sciences). The diluted supernatant was captured on an anti-mFcCM5 biosensor chip, and then 200 nM hAREG EGF domain-hFc in the mobile phase was passed through. Subclones with high affinity were amplified for RNA extraction. Cells were resuspended in TRIzol (Life Technologies), and total RNA was extracted according to the instruction manual. PrimeScript was used. TM Subcloned cDNA was synthesized using the RT master mix (TaKaRa). The VH and VL genes for each antibody were amplified using a set of PCR primers specifically targeting the variable genes of mouse antibodies. The PCR products were then cloned into a PCR sequencing vector for sequencing.

[0189] 3. Humanization of anti-hAREG EGF-like monoclonal antibody

[0190] To humanize the AREG monoclonal antibody, a sequence search was conducted using the murine monoclonal antibody to identify homologous human germline IgG genes (9C12, 23H8, and 1H9) that were highly homologous to the murine monoclonal antibody. These genes were then selected as templates for humanization. Humanization was performed via complementarity-determining region (CDR) transplantation, specifically by transplanting the CDR of the murine monoclonal antibody into the human receptor framework of the selected human germline gene template. This humanization process was also guided by a simulated 3D structure for each antibody, with human framework residues reverting to murine residues to maintain the overall antibody and CDR loop structure, as well as AREG binding affinity.

[0191] 4. Construction and selection of hu9C12v1 antibody subset libraries for enhancing affinity

[0192] To improve antibody affinity, two phage display libraries with random mutations in HCDR3 and LCDR1 of hu9C12v1 were constructed using NNK degenerate codons. The selection and screening of antibody sub-libraries were performed similarly to the screening of the AREG-targeted antibody library and the affinity enhancement of the E1L2 antibody described above. After screening, only hits with higher binding affinity than hu9C12v1 were retained.

[0193] 5. SPR measurement of monoclonal antibody affinity

[0194] To evaluate the affinity of different mouse hybridoma monoclonal antibodies or their humanized and engineered variants, SPR measurements were performed using a Biacore T200 instrument. Monoclonal antibodies were captured on the surface of an anti-human Fc CM5 biosensor chip. Serially diluted hAREG-EGFd-mFc, mARG-EGFd-mFc, or hAREG-98aa (purchased from PeproTech, catalog number 100-55B) were injected onto the antibody-bound surface, followed by a dissociation phase. The binding rate (Ka) and dissociation rate (Kd) were calculated using a 1:1 Langmuir binding model (BIA assessment software, GE Life Sciences). The equilibrium dissociation constant (KD) was calculated as the ratio of kd / ka.

[0195] 6. Results

[0196] Production of anti-hAREG EGF domain monoclonal antibodies

[0197] Anti-hAREG monoclonal antibodies were generated using conventional hybridoma fusion technology. Monoclonal antibodies exhibiting high binding activity in ELISA and SPR assays were selected for further characterization. Through screening thousands of hybridoma clones, a series of monoclonal antibodies with high binding affinity for hAREG were identified. Based on sequence uniqueness, binding affinity, and recombinant antibody production yield, the top three monoclonal antibodies—9C12, 23H8, and 1H9—were selected for further analysis. These antibodies were then recombinantly expressed to prepare mouse antibodies or chimeric antibodies (mIgG1 or mIgG2a isotypes transplanted onto the human IgG1 constant region) of mIgG1 or mIgG2a.

[0198] Humanization of 9C12 or obtaining enhanced binding affinity and improved physicochemical properties for hAREG or mAREG. Humanized antibody variants of a certain nature

[0199] CDR transplantation and structural modeling were used to generate the first humanized version of 9C12, hu9C12v1, which exhibits hAREG affinity comparable to the chimeric antibody ch9C12 (possessing the variable region of 9C12 and the constant region of human IgG1). To enhance the hAREG affinity of hu9C12v1, two phage display libraries with random mutations in the HCDR3 and LCDR3 regions were constructed, respectively. Following rigorous biopanning, a small subset of antibodies with enhanced affinity were obtained. Based on the sequences of these antibodies, three monoclonal antibodies, hu9C12v4, hu9C12v5, and hu9C12v6, were generated to improve binding affinity to either hAREG or mAREG and refine their physicochemical properties. Compared to hu9C12v1, hu9C12v4 has one amino acid difference in VH-CDR2, six amino acid differences in VK-CDR1, and two amino acid differences in VK-CDR2; hu9C12v5 has five amino acid differences in VH-CDR3, five amino acid differences in VK-CDR1, and one amino acid difference in VK-CDR2; hu9C12v6 has two amino acid differences in VH-CDR3, five amino acid differences in VK-CDR1, and one amino acid difference in VK-CDR2. A comparison of the CDRs of the three monoclonal antibodies with those of the murine antibody is shown in Table 3. The nucleotide and amino acid sequences of the VH and VL of the three monoclonal antibodies and the murine antibody are listed in Table 4. The binding affinities of the three monoclonal antibodies with hAREG or mARG determined by SPR are listed in Tables 5-6.

[0200] Table 3

[0201]

[0202] The difference between Ab is indicated by an underline.

[0203] Use the Kabat system to define CDRs.

[0204] Table 4

[0205] m9C12 SEQ ID NO:96 SEQ ID NO:60 SEQ ID NO:97 SEQ ID NO:73 hu9C12v1 SEQ ID NO:98 SEQ ID NO:61 SEQ ID NO:99 SEQ ID NO:74 hu9C12v4 SEQ ID NO:100 SEQ ID NO:62 SEQ ID NO:101 SEQ ID NO:75 hu9C12v5 SEQ ID NO:102 SEQ ID NO:63 SEQ ID NO:103 SEQ ID NO:76 hu9C12v6 SEQ ID NO:104 SEQ ID NO:64 SEQ ID NO:105 SEQ ID NO:77

[0206] Table 5

[0207]

[0208] Table 6

[0209]

[0210] Humanization of 23H8

[0211] We used CDR transplantation and structural modeling to generate humanized 23H8 mAbs. The human VH germline gene IGHV3-21 was used for VH-CDR transplantation. The human VK germline genes IGKV7-3, IGKV1-39, and IGKV4-1 were used for VK-CDR transplantation, resulting in three versions of the humanized 23H8 VK chain. By combining the humanized VH with the three humanized VKs, we generated monoclonal antibodies hu23H8v1, hu23H8v2, and hu23H8v3, respectively. These three humanized monoclonal antibodies exhibited hAREG affinity similar to that of chimeric 23H8 (mouse variable region and human IgG1 constant region), indicating that the transplantation of the 23H8 VK-CDR into the three different human VK germline backbones was successful. Several additional mutations were introduced into the humanized monoclonal antibodies to remove potentially undesirable post-translational modifications or immunogenicity, resulting in three additional variants: hu23H8v4, -v5, and -v6. The CDRs of these monoclonal antibodies are compared with those of the mouse antibody in Table 7. The nucleotide and amino acid sequences of the VH and VL CDRs of these monoclonal antibodies are shown in Table 8. The binding affinities of these monoclonal antibodies with hAREG determined by SPR are listed in Tables 9-10.

[0212] Table 7

[0213]

[0214] The difference between Ab is indicated by an underline.

[0215] Use the Kabat system to define CDRs.

[0216] Table 8

[0217] 23H8 SEQ ID NO:106 SEQ ID NO:65 SEQ ID NO:107 SEQ ID NO:78 hu23H8v1 SEQ ID NO:108 SEQ ID NO:66 SEQ ID NO:109 SEQ ID NO:79 hu23H8v2 SEQ ID NO:108 SEQ ID NO:66 SEQ ID NO:110 SEQ ID NO:80 hu23H8v3 SEQ ID NO:108 SEQ ID NO:66 SEQ ID NO:111 SEQ ID NO:81 hu23H8v4 SEQ ID NO:112 SEQ ID NO:67 SEQ ID NO:109 SEQ ID NO:79 hu23H8v5 SEQ ID NO:112 SEQ ID NO:67 SEQ ID NO:113 SEQ ID NO:82 hu23H8v6 SEQ ID NO:112 SEQ ID NO:67 SEQ ID NO:114 SEQ ID NO:83

[0218] Table 9

[0219]

[0220]

[0221] Table 10

[0222]

[0223] Humanization of 1H9

[0224] Similar to the humanization of 23H8, the human VH germline gene IGHV3-21 was used for VH-CDR transplantation; and the human VK germline genes IGKV7-3, IGKV1-39, and IGKV4-1 were used for VK-CDR transplantation. The CDRs of these monoclonal antibodies are compared with those of mouse antibodies in Table 11. The nucleotide and amino acid sequences of the VH and VL CDRs of these monoclonal antibodies are shown in Table 12. The binding affinity of these monoclonal antibodies to hAREG determined by SPR is listed in Table 13.

[0225] Table 11

[0226]

[0227] The difference between Ab is indicated by an underline.

[0228] Use the Kabat system to define CDRs.

[0229] Table 12

[0230]

[0231]

[0232] Table 13

[0233]

[0234] Example 3. Activity analysis of anti-AREG monoclonal antibody

[0235] 1. Preparation of AREG protein for in vitro activity analysis of anti-hAREG antibodies

[0236] A cDNA encoding the extracellular domain (ECD) of human or mouse EGFR, fused with His6 and Avi tags at the C-terminus, was co-transfected into 293F cells with a plasmid encoding BirA-hFc for biotinylation. 72 hours post-transfection, the cell culture supernatant was harvested and used to purify either the hEGFR ECD His6-Avi-Biotin fusion protein or the mEGFR ECD His6-Avi-Biotin fusion protein (hEFGR-ECD, mEGFR-ECD) by protein A affinity chromatography.

[0237] Human or mouse AREG EGF domains with four additional residues (DLLA) at the C-terminus were expressed as mFc fusion proteins in 293F cells. 72 hours post-transfection, cell culture supernatant was harvested for purification of hAREG-EGFd-DLLA-mFc (hAREG-DLLA) or mAREG-EGFd-DLLA-mFc (mAREG-DLLA) fusion proteins by protein A affinity chromatography.

[0238] 2. Analysis of the inhibition of hAREG binding to EGFR using competitive ELISA

[0239] In simple terms, streptavidin (Sigma, 5 μg / mL) is coated into 96-well U-bottom plates, and then 100 nM biotinylated hEGFR-ECD or mEGFR-ECD is captured on the plate at a rate of 100 μL per well. Different concentrations of antibodies with sequential dilutions are mixed with 5 nM hAREG-DLLA or 50 nM mAREG-DLLA protein and added to the ELISA plate. The binding of hAREG-DLLA to hEGFR-ECD or mAREG-DLLA to mEGFR-ECD is detected by HRP-conjugated mouse anti-mouse IgG Fc antibody (Thermo Fisher).

[0240] 3. Inhibition of EGFR receptor phosphorylation

[0241] A431 (human epidermal carcinoma cell line) cells were starved of serum for 1 hour, followed by treatment with hAREG-DLLA alone (2.5 nM) or a mixture of hAREG and anti-AREG antibody for 1 hour. Approximately 1-2 × 10⁶ cells were used in each treatment in a 6-well plate. 5 Cells / wells. Treated cells were washed twice with PBS and then lysed on ice using RIPA buffer. Cell lysates were then subjected to SDS-PAGE followed by Western blotting. Phosphorylated EGFR (Tyr1068) and total EGFR were detected using an anti-phosphotyrosine monoclonal antibody (Abcam, EP774Y) and a rabbit polyclonal antibody (Cell Signaling Technology, #2232), respectively. α-tubulin expression in cell lysates was detected using an anti-α-tubulin monoclonal antibody (clone B-5-1-2, Sigma-Aldrich) and served as a loading control for Western blotting analysis.

[0242] 4. Epitope identification

[0243] To identify the epitopes of our anti-AREG monoclonal antibody, five amino acids that differ from hAREG-EGFd and mAREG-EGFd and possess different physical properties were selected for mutation. Five hAREG-EGFd variants were generated by changing the amino acids at five different sites in hAREG-EGFd to their corresponding amino acids in mAREG-EGFd. Furthermore, two mAREG-EGFd variants were generated by changing the amino acids at two different sites in mAREG-EGFd to their corresponding amino acids in hAREG-EGFd. The binding of these variants to the anti-AREG monoclonal antibody was then examined using SPR (Biacore T200).

[0244] 5. Results

[0245] Anti-AREG monoclonal antibodies block the binding of AREG to EGFR.

[0246] Previous reports have shown that adding four amino acids (DLLA) to the C-terminus of the EGF domain of human AREG significantly enhances the bioactivity of recombinantly expressed EGF domains (Thompson et al., 1996). For ease of competitive ELISA assays, we used hAREG-EGFd-DLLA-mFc (hAREG-DLLA) or mAREG-EGFd-DLLA-mFc (mAREG-DLLA) as ligands for binding to EGFR in the assay. Results showed that E1H3L4, P7, and hu9C12v4 all competed with mAREG-DLLA for binding to mEGFR-ECD. Among these three monoclonal antibodies, hu9C12v4 exhibited the best activity. The hIgG1 forms hu9C12v4, hu9C12v6, hu23H8v5, hu23H8v6 and hu1H9v3 were also tested in competitive ELISA, and they all showed strong activity in competing with hAREG-DLLA for binding to hEGFR-ECD, with sub-nanomolar IC50.

[0247] In addition, we tested two previously reported antibodies in the form of human IgG1, huPAR34 (US Patent Application No. 2004 / 0210040) and AR558 (US20170002068A1). Both antibodies also showed potent activity in competing with hAREG-DLLA for binding to hEGFR-ECD.

[0248] Anti-AREG monoclonal antibodies inhibit EGFR phosphorylation

[0249] The inhibitory activity of anti-AREG monoclonal antibodies against EGFR phosphorylation was tested in hEGFR-expressing epidermal-like cancer cells A431. Low concentrations of the antibodies were sufficient to block AREG-induced EGFR phosphorylation in A431 cells. 1.2 nM of 23H8 or 1H9 completely blocked hAREG-induced EGFR phosphorylation. Compared with 23H8 and 1H9, 9C12 showed relatively weaker blocking activity. Figure 2 ).

[0250] Epitope identification

[0251] To identify the epitopes of our anti-AREG monoclonal antibody, five hAREG-EGFd variants were generated by altering the amino acids at five different sites of hAREG-EGFd to the corresponding amino acids of mAREG-EGFd. Figure 3 The binding of the variants to the anti-AREG monoclonal antibody was then examined using SPR (Biacore T200). Two amino acids (Glu149 and His164) were identified as key epitope residues for the binding of the monoclonal antibody to hAREG. As revealed by Biacore analysis, for the hu9C12v4, hu9C12v6, hu23H8, and hu1H9 monoclonal antibodies, the hAREG-H164N variant completely lost its ability to bind to the monoclonal antibody, the hAREG-E149K variant had slightly reduced binding activity, and the other three hAREG variants had no effect on binding to the monoclonal antibody, confirming that His164 is the most critical epitope residue for the binding of our anti-AREG monoclonal antibody. For huPAR34, the E149K and H164N variants had reduced binding activity, while changes to the other three residues had no effect or a very small effect. For AR558, the E149K variant completely loses its binding activity, and changes to the other four residues have little or no effect on the binding of hAREG to AR558, indicating that Glu149 is the most critical epitope residue for AR558.

[0252] Furthermore, using two mAAREG variants, we found that the mAAREG-K149E / N164H (using hAREG numbering) variant acquired full binding affinity to anti-hAREG antibodies that had no or very weak cross-reactivity with mAAREG; the mAAREG-N164H variant acquired partial binding ability to the antibodies. These results indicate that the amino acids Lys149 and Asn164 in mAAREG are the residues that cause the lack of cross-reactivity between monoclonal antibodies (hu9C12v6, hu23H8, hu1H9, huPAR34, and AR558) and mAAREG.

[0253] Example 4. Animal Research

[0254] 1. Establishing an animal model

[0255] Cdc42 AT2 knockout mice were generated by specifically knocking out the Cdc42 gene in alveolar type II cells (AT2 cells).

[0256] To specifically delete the Cdc42 gene in AT2 cells, mice carrying the Spc-CreER knock-in allele were compared with Cdc42 floxed (Cdc42 flox / flox Mouse hybridization Figure 4 A). In Cdc42 flox / flox In mice, exon 2 of the Cdc42 gene, which contains the translation initiation exon, has two loxp sites flanking it. In Spc-CreER;Cdc42 flox / flox In mice, exon 2 of the Cdc42 gene was specifically deleted in AT2 cells via Cre / loxp-mediated recombination after tamoxifen treatment. Figure 4 B). Spc-CreER; Cdc42 flox / flox The mice were named Cdc42 AT2 knockout mice. The Cdc42 DNA sequence fragments before and after exon 2 of the Cdc42 gene deletion are shown below. All these mice were maintained in a pathogen-free animal facility.

[0257] The Cdc42 sequence preceding exon 2 of the Cdc42 gene deletion is shown in SEQ ID NO:133. The Cdc42 sequence following exon 2 of the Cdc42 gene deletion is shown in SEQ ID NO:134.

[0258] Cdc42 AT2 knockout mice developed progressive fibrotic changes in their lungs after PNX treatment.

[0259] Left lobectomy (PNX) was performed on Cdc42 AT2 knockout mice and control mice. Lung tissue samples from Cdc42 AT2 knockout mice and control mice were analyzed at different time points after PNX treatment. Figure 5 A). We found that on day 21 after PNX, some Cdc42 AT2 knockout mice showed significant weight loss and increased respiratory rate. In fact, by day 60 after PNX, almost 50% of the PNX-treated Cdc42 AT2 knockout mice reached the predetermined health criteria for endpoint euthanasia. Figure 5 B), and by day 180 after PNX treatment, over 70% of the PNX-treated Cdc42 AT2 knockout mice (n=33) reached their endpoint ( Figure 5 B). H&E staining showed that the lungs of control mice treated with sham surgery and PNX did not show fibrotic changes (B). Figure 5 C). H&E staining showed that at the endpoint, the entire lung lobe of PNX-treated Cdc42 AT2 knockout mice exhibited dense fibrotic changes. Figure 5 D).

[0260] On day 21 after PNX, the lungs of Cdc42 AT2 knockout mice began to show fibrotic changes. The Cdc42 AT2 knockout lungs already showed dense fibrotic changes at the lung margins. Figure 5 D). H&E staining showed that the histological changes in the fibrotic regions of the Cdc42 AT2 knockout lung recapitulated the histological changes in human IPF lung.

[0261] Lungs collected from control and Cdc42 AT2 knockout mice on day 21 after PNX were stained with anti-collagen I antibody. Figure 5 E). Compared to control lungs, a stronger immunofluorescence signal of collagen I was detected in the dense fibrotic regions of the lungs of Cdc42 AT2 knockout mice. From day 21 to day 60 after PNX, the area of ​​dense collagen I in the lungs of Cdc42 AT2 knockout mice gradually increased (E). Figure 5 F). qPCR analysis showed that from day 21 to day 60 after PNX, the expression level of collagen I mRNA in the lungs of Cdc42 AT2 knockout mice gradually increased. Figure 5 G). Respiratory function analysis showed that lung compliance gradually decreased in Cdc42 AT2 knockout mice from day 21 to day 60 after PNX. Figure 5 H). *P<0.05, ***P<0.001; ****P<0.0001, Student's t-test.

[0262] This is the first mouse model that can highly mimic the pathogenesis and progression of IPF. Therefore, it will be referred to as the IPF-like pulmonary fibrosis mouse model hereafter. Using this animal model, we identified AREG as a potential therapeutic target for pulmonary fibrosis.

[0263] Bleomycin-induced pulmonary fibrosis mouse model

[0264] Bleomycin-induced pulmonary fibrosis is a commonly used experimental model for studying pulmonary fibrosis in humans. Wild-type FVB / N mice (Charles River) in each group were administered a single dose of BLM (1U / 1 kg body weight, H20055883, Hai Zheng Pfizer Inc.) via intratracheal infusion. Mice treated with bleomycin (BLM) in all groups were monitored in a closed environment at different time points following bleomycin administration.

[0265] This is an animal model that can recreate acute lung injury-induced pulmonary fibrosis, such as post-pneumonia fibrosis or ILD (interstitial lung disease). Bleomycin induces lung injury through oxidatively mediated DNA breaks, leading to alveolar epithelial cell death (1-3 days post-injury) and an acute inflammatory response (3-9 days post-injury). Pulmonary fibrosis then occurs in the lungs between days 10 and 21 post-injury.

[0266] We used our IPF-like mouse model and bleomycin-induced pulmonary fibrosis mouse model to explore the therapeutic efficacy of our AREG antibody. Furthermore, we compared the therapeutic effects of two drugs, nintedanib and pirfenidone, to comprehensively evaluate the potential therapeutic efficacy of the AREG antibody against existing FDA-approved drugs. 2. Animal Study Design and Analysis for the Treatment of Pulmonary Fibrosis in Mouse Models

[0267] 1) IPF-like pulmonary fibrosis mouse model: Three-month-old male Cdc42 AT2 knockout mice with similar body weight (~30g) were selected for the experiment. Mice were administered tamoxifen (75mg / kg) intraperitoneally every other day for a total of four times. Two weeks after the last injection, the mice were treated with PNX. The onset of fibrosis was observed on day 14 after PNX. Fourteen days after PNX, the PNX-treated mice were weighed and treated.

[0268] 2) Bleomycin-induced pulmonary fibrosis mouse model: Three-month-old male FVB / N mice with similar body weight (~30g) were selected for bleomycin treatment. Specifically, an endotracheal tube was inserted into the trachea of ​​anesthetized mice, and then bleomycin solution (dose: 1U / kg) was delivered. The mice were then weighed and treated one day after bleomycin delivery.

[0269] 3) Treatment Groups: Mice were divided into different groups: control group, anti-AREG antibody group, nintedanib group, and pirfenidone group. All mice in each group were age-matched and weight-matched. For the control group, mice were treated with isotype-matched anti-AREG antibodies. The control antibody or anti-AREG antibody was administered intraperitoneally every 5 days at a dose of 10-15 mg / kg. In addition, mice in the control group were treated once daily via oral feeding with 0.5% sodium methylcellulose solution. Mice in the nintedanib group were treated once daily via oral feeding with nintedanib (60 mg / kg). Mice in the pirfenidone treatment group were treated once daily via oral feeding with pirfenidone (100 mg / kg). Mice in the nintedanib and pirfenidone groups were also treated intraperitoneally every 5 days with PBS solution.

[0270] 4) Animal research

[0271] a) Monitor the body weight of mice in all groups every other day. Closely monitor the overall health status of mice in all groups twice daily.

[0272] b) The humane endpoint is defined by the reduction of total body weight (30% of initial body weight).

[0273] Animal studies were conducted under an approved Institutional Animal Care and Use Committee protocol. Lung tissue was collected at the study endpoint. Hydroxyproline content in the lungs of each mouse was measured using a hydroxyproline kit (Sigma, catalog number MAK008). Histological analysis was used to assess pulmonary fibrosis scales. Lung tissue was fixed with 4% PFA, sectioned, and stained with H&E. Final histological fibrosis scores were assigned by analyzing different regions of the lung.

[0274] 3. Results

[0275] 1) Anti-AREG antibody (P7): Our results showed that the anti-AREG (P7) antibody significantly slowed weight loss in Cdc42 AT2 knockout mice and prolonged their survival time. Figure 6 A-6C). Furthermore, the anti-AREG antibody (P7) significantly reduced the hydroxyproline content in the lungs of Cdc42 AT2 knockout mice. Figure 6 D). Figure 6 A shows a general outline of the treatment and sampling procedures. Figure 6 B shows that the anti-AREG antibody (P7) can prolong the survival time of Cdc42 AT2 knockout mice. Figure 6 C showed that the anti-AREG antibody (P7) significantly slowed down weight loss in Cdc42 AT2 knockout mice. Figure 6 D shows that, compared with mice treated with blank antibody, anti-AREG antibody (P7) significantly reduced the hydroxyproline content in the lungs of Cdc42 AT2 knockout mice (*, P<0.05, Student's t-test).

[0276] 2) Anti-AREG antibody (E1H3L4): Our results showed that in bleomycin-treated mice, anti-AREG antibody (E1H3L4) accelerated the resolution of fibrosis and promoted weight recovery. Figure 7 B). Figure 7 A shows that in a bleomycin-induced pulmonary fibrosis mouse model, there was no significant difference in survival between mice treated with a blank antibody and mice treated with an anti-AREG antibody (E1H3L4). However, Figure 7 B shows that mice in the anti-AREG antibody (E1H3L4) treatment group recovered better compared to mice in the blank antibody treatment group.

[0277] Furthermore, anti-AREG (E1H3L4) antibody treatment can significantly prolong the survival time of Cdc42 AT2 knockout mice. Figure 8 A-8B). H&E staining analysis showed that in the lungs of Cdc42 AT2 knockout mice, the area of ​​pulmonary fibrosis was significantly reduced in the anti-AREG antibody (E1H3L4) treatment group. Figure 8 C). Figure 8 A shows a general outline of the treatment and sampling procedures. Figure 8 B shows that the anti-AREG antibody (E1H3L4) can significantly prolong the survival time of Cdc42 AT2 knockout mice. Figure 8 H&E staining analysis showed that, compared with the control group, mice in the anti-AREG antibody (E1H3L4) treatment group had significantly reduced pulmonary fibrosis.

[0278] 3) Anti-AREG antibody (hu9C12v4): Our results showed that the anti-AREG antibody (hu9C12v4) significantly prolonged the survival time of Cdc42 AT2 knockout mice. Figure 9 A-9B), while nintedanib and pirfenidone did not significantly prolong the survival time of Cdc42AT2 knockout mice. H&E staining analysis showed that in the lungs of Cdc42AT2 knockout mice, the area of ​​pulmonary fibrosis in the anti-AREG antibody (hu9C12v4) treatment group was significantly reduced. Figure 9 C). Specifically... Figure 9 A shows a general outline of the treatment and sampling procedures. Figure 9 B showed that anti-AREG antibody (hu9C12v4) treatment significantly prolonged the survival time of Cdc42 AT2 knockout mice, and Figure 9 H&E staining analysis showed that, compared with mice in the control group, nintedanib group and pirfenidone group, mice in the anti-AREG antibody (hu9C12v4) treatment group had significantly reduced pulmonary fibrosis.

[0279] In summary, these results confirm the effectiveness of our anti-AREG monoclonal antibody in treating pulmonary fibrosis.

[0280] References:

[0281] 1. Barkauskas, CE and Noble, PW (2014), Cellular mechanisms of tissue fibrosis. 7. New insights into the cellular mechanisms of pulmonary fibrosis, American Journal of Physiology, Cell Physiology 306, C987-996.

[0282] 2Li,D.,He,W.,Liu,X.,Zheng,S.,Qi,Y.,Li,H.,Mao,F.,Liu,J.,Sun,Y.,Pan,L. et al., (2017), A potent human neutralizing antibody Fc-dependently reduces established HBV infections, eLife 6:e26738.

[0283] 3. Steele, MP and Schwartz, DA (2013), Molecular mechanisms in progressive idiopathic pulmonary fibrosis, Annual review of medicine 64, 265-276.

[0284] 4. Thompson, SA, Harris, A., Hoang, D., Ferrer, M. and Johnson, GR (1996), COOH-terminal extended recombinant amphiregulin with bioactivity comparable with naturally derived growth factor, The Journal of Biological Chemistry 271, 17927-17931.

Claims

1. An isolated anti-AREG antibody or its antigen-binding fragment thereof, having the ability to inhibit fibrosis, comprising: The heavy chain variable region containing heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region containing light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from: (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, LCDR2 shown in SEQ ID NO: 26, and 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, and 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, and 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, LCDR2 shown in SEQ ID NO: 34, and 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, and LCDR3 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, and 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: 136, LCDR1 shown in SEQ ID NO: 39, LCDR2 shown in SEQ ID NO: 40, and 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: 136, LCDR1 shown in SEQ ID NO: 41, LCDR2 shown in SEQ ID NO: 42, and 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: 136, LCDR1 shown in SEQ ID NO: 43, LCDR2 shown in SEQ ID NO: 44, and 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, and 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, and LCDR3 shown in SEQ ID NO: 46; (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, and LCDR3 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, and 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, and 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, and 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, and 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, and 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, and LCDR3 shown in SEQ ID NO:

55.

2. The anti-AREG antibody or its antigen-binding fragment according to claim 1, wherein the fibrosis is renal fibrosis, liver fibrosis or pulmonary fibrosis.

3. The anti-AREG antibody of claim 1 or its antigen-binding fragment, wherein the fibrosis is IPF.

4. The anti-AREG antibody or its antigen-binding fragment according to claim 1, which is capable of binding to AREG.

5. The anti-AREG antibody or its antigen-binding fragment according to claim 1, which is capable of binding to human AREG.

6. The anti-AREG antibody or its antigen-binding fragment according to claim 1, wherein it is a human anti-AREG antibody, a mouse anti-AREG antibody, a humanized anti-AREG antibody, or a chimeric anti-AREG antibody.

7. The anti-AREG antibody or its antigen-binding fragment according to claim 1 is a human monoclonal antibody (mAb), a mouse mAb, a humanized mAb, or a chimeric mAb.

8. The anti-AREG antibody or its antigen-binding fragment according to claim 1, which binds to AREG with high affinity and has a dissociation constant of less than 10 nM.

9. The anti-AREG antibody or its antigen-binding fragment according to claim 1, wherein the dissociation constant is less than 1 nM, 0.1 nM or 0.01 nM.

10. The anti-AREG antibody or its antigen-binding fragment according to claim 1, wherein the dissociation constant is in the range of 1 × 10⁻⁶. -8 ~1×10 -11 Within the range of M.

11. The anti-AREG antibody or its antigen-binding fragment according to claim 1, wherein the dissociation constant is in the range of 1 × 10⁻⁶. -9 ~1×10 -11 Within the range of M.

12. The anti-AREG antibody or its antigen-binding fragment according to claim 1, which is capable of binding to soluble form of AREG.

13. The anti-AREG antibody or its antigen-binding fragment according to claim 1, which is capable of binding to the epidermal growth factor-like domain of AREG in a soluble form.

14. The anti-AREG antibody or its antigen-binding fragment according to claim 1, which is capable of binding to residues 101-184 of human pro-AREG, and / or residues 171-184 of human pro-AREG, and / or residues 94-177 of mouse pro-AREG, and / or residues 135-177 of mouse pro-AREG.

15. The anti-AREG antibody of claim 1 or its antigen-binding fragment thereof, which is capable of binding the amino acid sequence represented by any one of SEQ ID NO: 123, 125, 127-129 and 132.

16. The anti-AREG antibody of claim 1 or its antigen-binding fragment thereof, which is capable of interacting with Glu149 and / or His164 of human pro-AREG.

17. The anti-AREG antibody of claim 1 or its antigen-binding fragment thereof, wherein the antigen-binding fragment is an antibody fragment that binds to AREG in a soluble form.

18. The anti-AREG antibody of claim 1 or its antigen-binding fragment thereof, wherein the antigen-binding fragment is a Fab fragment or an F(ab)2 fragment that binds to soluble form of AREG.

19. The anti-AREG antibody or its antigen-binding fragment according to claim 1, comprising a heavy chain variable region and a light chain variable region. The heavy chain variable region has an amino acid sequence selected from SEQ ID NO: 57-69 and an amino acid sequence that has at least 95% sequence identity with any of SEQ ID NO: 57-69 and retains epitope binding activity. The light chain variable region has an amino acid sequence selected from SEQ ID NO: 70-89 and an amino acid sequence that has at least 95% sequence identity with any of SEQ ID NO: 70-89 and retains epitope binding activity.

20. The anti-AREG antibody of claim 1 or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region have an amino acid sequence selected from the following: (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 that have at least 95% sequence identity with any one of (1) to (21) and retain epitope binding activity.

21. The anti-AREG antibody or its antigen-binding fragment according to claim 1, wherein it is an isotype of IgG, IgM, IgA, IgE or IgD.

22. The anti-AREG antibody or its antigen-binding fragment according to claim 1, wherein it is an isotype of IgG1, IgG2, IgG3 or IgG4.

23. The anti-AREG antibody or its antigen-binding fragment according to claim 1, which can block the binding of AREG to the epidermal growth factor receptor EGFR and / or inhibit EGFR phosphorylation.

24. An isolated polynucleotide encoding an anti-AREG antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 23.

25. The isolated polynucleotide of claim 24, wherein the heavy chain variable region and the light chain variable region are encoded on the same polynucleotide or on separate polynucleotides.

26. The isolated polynucleotide of claim 24, comprising: The DNA sequence encoding the variable region of the heavy chain is shown in any of the sequences SEQ ID NO: 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 112, 115, and 117; and The DNA sequence encoding the variable region of the light chain is shown in any of the sequences SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 110, 111, 113, 114, 116, 118, 119, 120, 121 and 122.

27. An isolated cell or vector comprising a polynucleotide encoding an anti-AREG antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 23.

28. The isolated cell or vector of claim 27, wherein the cell is a hybridoma cell.

29. A composition comprising an anti-AREG antibody or an antigen-binding fragment thereof according to any one of claims 1 to 23 and a pharmaceutically acceptable carrier.

30. Use of the anti-AREG antibody or antigen-binding fragment thereof according to any one of claims 1 to 23 in the preparation of a medicament for treating a subject with a disorder, wherein AREG is overexpressed, upregulated, or activated; the disorder is a fibrotic disease; and the disorder is renal fibrosis, hepatic fibrosis, or pulmonary fibrosis.

31. The use as claimed in claim 30, wherein the subject is a mammalian subject requiring diagnosis, prognosis, or treatment.

32. The use as described in claim 31, wherein, The mammalian subjects included dogs, cats, guinea pigs, rabbits, rats, mice, horses, and cattle.

33. The use as described in claim 31, wherein, The mammalian subjects included dairy cows.

34. The use as claimed in claim 30, wherein the barrier is an IPF.

35. Use of the anti-AREG antibody or antigen-binding fragment thereof according to any one of claims 1 to 23 in the preparation of a reagent for determining the presence of AREG protein, the determination comprising exposing cells suspected of containing AREG protein to the anti-AREG antibody or antigen-binding fragment thereof, and determining the binding of the anti-AREG antibody or antigen-binding fragment thereof to the cells.

36. The use of claim 35, which is for diagnosing a disorder in a subject, wherein AREG is overexpressed, upregulated, or activated in the disorder.

37. The use of claim 36, wherein the disorder is a fibrotic disease, including renal fibrosis, liver fibrosis, and pulmonary fibrosis.

38. The use according to claim 36, wherein the barrier is an IPF.

39. The use as claimed in claim 36, wherein the subject is a mammalian subject.

40. The use according to claim 39, wherein the mammalian subject includes dogs, cats, guinea pigs, rabbits, rats, mice, horses, and cattle.

41. The use as described in claim 39, wherein, The mammalian subjects included dairy cows.

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