Anti-connective tissue growth factor antibodies and their applications

Abstract

Anti-CTGF antibodies, including the variable regions of the light and heavy chains, and their uses as pharmaceuticals are provided. The antibodies can be used to manufacture medicaments for the treatment of CTGF-related diseases or conditions.

Description

[Technical field]

[0001] This application claims priority from Chinese Patent Application No. 201910480169.4 (Title: Anti-Connective Tissue Growth Factor Antibody and Its Application; Priority Date: June 4, 2019).

[0002] FIELD OF THEINVENTION The present disclosure is in the field of biomedical sciences, and in particular relates to antibodies that bind to connective tissue growth factor (CTGF) and applications thereof. [Background technology]

[0003] 2. Background of the Invention The statements herein are merely intended to provide background information related to the present disclosure and do not necessarily constitute prior art.

[0004] CTGF expression is induced by members of the transforming growth factor beta (TGFβ) superfamily, which includes TGFβ-1, -2, and -3, bone morphogenetic protein (BMP)-2, and activins. Many regulatory agents (including dexamethasone, thrombin, vascular endothelial growth factor (VEGF) and angiotensin II) and environmental stresses (including hyperglycemia and hypertension) also induce expression of CTGF (see, e.g., Franklin (1997) Int J Biochem Cell Biol 29:79-89; Wunderlich (2000) Graefes Arch Clin Exp Ophthalmol 238: 910-915; Denton and Abraham (2001) Curr Opin Rheumatol 13: 505-511; and Riewald (2001) Blood 97: 3109-3116; Riser et al. (2000) J Am SocNephrol 11: 25-38; and International Patent Application Publication No. WO 00 / 13706).

[0005] The stimulatory effect of TGFβ on CTGF expression is rapid and sustained, and does not require sustained application of TGFβ (Igarashi et al. (1993) Mol Biol Cell 4:637-645). TGFβ activates transcription through DNA regulatory elements present in the CTGF promoter, resulting in increased CTGF expression (Grotendorst et al. (1996) Cell Growth Differ 7: 469-480; Grotendorst and Bradham, U.S. Patent No. 6,069,006; Holmes et al. (2001) J Biol Chem 276: 10594-10601).

[0006] CTGF expression is upregulated in glomerulonephritis, IgA nephropathy, focal and segmental glomerulosclerosis, and diabetic nephropathy (see, e.g., Riser et al. (2000) J Am Soc Nephrol 11:25-38). Increased numbers of CTGF-expressing cells have also been observed at sites of chronic tubulointerstitial injury, with CTGF levels correlating with the extent of injury (Ito et al. (1998) Kidney Int 53:853-861). Furthermore, CTGF expression in glomeruli and tubulointerstitium is increased in various nephropathies that involve scarring and sclerosis of the renal parenchyma. High levels of CTGF are also associated with liver fibrosis, myocardial infarction, and pulmonary fibrosis. For example, CTGF is also significantly upregulated in cells derived from biopsies and bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis (IPF) (Ujike et al. (2000) Biochem Biophys Res Commun 277:448-454; Abou-Shady et al. (2000) Liver 20: 296-304; Williams et al. (2000) J Hepatol 32: 754-761). Therefore, CTGF is an effective target for the treatment of said diseases. Summary of the Invention [Problem to be solved by the invention]

[0007] However, there are currently no effective CTGF-targeting antibody drugs in clinical use, and there is still a need to develop new safe and effective CTGF antibody drugs. [Means for solving the problem]

[0008] Summary of the Invention The present disclosure provides anti-CTGF antibodies, including anti-human CTGF full-length antibodies and antigen-binding fragments thereof.

[0009] In some embodiments, the anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, wherein: i) the heavy chain variable region comprises the same HCDR1, HCDR2 and HCDR3 sequences as those of the heavy chain variable region set forth in SEQ ID NO:6, and the light chain variable region comprises the same LCDR1, LCDR2 and LCDR3 sequences as those of the light chain variable region set forth in SEQ ID NO:7; or ii) the heavy chain variable region comprises the same HCDR1, HCDR2 and HCDR3 sequences as those of the heavy chain variable region shown in SEQ ID NO:8, and the light chain variable region comprises the same LCDR1, LCDR2 and LCDR3 sequences as those of the light chain variable region shown in SEQ ID NO:9.

[0010] In some embodiments, the anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, wherein: ii-i) the heavy chain variable region comprises the same HCDR1, HCDR2 and HCDR3 sequences as those of the heavy chain variable region set forth in SEQ ID NO: 69, and the light chain variable region comprises the same LCDR1, LCDR2 and LCDR3 sequences as those of the light chain variable region set forth in SEQ ID NO: 70; or ii-ii) the heavy chain variable region comprises the same HCDR1, HCDR2 and HCDR3 sequences as those of the heavy chain variable region set forth in SEQ ID NO: 85, and the light chain variable region comprises the same LCDR1, LCDR2 and LCDR3 sequences as those of the light chain variable region set forth in SEQ ID NO: 70.

[0011] In some embodiments, the anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, wherein: iii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, respectively; or iv) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, respectively.

[0012] In some embodiments, the anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, wherein: iv-i) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:71, SEQ ID NO:72 and SEQ ID NO:73, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:74, SEQ ID NO:75 and SEQ ID NO:76, respectively; or iv-ii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 102, SEQ ID NO: 103 and SEQ ID NO: 104, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, respectively.

[0013] In some embodiments, the anti-CTGF antibody is a murine antibody, a chimeric antibody or a humanized antibody.

[0014] In some embodiments, the anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, wherein: (v-1) the amino acid sequence of the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 6, 27, 28, or 29, and the amino acid sequence of the light chain variable region has at least 90% sequence identity to SEQ ID NO: 7, 22, 23, 24, 25, or 26; (vi-1) the amino acid sequence of the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 8, 33, 34, 35, or 36, and the amino acid sequence of the light chain variable region has at least 90% sequence identity to SEQ ID NO: 9, 30, 31, or 32; or (vi-i) the amino acid sequence of the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 69, 81, 82, 83, 84 or 85, and the amino acid sequence of the light chain variable region has at least 90% sequence identity to SEQ ID NO: 70, 77, 78, 79 or 80.

[0015] In some embodiments, the anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, wherein: (v) the heavy chain variable region is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the heavy chain variable region set forth in SEQ ID NO:6, 27, 28 or 29 and the light chain variable region is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the light chain variable region set forth in SEQ ID NO:7, 22, 23, 24, 25 or 26; or (vi) the heavy chain variable region is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the heavy chain variable region set forth in SEQ ID NO:8, 33, 34, 35 or 36 and the light chain variable region is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the light chain variable region set forth in SEQ ID NO:9, 30, 31 or 32; or the heavy chain variable region is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the heavy chain variable region set forth in SEQ ID NO:6 and the light chain variable region is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the light chain variable region set forth in SEQ ID NO:7; the heavy chain variable region is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the heavy chain variable region set forth in SEQ ID NO:27, 28 or 29, and the light chain variable region is at least 90%, 91%, 92%, 93%, 94%, 95%, 96, 97%, 98%, 99% or 100% identical to the light chain variable region set forth in SEQ ID NO:22, 23, 24, 25 or 26; the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the heavy chain variable region set forth in SEQ ID NO:8 and the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the light chain variable region set forth in SEQ ID NO:9; The heavy chain variable region is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the heavy chain variable region set forth in SEQ ID NO: 33, 34, 35 or 36, and the light chain variable region is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the light chain variable region set forth in SEQ ID NO: 30, 31 or 32.

[0016] In some embodiments, the anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, wherein: (vi-ii) the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the heavy chain variable region set forth in SEQ ID NO: 69, 81, 82, 83, 84 or 85, and the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the light chain variable region set forth in SEQ ID NO: 70, 77, 78, 79 or 80.

[0017] In some embodiments of the anti-CTGF antibody, the anti-CTGF antibody is a humanized antibody comprising a framework region of a human antibody or a framework region variant thereof, wherein the framework region variant has up to 10 back mutations in each of the human antibody light chain framework region and / or heavy chain framework region.

[0018] In some embodiments, the anti-CTGF antibody comprises the heavy and light chain variable regions set forth as follows: (a) the light chain variable region comprises LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, respectively, and comprises one or more amino acid back mutations selected from the group consisting of 4L, 36F, 43S, 45K, 47W, 58V or 71Y, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively, and comprises one or more amino acid back mutations selected from the group consisting of 28S, 30N, 49A, 75E, 76S, 93V, 94E or 104D; or (b) the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively, and contains one or more back mutations selected from the group consisting of 36V, 44F, 46G or 49G, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively, and contains one or more back mutations selected from the group consisting of 44G, 49G, 27F, 48L, 67L, 71K, 78V or 80F.

[0019] In some embodiments, the anti-CTGF antibody comprises the heavy and light chain variable regions set forth as follows: (bi) the light chain variable region comprises LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NO:74, SEQ ID NO:75 and SEQ ID NO:76, respectively, and comprises one or more backmutations selected from the group consisting of 45P, 46W, 48Y, 69S or 70Y, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO:71, SEQ ID NO:72 and SEQ ID NO:73, respectively, and comprises one or more backmutations selected from the group consisting of 27F, 38K, 48I, 67K, 68A, 70L or 72F; or (b-ii) the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, respectively, and comprises one or more back mutations selected from the group consisting of 45P, 46W, 48Y, 69S or 70Y; and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 102, SEQ ID NO: 103 and SEQ ID NO: 104, respectively, and comprises one or more back mutations selected from the group consisting of 27F, 38K, 48I, 67K, 68A, 70L or 72F.

[0020] In some embodiments, the anti-CTGF antibody comprises the heavy and light chain variable regions set forth as follows: (vii) the sequence of the heavy chain variable region is set forth in SEQ ID NO:6 and the sequence of the light chain variable region is set forth in SEQ ID NO:7; (viii) the sequence of the heavy chain variable region is set forth in SEQ ID NO: 27, 28 or 29 and the sequence of the light chain variable region is set forth in SEQ ID NO: 22, 23, 24, 25 or 26; (ix) the sequence of the heavy chain variable region is set forth in SEQ ID NO: 8 and the sequence of the light chain variable region is set forth in SEQ ID NO: 9; or (x) the sequence of the heavy chain variable region is set forth in SEQ ID NO: 33, 34, 35 or 36, and the sequence of the light chain variable region is set forth in SEQ ID NO: 30, 31 or 32.

[0021] In some embodiments, the anti-CTGF antibody comprises the heavy and light chain variable regions set forth as follows: (xi) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 69 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 70; or (xii) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 81, 82, 83, 84 or 85, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 77, 78, 79 or 80.

[0022] In some embodiments, the anti-CTGF antibody comprises the heavy and light chain variable regions shown in Tables a, b, and c below:

[0023] TIFF0007627665000001.tif60148

[0024] TIFF0007627665000002.tif60126

[0025] TIFF0007627665000003.tif71149

[0026] In some embodiments, the anti-CTGF antibody comprises the heavy and light chain variable regions set forth as follows: (xiii) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 27 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 22; (xiv) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 34 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 30; or (xv) The amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 85, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 77.

[0027] In some embodiments of the anti-CTGF antibody, where the antibody further comprises an antibody heavy chain constant region and a light chain constant region, preferably the heavy chain constant region is selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4 constant regions and common variants thereof, and the light chain constant region is selected from the group consisting of human antibody kappa and lambda chain constant regions and common variants thereof; more preferably, the antibody comprises a heavy chain constant region set forth in SEQ ID NO: 37 or 38, and a light chain constant region set forth in SEQ ID NO: 39 or 40.

[0028] In some embodiments, the anti-CTGF antibody includes: (c) a heavy chain that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a heavy chain set forth in SEQ ID NO: 41, 43, 44, 45, 46, 47 or 48, and a light chain that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a light chain set forth in SEQ ID NO: 42, 49, 50, 51, 52 or 53; (d) a heavy chain that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a heavy chain set forth in SEQ ID NO: 54, 56, 57, 58, 59, 60, 61, 62 or 63, and a light chain that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a light chain set forth in SEQ ID NO: 55, 64, 65 or 66; (e) a heavy chain set forth in SEQ ID NO: 41, 43, 44, 45, 46, 47 or 48, and a light chain set forth in SEQ ID NO: 42, 49, 50, 51, 52 or 53; or (f) a heavy chain depicted in SEQ ID NO: 54, 56, 57, 58, 59, 60, 61, 62 or 63, and a light chain depicted in SEQ ID NO: 55, 64, 65 or 66.

[0029] In some embodiments, the anti-CTGF antibody includes: (g) a heavy chain that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a heavy chain set forth in SEQ ID NO: 86, 88, 89, 90, 91, 92, 93, 94, 95, 96, or 97, and a light chain that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a light chain set forth in SEQ ID NO: 87, 98, 99, 100, or 101; or (h) a heavy chain depicted in SEQ ID NO: 86, 88, 89, 90, 91, 92, 93, 94, 95, 96 or 97, and a light chain depicted in SEQ ID NO: 87, 98, 99, 100 or 101.

[0030] In some embodiments, the anti-CTGF antibody includes: (j) a heavy chain set forth in SEQ ID NO: 46, and a light chain set forth in SEQ ID NO: 49; (k) a heavy chain set forth in SEQ ID NO: 61, and a light chain set forth in SEQ ID NO: 64; or (l) a heavy chain as set forth in SEQ ID NO: 97, and a light chain as set forth in SEQ ID NO: 98.

[0031] In other aspects of the disclosure, there are provided anti-CTGF antibodies that compete with the anti-CTGF antibodies, or antigen-binding fragments thereof, for binding to human CTGF.

[0032] In other aspects of the disclosure, nucleic acid molecules encoding the anti-CTGF antibodies are provided.

[0033] In other aspects of the disclosure, a host cell is provided that contains the nucleic acid molecule.

[0034] In other aspects of the disclosure, there are provided pharmaceutical compositions comprising a therapeutically or prophylactically effective amount of the anti-CTGF antibody or the nucleic acid molecule and one or more pharma- ceutically acceptable carriers, diluents, buffers, or excipients.

[0035] In some specific embodiments, a therapeutically or prophylactically effective amount means that a unit dose of the composition contains 0.1-3000 mg or 1-1000 mg of the anti-CTGF antibody.

[0036] In other aspects of the disclosure, there are provided methods of immunoassay or determination of CTGF comprising applying said anti-CTGF antibody.

[0037] In some other aspects of the disclosure, methods of immunoassaying or determining CTGF are provided that include contacting a subject or a sample thereof with the anti-CTGF antibody.

[0038] In other aspects of the disclosure, kits are provided that include the anti-CTGF antibodies.

[0039] In other aspects of the disclosure, there is provided a method of treating a CTGF-related disease comprising administering to a subject a therapeutically effective amount of the anti-CTGF antibody or the nucleic acid molecule or the pharmaceutical composition, wherein the disease is preferably a fibrotic disease (the fibrotic disease is preferably idiopathic pulmonary fibrosis, diabetic nephropathy, diabetic retinopathy, osteoarthritis, scleroderma, chronic heart failure, liver cirrhosis, or renal fibrosis), hypertension, diabetes, myocardial infarction, arthritis, a CTGF-related cell proliferative disease, atherosclerosis, glaucoma, or cancer (the cancer is preferably acute lymphoblastic leukemia, dermatofibroma, breast cancer, angiolipoma, angioleiomyoma, connective tissue generating cancer, prostate cancer, ovarian cancer, colorectal cancer, pancreatic cancer, gastrointestinal cancer, or liver cancer).

[0040] In other aspects of the disclosure, there is provided a use of the anti-CTGF antibody or the nucleic acid molecule or the pharmaceutical composition in the manufacture of a medicament for treating a CTGF-related disease, wherein the CTGF-related disease includes a fibrotic disease (the fibrotic disease is preferably idiopathic pulmonary fibrosis, diabetic nephropathy, diabetic retinopathy, osteoarthritis, scleroderma, chronic heart failure, liver cirrhosis, or renal fibrosis), hypertension, diabetes, myocardial infarction, arthritis, a CTGF-related cell proliferative disease, atherosclerosis, glaucoma, or cancer (the cancer is preferably acute lymphoblastic leukemia, dermatofibroma, breast cancer, angiolipoma, angioleiomyoma, connective tissue generating cancer, prostate cancer, ovarian cancer, colorectal cancer, pancreatic cancer, gastrointestinal cancer, or liver cancer).

[0041] In other aspects of the disclosure, there is provided the anti-CTGF antibody or the nucleic acid molecule encoding the anti-CTGF antibody or the pharmaceutical composition for use in treating a CTGF-related disease, wherein the CTGF-related disease includes a fibrotic disease (the fibrotic disease is preferably idiopathic pulmonary fibrosis, diabetic nephropathy, diabetic retinopathy, osteoarthritis, scleroderma, chronic heart failure, liver cirrhosis, or renal fibrosis), hypertension, diabetes, myocardial infarction, arthritis, a CTGF-related cell proliferative disease, atherosclerosis, glaucoma, or cancer (the cancer is preferably acute lymphoblastic leukemia, dermatofibroma, breast cancer, angiolipoma, angioleiomyoma, connective tissue generating cancer, prostate cancer, ovarian cancer, colorectal cancer, pancreatic cancer, gastrointestinal cancer, or liver cancer). [Brief description of the drawings]

[0042] [Figure 1] Affinity of humanized antibodies derived from mab164 to human CTGF as detected by ELISA. [Diagram 2] Results of SU86.86 mouse xenograft tumor suppression experiments with humanized antibodies derived from mab147 or mab164. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Detailed Description of the Invention term In order to more readily understand this disclosure, certain technical and scientific terms are defined below. All other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs, unless otherwise defined herein.

[0044] The three-letter and one-letter codes for amino acids used in this disclosure are those described in J. biol. chem, 243, p3558 (1968).

[0045] Connective tissue growth factor (CTGF) is a cysteine-rich, 36 kD, secreted, heparin-binding glycoprotein that was first isolated from human umbilical vein endothelial cells (see, e.g., Bradham et al. (1991) J Cell Biol 114: 1285-1294; Grotendorst and Bradham, U.S. Patent No. 5,408,040). CTGF belongs to the CCN (CTGF, Cyr61, Nov) family of proteins (secreted glycoproteins), which includes the serum-inducible immediate-early gene product Cyr61, the putative oncogene Nov, the ECM-associated protein FISP-12, the src-inducible gene CEF-10, the Wnt-inducible secreted protein WISP-3, and the growth-suppressing protein HICP / rCOP (Brigstock (1999) Endocr Rev 20: 189-206; O'Brian et al. (1990) Mol Cell Biol 10: 3569-3577; Joliot et al. (1992) Mol Cell Biol 12: 10-21; Ryseck et al. (1990) Cell Growth and Diff 2: 225-233; Simmons et al. (1989) Proc Natl Acad Sci USA 86: 1178-1182; Pennica et al. (1998) Proc Natl Acad Sci USA, 95: 14717-14722; and Zhang et al. (1998) Mol Cell Biol 18: 6131-6141). CCN proteins are characterized by 38 conserved cysteine ​​residues, which constitute more than 10% of the total amino acids and form a modular structure with N- and C-terminal domains. The modular structure of CTGF contains conserved motifs of insulin-like growth factor binding protein (IGF-BP) and von Willebrand factor (VWC) in the N-terminal domain and thrombospondin (TSP1) and cysteine ​​knot motifs in the C-terminal domain.

[0046] In this document, "antibody" refers to an immunoglobulin, and a full-length antibody is a four-peptide chain structure connected by interchain disulfide bonds between two identical heavy chains and two identical light chains. The immunoglobulin heavy chain constant regions exhibit different amino acid compositions and sequences, and therefore different antigenicity. Thus, immunoglobulins can be classified into five types, which can be called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE, with the corresponding heavy chains being μ, δ, λ, α, and ε. Depending on the amino acid composition of the hinge region and the number and position of the heavy chain disulfide bonds, the same type of Ig can be further classified into different subtypes, for example, IgG can be classified into IgG1, IgG2, IgG3, and IgG4. The light chain can be classified into κ or λ chains based on the differences in the constant region. Each of the five Ig types can have κ or λ chains.

[0047] The sequences of about 110 amino acids from the N-terminus of antibody heavy and light chains are highly variable and known as the variable region (Fv region), while the remaining C-terminal amino acid sequences are relatively constant and known as the constant region. The variable region contains three hypervariable regions (HVRs) and four framework regions (FRs) with relatively conserved sequences. The three hypervariable regions that determine the specificity of the antibody are also called complementarity determining regions (CDRs). Each light chain variable region (VL) and each heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2 and LCDR3, and the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3.

[0048] The antibodies of the present disclosure include murine antibodies, chimeric antibodies, and humanized antibodies.

[0049] As used herein, the term "mouse antibody" refers to a monoclonal antibody against human CTGF produced by the knowledge and techniques in the art. In production, a test subject is injected with CTGF antigen, and then hybridomas expressing antibodies with the desired sequence or functional characteristics are isolated. In a preferred embodiment of the present disclosure, the mouse anti-CTGF antibody or antigen-binding fragment thereof can further comprise a mouse kappa, lambda chain light chain constant region or variants thereof, or a mouse IgG1, IgG2, IgG3 heavy chain constant region or variants thereof.

[0050] The term "chimeric antibody" refers to an antibody that is produced by fusing the variable region of a mouse antibody to the constant region of a human antibody, and such an antibody can reduce the immune response elicited by mouse antibodies. To produce a chimeric antibody, first prepare a hybridoma that secretes a specific mouse monoclonal antibody, and then clone the variable region gene from the mouse hybridoma. Then, if necessary, clone the constant region gene from a human antibody. The mouse variable region gene and the human constant region gene can be linked to form a chimeric gene, which can then be inserted into an expression vector. Finally, the chimeric antibody molecule can be expressed in eukaryotic or prokaryotic systems. In a preferred embodiment of the present disclosure, the antibody light chain of the chimeric antibody further comprises a human kappa, lambda chain light chain constant region or a variant thereof. The antibody heavy chain of the CTGF chimeric antibody further comprises a human IgG1, IgG2, IgG3, IgG4 heavy chain constant region or a variant thereof, preferably a human IgG1, IgG2 or IgG4 heavy chain constant region, or an IgG1, IgG2 or IgG4 variant having amino acid mutations (e.g., an L234A and / or an L235A mutation, and / or an S228P mutation).

[0051] The term "humanized antibody", also called CDR-grafted antibody, refers to an antibody made by grafting mouse CDR sequences into a human antibody variable region framework, i.e., an antibody made into various kinds of human germline antibody framework sequences. Humanized antibodies can overcome the heterologous reaction elicited by chimeric antibodies containing many mouse protein components. Such framework sequences can be obtained from public DNA databases or publications that contain germline antibody gene sequences. For example, germline human heavy and light chain variable region gene DNA sequences can be found in the "VBase" human germline sequence database (available at www.mrccpe.com.ac.uk / vbase), and in Kabat, EA, et al. 1991 Sequences of Proteins of Immunological Interest, 5th Ed. To avoid the activity loss caused by reduced immunogenicity, the framework sequences in the human antibody variable regions can be subjected to minimal back mutations to maintain or enhance activity. The humanized antibodies of the present disclosure also include human antibodies that have been subjected to CDR affinity maturation by yeast display.

[0052] Due to the contact of the residues with the antigen, the grafting of the CDRs may result in a decrease in the affinity of the antibody or its antigen-binding fragment to the antibody due to the contact of the framework residues with the antigen. Such interactions can be caused by a high degree of somatic mutation. Therefore, it may still be necessary to graft such donor framework amino acids into the humanized antibody framework. The amino acid residues involved in antigen binding from the non-human antibody or its antigen-binding fragment can be identified by examining the sequence and structure of the animal monoclonal antibody variable region. Donor CDR framework amino acid residues that are different from the germline can be considered relevant. If it is not possible to determine the most relevant germline, the sequence can be compared against sequences common to the subtype or against animal antibody sequences with a high percentage of similarity. Rare framework residues are considered to be the result of a high degree of somatic mutation and play an important role in binding.

[0053] In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human or mouse Kappa, Lambda chain or a variant thereof, or further comprises a heavy chain constant region derived from a human or mouse IgG1, IgG2, IgG3, IgG4 or a variant thereof; it may comprise a heavy chain constant region derived from human IgG1, IgG2 or IgG4, or from an IgG1, IgG2 or IgG4 variant with amino acid mutations (e.g., L234A and / or L235A mutations, and / or S228P mutations).

[0054] As used herein, "conventional variants" of human antibody heavy chain constant regions and human antibody light chain constant regions refer to human heavy or light chain constant region variants disclosed in the prior art that do not change the structure and function of the antibody variable region. Examples of variants include IgG1, IgG2, IgG3 or IgG4 heavy chain constant region variants with site-specific modifications and amino acid substitutions in the heavy chain constant region. Specific examples of substitutions include YTE mutations, L234A and / or L235A mutations, S228P mutations, or mutations that result in a knob-into-hole structure (allowing the antibody heavy chain to have a combination of knob-Fc and hole-Fc). These mutations have been shown to confer new properties to antibodies without changing the function of the antibody variable region.

[0055] "Human antibody (HuMAb)", "human-derived antibody" and "fully human antibody" can be used interchangeably and can be or be produced from an antibody derived from a human or from a genetically modified organism that has been "modified" in any manner known in the art to produce specific human antibodies in response to antigenic stimulation. In some techniques, elements of human heavy and light chain loci are introduced into a cell line of an organism derived from a germline stem cell line, and the endogenous heavy and light chain loci of the cell line are targeted and disrupted. The targeted endogenous heavy and light chain loci contained in the cell line are disrupted. The transgenic organism can synthesize human antibodies specific to human antigens, and the organism can be used to produce hybridomas that secrete human antibodies. Human antibodies can also be antibodies whose heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources. Fully human antibodies can also be produced by genetic or chromosomal transfection methods and phage display techniques, or from in vitro activated B cells, both of which are known in the art.

[0056] The terms "full length antibody", "complete antibody" and "whole antibody" are used interchangeably herein and refer to an antibody in a substantially intact form, as distinguished from an antigen-binding fragment, as defined below. The terms particularly refer to antibodies comprising the constant regions in the light and heavy chains.

[0057] An "antibody" of this disclosure includes a "full-length antibody" and an antigen-binding fragment thereof.

[0058] In some embodiments, the full-length antibody of the present disclosure encompasses a full-length antibody formed by linking a light chain variable region with a light chain constant region, and linking a heavy chain variable region with a heavy chain constant region, as shown in the combinations of light chains and heavy chains listed in Tables 1, 2 and 3 below. Those skilled in the art can select light chain constant regions and heavy chain constant regions from various antibody sources according to actual needs, such as light chain constant regions and heavy chain constant regions derived from human antibodies. In addition, various combinations of light chain and heavy chain variable regions listed in Tables 1, 2 and 3 can form single chain antibodies (scFv), Fab, or other forms of antigen-binding fragments including scFv or Fab.

[0059] [Table 1]

[0060] [Table 2]

[0061] [Table 3]

[0062] NOTE: Hu164-57 means that the humanized antibody Hu164-57 derived from mab164 has a heavy chain variable region as set forth in Hu164-VH5 and a light chain variable region as set forth in Hu164-VL7; others can be interpreted in the same manner.

[0063] The term "antigen-binding fragment" or "functional fragment" refers to one or more fragments of an antibody that maintain the ability to specifically bind to an antigen (e.g., CTGF). It has been shown that fragments of full-length antibodies can be used to achieve the function of binding to a specific antigen. Examples of binding fragments for the term "antigen-binding fragment" of an antibody include: (i) Fab fragments, which are monovalent fragments consisting of the VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting of the VH and CH1 domains; (iv) Fv fragments consisting of the VH and VL domains of one arm of the antibody; (v) dsFv, which is a stable antigen-binding fragment formed by interchain disulfide bonds of VH and VL; and (vi) diabodies, bispecific antibodies and multispecific antibodies, including fragments such as scFv, dsFv and Fab. Furthermore, the two domains of the Fv fragment, the VL and VH domains, are encoded by two separate genes, but can be linked by a synthetic linker using recombinant techniques to generate a single-chain protein in which the VL and VH domains are combined to form a monovalent molecule (referred to as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single-chain antibodies are also encompassed by the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known in the art, and screened for functional fragments using the same methods as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. The antibodies can be of the various isotypes, for example IgG (e.g. IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0064] Fab is an antibody fragment obtained by treating an IgG antibody molecule with papain (which cleaves the amino acid residue at position 224 of the H chain). This antibody fragment, in which approximately the N-terminal half of the H chain and the entire L chain are linked by a disulfide bond, has a molecular weight of approximately 50,000 and has antigen-binding activity.

[0065] "F(ab')2" is an antibody fragment having an antigen-binding activity and a molecular weight of about 100,000, which can be obtained by digesting the downstream part of the two disulfide bonds in the hinge region of IgG with pepsin. F(ab')2 contains two Fabs linked by the hinge region.

[0066] Fab' is an antibody fragment having an antigen-binding activity and a molecular weight of about 50,000, and can be obtained by cleaving the disulfide bond in the hinge region of the F(ab')2. The Fab' of the present disclosure can be prepared by treating the F(ab')2 of the present disclosure, which specifically recognizes CTGF and binds to its extracellular domain amino acid sequence or three-dimensional structure, with a reducing agent such as dithiothreitol.

[0067] Fab' can also be prepared by inserting DNA encoding the Fab' of an antibody into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express Fab'.

[0068] The term "single chain antibody", "single chain Fv" or "scFv" refers to a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) linked by a linker. Such scFv molecules have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable linkers in the prior art consist of a repeating GGGGS amino acid sequence or variants thereof, such as 1-4 repeat variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that may be used in the present disclosure are described in Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

[0069] Diabodies are antibody fragments in which scFv or Fab are dimerized, which are antibody fragments with bivalent antigen-binding activity, in which the two antigens can be the same or different.

[0070] Bispecific and multispecific antibodies refer to antibodies that can bind two or more antigens or antigenic determinants simultaneously, including scFv or Fab fragments capable of binding CTGF.

[0071] The diabody of the present disclosure can be produced by the following steps: obtaining cDNA encoding the VL and VL of a monoclonal antibody of the present disclosure that specifically recognizes human CTGF and binds to its extracellular domain or three-dimensional structure; generating DNA encoding the scFv such that the linker peptide is less than 8 amino acid residues in length; inserting the DNA into a prokaryotic or eukaryotic expression vector; and then introducing the expression vector into a prokaryotic or eukaryotic organism to express the diabody.

[0072] dsFv is obtained by substituting one amino acid residue in each of VH and VL with a cysteine ​​residue, and then linking the substituted polypeptides by a disulfide bond between the two cysteine ​​residues. The amino acid residue to be substituted with a cysteine ​​residue can be selected based on the three-dimensional structure prediction of an antibody by a known method (Protein Engineering, 7, 697 (1994)).

[0073] A full-length antibody or antigen-binding fragment thereof of the present disclosure can be produced by the following steps: obtaining a cDNA encoding an antibody of the present disclosure that specifically recognizes and binds to human CTGF and its extracellular domain amino acid sequence or three-dimensional structure; producing DNA encoding a dsFv; inserting the DNA into a prokaryotic or eukaryotic expression vector; and then introducing the expression vector into a prokaryote or eukaryote to express the dsFv.

[0074] The term "amino acid change" or "amino acid mutation" refers to an amino acid change or mutation in a protein or polypeptide variant compared to an original protein or polypeptide, and includes one, two, three or more amino acid insertions, deletions or substitutions based on the original protein or polypeptide.

[0075] The term "antibody framework" or "FR region" refers to the portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. This is essentially the variable domain minus the CDRs.

[0076] The term "complementarity determining region", "CDR" or "hypervariable region" refers to one of the six hypervariable regions present in an antibody variable domain that primarily contributes to antigen binding. Generally, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. The amino acid sequence boundaries of the CDRs can be determined according to any of a variety of well-known schemes, including the "Kabat" numbering standard (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering standard (Al-Lazikani et al., (1997) JMB 273:927-948), and the ImmunoGenTics (IMGT) numbering standard (Lefranc MP, Immunologist, 7, 132- 136 (1999); Lefranc, MP, etc., Dev. Comp. Immunol., 27, 55-77 (2003)). For example, in the traditional format, according to the Kabat criteria, the CDR amino acid residues of the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3), and the CDR amino acid residues of the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). According to the Chothia criteria, the CDR amino acid residues of the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3), and the amino acid residues in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3).Combining Kabat and Chothia to define the CDRs, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) in human VL. According to the IMGT standard, the CDR amino acid residues of the VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residues of the VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2) and 89-97 (CDR3). According to the IMGT standard, the CDR regions of the antibody can be determined by using the IMGT / DomainGap Align Program. Unless otherwise specified, antibody variable region and CDR sequences related to embodiments of the present disclosure are applicable to the "Kabat" numbering system.

[0077] The term "epitope" or "antigenic determinant" refers to a portion on an antigen (e.g., a particular portion on a CTGF molecule) to which an immunoglobulin or antibody specifically binds. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids that form a unique tertiary conformation. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996).

[0078] "Specifically binds to" or "selectively binds to" refers to an antibody that binds to a predetermined epitope on an antigen. Typically, an antibody binds to a given epitope on an antigen at a concentration of about 10 -8 Less than M, e.g., about 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 Binds with an affinity (KD) less than or equal to M.

[0079] The term "KD" refers to the dissociation equilibrium constant for a particular antibody-antigen interaction. Antibodies of the present disclosure generally bind to CTGF with an affinity of about 10 -7 Less than M, e.g., about 10 -8 M or 10 -9 It binds with a dissociation equilibrium constant (KD) less than M, for example, the affinity of the antibodies of the present disclosure for a cell surface antigen is determined by measuring the KD value by FACS.

[0080] The term "competition," when used in the context of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope, means that competition occurs between the antigen binding proteins as measured by an assay in which the antigen binding protein being tested (e.g., an antibody or immunologically functional fragment thereof) inhibits or inhibits (e.g., reduces) the specific binding of a reference antigen binding protein (e.g., a ligand or reference antibody) to a common antigen (e.g., the CTGF antigen or a fragment thereof). Many types of competitive binding assays are available for determining whether antigen binding proteins compete with each other. Such assays include, for example, solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9: 242-253); solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137: 3614-3619), solid-phase direct label assays, solid-phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct label RIA using I-125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25: 7-15); solid-phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176: 546-552); and direct labeling RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32: 77-82). Typically, the assay involves the use of purified antigen that is capable of binding to a solid phase or cells that are loaded with both an unlabeled test antigen-binding protein and a labeled reference antigen-binding protein. Competitive inhibition is measured by measuring the amount of label bound to the solid phase or cells in the presence of the test antigen-binding protein. Usually, the test antigen-binding protein is present in excess.Antigen binding proteins identified by competitive assays (competition with antigen binding proteins) include: antigen binding proteins that bind to the same epitope as a reference antigen binding protein; and antigen binding proteins that bind to an epitope sufficiently close to that bound by the reference antigen binding protein, where the two epitopes sterically hinder each other to prevent binding). Further details on how to measure competitive binding are described in the Examples herein. Typically, when a competing antigen binding protein is present in excess, it can inhibit (e.g., reduce) specific binding of the reference antigen binding protein to a common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0081] As used herein, the term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and are preferably double-stranded DNA or single-stranded or modified mRNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0082] "Identity" of amino acid sequence refers to the percentage of identical amino acid residues between the first and second sequences when the amino acid sequences are aligned to maximize sequence identity (gap insertions, if necessary), and any conservative substitutions are not considered to constitute sequence identity. For the purpose of determining amino acid sequence identity percentage, alignment can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for alignment comparison, including any algorithms required to achieve optimal alignment over the entire length of the sequences to be compared.

[0083] The term "expression vector" refers to a nucleic acid molecule that can carry another nucleic acid linked thereto. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA fragments can be linked. In another embodiment, the vector is a viral vector, into whose viral genome additional DNA fragments can be linked. The vectors of the present disclosure can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication, and episomal mammalian vectors), or can integrate into the genome of the host cell after introduction into the host cell, thereby replicating together with the host genome (e.g., non-episomal mammalian vectors).

[0084] Methods for making and purifying antibodies and antigen-binding fragments are well known in the art. For example, see A Laboratory Manual for Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, chapters 5-8 and 15. For example, mice can be immunized with human CTGF or a fragment thereof, and the resulting antibodies can then be renatured, purified, and sequenced using conventional methods well known in the art. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments of the present disclosure are modified to incorporate one or more human framework regions into the CDR regions derived from a non-human antibody. Human FR germline sequences can be obtained from ImMunoGeneTics (IMGT) via the website http: / / imgt.cines.fr, or from The Immunoglobulin Facts Book, 2001, ISBN 012441351, by aligning with the IMGT human antibody variable germline gene database using MOE software.

[0085] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacteria, microorganisms, plant or animal cells. Bacteria that are amenable to introduction may include members of the Enterobacteriaceae family, such as Escherichia coli or Salmonella strains; Bacillaceae family, such as Bacillus subtilis; Streptococcus pneumoniae; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia yeast. Suitable animal host cell lines include CHO (Chinese Hamster Ovary bacterial strain), 293 bacteria, and NS0 cells.

[0086] The recombinant antibody or antigen-binding fragment of the present disclosure can be produced and purified in a conventional manner. For example, the cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector. The vector expressing the recombinant immunoglobulin can then be stably transfected into CHO cells. As is well known in the art and more recommended, mammalian expression systems can provide glycosylation, typically at the highly conserved N-terminal site of the Fc region. Appropriate clones can be obtained by expression of an antibody that specifically binds to human CTGF. Positive clones can be amplified in serum-free culture medium in an antibody production bioreactor. The culture medium into which the antibody is secreted can be purified in a conventional manner. For example, purification can be performed on a buffer-conditioned Protein A or G Sepharose FF column. Non-specific binding components are washed off. Bound antibody is eluted by a pH gradient, and antibody fractions are detected by SDS-PAGE and pooled. The antibody is filtered and concentrated in a conventional manner. Soluble mixtures and multimers can be effectively removed by conventional methods such as size exclusion or ion exchange. The resulting product should be immediately frozen, for example frozen at -70°C, or lyophilized.

[0087] "Administer", "medicate" or "treat", as applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to contacting an exogenous pharmaceutical, therapeutic, diagnostic or composition with an animal, human, subject, cell, tissue, organ or biological fluid. "Administer", "medicate" or "treat" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of a cell includes contacting an agent with a cell, and contacting an agent with a fluid that is in contact with the cell. "Administer", "medicate" or "treat" can also mean treating, for example, a cell with a drug, diagnostic, binding compound or another cell, in vitro or ex vivo. "Treat", as applied to a human, animal or experimental subject, refers to therapeutic treatment, prophylactic or suppressive measures, research and diagnostic applications.

[0088] "Treatment" refers to administering a therapeutic agent, such as a composition comprising any of the binding compounds of the present disclosure, internally or externally to a patient with one or more disease symptoms for which it is known to exhibit therapeutic activity. Typically, the therapeutic agent is administered in an amount effective to clinically measurably alleviate, induce regression of, or inhibit the progression of one or more disease symptoms in the patient or population being treated. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "treatment effective amount") may vary depending on various factors, such as the disease state, age, and weight of the patient, as well as the ability of the therapeutic agent to induce a desired response in the patient. The alleviation of disease symptoms can be evaluated by any clinical measurement typically used by a physician or other skilled health care provider to assess the severity or progression of the condition. While certain embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may not be effective in all patients in alleviating the target disease symptoms, they should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, Mann-Whitney U test, Kruskal-Wallis test (H test), Jonckheer-Terpstra test, and Wilcoxon test.

[0089] "Conservative modification" or "conservative substitution" refers to the replacement of amino acids in a protein with other amino acids having similar properties (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.), often without altering the biological activity of the protein. Those skilled in the art will appreciate that single amino acid substitutions in non-essential regions of a polypeptide generally do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., Page 224, (4th edition)). Furthermore, substitutions with amino acids that are structurally or functionally similar are unlikely to disrupt biological activity. Examples of conservative substitutions are given below:

[0090] TIFF0007627665000007.tif10658

[0091] "Effective amount" or "effective dose" refers to the amount of a medicament, compound or pharmaceutical composition required to obtain any one or more beneficial or desired results. In prophylactic applications, beneficial or desired results include eliminating or reducing the risk of disease, reducing the severity, or delaying the onset of disease, including biochemical, histological, and behavioral manifestations of disease states, their complications, and intermediate pathological phenotypes in the course of the condition's development. In therapeutic applications, beneficial or desired results include clinical results such as reducing the onset of various conditions associated with the target antigen of the present disclosure, or ameliorating one or more symptoms of the condition, reducing the dosage / administration of other drugs required to treat the condition, enhancing the effects of other drugs, and / or delaying the progression of the condition associated with the target antigen of the present disclosure in a patient.

[0092] "Exogenous" refers to a substance that is produced outside an organism, cell, or human, depending on the circumstances. "Endogenous" refers to a substance that is produced within a cell, organism, or human, depending on the circumstances.

[0093] "Homology" refers to the sequence similarity between two polynucleotide sequences or two polypeptide sequences. When the same base or amino acid monomer subunit occupies a position in both of the two sequences being compared, for example, when a position is occupied by adenine in each of the two DNA molecules, the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of positions that match or are homologous between the two sequences divided by the number of positions compared, multiplied by 100. For example, when two sequences are optimally aligned, if 6 out of 10 positions of the two sequences are matched or homologous, the homology of the two sequences is 60%; if 95 out of 100 positions of the two sequences are matched or homologous, the homology of the two sequences is 95%. In general, when two sequences are aligned, the comparison is performed to maximize the percentage of homology. For example, comparison can be performed by BLAST algorithm, where the parameters of the algorithm are selected to maximize the match between each sequence over the entire length of each reference sequence.The following references refer to the BLAST algorithm that is often used for sequence analysis: BLAST ALGORITHMS: Altschul, SF et al., (1990) J. Mol. Biol. 215:403-410; Gish, W. et al., (1993) Nature Genet. 3:266-272; Madden, TL et al., (1996) Meth. Enzymol. 266:131-141; Altschul, SF et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J. et al. (1997) Genome Res. 7:649-656. Other conventional BLAST algorithms, such as that available from NCBI BLAST, are also familiar to those of skill in the art.

[0094] The terms "cell", "cell line" and "cell culture" used herein are used interchangeably, and all such terms include progeny. Thus, the terms "transformed cell" and "cells transformed" include the primary subject cell and cultures derived therefrom, regardless of the number of passages. It should also be understood that not all progeny may be exactly identical in DNA content due to intentional or unintentional mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where a clear designation is intended, it will be clearly understood from the context.

[0095] As used herein, "polymerase chain reaction" or "PCR" refers to a method or technique for amplifying specific portions of minute amounts of nucleic acid, RNA and / or DNA, as described, for example, in U.S. Pat. No. 4,683,195. Generally, sequence information at or beyond the region of interest must be available so that oligonucleotide primers can be designed; the primers can be identical or similar in sequence to opposite strands of the template to be amplified. The 5' terminal nucleotides of the two primers can match the ends of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences derived from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage, or plasmid sequences. See, for example, generally, Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51:263; Erlich editors, (1989) PCR TECHNOLOGY (Stockton Press, NY). As used in this disclosure, PCR is one example, but is not believed to be the only example, of a polymerase reaction method for amplifying a test nucleic acid sample. The method involves the use of nucleic acid sequences known as primers in conjunction with a nucleic acid polymerase to amplify or create specific portions of a nucleic acid.

[0096] "Isolated" refers to a pure state in which the molecule concerned is substantially separated from other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other substances, such as cell debris and growth medium. The term "isolated" is not generally intended to mean completely free of such substances, or free of water, buffers, or salts, unless present in amounts that would substantially interfere with the experimental or therapeutic use of the compounds described herein.

[0097] "Optional" or "optionally" means that the event or circumstance to which it pertains may occur, but does not necessarily occur, and the description encompasses cases where the event or circumstance occurs or does not occur.

[0098] A "pharmaceutical composition" refers to a mixture containing one or more compounds of the present disclosure or physiologically / pharmaceutical acceptable salts or prodrugs thereof, and other chemical compounds, such as physiologically / pharmaceutical acceptable carriers and excipients. Pharmaceutical compositions are intended to facilitate administration to a living organism, facilitate absorption of the active ingredient, and thereby exert a biological effect.

[0099] The term "pharmaceutical acceptable carrier" refers to any inert substance suitable for use in a formulation for delivery of an antibody or antigen-binding fragment. Carriers can be anti-adhesives, adhesives, coatings, disintegrants, fillers or diluents, preservatives (e.g., antioxidants, antibacterial or antifungal agents), sweeteners, absorption delaying agents, wetting agents, emulsifiers, and the like. Examples of suitable pharmaceutical acceptable carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like), dextrose, vegetable oils (e.g., olive oil), saline, buffers, buffered saline, and isotonicity agents (e.g., sugars, polyols, sorbitol, and sodium chloride).

[0100] The present disclosure further encompasses a pharmaceutical agent for treating a disease associated with target antigen (e.g., CTGF)-positive cells, comprising an anti-CTGF antibody or antigen-binding fragment thereof of the present disclosure as an active ingredient. The active ingredient is administered to a subject in a therapeutically effective amount to treat a disease associated with CTGF-positive cells in the subject. By therapeutically effective amount, it is meant that a unit dose of the composition contains 0.1-3000 mg of the full-length antibody or antigen-binding fragment thereof that specifically binds to human CTGF.

[0101] The CTGF-related disease of the present disclosure is not limited as long as it is a disease related to CTGF. For example, the treatment response induced by the molecules of the present disclosure can play a role by binding to human CTGF and blocking CTGF from binding to its receptor / ligand, or by killing tumor cells that overexpress CTGF. Thus, the molecules of the present disclosure, when present in a formulation or formulation suitable for treatment applications, are highly useful in such subjects suffering from tumors or cancer, preferably pancreatic cancer, pulmonary fibrosis and renal fibrosis, optionally including pulmonary fibrosis, renal fibrosis, tumor development and proliferation, glaucoma, cell proliferative diseases, cataracts, choroidal neovascularization, retinal detachment, proliferative vitreoretinopathy, macular degeneration, diabetic retinopathy, corneal scarring and opacification, cysts, vascular hypocalcification, pancreatic ductal adenocarcinoma, pancreatic cancer, melanoma, radiation-induced fibrosis (RIF), idiopathic pulmonary fibrosis, pulmonary remodeling diseases selected from the group consisting of asthma, chronic bronchitis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, emphysema, etc.

[0102] The present disclosure further relates to methods for immunoassaying or measuring a target antigen (e.g., CTGF), reagents for immunoassaying or measuring a target antigen (e.g., CTGF), methods for immunoassaying or measuring a target antigen (e.g., CTGF), and diagnostic agents for diagnosing diseases involving target antigen (e.g., CTGF)-positive cells, comprising (as an active ingredient) an antibody or antigen-binding fragment of the present disclosure that specifically recognizes the target antigen (e.g., human CTGF) and binds to its extracellular amino acid sequence or three-dimensional structure.

[0103] In the present disclosure, the method of detecting or measuring the amount of a target antigen (e.g., CTGF) can be any known method, including, for example, immunoassays or measurement methods.

[0104] The immunoassay or measurement method may be a method for detecting or measuring the amount of an antibody or antigen using a labeled antigen or antibody. Examples of the immunoassay or measurement method include radioactive substance labeled immunoassay (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescence immunoassay, Western blotting, physicochemical methods, etc.

[0105] The above-mentioned diseases involving CTGF-positive cells can be diagnosed by detecting or measuring CTGF-expressing cells using the antibodies or antibody fragments of the present disclosure.

[0106] Polypeptide-expressing cells can be detected by known immunodetection methods, preferably by immunoprecipitation, fluorescent cell staining, immunohistochemical staining, etc. Furthermore, methods such as fluorescent antibody staining using the FMAT8100HTS system (Applied Biosystem) can be used.

[0107] In the present disclosure, there is no particular limitation on the sample in which the target antigen (e.g., CTGF) is detected or measured, such as tissue, cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture medium, so long as it may contain cells expressing the target antigen (e.g., CTGF).

[0108] Depending on the required diagnostic method, the diagnostic agent containing the monoclonal antibody or its antibody fragment of the present disclosure may also include an agent for carrying out an antigen-antibody reaction or an agent for detecting the reaction. Agents for carrying out an antigen-antibody reaction include buffers, salts, etc. Reagents used for detection include agents commonly used in immunoassays or immunodetection methods, such as a labeled secondary antibody that recognizes the monoclonal antibody, the antibody fragment, or a conjugate thereof, and a substance corresponding to the label.

[0109] Aspects of the present disclosure are further described in the following sections: [Section 1] 1. An anti-CTGF antibody comprising a heavy chain variable region and a light chain variable region, i) the heavy chain variable region comprises the same HCDR1, HCDR2 and HCDR3 sequences as those of the heavy chain variable region set forth in SEQ ID NO:6, and the light chain variable region comprises the same LCDR1, LCDR2 and LCDR3 sequences as those of the light chain variable region set forth in SEQ ID NO:7; ii) the heavy chain variable region comprises the same HCDR1, HCDR2 and HCDR3 sequences as those of the heavy chain variable region set forth in SEQ ID NO:8, and the light chain variable region comprises the same LCDR1, LCDR2 and LCDR3 sequences as those of the light chain variable region set forth in SEQ ID NO:9; ii-i) the heavy chain variable region comprises the same HCDR1, HCDR2 and HCDR3 sequences as those of the heavy chain variable region set forth in SEQ ID NO: 69, and the light chain variable region comprises the same LCDR1, LCDR2 and LCDR3 sequences as those of the light chain variable region set forth in SEQ ID NO: 70; or ii-ii) the heavy chain variable region comprises the same HCDR1, HCDR2 and HCDR3 sequences as those of the heavy chain variable region set forth in SEQ ID NO: 85, and the light chain variable region comprises the same LCDR1, LCDR2 and LCDR3 sequences as those of the light chain variable region set forth in SEQ ID NO: 70; Anti-CTGF antibody. [Section 2] iii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, respectively; iv) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, respectively; iv-i) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:71, SEQ ID NO:72 and SEQ ID NO:73, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:74, SEQ ID NO:75 and SEQ ID NO:76, respectively; or iv-ii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 102, SEQ ID NO: 103 and SEQ ID NO: 104, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, respectively; 2. The anti-CTGF antibody of claim 1, comprising a heavy chain variable region and a light chain variable region. [Section 3] 3. The anti-CTGF antibody according to item 1 or 2 above, wherein the anti-CTGF antibody is a mouse antibody, a chimeric antibody or a humanized antibody. [Section 4] (v) the amino acid sequence of the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 6, 27, 28, or 29, and the amino acid sequence of the light chain variable region has at least 90% sequence identity to SEQ ID NO: 7, 22, 23, 24, 25, or 26; (vi) the amino acid sequence of the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 8, 33, 34, 35, or 36, and the amino acid sequence of the light chain variable region has at least 90% sequence identity to SEQ ID NO: 9, 30, 31, or 32; or (vi-i) the amino acid sequence of the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 69, 81, 82, 83, 84 or 85, and the amino acid sequence of the light chain variable region has at least 90% sequence identity to SEQ ID NO: 70, 77, 78, 79 or 80; 4. The anti-CTGF antibody according to any one of items 1 to 3 above, which comprises a heavy chain variable region and a light chain variable region. [Section 5] the anti-CTGF antibody is a humanized antibody, the humanized antibody comprises a framework region of a human antibody or a variant of that framework region, the variant having up to 10 back mutations in each of the human antibody light chain framework region and / or heavy chain framework region; Preferably, the humanized antibody is an anti-CTGF antibody according to item 2, comprising a light chain variable region and a heavy chain variable region selected from the group consisting of the following (a), (b) or (bi): (a) a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, respectively, and one or more backmutations selected from the group consisting of 4L, 36F, 43S, 45K, 47W, 58V and 71Y; and a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively, and one or more back mutations selected from the group consisting of 28S, 30N, 49A, 75E, 76S, 93V, 94E and 104D; (b) a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, respectively, and one or more backmutations selected from the group consisting of 36V, 44F, 46G and 49G; and a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, respectively, and one or more back mutations selected from the group consisting of 44G, 49G, 27F, 48L, 67L, 71K, 78V and 80F; (bi) a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, respectively, and one or more backmutations selected from the group consisting of 45P, 46W, 48Y, 69S and 70Y; and a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:71, SEQ ID NO:72 and SEQ ID NO:73, respectively, and one or more backmutations selected from the group consisting of 27F, 38K, 48I, 67K, 68A, 70L and 72F; or (b-ii) a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, respectively, and one or more backmutations selected from the group consisting of 45P, 46W, 48Y, 69S and 70Y; and A heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:102, SEQ ID NO:103 and SEQ ID NO:104, respectively, and one or more backmutations selected from the group consisting of 27F, 38K, 48I, 67K, 68A, 70L and 72F. [Section 6] (vii) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:6 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:7; (viii) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 27, 28 or 29 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 22, 23, 24, 25 or 26; (ix) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 8 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 9; (x) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 33, 34, 35 or 36 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 30, 31 or 32; (xi) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 69 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 70; or (xii) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 81, 82, 83, 84 or 85, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 77, 78, 79 or 80; Preferably, the heavy chain variable region and the light chain variable region are (xiii) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 27 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 22; (xiv) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 34 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 30; or (xv) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 85 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 77; 6. The anti-CTGF antibody of item 5, comprising a heavy chain variable region and a light chain variable region. [Section 7] The anti-CTGF antibody of any one of items 1 to 6 above, wherein the antibody further comprises an antibody heavy chain constant region and a light chain constant region, and preferably the heavy chain constant region is selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4 constant regions and variants thereof, and the light chain constant region is selected from the group consisting of human antibody κ and λ chain constant regions and variants thereof; more preferably the antibody comprises a heavy chain constant region as shown in SEQ ID NO: 37 or 38, and a light chain constant region as shown in SEQ ID NO: 39 or 40. [Section 8] (c) a heavy chain that has at least 85% sequence identity to SEQ ID NO: 41, 43, 44, 45, 46, 47, or 48, and a light chain that has at least 85% sequence identity to SEQ ID NO: 42, 49, 50, 51, 52, or 53; (d) a heavy chain that has at least 85% sequence identity to SEQ ID NO: 54, 56, 57, 58, 59, 60, 61, 62, or 63, and a light chain that has at least 85% sequence identity to SEQ ID NO: 55, 64, 65, or 66; (e) a heavy chain set forth in SEQ ID NO: 41, 43, 44, 45, 46, 47 or 48, and a light chain set forth in SEQ ID NO: 42, 49, 50, 51, 52 or 53; (f) a heavy chain depicted in SEQ ID NO: 54, 56, 57, 58, 59, 60, 61, 62 or 63, and a light chain depicted in SEQ ID NO: 55, 64, 65 or 66; (g) a heavy chain that has at least 85% sequence identity to SEQ ID NO: 86, 88, 89, 90, 91, 92, 93, 94, 95, 96, or 97, and a light chain that has at least 85% sequence identity to SEQ ID NO: 87, 98, 99, 100, or 101; or (h) a heavy chain as set forth in SEQ ID NO: 86, 88, 89, 90, 91, 92, 93, 94, 95, 96 or 97, and a light chain as set forth in SEQ ID NO: 87, 98, 99, 100 or 101. Preferably (j) a heavy chain set forth in SEQ ID NO: 46, and a light chain set forth in SEQ ID NO: 49; (k) a heavy chain set forth in SEQ ID NO: 61, and a light chain set forth in SEQ ID NO: 64; or (l) a heavy chain as set forth in SEQ ID NO: 97, and a light chain as set forth in SEQ ID NO: 98. 8. The anti-CTGF antibody according to any one of items 1 to 7 above, [Section 9] An isolated anti-CTGF antibody that competes with the anti-CTGF antibody of any one of items 1 to 8 above for binding to human CTGF. [Section 10] A nucleic acid molecule encoding the anti-CTGF antibody according to any one of items 1 to 9 above. [Section 11] A host cell comprising the nucleic acid molecule according to paragraph 10 above. [Section 12] A pharmaceutical composition comprising a therapeutically effective amount of the anti-CTGF antibody according to any one of items 1 to 9 above or the nucleic acid molecule according to item 10 above, and one or more pharma- ceutically acceptable carriers, diluents, buffers or excipients. [Section 13] A method for immunoassay or determination of CTGF, comprising the step of contacting a subject or a sample thereof with the anti-CTGF antibody according to any one of items 1 to 9 above. [Section 14] A kit comprising the anti-CTGF antibody according to any one of items 1 to 9 above. [Section 15] A method for treating or preventing a CTGF-related disease, the method comprising administering to a subject a therapeutically or prophylactically effective amount of the anti-CTGF antibody according to any one of items 1 to 9 above, or the nucleic acid molecule according to item 10 above, or the pharmaceutical composition according to item 12 above, wherein the CTGF-related disease is preferably a fibrotic disease, hypertension, diabetes, myocardial infarction, arthritis, a CTGF-related cell proliferative disease, atherosclerosis, glaucoma, or cancer; The fibrotic disease is preferably selected from the group consisting of idiopathic pulmonary fibrosis, diabetic nephropathy, diabetic retinopathy, osteoarthritis, scleroderma, chronic heart failure, liver cirrhosis or renal fibrosis; The cancer is preferably selected from the group consisting of acute lymphoblastic leukemia, dermatofibroma, breast cancer, angiolipoma, angioleiomyoma, connective tissue generating cancer, prostate cancer, ovarian cancer, colorectal cancer, pancreatic cancer, gastrointestinal cancer or liver cancer; method. The details of one or more aspects of the present disclosure are described above. Preferred methods and materials are described below, although any methods and materials similar or identical to those described herein can be used to practice or test the present disclosure. Other features, objects and advantages of the present disclosure may become apparent throughout the present specification and claims. In the present specification and claims, the singular form is intended to encompass the plural form unless otherwise clear from the context. All technical and scientific terms used herein have the meanings that are commonly understood by those skilled in the art to which the present disclosure belongs, unless otherwise clearly defined herein. All patents and publications cited herein are incorporated by reference. The following examples are provided to more specifically describe the preferred aspects of the present disclosure. The examples should not be construed as limiting the scope of the present disclosure in any way, which is defined by the claims. EXAMPLES

[0110] Example 1: Generation of CTGF antigen and antibodies I. Antigen Design and Expression Human CTGF protein (Genbank number: NM_001901.2) was used as a template to design the amino acid sequences of antigens and detection proteins. CTGF protein or fragments thereof were optionally fused with various tags and cloned separately into pTT5 or pXC vectors and expressed transiently in 293 cells or stably in LONZA CHO cells to obtain the antigens and detection proteins of the present disclosure. Unless otherwise indicated, the following CTGF antigens are referred to as human CTGF:

[0111] Human CTGF full length protein (for immunization); SEQ ID NO:1: TIFF0007627665000008.tif43165

[0112] CTGF-his (a fusion protein of human CTGF mature protein and his tag) can be used for detection; SEQ ID NO:2: TIFF0007627665000009.tif44168 (Note: Single underline indicates signal peptide, italics indicates extracellular domain of hCTGF, double underline indicates his tag.)

[0113] mCTGF-mFc (a fusion protein of mouse CTGF coding region and mouse Fc tag that can be used for detection); SEQ ID NO:3: TIFF0007627665000010.tif67165 (Note: single underline indicates signal peptide, italics indicates mouse CTGF coding region, double underline indicates mFc tag.)

[0114] Mouse CTGF protein-His tag; SEQ ID NO:4: TIFF0007627665000011.tif43165 (Note: single underline indicates signal peptide, italics indicates mouse CTGF coding region, double underline indicates his tag.)

[0115] Cynomolgus CTGF-Fc (a fusion protein of the cynomolgus CTGF coding region and Fc, which can be used for detection); SEQ ID NO:5: TIFF0007627665000012.tif68167 (Note: single underline indicates signal peptide, italics indicates cyno CTGF coding region, double underline indicates Fc tag.)

[0116] II. Purification of CTGF-Related Recombinant Proteins and Anti-Human CTGF Hybridoma and Recombinant Antibodies 1. Isolation and purification of hybridoma supernatants by protein G affinity chromatography For the purification of mouse hybridoma supernatant, affinity chromatography using protein G was suitable. The hybridoma resulting from the culture was centrifuged, the supernatant was taken, and the pH of the supernatant was adjusted to neutral by adding 10-15% by volume of 1M Tris-HCl (pH 8.0-8.5) based on the volume of the supernatant. The protein G column was washed with 3-5 column volumes of 6M guanidine hydrochloride, then with 3-5 column volumes of pure water; the column was equilibrated with 3-5 column volumes of 1x PBS (pH 7.4); the cell supernatant was loaded at a low flow rate for binding, and the flow rate was adjusted so that the retention time was about 1 minute or more; the column was washed with 3-5 column volumes of 1x PBS (pH 7.4) until the UV absorption decreased to the baseline; the sample was eluted with 0.1M acetic acid / sodium acetate (pH 3.0) buffer, and the elution peak was pooled according to UV detection. The eluate was immediately adjusted to pH 5-6 with 1M Tris-HCl (pH 8.0). The eluate could be subjected to solution exchange according to methods well known to those skilled in the art, for example, by ultrafiltration-concentration using ultrafiltration tubing to exchange the solution into the desired buffer system, or by molecular exclusion such as G-25 desalting to exchange the solution into the desired buffer system, or by using a high resolution molecular exclusion column such as Superdex 200 to remove aggregates from the eluate and improve sample purity.

[0117] 2. Purification of antibodies by Protein A affinity chromatography First, the cell culture supernatant expressing the antibody was centrifuged at high speed, and the supernatant was collected. The Protein A affinity column was washed with 3-5 column volumes of 6 M guanidine hydrochloride, and then with 3-5 column volumes of pure water. The column was equilibrated with 3-5 column volumes, e.g., 1× PBS (pH 7.4). The cell supernatant was loaded at a low flow rate for binding, and the flow rate was adjusted so that the retention time was about 1 min or more; when binding was completed, the column was washed with 3-5 column volumes of 1× PBS (pH 7.4) until the UV absorption dropped to the baseline. The sample was eluted with 0.1 M acetic acid / sodium acetate (pH 3.0-3.5) buffer, and the elution peak was pooled according to UV detection. The eluate was quickly adjusted to pH 5-6 with 1 M Tris-HCl (pH 8.0). The eluate can be subjected to solution exchange according to methods well known to those skilled in the art, for example, by ultrafiltration-concentration using ultrafiltration tubing to exchange the solution into the desired buffer system, or by molecular exclusion such as G-25 desalting to exchange the solution into the desired buffer system, or by use of a high resolution molecular exclusion column such as Superdex 200 to remove aggregates from the eluate and improve sample purity.

[0118] 3. Purification of CTGF-related recombinant proteins (e.g., human CTGF-his) using nickel columns The cell expression supernatant samples were centrifuged at high speed to remove impurities, and the buffer was exchanged with PBS, and imidazole was added at a final concentration of 5 mM. The nickel column was equilibrated with PBS solution containing 5 mM imidazole and washed with 2-5 times the column volume. The exchanged supernatant sample was loaded onto the column for binding, and nickel columns available from different companies can be selected as the medium. The column was washed with PBS solution containing 5 mM imidazole until the A280 reading dropped to baseline. The chromatography column was washed with PBS + 10 mM imidazole to remove non-specifically bound protein impurities, and the flow-through was collected. The target protein was eluted with PBS solution containing 300 mM imidazole, and the elution peak was collected.

[0119] The collected eluate was concentrated, which can then be further purified by gel chromatography Superdex200 (GE) with PBS as the mobile phase to remove aggregates and impurity protein peaks and collect the target product elution peak. The resulting proteins were identified by electrophoresis, peptide mapping and LC-MS, and the correct proteins were aliquoted for use.

[0120] 4. Purification of CTGF-related recombinant proteins (e.g., human CTGF-Fc, cynomolgus CTGF-Fc) by Protein A affinity chromatography First, the culture supernatant was centrifuged at high speed to collect the supernatant. The Protein A affinity column was washed with 6 M guanidine hydrochloride, 3-5 times the column volume, and then with pure water, 3-5 times the column volume. The column was equilibrated with 3-5 times the column volume, e.g., 1× PBS (pH 7.4). The cell supernatant was loaded at a low flow rate for binding, and the flow rate was adjusted so that the retention time was about 1 min or more; when binding was completed, the column was washed with 1× PBS (pH 7.4), 3-5 times the column volume, until the UV absorption dropped to the baseline. The sample was eluted with 0.1 M acetic acid / sodium acetate (pH 3.0-3.5) buffer, and the elution peak was pooled according to UV detection.

[0121] The collected eluate was concentrated, which can then be further purified by gel chromatography Superdex200 (GE) with PBS as the mobile phase to remove aggregates and impurity protein peaks and collect the target product elution peak. The resulting proteins were identified by electrophoresis, peptide mapping and LC-MS, and the correct proteins were aliquoted for use. EXAMPLES

[0122] Example 2: Generation of anti-human CTGF monoclonal antibodies I. Immunity Anti-human CTGF monoclonal antibodies were produced by immunizing mice. 6-8 week-old female SJL white mice were used in the experiment (Beijing Charles River Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2012-0001). Rearing environment: SPF level. After purchasing the mice, they were kept in a laboratory environment with a 12 / 12 hour light / dark cycle, temperature of 20-25°C, and humidity of 40-60% for one week. Mice that had adapted to the environment were immunized according to the following scheme. CTGF (R&D) and mCTGF-mFc were used as immunization antigens.

[0123] Immunization scheme: Mice were immunized by intraperitoneal injection of CTGF protein at 25 μg / mouse / time. 100 μl / mouse was injected intraperitoneally (IP) on day 0, and one booster was administered every 14 days. Blood samples were taken on days 21, 35, 49, and 63, and antibody titers in mouse serum were measured by ELISA. After 7 to 9 immunizations, mice with high serum antibody titers approaching a plateau were selected for splenocyte fusion. Three days before splenocyte fusion, CTGF protein antigen solution prepared in saline was injected intraperitoneally (IP) at 25 μg / mouse for booster immunization.

[0124] II. Splenocyte fusion Hybridoma cells were obtained by fusing splenic lymphocytes with myeloma Sp2 / 0 (ATCC® CRL-8287™) using the PEG-mediated fusion method. The fused hybridoma cells were cultured in complete medium (IMDM medium containing 20% ​​FBS, 1x HAT, and 1x OPI) at 0.5–1x10 6The cells were resuspended at a density of 100 μl / well and seeded in a 96-well plate at 100 μl / well, incubated at 37°C and 5% CO2 for 3-4 days, supplemented with 100 μl / well of HAT complete medium, and continued to be cultured for 3-4 days until pinpoint clones were formed. The supernatant was removed, and 200 μl / well of HT complete medium (IMDM medium containing 20% ​​FBS, 1×HT, 1×OPI) was added, incubated at 37°C and 5% CO2 for 3 days, and then subjected to ELISA detection.

[0125] III. Screening of Hybridoma Cells According to the proliferation density of hybridoma cells, the hybridoma culture supernatant was detected by binding ELISA. The cell supernatant of the positive well was detected by ELISA assay for binding with monkey CTGF / mouse CTGF / human CTGF protein (the specific method was the same as that of Example 3). The cells of the wells positive in protein ELISA binding assay were expanded in a timely manner and cryopreserved; they were subcloned 2-3 times until a single cell clone was obtained.

[0126] The cells after each subcloning were also tested by CTGF binding ELISA. Hybridoma clones were obtained by screening with the above assay. Antibodies were further prepared by serum-free cell culture method. Antibodies were purified according to the purification example for use in the test example.

[0127] IV. Sequencing of positive hybridoma clones The procedure for cloning sequences from positive hybridomas was as follows: Hybridoma cells in logarithmic growth phase were collected. RNA was extracted using Trizol (Invitrogen, Cat No. 15596-018) according to the kit's instructions, and reverse transcribed using PrimeScript™ Reverse Transcription Kit (Takara, Cat No. 2680A). The cDNA generated by reverse transcription was amplified by PCR using a mouse Ig primer set (Novagen, TB326 Rev. B 0503), and the amplified product was subjected to sequencing. After sequencing, mouse anti-CTGF antibodies mab147, mab164, and mab95 were obtained. The amino acid sequences of the variable regions are as follows:

[0128] The VH amino acid sequence of mab147 is SEQ ID NO:6: Represented by TIFF0007627665000013.tif18164. The VL amino acid sequence of mab147 is SEQ ID NO:7: Represented by TIFF0007627665000014.tif13164.

[0129] The VH amino acid sequence of mab164 is SEQ ID NO:8: Represented by TIFF0007627665000015.tif13165. The VL amino acid sequence of mab164 is SEQ ID NO:9: Represented by TIFF0007627665000016.tif13166.

[0130] The VH amino acid sequence of mab95 is SEQ ID NO:69: Represented by TIFF0007627665000017.tif18166. The VL amino acid sequence of mab95 is SEQ ID NO:70: Represented by TIFF0007627665000018.tif12166.

[0131] In the above mab147, mab164 and mab95 antibody variable region sequences, the italics represent FR sequences, the underlined italics represent CDR sequences, and the sequence order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0132] [Table 4]

[0133] V. Generation of Human IgG1 Chimeric Antibodies Candidate molecules mab147, mab164 and mab95 were amplified and sequenced to obtain gene sequences encoding variable regions. Based on the sequences obtained by sequencing, forward and reverse primers were designed, and the sequenced genes were used as templates to generate VH / VK gene fragments of each antibody by PCR, which were then inserted into expression vector pHr (with signal peptide and hIgG1 / hκ / hλ constant region gene (CH1-Fc / CL) fragment) by homologous recombination to generate expression plasmids VH-CH1-Fc-pHr / VL-CL-pHr of full-length recombinant chimeric antibodies, resulting in three chimeric antibodies Ch147, Ch164 and Ch95.

[0134] VI. Humanization of Mouse Anti-Human CTGF Antibody Highly homologous heavy and light chain variable region germline genes with mouse antibodies were selected as templates by aligning them against the IMGT (http: / / imgt.cines.fr) human antibody heavy and light chain variable germline gene database using MOE software (Molecular Operating Environment) software. Mouse antibody CDRs were grafted into the corresponding human templates to form variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0135] 1. Humanization of mab147 For the mouse antibody mab147, the light chain templates for humanization were IGKV3-11*01 and IGKJ2*01, or IGKV1-39*01 and IGKJ2*01, and the heavy chain templates for humanization were IGHV3-72*01 and IGHJ1*01. Each CDR of the mouse antibody mab147 was grafted onto its human template, and then the amino acids of the FR of the humanized antibody were subjected to back mutation. Among them, the back mutations in the light chain variable region included one or more selected from the group consisting of 4L, 36F, 43S, 45K, 47W, 58V or 71Y, and the back mutations in the heavy chain variable region included one or more selected from the group consisting of 28S, 30N, 49A, 75E, 76S, 93V, 94E or 104D (the positions of the back mutations in the light and heavy chain variable regions were determined according to the Kabat numbering standard), resulting in light and heavy chain variable regions of different sequences. A humanized antibody was obtained containing the CDRs of mab147, whose variable region sequence is as follows:

[0136] The specific variable region sequences of the humanized antibody of mAb147 are as follows: The amino acid sequence of Hu147-VL1 is SEQ ID NO:22: Represented by TIFF0007627665000020.tif13164. The amino acid sequence of Hu147-VL2 is SEQ ID NO:23: Represented by TIFF0007627665000021.tif14164. The amino acid sequence of Hu147-VL3 is SEQ ID NO:24: Represented by TIFF0007627665000022.tif14165. The amino acid sequence of Hu147-VL4 is SEQ ID NO:25: Represented by TIFF0007627665000023.tif14165. The amino acid sequence of Hu147-VL5 is SEQ ID NO:26: Represented by TIFF0007627665000024.tif13165. The amino acid sequence of Hu147-VH1 is SEQ ID NO:27: Represented by TIFF0007627665000025.tif19165. The amino acid sequence of Hu147-VH2 is SEQ ID NO:28: Represented by TIFF0007627665000026.tif19165. The amino acid sequence of Hu147-VH3 is SEQ ID NO:29: Represented by TIFF0007627665000027.tif19167.

[0137] Note: The CDR sequences determined according to the Kabat criteria are underlined and the sequences are arranged in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0138] 2. Humanization of mab164 For the mouse antibody mab164, the light chain templates for humanization consisted of IGLV7-43*01 and IGLJ2*01, IGLV8-61*01 and IGLJ2*01, or IGLV1-40*02 and IGLJ2*01, respectively, and the heavy chain templates for humanization consisted of IGHV2-26*01 and IGHJ6*01, or IGHV4-31*02 and IGHJ6*01, respectively. Each CDR of the mouse antibody mab164 was grafted onto its human template, and then the amino acids of the FR of the humanized antibody were subjected to back mutation. wherein the backmutations in the light chain variable region included one or more selected from the group consisting of 36V, 44F, 46G or 49G, and the backmutations in the heavy chain variable region included one or more selected from the group consisting of 44G, 49G, 27F, 48L, 67L, 71K, 78V or 80F (the backmutation positions in the light and heavy chain variable regions were determined according to the Kabat numbering standard), resulting in humanized heavy and light chains of mab164 having the following variable region sequences:

[0139] The specific variable region sequences of the humanized antibody of mAb164 are as follows: The amino acid sequence of Hu164-VL7 is SEQ ID NO:30: Represented by TIFF0007627665000028.tif13164. The amino acid sequence of Hu164-VL8 is SEQ ID NO:31: Represented by TIFF0007627665000029.tif13166. The amino acid sequence of Hu164-VL9 is SEQ ID NO:32: Represented by TIFF0007627665000030.tif12166. The amino acid sequence of Hu164-VH5 is SEQ ID NO:33: Represented by TIFF0007627665000031.tif18167. The amino acid sequence of Hu164-VH6 is SEQ ID NO:34: Represented by TIFF0007627665000032.tif19166. The amino acid sequence of Hu164-VH7 is SEQ ID NO:35: Represented by TIFF0007627665000033.tif18165. The amino acid sequence of Hu164-VH8 is SEQ ID NO:36: Represented by TIFF0007627665000034.tif19165.

[0140] Note: In the above Hu164 antibody sequence, the underlines indicate the CDR sequences and the sequence order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where the CDR sequences are defined according to the Kabat criteria.

[0141] 3. Humanization of mab95 For the mouse antibody mab95, the light chain templates for humanization were IGKV3-20*02 and IGKV1-40*01, and the heavy chain template for humanization was IGHV1-3*01. Each CDR of the mouse antibody mab95 was grafted onto its human template, and then the amino acids of the FR of the humanized antibody were subjected to back mutation. Among them, the back mutations in the light chain variable region included one or more selected from the group consisting of 45P, 46W, 48Y, 69S or 70Y, and the back mutations in the heavy chain variable region included one or more selected from the group consisting of 27F, 38K, 48I, 67K, 68A, 70L or 72F (the back mutation positions in the light and heavy chain variable regions were determined according to the Kabat numbering standard), resulting in light and heavy chain variable regions with different sequences. A humanized antibody containing the CDRs of mab95 was obtained, and its variable region sequence is as follows:

[0142] The specific variable region sequences of the humanized antibody of mAb95 are as follows: The amino acid sequence of Hu95-VL1 is SEQ ID NO:77: Represented by TIFF0007627665000035.tif13165. The amino acid sequence of Hu95-VL2 is SEQ ID NO:78: Represented by TIFF0007627665000036.tif13167. The amino acid sequence of Hu95-VL3 is SEQ ID NO:79: Represented by TIFF0007627665000037.tif14168. The amino acid sequence of Hu95-VL4 is SEQ ID NO:80: Represented by TIFF0007627665000038.tif13166. The amino acid sequence of Hu95-VH1 is SEQ ID NO:81: Represented by TIFF0007627665000039.tif19166. The amino acid sequence of Hu95-VH2 is SEQ ID NO:82: Represented by TIFF0007627665000040.tif19167. The amino acid sequence of Hu95-VH3 is SEQ ID NO:83: Represented by TIFF0007627665000041.tif20166. The amino acid sequence of Hu95-VH4 is SEQ ID NO:84: Represented by TIFF0007627665000042.tif20166. The amino acid sequence of Hu95-VH5 is SEQ ID NO:85: Represented by TIFF0007627665000043.tif19166.

[0143] Note: The CDR sequences determined according to the Kabat criteria are underlined and the sequences are arranged in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0144] Of the above, the CDR regions of Hu95-VH5 have been further modified, with HCDR1 being shown in SEQ ID NO: 102 (NYAIH), HCDR2 being shown in SEQ ID NO: 103 (LVYPYTGGTAYNQKFKD), and HCDR3 being shown in SEQ ID NO: 104 (WGMIPGTNSYFDV).

[0145] 4. Generation and expression of anti-CTGF humanized antibodies Primers were designed, and VH / VL gene fragments of each humanized antibody were generated by PCR, and inserted into expression vector pHr (with signal peptide and constant region gene (CH / CL) fragments) by homologous recombination to generate full-length antibody expression vector VH-CH-pHr / VL-CL-pHr. For humanized antibodies, the constant region can be selected from the group consisting of human kappa, lambda light chain constant regions, and IgG1, IgG2, IgG3, or IgG4 heavy chain constant regions or variants thereof. Non-limiting examples include optimizing the constant region of human IgG1, IgG2, or IgG4 to improve antibody function. For example, point mutations in the constant region, such as mutations at M252Y / S254T / T256E (YTE) sites, and mutations in the constant region, such as S228P, F234A, and L235A, can extend the half-life of the antibody.

[0146] An example of an anti-CTGF humanized antibody constant region sequence is as follows: The amino acid sequence of the heavy chain constant region is SEQ ID NO:37: The full length antibody heavy chain is indicated by a w at the end of the name.

[0147] The amino acid sequence of the heavy chain constant region of the YTE modified antibody is SEQ ID NO:38: The full length antibody heavy chain is indicated by a y at the end of the name.

[0148] The amino acid sequence of the light chain kappa constant region of the anti-CTGF humanized antibody is SEQ ID NO:39: The full length antibody heavy chain is indicated by a k at the end of the name.

[0149] The amino acid sequence of the light chain lambda constant region of the anti-CTGF humanized antibody is SEQ ID NO:40: The full length antibody heavy chain is indicated by an l at the end of the name.

[0150] The heavy chain variable region of the humanized antibody was linked to the heavy chain constant region shown in SEQ ID NO:37 and the light chain variable region of the humanized antibody was linked to the light chain kappa constant region shown in SEQ ID NO:39 to obtain a full-length humanized antibody derived from mab147 that includes: [Table 5]

[0151] The heavy chain variable region of the humanized antibody was linked to the heavy chain constant region YTE variant shown in SEQ ID NO:38 and the light chain variable region of the humanized antibody was linked to the light chain kappa constant region shown in SEQ ID NO:39 to obtain a full-length humanized antibody derived from mab147 that includes: [Table 6]

[0152] The heavy chain variable region of the humanized antibody was linked to the heavy chain constant region shown in SEQ ID NO:37 and the light chain variable region of the humanized antibody was linked to the light chain lambda constant region shown in SEQ ID NO:40 to obtain a full-length humanized antibody derived from mab164 that includes: [Table 7]

[0153] The heavy chain variable region of the humanized antibody was linked to the heavy chain constant region YTE variant shown in SEQ ID NO:38, and the light chain variable region of the humanized antibody was linked to the light chain lambda constant region shown in SEQ ID NO:40 to obtain a full-length humanized antibody derived from mab164 that includes: [Table 8]

[0154] The heavy chain variable region of the humanized antibody was linked to the heavy chain constant region shown in SEQ ID NO:37 and the light chain variable region of the humanized antibody was linked to the light chain kappa constant region shown in SEQ ID NO:39 to obtain a full-length humanized antibody derived from mab95 that includes: [Table 9]

[0155] The heavy chain variable region of the humanized antibody was linked to the heavy chain constant region YTE variant shown in SEQ ID NO:38 and the light chain variable region of the humanized antibody was linked to the light chain kappa constant region shown in SEQ ID NO:39 to obtain a full length humanized antibody derived from mab95 that includes: [Table 10]

[0156] An example of the amino acid sequence of a full-length anti-CTGF antibody is as follows: [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4] [Table 11-5] [Table 11-6] [Table 11-7] [Table 11-8] [Table 11-9]

[0157] The sequence of the positive control CTGF antibody mAb1 (from CN1829740B; antibody name pamrevlumab, also known as FG-3019) is as follows: Heavy Chain (SEQ ID NO:67): TIFF0007627665000063.tif55160 Light Chain (SEQ ID NO:68): TIFF0007627665000064.tif29158 EXAMPLES

[0158] Example 3: Detection of CTGF antibody binding activity I. ELISA Assay of Binding of CTGF Antibodies to Human CTGF Protein The binding activity of anti-human CTGF antibody to human CTGF protein was tested by ELISA assay and Biacore. CTGF recombinant protein was directly coated on a 96-well microtiter plate. The binding activity of the antibody to CTGF was measured using the signal intensity after the addition of the antibody. The specific experimental procedure was as follows:

[0159] CTGF protein (R&D, Cat No. 9190-CC) was diluted to a concentration of 0.2 μg / ml in PBS (pH 7.4) (Shanghai Yuanpei, Cat No. B320) buffer, and 50 μl / well was added to a 96-well microtiter plate (Corning, Cat No. CLS3590-100EA) and incubated for 2 hours in a 37°C incubator or overnight (16-18 hours) at 4°C. The liquid was removed and 250 μl / well of blocking solution (5% skim milk (BD skim milk, Cat No. 232100) diluted in PBS) was added and incubated for 3 hours in a 37°C incubator or overnight (16-18 hours) at 4°C for blocking. After blocking, the blocking solution was removed, the plate was washed five times with PBST buffer (PBS containing 0.1% Tween-20, pH 7.4), and various concentrations of each test antibody (hybridoma purified antibody or humanized antibody) diluted in sample dilution solution were added at 50 μl / well and incubated for 1 hour in an incubator at 37°C. After incubation, the plate was washed three times with PBST, and HRP-labeled goat anti-mouse secondary antibody (Jackson Immuno Research, Cat No. 115-035-003) or goat anti-human secondary antibody (Jackson Immuno Research, Cat No. 109-035-003) diluted in sample dilution solution was added at 50 μl / well and incubated for 1 hour at 37°C. The plates were washed three times with PBST, TMB chromogenic substrate (KPL, Cat No. 52-00-03) was added at 50 μl / well, incubated at room temperature for 5-10 min, and the reaction was stopped by adding 50 μl / well of 1M H2SO4. The absorbance values ​​at 450 nm wavelength were read using a microplate reader (Molecular Devices, VERSA max) and the data were analyzed using GraphPad Prism 5. The binding of the CTGF antibody to the human CTGF protein was calculated as the EC50 value. The experimental results are shown in Figure 1 and Table 12.

[0160] [Table 12]

[0161] Experimental results demonstrate that all of the humanized full-length anti-CTGF antibodies disclosed herein have excellent binding activity to human CTGF protein.

[0162] II. ELISA Assay of Binding of CTGF Antibodies to Cynomolgus CTGF Protein The binding activity of the anti-monkey CTGF antibody to the monkey CTGF protein was tested by ELISA assay. Anti-mFc protein was directly coated on a 96-well microtiter plate, and then Fc-tagged cynomolgus CTGF protein was bound to the plate, and then the antibody was added. The signal intensity after the addition of the antibody was used to measure the binding activity of the antibody to the cynomolgus CTGF. The specific experimental procedure was as follows:

[0163] Anti-mFc protein (Sigma, Cat No. M4280) was diluted to 2 μg / ml in PBS (pH 7.4) (Shanghai Yuanpei, Cat No. B320) buffer, 50 μl / well was placed in a 96-well microtiter plate (Corning, Cat No. CLS3590-100EA) and incubated for 2 hours in a 37°C incubator. The liquid was removed and 250 μl / well of blocking solution (5% skim milk (BD skim milk, Cat No. 232100) diluted in PBS) was added and incubated for 3 hours in a 37°C incubator or overnight (16-18 hours) at 4°C for blocking. After blocking, the blocking solution was removed, the plate was washed three times with PBST buffer (PBS containing 0.1% Tween-20, pH 7.4), 50 μl of 1 μg / ml cynomolgus monkey CTGF-mFc protein was added to each well and incubated for 1 hour in an incubator at 37° C., and then the plate was washed three times with PBST buffer (PBS containing 0.1% Tween-20, pH 7.4). Various concentrations of each test antibody (hybridoma purified antibody or humanized antibody) diluted with sample dilution solution were added at 50 μl / well and incubated for 1 hour in an incubator at 37° C. After the incubation, the plate was washed three times with PBST, and 50 μl / well of HRP-conjugated goat anti-mouse secondary antibody (Jackson Immuno Research, Cat No. 115-035-003) or goat anti-human secondary antibody (Jackson Immuno Research, Cat No. 109-035-003) diluted in sample diluent was added and incubated at 37°C for 1 h. The plate was washed five times with PBST, and 50 μl / well of TMB chromogenic substrate (KPL, Cat No. 52-00-03) was added and incubated at room temperature for 5–10 min, and the reaction was stopped by adding 50 μl / well of 1M H2SO4. The absorbance values ​​at 450 nm were read using a microplate reader (Molecular Devices, VERSA max), and the data were analyzed using GraphPad Prism 5.Binding of CTGF antibodies to cynomolgus CTGF protein was calculated as an EC50 value. The experimental results are shown in Table 13.

[0164] [Table 13]

[0165] Experimental results demonstrate that all of the humanized full-length anti-CTGF antibodies disclosed herein have potent binding activity to monkey CTGF protein.

[0166] III. ELISA Assay of Binding of CTGF Antibodies to Mouse CTGF Protein The binding activity of anti-mouse CTGF antibody to mouse CTGF protein was tested by ELISA assay. Mouse CTGF fusion protein was directly coated on a 96-well microtiter plate. The binding activity of the antibody to mouse CTGF was measured using the signal intensity after the addition of the antibody. The specific experimental procedure was as follows:

[0167] Mouse CTGF-his protein was diluted to 0.5 μg / ml in PBS (pH 7.4) (Shanghai Yuanpei, Cat No. B320) buffer, 50 μl / well was placed in a 96-well microtiter plate (Corning, Cat No. CLS3590-100EA) and incubated for 2 hours in a 37°C incubator. The liquid was removed and 250 μl / well of blocking solution (5% skim milk (BD skim milk, Cat No. 232100) diluted in PBS) was added and incubated for 3 hours in a 37°C incubator or overnight (16-18 hours) at 4°C for blocking. After blocking, the blocking solution was removed, the plate was washed three times with PBST buffer (PBS containing 0.1% Tween-20, pH 7.4), and various concentrations of each test antibody (hybridoma purified antibody or humanized antibody) diluted in sample dilution solution were added at 50 μl / well and incubated for 1 hour in an incubator at 37°C. After incubation, the plate was washed three times with PBST, and HRP-labeled goat anti-mouse secondary antibody (Jackson Immuno Research, Cat No. 115-035-003) or goat anti-human secondary antibody (Jackson Immuno Research, Cat No. 109-035-003) diluted in sample dilution solution was added at 50 μl / well and incubated for 1 hour at 37°C. The plates were washed five times with PBST, TMB chromogenic substrate (KPL, Cat No. 52-00-03) was added at 50 μl / well, incubated at room temperature for 5-10 min, and the reaction was stopped by adding 50 μl / well of 1M H2SO4. The absorbance values ​​at 450 nm wavelength were read using a microplate reader (Molecular Devices, VERSA max) and the data were analyzed using GraphPad Prism 5. The binding of the CTGF antibody to mouse CTGF protein was calculated as EC50 value. The experimental results are shown in Table 14.

[0168] [Table 14]

[0169] Experimental results demonstrate that all of the humanized full-length anti-CTGF antibodies disclosed herein have excellent binding activity to mouse CTGF protein. EXAMPLES

[0170] Example 4: Functional Inhibition Assay of CTGF Antibodies In this assay, a Biacore instrument was used to detect the activity of the tested humanized anti-CTGF antibodies to inhibit human TGF-β1 function.

[0171] First, the antigen human CTGF was immobilized on a CM5 biosensor chip (Cat. # BR-1005-30, GE) by amino coupling at an immobilization level of 1500RU, then a high concentration of CTGF antibody (150μg / ml) was flowed over the chip surface for 150 seconds; 50nM TGF-β1 protein was injected for 120 seconds, and the reaction signal was detected in real time by a Biacore T200 instrument to generate binding and dissociation curves. After the dissociation was completed in each experimental cycle, the biosensor chip was washed and regenerated with regeneration buffer.

[0172] The experimental data obtained were statistically analyzed by GE Biacore T200 Evaluation version 3.0 software.

[0173] Inhibition efficiency=[1-(sample response value / blank control response value)]×100%. Specific results are shown in Table 15.

[0174] [Table 15]

[0175] Experimental results demonstrate that all of the humanized CTGF antibodies disclosed herein have high functional inhibitory activity.

[0176] Measurement of anti-CTGF antibody affinity by Biacore In this experiment, a Biacore instrument was used to measure the affinity of the humanized anti-CTGF antibodies being tested for human and mouse CTGF.

[0177] A certain amount of each test antibody was affinity captured onto a Protein A biosensor chip (Cat. # 29127556, GE), and then a certain concentration of human or mouse CTGF was flowed over the chip surface. The reaction signal was detected in real time by a Biacore instrument to generate binding and dissociation curves. After dissociation was completed in each experimental cycle, the biosensor chip was washed and regenerated with glycine-HCl regeneration solution (pH 1.5) (Cat. # BR-1003-54, GE). The running buffer for the assay was 1×HBS-EP buffer solution (Cat. # BR-1001-88, GE).

[0178] The experimental data obtained was fitted to a (1:1) Langmuir model using GE Biacore T200 Evaluation version 3.0 software to obtain affinity values ​​as detailed in Tables 16 and 17.

[0179] [Table 16]

[0180] [Table 17]

[0181] Experimental results show that both chimeric antibodies Ch147 and Ch164 derived from mouse antibodies mAb147 and mAb164 and humanized anti-CTGF antibodies can bind human and mouse CTGF with high affinity. Neither substitutions nor back mutations in the human germline antibody FR regions impair the affinity of the humanized antibodies, rather some modifications can increase the affinity of the antibody for the antigen. EXAMPLES

[0182] Example 5: Assay of inhibition of TGFβ-induced C2C12 cell migration in vitro by CTGF antibodies C2C12 cells (mouse myoblasts, Cell Bank of Chinese Academy of Sciences, #GNM26) were digested with 0.25% trypsin (Gibco, #25200-072), centrifuged, and resuspended in serum-free DMEM medium (Gibco, #10564-029). Cell-antibody mixtures and TGFβ1-antibody mixtures were prepared in serum-free DMEM medium, where the cell density was 2 × 10 5 / ml, the concentration of recombinant human TGFβ1 (Cell signaling Technology, #8915LC) was 10 ng / ml, and the concentration of the tested antibodies was 30 μg / ml.

[0183] The cover and filter membrane of ChemoTx® Disposable Chemotaxis System (Neuro Probe, #106-8) were opened, and the TGFβ-antibody mixture was placed in the lower chamber at 30 μl / well, with 4 pairs of wells used for each group. The filter membrane was covered, and the cell-antibody mixture was added onto the membrane at 50 μl / well, with 4 pairs of wells used for each group. The cover was closed and placed in an incubator (37° C., 5% CO2).

[0184] At 48 hours after incubation, the liquid on the filter membrane was removed with a clean paper towel, the filter membrane was opened, and 10 μl of pre-chilled 0.25% trypsin was added to each well into the lower chamber and covered with the filter membrane. Five minutes after digestion, the samples were centrifuged at 1000 rpm for 1 minute. The filter membrane was opened, and 20 μl of Cell Titer-Glo solution (Promega, #G7573) was added to each well into the lower chamber and incubated at room temperature for 10 minutes. The liquid was transferred to a 384-well white bottom plate (Thermo Scientific, #267462) and the plate was read by chemiluminescence using a microplate reader (BMG, #PheraStar). Data was analyzed and processed by Graphpad Prism 6.

[0185] Inhibition rate = [(mean value of TGFβ - mean value of sample group) / (mean value of TGFβ group - mean value of untreated group)] x 100%

[0186] The experimental results are shown in Table 18. [Table 18]

[0187] The results show that the humanized anti-CTGF antibody of the present disclosure can inhibit the migration ability of C2C12 cells induced by TGFβ1 in vitro, and shows a stronger inhibitory effect on the migration ability of C2C12 cells compared to the positive control antibody mAb1. EXAMPLES

[0188] Example 6: Assay demonstrating the inhibitory effect of CTGF antibodies on TGFβ-induced PANC1 cell migration in vitro PANC1 cells (human pancreatic cancer cells, ATCC, #CRL-1469) were digested with 0.25% trypsin (Gibco, #25200-072), centrifuged, and resuspended in DMEM medium (Gibco, #10564-029) containing 0.1% fetal bovine serum (Gibco, #10099-141). Cell-antibody mixtures and TGFβ-antibody mixtures were prepared in DMEM medium containing 0.1% fetal bovine serum. Here, the cell density was 4 × 10 5 / ml, the concentration of recombinant human TGFβ (Cell signaling Technology, #8915LC) was 10 ng / ml, and the concentration of the antibody being tested was 30 μg / ml.

[0189] The cover and filter membrane of ChemoTx® Disposable Chemotaxis System (Neuro Probe, #106-8) were opened, and the TGFβ-antibody mixture was placed in the lower chamber at 30 μl / well, with 4 pairs of wells used for each group. The filter membrane was covered, and the cell-antibody mixture was added onto the membrane at 50 μl / well, with 4 pairs of wells used for each group. The cover was closed and placed in an incubator (37° C., 5% CO2).

[0190] 48 hours after incubation, the liquid on the filter membrane was removed with a clean paper towel, the filter membrane was opened, and 10 μl of pre-chilled 0.25% trypsin was added to each well into the lower chamber and covered with the filter membrane. Five minutes after digestion, the samples were centrifuged at 1000 rpm for 1 minute. The cells attached to the plate bottom were counted under an inverted microscope (Leica, #DMIL LED). The data were analyzed and processed by Graphpad Prism 6. Inhibition rate=[(mean value of TGFβ-mean value of sample group) / (mean value of TGFβ-mean value of untreated group)]×100%.

[0191] The experimental results are shown in Table 19. [Table 19]

[0192] The results demonstrate that all of the humanized anti-CTGF antibodies disclosed herein are capable of inhibiting the migration ability of PANC1 cells induced by TGFβ1 in vitro. EXAMPLES

[0193] Example 7: Assay of the inhibitory effect of CTGF antibody on PANC1 proliferation in soft agar in vitro A 1.4% gel solution was prepared by adding deionized water to agar (BD, #214220) and heating it. 2x DMEM medium (Thermo, #12100046) was mixed with the gel solution in a 1:1 volume ratio, and 500 μl was placed in each well of a 24-well plate (Costar, #3524) and placed at 4 degrees to solidify.

[0194] PANC-1 cells (human pancreatic cancer cells, ATCC, #CRL-1469) were digested with 0.25% trypsin (Gibco, #25200-072), centrifuged, and cultured at a cell density of 5 × 10 in DMEM medium (GE, #SH30243.01) containing 4% fetal bovine serum (Gibco, #10099-141). 4 The antibody was adjusted to 2 mg / ml in 2x DMEM medium. The cells, antibody and 1.4% gel solution were mixed in a volume ratio of 2:1:1, and 200 μl was placed into each well of a 24-well plate covered with the lower gel layer. After the upper gel solidified, 200 μl of DMEM medium containing 2% fetal bovine serum was added to the 24-well plate. After 28 days of incubation in an incubator (37°C, 5% CO2), images were taken using a high content analysis system (Molecular Devices, ImageXpress) to analyze the area of ​​the generated cell clones. Inhibition rate = (1 - mean value of sample group / mean value of untreated group) x 100%. The experimental results are shown in Table 20.

[0195] [Table 20]

[0196] The results show that the humanized antibodies derived from the mAb147 and mAb164 clones have significant inhibitory effects on human pancreatic cancer cell (PANC-1 cell) proliferation in vitro. EXAMPLES

[0197] Example 8: In vivo biological activity of CTGF antibodies in animals I. Assay of the inhibitory effect of CTGF antibody on bleomycin-induced pulmonary fibrosis in mice Experimental protocol: 40 mice were randomly divided into 4 groups according to body weight: normal control group (PBS, ip, qod), mAb1 group (10mg / kg, ip, qod), Hu164-67wl group (10mg / kg, ip, qod), and Hu164-67yl group (10mg / kg, ip, qod). There were 10 mice in each group, and the group details are shown in Table 21 below. On the first day of the experiment, vehicle and antibody were intraperitoneally injected according to body weight (10ml / kg; normal control and model groups were administered with PBS); 2 hours later, aerosol bleomycin (50mg / 8ml nebulization for 30 minutes and retention for 5 minutes) was used to establish the model. Then, vehicle or antibody was intraperitoneally injected every other day according to the above groups. The experiment period was 21 days, and a total of 11 intraperitoneal injections were performed. On the 22nd day of the experiment, the mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (10 ml / kg) and fixed in a supine position on the operating table. A 1 cm skin incision was made along the midline of the neck, with the lower end of the incision reaching the inlet of the chest. The subcutaneous connective tissue and muscle were bluntly separated with forceps to expose the trachea. The connective tissue on both ends of the trachea and between the trachea and the esophagus were separated to free the trachea. The intravenous needle was inserted and connected where the cannula entered the trachea and secured with sutures. A 1 ml syringe filled with 0.8 ml of PBS was connected to the inlet end of the intravenous needle, and the PBS was slowly injected into the trachea and allowed to dwell for 30 seconds, after which the PBS was slowly withdrawn. A milky foamy liquid could be observed during withdrawal. The washing was repeated three times, the washing liquid was transferred to an EP tube, and then 0.5 ml of PBS was taken and the above steps were repeated. The two lavage fluids were mixed and centrifuged at 1500 rpm for 5 min, and the supernatant was stored at -20°C for testing. The BALF supernatant was centrifuged again at 10000 rpm for 10 min, and the supernatant was taken to detect soluble collagen (kit: Biocolor, Batch No. AA883).

[0198] [Table 21]

[0199] Excel statistical software was used: mean values ​​were calculated as avg; SD values ​​were calculated as STDEV; SEM values ​​were calculated as STDEV / SQRT (square root) (number of animals in each group); GraphPad Prism software was used for plotting and two-way ANOVA or one-way ANOVA were used for statistical analysis of data.

[0200] The experimental results are shown in Table 22. [Table 22]

[0201] The results show that Hu164-67wl or Hu164-67yl exhibits strong inhibitory effects on pulmonary fibrosis in mice.

[0202] II. Assay of CTGF antibody inhibition of subcutaneously transplanted Su86.86 human pancreatic cancer cells Experimental protocol: 200 μl of SU86.86 cells (ATCC, CRL-1837, 3.0 × 10 6 The tumor volume was approximately 140 mm. 3 Mice with too large or small body weight or tumor volume were excluded 11 days after inoculation when the tumor volume reached 10. Mice were randomly divided into 5 groups of 10 mice per group according to tumor volume, and treatment was started on the same day. Antibodies were intraperitoneally injected twice a week for a total of 18 days. Tumor volume and body weight were measured once or twice a week, and the data were recorded. The grouping and treatment regimen are shown in Table 23.

[0203] [Table 23]

[0204] Excel statistical software was used: mean values ​​were calculated as avg; SD values ​​were calculated as STDEV; SEM values ​​were calculated as STDEV / SQRT (number of animals in each group); GraphPad Prism software was used for plotting and two-way ANOVA or one-way ANOVA was used for statistical analysis of data.

[0205] Tumor volume (V) was calculated according to the following formula: V=1 / 2×L long ×L short 2

[0206] Relative tumor growth rate T / C(%)=(T t -T0) / (C t -C0) x 100 Here, T t and C t are the tumor volumes of the treated and control groups at the end of the experiment; T0 and C0 are the tumor volumes at the start of the experiment.

[0207] Tumor inhibition rate TGI(%)=1-T / C(%)

[0208] Test Results: From D1 to D18, subcutaneous injection was performed twice a week to detect the effect of CTGF antibody on SU86.86 tumor growth inhibition. The experimental results are shown in Table 24 and Figure 2. Compared with the blank control group of the same isotype human IgG, the tumor inhibition rates of mAb1-40mg / kg, Hu164-67yl-40mg / kg, Hu164-67yl-20mg / kg and Hu147-33wk(40mg / kg) groups were 45%, 53%, 47% and 54%, respectively. mAb1-40mg / kg, Hu164-67yl-40mg / kg, Hu164-67yl-20mg / kg and Hu147-33wk(40mg / kg) groups could significantly inhibit the growth of Su86.86 tumor (p<0.001), and the inhibitory effect on SU86.86 tumor is dose-dependent. Furthermore, there was no significant change in the body weight of the mice in each group.

[0209]

Table 24

Claims

1. An anti-CTGF antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively.

2. The anti-CTGF antibody of claim 1 , wherein the anti-CTGF antibody is a murine antibody, a chimeric antibody or a humanized antibody.

3. The anti-CTGF antibody of claim 1 or 2, comprising a heavy chain variable region and a light chain variable region, the amino acid sequence of which is shown in SEQ ID NO: 8, 33, 34, 35 or 36, and the amino acid sequence of which is shown in SEQ ID NO: 9, 30, 31 or 32.

4. the anti-CTGF antibody is a humanized antibody, the humanized antibody comprises a framework region of a human antibody or a variant of that framework region, the variant having up to 10 back mutations in each of the human antibody light chain framework region and / or heavy chain framework region; Where: the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, respectively, and one or more backmutations selected from the group consisting of 36V, 44F, 46G and 49G; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, respectively, and one or more back mutations selected from the group consisting of 44G, 49G, 27F, 48L, 67L, 71K, 78V and 80F; The anti-CTGF antibody according to any one of claims 1 to 3, wherein the backmutation positions in the light chain variable region and the heavy chain variable region are determined according to the Kabat numbering standard.

5. (ix) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 8 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 9; or (x) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 33, 34, 35 or 36, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 30, 31 or 32; The anti-CTGF antibody according to any one of claims 1 to 3, comprising a heavy chain variable region and a light chain variable region.

6. (xiv) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 34, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 30; The anti-CTGF antibody of claim 4 , comprising a heavy chain variable region and a light chain variable region.

7. The anti-CTGF antibody of any one of claims 1 to 6, wherein the antibody further comprises an antibody heavy chain constant region and a light chain constant region, the heavy chain constant region being selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4 constant regions and variants thereof, and the light chain constant region being selected from the group consisting of human antibody κ and λ chain constant regions and variants thereof.

8. The anti-CTGF antibody of any one of claims 1 to 6, wherein the antibody comprises a heavy chain constant region shown in SEQ ID NO: 37 or 38, and a light chain constant region shown in SEQ ID NO: 39 or 40.

9. 9. An anti-CTGF antibody according to any one of claims 1 to 8, comprising a heavy chain as shown in SEQ ID NO: 54, 56, 57, 58, 59, 60, 61, 62 or 63, and a light chain as shown in SEQ ID NO: 55, 64, 65 or 66.

10. 9. An anti-CTGF antibody according to any one of claims 1 to 8, comprising a heavy chain as shown in SEQ ID NO:61 and a light chain as shown in SEQ ID NO:

64.

11. A nucleic acid molecule encoding the anti-CTGF antibody of any one of claims 1 to 10.

12. A host cell comprising the nucleic acid molecule of claim 11.

13. A pharmaceutical composition comprising a therapeutically effective amount of an anti-CTGF antibody according to any one of claims 1 to 10 or a nucleic acid molecule according to claim 11, and one or more pharma- ceutically acceptable carriers, diluents, buffers or excipients.

14. A pharmaceutical composition for treating or preventing a CTGF-related disease in a subject, the pharmaceutical composition comprising an anti-CTGF antibody according to any one of claims 1 to 10 or a nucleic acid molecule according to claim 11, wherein the CTGF-related disease is a fibrotic disease, hypertension, diabetes, myocardial infarction, arthritis, a CTGF-related cell proliferative disease, atherosclerosis, glaucoma or cancer.

15. the fibrotic disease is selected from the group consisting of idiopathic pulmonary fibrosis, diabetic nephropathy, diabetic retinopathy, osteoarthritis, scleroderma, chronic heart failure, liver cirrhosis or renal fibrosis; The cancer is selected from the group consisting of acute lymphoblastic leukemia, dermatofibroma, breast cancer, angiolipoma, angioleiomyoma, connective tissue generating cancer, prostate cancer, ovarian cancer, colorectal cancer, pancreatic cancer, gastrointestinal cancer or liver cancer; 15. The pharmaceutical composition of claim 14.

Images