Anti-connective tissue growth factor antibodies and uses thereof

Abstract

Provided is an anti-CTGF antibody comprising a light chain and a heavy chain variable region and use thereof as a medicament, which is useful for the manufacture of a medicament for treating a CTGF-related disease or disorder.

Description

[0001] This application is a divisional application of Chinese patent application No. 202080036744.2, filed on June 3, 2020, entitled "Anti-connective tissue growth factor antibody and its application".

[0002] This application claims priority to Chinese patent application No. 201910480169.4 (titled: Anti-connective tissue growth factor antibody and its application, priority date June 4, 2019). Technical Field

[0003] This disclosure pertains to the field of biomedicine, specifically relating to antibodies that bind to connective tissue growth factor (CTGF) and their applications. Background Technology

[0004] The statements herein are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

[0005] CTGF expression is induced by members of the transforming growth factor β (TGFβ) superfamily. This superfamily includes TGFβ-1, -2, and -3, bone morphogenetic protein (BMP)-2, and activin. Many modulators, including dexamethasone, thrombin, vascular endothelial growth factor (VEGF), and angiotensin II, as well as environmental stimuli, including hyperglycemia and hypertension, also induce CTGF expression (see, for example, Franklin (1997) Int J Biochem Cell Biol 29:79-89; Wunderlich (2000) GraefesArch 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 Soc Nephrol 11:25-38; and International Publication WO 00 / 13706).

[0006] TGFβ stimulates CTGF expression rapidly and for a long time without requiring persistent application of TGFβ (Igarashi et al. (1993) Mol Biol Cell 4: 637-645). TGFβ leads to enhanced CTGF expression through transcriptional activation of DNA regulatory elements in the CTGF promoter (Grotendorst et al. (1996) Cell Growth Differ 7: 469-480; Grotendorst and Bradham, US Patent No. 6,069,006; Holmes et al. (2001) J Biol Chem 276: 10594-10601).

[0007] CTGF expression is upregulated in glomerulonephritis, IgA nephropathy, focal and segmental glomerulosclerosis, and diabetic nephropathy (see, for example, 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, and CTGF levels correlate with the degree of injury (Ito et al. (1998) Kidney Int 53:853-861). Furthermore, CTGF expression is increased in the glomeruli and tubulointerstitium in various nephropathies associated with renal parenchymal scarring and sclerosis. Elevated CTGF levels are also associated with liver fibrosis, myocardial infarction, and pulmonary fibrosis. For example, in patients with spontaneous pulmonary fibrosis (IPF), CTGF is strongly upregulated in biopsy and bronchoalveolar lavage fluid cells (Ujike et al. (2000) Biochem Biophys ResCommun 277:448-454; Abou-Shady et al. (2000) Liver 20:296-304; Williams et al. (2000) J Hepatol 32:754-761). Therefore, in the diseases described above, CTGF represents a potential therapeutic target.

[0008] However, there are currently no effective antibody drugs targeting CTGF that are used in clinical practice, and new safe and effective CTGF antibody drugs still need to be developed. Summary of the Invention

[0009] This disclosure provides an anti-CTGF antibody. The anti-CTGF antibody comprises a full-length anti-human CTGF antibody and its antigen-binding fragment.

[0010] In some embodiments, the anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0011] i) The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with the same sequence as the heavy chain variable region shown in SEQ ID NO:6, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with the same sequence as the light chain variable region shown in SEQ ID NO:7; or

[0012] ii) The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 with the same sequence as the heavy chain variable region shown in SEQ ID NO:8, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 with the same sequence as the light chain variable region shown in SEQ ID NO:9.

[0013] In some embodiments, the anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0014] ii-i) The heavy chain variable region contains HCDR1, HCDR2, and HCDR3 with the same sequence as the heavy chain variable region shown in SEQ ID NO:69, and the light chain variable region contains LCDR1, LCDR2, and LCDR3 with the same sequence as the light chain variable region shown in SEQ ID NO:70; or

[0015] ii-ii) The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 with the same sequence as the heavy chain variable region shown in SEQ ID NO:85, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 with the same sequence as the light chain variable region shown in SEQ ID NO:70.

[0016] In some embodiments, the anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0017] iii) The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown 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 shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; or

[0018] iv) The heavy chain variable region includes 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 includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21 respectively.

[0019] In some embodiments, the anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0020] iv-i) The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown 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 shown in SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:76, respectively; or

[0021] (iv-ii) The heavy chain variable region includes 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 includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:74, SEQ ID NO:75 and SEQ ID NO:76 respectively.

[0022] In some implementations, the anti-CTGF antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

[0023] In some embodiments, the anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0024] (v-1) The heavy chain variable region, whose amino acid sequence has at least 90% sequence identity with SEQ ID NO:6, 27, 28 or 29, and the light chain variable region, whose amino acid sequence has at least 90% sequence identity with SEQ ID NO:7, 22, 23, 24, 25 or 26:

[0025] (vi-1) The heavy chain variable region, having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 8, 33, 34, 35 or 36, and the light chain variable region, having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 9, 30, 31 or 32; or

[0026] (vi-i) The heavy chain variable region having at least 90% sequence identity with SEQ ID NO:69, 81, 82, 83, 84 or 85, and the light chain variable region having at least 90% sequence identity with SEQ ID NO:70, 77, 78, 79 or 80.

[0027] In some embodiments, the anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0028] (v) The heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the heavy chain variable region shown in SEQ ID NO: 6, 27, 28, or 29, and the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the light chain variable region shown in SEQ ID NO: 7, 22, 23, 24, 25, or 26; or

[0029] (vi) The heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the heavy chain variable region shown in SEQ ID NO: 8, 33, 34, 35, or 36, and the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the light chain variable region shown in SEQ ID NO: 9, 30, 31, or 32; or

[0030] The heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the heavy chain variable region shown in SEQ ID NO:6, and the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the light chain variable region shown in SEQ ID NO:7;

[0031] The heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the heavy chain variable region shown in SEQ ID NO:27, 28, or 29, and the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the light chain variable region shown in SEQ ID NO:22, 23, 24, 25, or 26;

[0032] The heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the heavy chain variable region shown 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% identity with the light chain variable region shown in SEQ ID NO:9;

[0033] The heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the heavy chain variable region shown in SEQ ID NO:33, 34, 35, or 36, and the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the light chain variable region shown in SEQ ID NO:30, 31, or 32.

[0034] In some embodiments, the anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0035] (vi-ii) The heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the heavy chain variable region shown 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 with the light chain variable region shown in SEQ ID NO: 70, 77, 78, 79, or 80.

[0036] In some embodiments, the anti-CTGF antibody is a humanized antibody comprising a framework region of a human antibody or a variant thereof, the framework region variant having up to 10 reversion mutations in the light chain framework region and / or the heavy chain framework region of the human antibody, respectively.

[0037] In some embodiments, the anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region as described below:

[0038] (a) The light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively, and comprising one or more amino acid reversion mutations selected from 4L, 36F, 43S, 45K, 47W, 58V, or 71Y, and / or the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, and comprising one or more amino acid reversion mutations selected from 28S, 30N, 49A, 75E, 76S, 93V, 94E, or 104D; or

[0039] (b) The light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively, containing one or more reversion mutations selected from 36V, 44F, 46G, or 49G, and / or the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively, and containing one or more reversion mutations selected from 44G, 49G, 27F, 48L, 67L, 71K, 78V, or 80F.

[0040] In some embodiments, the anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region as described below:

[0041] (bi) A light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:76, respectively, and one or more reversion mutations selected from 45P, 46W, 48Y, 69S, or 70Y; and a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73, respectively, and one or more reversion mutations selected from 27F, 38K, 48I, 67K, 68A, 70L, or 72F; or

[0042] (b-ii) Light chain variable regions comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:76, respectively, and containing one or more reversion mutations selected from 45P, 46W, 48Y, 69S, or 70Y; and heavy chain variable regions comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:102, SEQ ID NO:103, and SEQ ID NO:104, respectively, and containing one or more reversion mutations selected from 27F, 38K, 48I, 67K, 68A, 70L, or 72F.

[0043] In some embodiments, the anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region as shown below:

[0044] (vii) The heavy chain variable region sequence is shown in SEQ ID NO:6 and the light chain variable region sequence is shown in SEQ ID NO:7;

[0045] (viii) The heavy chain variable region sequence is shown in SEQ ID NO:27, 28 or 29 and the light chain variable region sequence is shown in SEQ ID NO:22, 23, 24, 25 or 26;

[0046] (ix) The heavy chain variable region sequence is as shown in SEQ ID NO:8 and the light chain variable region sequence is as shown in SEQ ID NO:9; or

[0047] (x) The heavy chain variable region sequence is shown as SEQ ID NO:33, 34, 35 or 36 and the light chain variable region sequence is shown as SEQ ID NO:30, 31 or 32.

[0048] In some embodiments, the anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region as shown below:

[0049] (xi) the heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO:69; and the light chain variable region, the amino acid sequence of which is shown in SEQ ID NO:70; or

[0050] (xii) the heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO:81, 82, 83, 84 or 85; and the light chain variable region, the amino acid sequence of which is shown in SEQ ID NO:77, 78, 79 or 80.

[0051] In some embodiments, the anti-CTGF antibody comprises heavy chain variable regions and light chain variable regions as shown in Tables a, b, and c below:

[0052] Table a: Combination of light chain variable regions and heavy chain variable regions of humanized antibodies derived from mab147

[0053]

[0054]

[0055] Table b: Combination of light chain variable region and heavy chain variable region of mab164 humanized antibody

[0056]

[0057] Table c: Combination of light chain variable regions and heavy chain variable regions of mab95 humanized antibody

[0058]

[0059] In some embodiments, the anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region as shown below:

[0060] (xiii) The heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO:27; and the light chain variable region, the amino acid sequence of which is shown in SEQ ID NO:22;

[0061] (xiv) the heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO:34; and the light chain variable region, the amino acid sequence of which is shown in SEQ ID NO:30; or

[0062] (xv) The heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO:85; and the light chain variable region, the amino acid sequence of which is shown in SEQ ID NO:77.

[0063] In some embodiments, the anti-CTGF antibody further comprises an antibody heavy chain constant region and a light chain constant region; preferably, the heavy chain constant region is selected from human IgG1, IgG2, IgG3 and IgG4 constant regions and their conventional variants, and the light chain constant region is selected from human antibody κ and λ chain constant regions and their conventional variants; more preferably, the antibody comprises a heavy chain constant region with a sequence as shown in SEQ ID NO:37 or 38 and a light chain constant region with a sequence as shown in SEQ ID NO:39 or 40.

[0064] In some embodiments, the anti-CTGF antibody comprises:

[0065] (c) A heavy chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the heavy chain shown in SEQ ID NO:41, 43, 44, 45, 46, 47, or 48, and a light chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the light chain shown in SEQ ID NO:42, 49, 50, 51, 52, or 53;

[0066] (d) Heavy chains having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the heavy chains shown in SEQ ID NO: 54, 56, 57, 58, 59, 60, 61, 62, or 63, and light chains having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the light chains shown in SEQ ID NO: 55, 64, 65, or 66;

[0067] (e) Heavy chains with sequences as shown in SEQ ID NO:41, 43, 44, 45, 46, 47 or 48 and light chains with sequences as shown in SEQ ID NO:42, 49, 50, 51, 52 or 53; or

[0068] (f) Heavy chains with sequences as shown in SEQ ID NO:54, 56, 57, 58, 59, 60, 61, 62 or 63 and light chains with sequences as shown in SEQ ID NO:55, 64, 65 or 66.

[0069] In some embodiments, the anti-CTGF antibody comprises:

[0070] (g) a heavy chain having at least 85%, 90%, 91%, 92%, 93%, 94, 95, 96, or 97 sequence identity, and a light chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 87, 98, 99, 100, or 101 sequence identity; or

[0071] (h) Heavy chains as shown in SEQ ID NO:86, 88, 89, 90, 91, 92, 93, 94, 95, 96 or 97 and light chains as shown in SEQ ID NO:87, 98, 99, 100 or 101.

[0072] In some embodiments, the anti-CTGF antibody comprises:

[0073] (j) Heavy chains as shown in SEQ ID NO:46 and light chains as shown in SEQ ID NO:49;

[0074] (k) a heavy chain as shown in SEQ ID NO:61 and a light chain as shown in SEQ ID NO:64; or

[0075] (l) The heavy chain as shown in SEQ ID NO:97 and the light chain as shown in SEQ ID NO:98.

[0076] Other aspects of this disclosure provide an anti-CTGF antibody that competitively binds to human CTGF with an anti-CTGF antibody as described above or an antigen-binding fragment thereof.

[0077] Other aspects of this disclosure provide a nucleic acid molecule that encodes the anti-CTGF antibody as described above.

[0078] Other aspects of this disclosure provide a host cell comprising the nucleic acid molecules as described above.

[0079] Other aspects of this disclosure provide a pharmaceutical composition comprising a therapeutically effective or preventatively effective amount of the anti-CTGF antibody as described above, or a nucleic acid molecule as described above, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.

[0080] In some specific embodiments, the therapeutic or preventive effective amount is 0.1-3000 mg or 1-1000 mg of the anti-CTGF antibody as described above in a unit dose of the composition.

[0081] Other aspects of this disclosure provide a method for immune detection or determination of CTGF, the method comprising the step of using an anti-CTGF antibody as described above.

[0082] Other aspects of this disclosure provide a method for immune detection or determination of CTGF, the method comprising the step of contacting a subject or a sample thereof with an anti-CTGF antibody as described above.

[0083] Other aspects of this disclosure provide a kit comprising the anti-CTGF antibody as described above.

[0084] Other aspects of this disclosure provide a method for treating CTGF-related diseases, the method comprising administering to a subject a therapeutically effective amount of an anti-CTGF antibody as described above, or a nucleic acid molecule as described above, or a pharmaceutical composition as described above, wherein the disease is preferably a fibrotic disease (preferably spontaneous pulmonary fibrosis, diabetic nephropathy, diabetic retinopathy, osteoarthritis, scleroderma, chronic heart failure, cirrhosis, or renal fibrosis), hypertension, diabetes, myocardial infarction, arthritis, CTGF-related proliferative disorders, atherosclerosis, glaucoma, or cancer (preferably acute lymphoblastic leukemia, dermatofibroma, breast cancer, angiolipoma, angioleiomyoma, connective tissue proliferative cancer, prostate cancer, ovarian cancer, colorectal cancer, pancreatic cancer, gastrointestinal cancer, or liver cancer).

[0085] Other aspects of this disclosure provide the use of the anti-CTGF antibody as described above, or the nucleic acid molecule as described above, or the pharmaceutical composition as described above in the preparation of a medicament for treating CTGF-related diseases, including fibrotic diseases (preferably spontaneous pulmonary fibrosis, diabetic nephropathy, diabetic retinopathy, osteoarthritis, scleroderma, chronic heart failure, cirrhosis, or renal fibrosis), hypertension, diabetes, myocardial infarction, arthritis, CTGF-related proliferative disorders, atherosclerosis, glaucoma, or cancer (preferably acute lymphoblastic leukemia, dermatofibroma, breast cancer, angiolipoma, angioleiomyoma, connective tissue proliferative cancer, prostate cancer, ovarian cancer, colorectal cancer, pancreatic cancer, gastrointestinal cancer, or liver cancer).

[0086] Other aspects of this disclosure provide anti-CTGF antibodies as described above, or nucleic acid molecules encoding anti-CTGF antibodies as described above, or pharmaceutical compositions as described above for treating CTGF-related diseases; said CTGF-related diseases include fibrotic diseases (preferably spontaneous pulmonary fibrosis, diabetic nephropathy, diabetic retinopathy, osteoarthritis, scleroderma, chronic heart failure, cirrhosis, or renal fibrosis), hypertension, diabetes, myocardial infarction, arthritis, CTGF-related proliferative disorders, atherosclerosis, glaucoma, or cancer (preferably acute lymphoblastic leukemia, dermatofibroma, breast cancer, angiolipoma, angioleiomyoma, connective tissue proliferative cancer, prostate cancer, ovarian cancer, colorectal cancer, pancreatic cancer, gastrointestinal cancer, or liver cancer). Attached Figure Description

[0087] Picture 1 The affinity of the humanized antibody derived from mab164 to human CTGF was detected by ELISA.

[0088] Picture 2Results of experiments on the inhibition of xenograft tumors in SU86.86 mice by humanized antibodies derived from mab147 or mab164. Detailed Implementation Invention Details

[0090] the term

[0091] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0092] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0093] Connective tissue growth factor (CTGF) is a 36kD cysteine-rich heparin-bound secretory glycoprotein originally isolated from human umbilical vein endothelial cells (see, for example, 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 (secretory glycoproteins). This family includes serum-induced immediate early gene product Cyr61, the inferred oncogene Nov, the ECM-associated protein FISP-12, src-induced gene CEF-10, Wnt-induced secretory protein WISP-3, and the antiproliferative 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) ProcNatl 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). The CCN protein is characterized by 38 conserved cysteine ​​residues, which constitute more than 10% of the total amino acid content and form a modular structure with N-terminal and C-terminal domains. The CTGF modular structure includes conserved motifs targeting insulin-like growth factor binding protein (IGF-BP) and von Willebrand factor (VWC) in the N-terminal domain, and platelet-reactive protein (TSP1) and cysteine ​​knot motifs in the C-terminal domain.

[0094] The "antibody" described in this disclosure refers to immunoglobulins. A full-length antibody is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in its heavy chain, it can be further divided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ chain or a λ chain.

[0095] The sequence of approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains varies considerably and is known as the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are called the constant region. The variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDR). Each light chain variable region (VL) and 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, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0096] The antibodies disclosed herein include murine antibodies, chimeric antibodies, and humanized antibodies.

[0097] The term "mouse antibody" in this disclosure refers to a monoclonal antibody against human CTGF prepared in accordance with the knowledge and skills in the art. Preparation involves injecting the test subject with CTGF antigen, followed by isolation of hybridomas expressing antibodies with the desired sequence or functional characteristics. In a preferred embodiment of this disclosure, the mouse anti-CTGF antibody or its antigen-binding fragment may further comprise a light chain constant region of a mouse κ, λ chain or a variant thereof, or further comprise a heavy chain constant region of mouse IgG1, IgG2, IgG3 or a variant thereof.

[0098] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by murine antibodies. To create a chimeric antibody, a hybridoma secreting a murine-specific monoclonal antibody is first established. Then, the variable region gene is cloned from the murine hybridoma cells, followed by the cloning of the constant region gene of the human antibody as needed. The murine variable region gene and the human constant region gene are then linked to form a chimeric gene, which is inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system. In a preferred embodiment of this disclosure, the antibody light chain of the chimeric antibody further comprises a light chain constant region of a human κ, λ chain, or a variant thereof. The antibody heavy chain of the CTGF chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, preferably comprising a heavy chain constant region of human IgG1, IgG2 or IgG4, or using an IgG1, IgG2 or IgG4 variant with amino acid mutations (e.g., L234A and / or L235A mutations, and / or S228P mutations).

[0099] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by grafting a mouse CDR sequence into a human antibody variable region framework, i.e., a human germline antibody framework sequence of different types. This overcomes the heterologous response induced by chimeric antibodies carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or publicly available references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available at www.mrccpe.com.ac.uk / vbase) and in Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse or reversion mutations can be performed on the said human antibody variable region framework sequence to maintain or enhance activity. The humanized antibodies disclosed herein also include humanized antibodies further demonstrated by yeast with affinity maturation mutations on the CDR.

[0100] Due to contact residues with the antigen, CDR transplantation can lead to a decrease in the affinity of the resulting antibody or its antigen-binding fragment for the antigen due to the framework residues in contact with the antigen. Such interactions may be a result of high somatic mutations. Therefore, it may still be necessary to transplant such donor framework amino acids into the framework of humanized antibodies. The amino acid residues involved in antigen binding from non-human antibodies or their antigen-binding fragments can be identified by examining the variable region sequence and structure of animal monoclonal antibodies. Residues in the CDR donor framework that differ from the species can be considered relevant. If the closest species cannot be determined, the sequence can be compared with a subclass common sequence or a common sequence of animal antibody sequences with a high percentage of similarity. Rare framework residues are thought to be a result of high somatic mutations, thus playing an important role in binding.

[0101] In one embodiment of this disclosure, the antibody or its antigen-binding fragment may further comprise a light chain constant region of a human or mouse κ, λ chain or a variant thereof, or further comprise a heavy chain constant region of a human or mouse IgG1, IgG2, IgG3, IgG4 or a variant thereof; it may comprise a heavy chain constant region of a human IgG1, IgG2 or IgG4, or use an IgG1, IgG2 or IgG4 variant with an amino acid mutation (e.g., L234A and / or L235A mutation, and / or S228P mutation).

[0102] The “conventional variants” of the human antibody heavy chain constant region and human antibody light chain constant region described in this disclosure refer to variants of the heavy chain constant region or light chain constant region derived from humans that do not alter the structure and function of the antibody variable region, as disclosed in the prior art. Exemplary variants include IgG1, IgG2, IgG3, or IgG4 heavy chain constant region variants that involve site-specific modifications and amino acid substitutions to the heavy chain constant region. Specific substitutions include YTE mutations, L234A and / or L235A mutations, S228P mutations, and / or mutations that obtain a knock-in-hole structure (resulting in a knock-Fc and hole-Fc combination in the antibody heavy chain). These mutations have been shown to give antibodies new properties without altering the function of the antibody variable region.

[0103] The terms "human antibody" (HuMAb), "human-derived antibody," "fully human antibody," and "completely human antibody" are used interchangeably. A human antibody can be an antibody derived from a genetically modified organism (GMO) that is "engineered" to produce a specific human antibody in response to antigenic stimulation and can be produced by any method known in the art. In some techniques, elemental elements of human heavy and light chain loci are introduced into cell lines derived from embryonic stem cell lines, where endogenous heavy and light chain loci in these cell lines are targeted for disruption. The GMO can synthesize human antibodies specific to human antigens, and the organism can be used to produce human antibody-secreting hybridomas. A human antibody can also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from the DNA of one or more individuals. Completely human antibodies can also be constructed using gene or chromosome transfection methods and phage display technology, or from in vitro activated B cells, all of which are known in the art.

[0104] The terms “full-length antibody,” “intact antibody,” “complete antibody,” and “all antibody” are used interchangeably herein to refer to an antibody in essentially its complete form, as defined below as an antigen-binding fragment. This term specifically refers to antibodies in which the light and heavy chains contain constant regions.

[0105] The “antibody” disclosed herein includes a “full-length antibody” and its “antigen-binding fragment”.

[0106] In some embodiments, the full-length antibody of this disclosure comprises a full-length antibody formed by linking a light chain variable region to a light chain constant region and a heavy chain variable region to a heavy chain constant region in the combinations of light and heavy chain variable regions in Tables 1, 2, and 3 below. Those skilled in the art can select different antibody sources for the light and heavy chain constant regions, such as light and heavy chain constant regions derived from human antibodies, according to actual needs. Furthermore, different combinations of light and heavy chain variable regions in Tables 1, 2, and 3 can form single-chain antibodies (scFv), Fab, or other antigen-binding fragments containing scFv or Fab.

[0107] Table 1. Combinations of light chain and heavy chain variable regions of humanized antibodies derived from mab147

[0108] Hu147-VH1 Hu147-11 Hu147-12 Hu147-13 Hu147-14 Hu147-15 Hu147-VH2 Hu147-21 Hu147-22 Hu147-23 Hu147-24 Hu147-25 Hu147-VH3 Hu147-31 Hu147-32 Hu147-33 Hu147-34 Hu147-35

[0109] Table 2. Combinations of light chain and heavy chain variable regions of the humanized mab164 antibody

[0110] Hu164-VL7 Hu164-57 Hu164-67 Hu164-77 Hu164-87 Hu164-VL8 Hu164-58 Hu164-68 Hu164-78 Hu164-88 Hu164-VL9 Hu164-59 Hu164-69 Hu164-79 Hu164-89

[0111] Table 3. Combinations of light chain and heavy chain variable regions of the mab95 humanized antibody

[0112] Hu95-VL1 Hu95-11 Hu95-21 Hu95-31 Hu95-41 Hu95-51 Hu95-VL2 Hu95-12 Hu95-22 Hu95-32 Hu95-42 Hu95-52 Hu95-VL3 Hu95-13 Hu95-23 Hu95-33 Hu95-43 Hu95-53 Hu95-VL4 Hu95-14 Hu95-24 Hu95-34 Hu95-44 Hu95-54

[0113] Note: Hu164-57 indicates that the humanized mab164 antibody Hu164-57 has a heavy chain variable region as described in Hu164-VH5 and a light chain variable region as described in Hu164-VL7, and so on.

[0114] 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 for antigen-binding function. Examples of binding fragments included in the term "antigen-binding fragment" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments connected by disulfide bridges on the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VH and VL domains on a single arm of the antibody; (v) dsFv, stable antigen-binding fragments formed by interchain disulfide bonds between VH and VL; and (vi) bispecific, bispecific, and multispecific antibodies containing fragments such as scFv, dsFv, and Fab. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked by synthetic linkers using recombinant methods, thereby enabling the production of a single protein chain (called a single-chain Fv (scFv) in which the VL and VH regions pair to form a monovalent molecule; 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 included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding moieties can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0115] Fab is an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity obtained by treating IgG antibody molecules with the protease papain (which cleaves the amino acid residue at position 224 of the H chain). Approximately half of the N-terminal side of the H chain and the entire L chain are linked together by disulfide bonds.

[0116] F(ab')2 is an antibody fragment with a molecular weight of approximately 100,000, possessing antigen-binding activity, and containing two Fab regions connected at the hinge position, obtained by digesting the portion below the two disulfide bonds in the hinge region of IgG with the enzyme pepsin.

[0117] Fab' is an antibody fragment with a molecular weight of about 50,000 and antigen-binding activity obtained by cleaving the disulfide bonds in the hinge region of the aforementioned F(ab')2. The Fab' of this disclosure can be produced by treating the F(ab')2 of this disclosure, which specifically recognizes CTGF and binds to the amino acid sequence or its three-dimensional structure in the extracellular region, with a reducing agent such as dithiothreitol.

[0118] In addition, the Fab' can be produced by inserting DNA encoding the Fab' fragment of an antibody into a prokaryotic or eukaryotic expression vector and then introducing the vector into a prokaryote or eukaryote to express the Fab'.

[0119] The terms “single-chain antibody,” “single-chain Fv,” or “scFv” refer to molecules containing a variable domain (or region; VH) of the antibody heavy chain and a variable domain (or region; VL) of the antibody light chain linked by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof, for example, using variants with 1–4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444–6448). Other connectors that may be used in this disclosure are described by 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.

[0120] A biantibody is an antibody fragment in which scFv or Fab is dimerized, and it is an antibody fragment with bivalent antigen-binding activity. In bivalent antigen-binding activity, the two antigens can be the same or different.

[0121] Bispecific and multispecific antibodies are antibodies that can bind to two or more antigens or antigenic determinants simultaneously, including scFv or Fab fragments that can bind to CTGF.

[0122] The biantibody of this disclosure can be produced by the following steps: obtaining the encoding cDNAs of VH and VL of the monoclonal antibody that specifically recognizes human CTGF and binds to the amino acid sequence or its three-dimensional structure in the extracellular region; constructing DNA encoding scFv such that the amino acid sequence of the peptide linker is 8 residues or less in length; inserting the DNA into a prokaryotic or eukaryotic expression vector; and then introducing the expression vector into a prokaryote or eukaryote to express the biantibody.

[0123] dsFv is obtained by linking polypeptides in which one amino acid residue in each VH and VL is replaced by a cysteine ​​residue via disulfide bonds between cysteine ​​residues. The amino acid residues to be replaced by cysteine ​​residues can be selected based on the prediction of the antibody's three-dimensional structure using known methods (Protein Engineering, 7, 697 (1994)).

[0124] The full-length antibody or antigen-binding fragment of this disclosure can be produced by the following steps: obtaining the cDNA encoding an antibody that specifically recognizes human CTGF and binds to the amino acid sequence or its three-dimensional structure in the extracellular region; constructing DNA encoding dsFv; inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector; and then introducing the expression vector into a prokaryote or eukaryote to express dsFv.

[0125] The term "amino acid difference" or "amino acid mutation" refers to an amino acid change or mutation in a variant protein or polypeptide compared to the original protein or polypeptide, including the insertion, deletion, or substitution of one, two, three, or more amino acids.

[0126] The term "antibody framework" or "FR region" refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain. Essentially, it is a variable domain without a CDR.

[0127] The terms “complementarity-determining region,” “CDR,” or “hypervariant region” refer to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitates antigen binding. Typically, three CDRs (HCDR1, HCDR2, HCDR3) exist in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, LCDR3) exist in each light chain variable region. The amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the “Kabat” numbering rule (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the “Chothia” numbering rule (see Al-Lazikani et al., (1997) JMB 273: 927-948), and the ImMunoGenTics (IMGT) numbering rule (Lefranc). MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003), etc. For example, for the classical format, following Kabat rules, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following Chothia rules, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acids in VL are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acids in VL are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acids in VL are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3). The amino acid residues are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). Combining the CDR definitions from Kabat and Chothia, the CDR consists of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) from human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) from human VL. Following the IMGT rules, the CDR amino acid residues in VH are approximately numbered 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), while those in VL are approximately numbered 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3).Following the IMGT rules, the CDR region of an antibody can be determined using the IMGT / DomainGap Align procedure. Unless otherwise stated, the antibody variable regions and CDR sequences involved in the embodiments of this disclosure are subject to the "Kabat" numbering rules.

[0128] The term "epitope" or "antigenic determinant" refers to a site on an antigen where an immunoglobulin or antibody specifically binds (e.g., a specific site on a CTGF molecule). Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a distinctive spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996).

[0129] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to a pre-defined epitope on an antigen. Typically, antibodies bind at a concentration of approximately less than 10... -8 M, for example, approximately less than 10 -9 M, 10 - 10 M, 10 -11 M, 10 -12 M or lower affinity (KD) binding.

[0130] The term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Typically, the antibodies of this disclosure have a dissociation equilibrium constant of less than approximately 10. -7 M, for example, less than approximately 10 -8 M or 10 -9 The dissociation equilibrium constant (KD) of M binds to CTGF. For example, in this disclosure, the affinity of the antibody for the cell surface antigen is determined by the FACS method to determine the KD value.

[0131] When the term "competition" is used in cases where antigen-binding proteins (e.g., neutralizing antigen-binding proteins or neutralizing antibodies) compete for the same epitope, it refers to competition between antigen-binding proteins, which is determined by an assay in which the antigen-binding protein to be detected (e.g., an antibody or an immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein (e.g., a ligand or a reference antibody) to a common antigen (e.g., a CTGF antigen or a fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another. These assays include: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, for example, Stahli et al., 1983, Methods in Enzymology 9: 242-253); solid-phase direct biotin-avidin EIA (see, for example, Kirkland et al., 1986, J. Immunol. 137: 3614-3619), solid-phase direct labeling assay, and solid-phase direct labeling sandwich assay (see, for example, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor). Press); solid-phase direct labeling of RIA with I-125 label (see, for example, Morel et al., 1988, Molec. Immunol. 25: 7-15); solid-phase direct biotin-avidin EIA (see, for example, Cheung et al., 1990, Virology 176: 546-552); and directly labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32: 77-82). The assay typically involves using a solid surface or cell to bind purified antigen loaded with either an unlabeled detection antigen-binding protein or a labeled reference antigen-binding protein. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cell in the presence of the detection antigen-binding protein. Typically, the detection antigen-binding protein is present in excess. Antigen-binding proteins identified by competitive assays (competitive 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 a neighboring epitope sufficiently close to the binding epitope of the reference antigen-binding protein, wherein the two epitopes spatially prevent each other from binding. Further details regarding methods for determining competitive binding are provided in the embodiments herein. Typically, when an excess of a competing antigen-binding protein is present, it will inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more of the specific binding of the reference antigen-binding protein to the common antigen.In some cases, the binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0132] As used herein, the term "nucleic acid molecule" refers to DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, preferably double-stranded DNA or single-stranded mRNA or modified mRNA. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0133] Amino acid sequence “identity” refers to the percentage of amino acid residues in a first sequence that are identical to those in a second sequence, after aligning the amino acid sequences and, where necessary, introducing gaps to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. For the purpose of determining the percentage of amino acid sequence identity, alignment can be performed in a variety of ways within the scope of the art, such as 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 parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0134] The term "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop to which an additional DNA segment can be linked. In another embodiment, the vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. The vectors disclosed herein are capable of autonomous replication in host cells that have been introduced into them (e.g., bacterial vectors with bacterial origins of replication and episodic mammalian vectors) or can be integrated into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome (e.g., non-episodic mammalian vectors).

[0135] Methods for producing and purifying antibodies and antigen-binding fragments are well-known in the prior art, such as those described in Cold Spring Harbor's Guide to Antibody Experimentation, Chapters 5-8 and 15. For example, mice can be immunized with human CTGF or fragments thereof, and the resulting antibodies can be refolded, purified, and sequenced amino acids using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The disclosed antibodies or antigen-binding fragments have one or more human FR regions added to a non-human CDR region using genetic engineering methods. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr by comparing with the IMGT Human Antibody Variable Region Germplasm Database and MOE software, or from the Immunoglobulin Journal, 2001 ISBN012441351.

[0136] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; members of the Bacillaceae family, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line), 293 cells, and NSO cells.

[0137] The engineered antibody or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more preferred prior art, mammalian expression systems lead to glycosylation of the antibody, particularly at the highly conserved N-terminal site in the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human CTGF. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. The culture medium secreting the antibody can be purified using conventional techniques, such as using an A or G Sepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody is then eluted using a pH gradient, and the antibody fragments are detected by SDS-PAGE and collected. The antibody can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, e.g., at -70°C, or lyophilized.

[0138] "Administration," "giving," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "giving," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Administration," "giving," and "treatment" also mean, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo, such as cells. "Treatment," when applied to humans, veterinary, or research subjects, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0139] "Treatment" means administering an oral or topical therapeutic agent, such as a composition comprising any of the compounds disclosed herein, to a patient who has symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more symptoms of the disease, inducing the regression of such symptoms or inhibiting their progression to any clinically measured extent. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical test method commonly used by a physician or other healthcare professional to assess the severity or progression of the symptoms. Although the embodiments of this disclosure (e.g., treatment methods or products) may be ineffective in alleviating symptoms of each target disease, they should reduce symptoms of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0140] "Conservative modification" or "conservative substitution" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity), allowing for frequent alterations without changing the protein's biological activity. Those skilled in the art will appreciate that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to disrupt biological activity. Exemplary conserved substitutions are described below.

[0141]

[0142] "Effective amount" or "effective dose" means the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic outcome. For prophylactic use, beneficial or desired outcomes include eliminating or reducing risk, mitigating severity, or delaying the onset of a condition, including the condition itself, its complications, and the biochemical, histological, and / or behavioral symptoms of intermediate pathological phenotypes present during the development of the condition. For therapeutic use, beneficial or desired outcomes include clinical outcomes such as reducing the incidence of various target antigen-related conditions of this disclosure or improving one or more symptoms of said conditions, reducing the dosage of other agents required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of the target antigen-related condition of this disclosure in a patient.

[0143] "Exogenous" refers to substances that are produced outside of an organism, cell, or human body, depending on the circumstances.

[0144] "Endogenous" refers to substances that are produced in cells, organisms, or the human body, depending on the circumstances.

[0145] "Homology" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. Two compared sequences are homologous at positions occupied by the same bases or amino acid monomer subunits; for example, if every position in two DNA molecules is occupied by adenine. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared multiplied by 100. For example, in optimal sequence alignment, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in two sequences match or are homologous, then the two sequences are 95% homologous. Typically, comparisons are made when aligning two sequences to give the maximum percentage of homology. For example, comparisons can be performed using the BLAST algorithm, where the algorithm's parameters are chosen to give the maximum match between the sequences over the entire length of each reference sequence. The following references relate to the BLAST algorithm commonly used in 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 common BLAST algorithms, such as those provided by NCBI BLAST, are also well-known to those skilled in the art.

[0146] The terms “cell,” “cell line,” and “cell culture” used herein are used interchangeably, and all such names include progeny. Therefore, the terms “transformation” and “transformed cell” include primary test cells and cultures derived from them, regardless of the number of transfections. It should also be understood that, due to intentional or unintentional mutations, all progeny cannot be exactly identical in terms of DNA content. This includes mutant progeny with the same function or biological activity as those screened from the original transformed cells. Where different names are intended, the context will be clear.

[0147] As used herein, “polymerase chain reaction” or “PCR” refers to a procedure or technique in which trace amounts of specific portions of nucleic acids, RNA, and / or DNA are amplified as described, for example, in U.S. Patent No. 4,683,195. Generally, sequence information from the ends or beyond of the target region is required to allow the design of oligonucleotide primers; these primers are identical or similar in sequence to the corresponding strand of the template to be amplified. The 5' nucleotides of both primers may be congruent to the ends of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage, or plasmid sequences transcribed from total cellular RNA, etc. See generally Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51:263; Erlich, ed., (1989) PCR TECHNOLOGY (Stockton Press, NY). The PCR used herein is considered one example, but not the only example, of a nucleic acid polymerase reaction method for amplifying nucleic acid test samples, which involves using known nucleic acids as primers and a nucleic acid polymerase to amplify or produce specific portions of nucleic acids.

[0148] "Separated" refers to a purified state, and in this context means that the specified molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth media. Generally, the term "separated" is not intended to mean the complete absence of these materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compound as described herein.

[0149] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, and the description includes the circumstances under which the event or circumstances may or may not occur.

[0150] "Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and its biological activity.

[0151] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for delivering antibody or antigen-binding fragments. Carriers can be anti-adhesion agents, adhesives, coatings, disintegrants, fillers or diluents, preservatives (such as antioxidants, antibacterial agents, or antifungal agents), sweeteners, absorption delay agents, wetting agents, emulsifiers, buffers, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (such as olive oil), saline, buffer solutions, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

[0152] Furthermore, this disclosure includes pharmaceutical agents for treating diseases associated with target antigen (e.g., CTGF)-positive cells, said pharmaceutical agents comprising an anti-CTGF antibody of this disclosure or an antigen-binding fragment thereof as an active ingredient. The active ingredient is administered to a subject in a therapeutically effective amount to treat the subject's CTGF-positive cell-related disease. The therapeutically effective amount is 0.1-3000 mg per unit dose of the composition containing the aforementioned full-length antibody specifically binding to human GCGR or an antigen-binding fragment thereof.

[0153] There are no limitations on the diseases related to CTGF in this disclosure, as long as they are CTGF-related diseases. For example, the molecularly induced therapeutic response of this disclosure can be achieved by binding to human CTGF and then inhibiting the binding of CTGF to its receptor / ligand, or by killing tumor cells that overexpress CTGF. Therefore, when in preparations and formulations suitable for therapeutic applications, the molecules of this disclosure are of great use to individuals suffering from tumors or cancers, optionally including pulmonary fibrosis, renal fibrosis, tumor formation and growth, glaucoma, proliferative cellular diseases, cataracts, choroidal neovascularization, retinal detachment, proliferative vitreoretinopathy, macular degeneration, diabetic retinopathy, corneal scarring and opacity, cysts, reduced vascular calcification, pancreatic ductal adenocarcinoma, pancreatic cancer, melanoma, radiation-induced fibrosis (RIF), idiopathic pulmonary fibrosis, lung remodeling diseases selected from the group consisting of asthma, chronic bronchitis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, emphysema, etc., preferably pancreatic cancer, pulmonary fibrosis, and renal fibrosis.

[0154] Furthermore, this disclosure relates to methods for immunodetection or determination of target antigens (e.g., CTGF), reagents for immunodetection or determination of target antigens (e.g., CTGF), methods for immunodetection or determination of cells expressing target antigens (e.g., CTGF), and diagnostic agents for diagnosing diseases associated with target antigen (e.g., CTGF) positive cells, comprising an antibody or antibody fragment thereof that specifically recognizes the target antigen (e.g., human CTGF) and binds to the amino acid sequence or three-dimensional structure of the extracellular region as an active ingredient.

[0155] In this disclosure, the method for detecting or determining the amount of a target antigen (e.g., CTGF) can be any known method. For example, it includes immunoassay or assay methods.

[0156] Immunological detection or assay methods are methods that use labeled antigens or antibodies to detect or measure the amount of antibody or antigen. Examples of immunological detection or assay methods include radiolabeled antibody methods (RIA), enzyme immunoassays (EIA or ELISA), fluorescence immunoassays (FIA), luminescent immunoassays, Western blotting, physicochemical methods, etc.

[0157] The aforementioned diseases associated with CTGF-positive cells can be diagnosed by detecting or measuring cells expressing CTGF using antibodies or antibody fragments disclosed herein.

[0158] To detect cells expressing peptides, known immunoassay methods can be used, with immunoprecipitation, fluorescent cell staining, and immunohistochemical staining being preferred. Alternatively, fluorescent antibody staining using the FMAT8100HTS (Applied Biosystem) can be employed.

[0159] In this disclosure, there are no particular limitations on the live sample used for detecting or measuring the target antigen (e.g., CTGF), as long as it has the potential to contain cells expressing the target antigen (e.g., CTGF), such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture medium.

[0160] Depending on the required diagnostic method, diagnostic reagents containing the monoclonal antibody or antibody fragment thereof of this disclosure may also contain reagents for performing an antigen-antibody reaction or for detecting the reaction. Reagents for performing the antigen-antibody reaction include buffers, salts, etc. Reagents for detection include those commonly used in immunoassay or assay methods, such as labeled second antibodies that recognize the monoclonal antibody, its antibody fragment, or conjugates, and substrates corresponding to the labeled antibodies.

[0161] Details of one or more embodiments of this disclosure are set forth in the foregoing description. While this disclosure may be practiced or tested using any methods and materials similar to or the same as those described herein, preferred methods and materials are described below. Other features, objects, and advantages of this disclosure will become apparent from the description and claims. In the description and claims, the singular form includes plural references unless clearly indicated otherwise in the context. Unless otherwise defined, all technical and scientific terms used herein have their general meaning as understood by one of ordinary skill in the art to which this disclosure pertains. All patents and publications referenced in the description are incorporated herein by reference. The following embodiments are presented to illustrate preferred embodiments of this disclosure more fully. These embodiments should not be construed in any way as limiting the scope of this disclosure, which is defined by the claims.

[0162] Example

[0163] Example 1. Preparation of CTGF antigen and antibody

[0164] I. Antigen Design and Expression

[0165] Using human CTGF protein (Genbank ID: NM_001901.2) as a template, the amino acid sequences of antigens and detection proteins were designed. Different tags could be optionally fused to the CTGF protein or fragments, and the sequences were cloned into pTT5 or pXC vectors, respectively. The antigens were transiently expressed in 293 cells or stably expressed in LONZA CHO cells to obtain the antigens and detection proteins encoding this disclosure. Unless otherwise specified, all CTGF antigens referred to below are human CTGF.

[0166] Human CTGF full-length protein (for immunization), SEQ ID NO:1

[0167] QNCSGPCRCPDEPAPRCPAGVSLVLDGCGCCRVCAKQLGELCTERDPCDPHKGLFCDFGSPANRKIGVCTAKDGAPCIFGGTVYRSGESFQSSCKYQCTCLDGAVGCMPLCSMDVRLPSPDCPFPRRVKLPGKCCEEWVCDEPKDQTVVGPALAAYRLEDT FGPDPTMIRANCLVQTTEWSACSKTCGMGISTRVTNDNASCRLEKQSRLCMVRPCEADLEENIKKGKKCIRTPKISKPIKFELSGCTSMKTYRAKFCGVCTDGRCCTPHRTTTLPVEFKCPDGEVMKKNMMFIKTCACHYNCPGDNDIFESLYYRKMYGDMA

[0168] CTGF-his (a fusion protein of mature human CTGF protein and his tag) can be used for detection, SEQ ID NO:2

[0169]

[0170] (Note: Single underscores indicate signal peptides, italics indicate the human CTGF coding region, and double underscores indicate the His tag.)

[0171] mCTGF-mFc (a fusion protein of the mouse CTGF coding region and the mouse Fc tag, which can be used for detection) SEQ ID NO:3

[0172]

[0173] (Note: Single underline indicates signal peptide, italics indicate mouse CTGF coding region, and double underline indicates mFc tag.)

[0174] Mouse CTGF protein-His tag, SEQ ID NO:4

[0175]

[0176] (Note: Single underline indicates signal peptide, italics indicate mouse CTGF coding region, and double underline indicates his tag.)

[0177] Cryocyena monkey CTGF-Fc (a fusion protein of the CTGF coding region and Fc in cynocyena monkeys, which can be used for detection), SEQ ID NO:5

[0178]

[0179] (Note: Single underscores indicate signal peptides, italics indicate the CTGF coding region of cynomolgus monkeys, and double underscores indicate Fc tags.)

[0180] II. Purification of CTGF-related recombinant proteins, and purification of anti-human CTGF hybridoma antibodies and recombinant antibodies.

[0181] 1. Protein G affinity chromatography was used to separate and purify the hybridoma supernatant.

[0182] For the purification of mouse hybridoma supernatant, Protein G is the preferred affinity chromatography method. After centrifuging the cultured hybridomas, collect the supernatant and adjust its pH to neutral by adding 10-15% of the supernatant volume of 1M Tris-HCl (pH 8.0-8.5). Wash the Protein G column 3-5 column volumes with 6M guanidine hydrochloride, followed by 3-5 column volumes of pure water. Equilibrate the column with 3-5 column volumes of 1×PBS (pH 7.4). Load the cell supernatant at a low flow rate, controlling the flow rate to maintain a retention time of approximately 1 min or longer. Wash the column with 1×PBS (pH 7.4) for 3-5 column volumes until the UV absorbance returns to baseline. Elute the sample using 0.1M acetate / sodium acetate (pH 3.0) buffer, collecting the elution peak based on UV detection. Quickly adjust the pH of the elution product to 5-6 using 1M Tris-HCl (pH 8.0). The elution products can be replaced with solutions using methods well known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube and replacement of the solution with the desired buffer system, or replacement with the desired buffer system using size exclusion columns such as G-25 desalting, or removal of polymeric components from the elution products using high-resolution size exclusion columns such as Superdex200 to improve sample purity.

[0183] 2. Protein A affinity chromatography to purify antibodies

[0184] First, the supernatant from the cell culture expressing the antibody was collected by high-speed centrifugation. The Protein A affinity column was washed 3-5 column volumes with 6M guanidine hydrochloride, followed by 3-5 column volumes of pure water. The column was then equilibrated with 3-5 column volumes of 1×PBS (pH 7.4). The cell supernatant was loaded for binding at a low flow rate, controlling the flow rate to maintain a retention time of approximately 1 minute or longer. After binding, the column was washed with 3-5 column volumes of 1×PBS (pH 7.4) until the UV absorbance returned to baseline. The sample was eluted using 0.1M acetate / sodium acetate (pH 3.0-3.5) buffer, and the elution peak was collected based on UV detection. The pH of the elution product was rapidly adjusted to 5-6 using 1M Tris-HCl (pH 8.0). The elution products can be replaced with solutions using methods well known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube and replacement of the solution with the desired buffer system, or desalting using size exclusion columns such as G-25 to replace the buffer system with the desired buffer system, or high-resolution size exclusion columns such as Superdex 200 to remove aggregate components from the elution products to improve sample purity.

[0185] 3. Purification of CTGF-related recombinant proteins such as human CTGF-his using nickel column chromatography.

[0186] Cell expression supernatant samples were centrifuged at high speed to remove impurities, and the buffer was replaced with PBS. Imidazole was added to a final concentration of 5 mM. The nickel column was equilibrated with PBS containing 5 mM imidazole and washed 2-5 column volumes. The supernatant sample was then loaded onto the column for binding. Nickel columns from different companies can be used as media. The column was washed with PBS containing 5 mM imidazole until the A280 reading dropped to baseline. The column was then washed with PBS + 10 mM imidazole to remove non-specifically bound proteins, and the eluent was collected. The target protein was then eluted with PBS containing 300 mM imidazole, and the elution peak was collected.

[0187] The collected elution product was concentrated and further purified by gel chromatography using a Superdex 200 (GE) with PBS as the mobile phase. Aggregates and other protein peaks were removed, and the elution peak of the target product was collected. The obtained protein was then identified by electrophoresis, peptide mapping, and LC-MS before being aliquoted and used.

[0188] 4. Protein A affinity chromatography was used to purify human CTGF-Fc, cynomolgus monkey CTGF-Fc, and other CTGF-related recombinant proteins.

[0189] First, collect the culture supernatant by high-speed centrifugation. Wash the Protein A affinity column with 3-5 column volumes of 6M guanidine hydrochloride, followed by 3-5 column volumes of pure water. Equilibrate the column with 3-5 column volumes of 1×PBS (pH 7.4). Load the cell supernatant at a low flow rate to bind, controlling the flow rate to maintain a retention time of approximately 1 minute or longer. After binding, wash the column with 3-5 column volumes of 1×PBS (pH 7.4) until the UV absorbance returns to baseline. Elute the sample using 0.1M acetate / sodium acetate (pH 3.0-3.5) buffer, and collect the elution peak based on UV detection.

[0190] The collected elution product was concentrated and further purified by gel chromatography using a Superdex 200 (GE) with PBS as the mobile phase. Aggregates and other protein peaks were removed, and the elution peak of the target product was collected. The obtained protein was then identified by electrophoresis, peptide mapping, and LC-MS before being aliquoted and used.

[0191] Example 2. Preparation of anti-human CTGF monoclonal antibody

[0192] I. Immunity

[0193] Anti-human CTGF monoclonal antibodies are generated by immunizing mice. SJL white female mice aged 6-8 weeks are used in the experiment (Vital River Laboratory Animal Technology Co., Ltd. Beijing, Animal Production License No.: SCXK (Beijing) 2012-0001). Feeding environment: SPF grade. After the mice are purchased, they are reared in the laboratory environment for 1 week, with a 12 / 12-hour light / dark cycle, a temperature of 20-25°C, and a humidity of 40-60%. The acclimatized mice are immunized according to the following protocol. The immunizing antigens are CTGF (R&D) and mCTGF-mFc.

[0194] Immunization protocol: Mice are immunized with CTGF protein at a dose of 25 micrograms per mouse per injection via intraperitoneal injection. On day 0, 100 μl per mouse is injected intraperitoneally (IP), followed by booster immunizations every 14 days. Blood is collected on days 21, 35, 49, and 63, and the antibody titer in the mouse serum is determined by ELISA. After 7-9 immunizations, mice with high serum antibody titers that have reached a plateau are selected for splenocyte fusion. Three days before splenocyte fusion, a booster immunization is performed, and 25 μg per mouse of CTGF protein antigen solution prepared in normal saline is injected intraperitoneally (IP).

[0195] II. Splenocyte Fusion

[0196] Splenic lymphocytes are fused with myeloma cells Sp2 / 0 via a PEG-mediated fusion procedure CRL-8287 TM ) to obtain hybridoma cells. The fused hybridoma cells are resuspended in complete medium (IMDM medium containing 20% FBS, 1×HAT, 1×OPI) at a density of 0.5-1×10 6 cells / ml, and seeded into 96-well plates at 100 μl per well. After incubation at 37°C with 5% CO₂ for 3-4 days, 100 μl of HAT complete medium is supplemented per well, and the culture is continued for 3-4 days until pinhead-sized clones form. The supernatant is removed, 200 μl of HT complete medium (IMDM medium containing 20% FBS, 1×HT and 1×OPI) is added per well, and the cells are cultured at 37°C with 5% CO₂ for 3 days before ELISA detection.

[0197] III. Screening of Hybridoma Cells

[0198] According to the growth density of the hybridoma cells, the hybridoma culture supernatant is detected by a binding ELISA method. The supernatant of the positive wells detected by binding ELISA is then subjected to a binding assay with monkey CTGF / mouse CTGF / human CTGF proteins (the specific method is the same as that in Example 3). The cells in the wells that test positive in the protein ELISA binding assay are promptly expanded, frozen for storage, and subjected to 2 to 3 rounds of subcloning until single-cell clones are obtained.

[0199] Each subcloned cell was also subjected to CTGF binding ELISA. Hybridoma clones were obtained through the above experiments, and antibodies were further prepared using serum-free cell culture. The antibodies were purified according to the purification examples for use in the detection cases.

[0200] IV. Hybridoma Positive Clonal Sequence Determination

[0201] The procedure for cloning sequences from positive hybridomas is as follows: Hybridoma cells in logarithmic growth phase are collected, and RNA is extracted using Trizol (Invitrogen, Cat No. 15596-018) according to the kit instructions. RNA is then cloned using PrimeScript. TM Reverse transcription was performed using a Reverse Transcriptase kit (Takara, Cat No. 2680A). The cDNA obtained from reverse transcription was amplified by PCR using a mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503), and the amplified products were sent to a sequencing company for sequencing. The variable region amino acid sequences of the mouse anti-CTGF antibodies mab147, mab164, and mab95 obtained by sequencing are as follows:

[0202] The amino acid sequence of mab147 VH is shown in SEQ ID NO:6.

[0203]

[0204] The amino acid sequence of mab147 VL is shown in SEQ ID NO:7.

[0205]

[0206] The amino acid sequence of mab164 VH is shown in SEQ ID NO:8.

[0207]

[0208] The amino acid sequence of mab164 VL is shown in SEQ ID NO:9.

[0209]

[0210] The amino acid sequence of mab95 VH is shown in SEQ ID NO:69.

[0211]

[0212] The amino acid sequence of mab95 VL is shown in SEQ ID NO:70.

[0213]

[0214] In the variable region sequences of the above mab147, mab164, and mab95 antibodies, the italicized sequences are FR sequences, and the underlined italicized sequences are CDR sequences. The sequence order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0215] Table 4. Amino acid sequence listing of the CDR regions of the heavy and light chains of anti-CTGF antibodies*

[0216]

[0217] *The CDRs in the table are determined by the Kabat numbering rules.

[0218] V. Preparation of Human IgG1 Chimeric Antibody

[0219] The variable region coding gene sequences of candidate molecules mab147, mab164, and mab95 were obtained through amplification and sequencing. Primers were designed based on the sequencing sequences, and the sequenced genes were used as templates to construct the VH / VK gene fragments of each antibody via PCR. These fragments were then homologously recombinated with the expression vector pHr (containing the signal peptide and the hIgG1 / hkappa / hlambda constant region gene (CH1-Fc / CL) fragment) to construct the full-length expression plasmids VH-CH1-Fc-pHr / VL-CL-pHr for the recombinant chimeric antibodies, forming three chimeric antibodies: Ch147, Ch164, and Ch95.

[0220] VI. Humanization of mouse-derived anti-human CTGF antibodies

[0221] By comparing the germline gene database of human antibody heavy and light chain variable regions (IMGT, http: / / imgt.cines.fr) with MOE (Molecular Operating Environment) software, germline genes of heavy and light chain variable regions with high homology to murine antibodies were selected as templates. The CDRs of murine antibodies were then transplanted into the corresponding human templates to form variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0222] 1. Humanization of mab147

[0223] The humanized light chain templates for the murine antibody mab147 are IGKV3-11*01 and IGKJ2*01 or IGKV1-39*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV3-72*01 and IGHJ1*01. The CDR of the murine antibody mab147 is transplanted into its humanized templates. Then, the FR amino acid portion of the humanized antibody is modified by reverse mutation. The reverse mutations in the light chain variable region include one or more of 4L, 36F, 43S, 45K, 47W, 58V or 71Y, and the reverse mutations in the heavy chain variable region include one or more of 28S, 30N, 49A, 75E, 76S, 93V, 94E or 104D (the positions of the reverse mutation sites in the light chain variable region and the heavy chain variable region are determined according to the Kabat numbering rules), resulting in light chain variable regions and heavy chain variable regions with different sequences. This yields a humanized antibody containing mab147 in the CDR, with the variable region sequence as follows:

[0224] The specific sequence of the variable region of the mAb147 humanized antibody is as follows:

[0225] The amino acid sequence of Hu147-VL1 is shown in SEQ ID NO:22:

[0226]

[0227] The amino acid sequence of Hu147-VL2 is shown in SEQ ID NO:23:

[0228]

[0229] The amino acid sequence of Hu147-VL3 is shown in SEQ ID NO:24:

[0230]

[0231] The amino acid sequence of Hu147-VL4 is shown in SEQ ID NO:25:

[0232]

[0233] The amino acid sequence of Hu147-VL5 is shown in SEQ ID NO:26:

[0234]

[0235] The amino acid sequence of Hu147-VH1 is shown in SEQ ID NO:27:

[0236]

[0237] The amino acid sequence of Hu147-VH2 is shown in SEQ ID NO:28:

[0238]

[0239] The amino acid sequence of Hu147-VH3 is shown in SEQ ID NO:29:

[0240]

[0241] Note: The underlined part is the CDR sequence defined by the Kabat rule, and the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0242] 2. Humanization of mab164

[0243] The humanized light chain templates of the murine antibody mab164 were composed of IGLV7-43*01 and IGLJ2*01, IGLV8-61*01 and IGLJ2*01, or IGLV1-40*02 and IGLJ2*01, respectively. The humanized heavy chain templates were composed of IGHV2-26*01 and IGHJ6*01, or IGHV4-31*02 and IGHJ6*01, respectively. The CDR of the murine antibody mab164 was transplanted into its human templates, and then... The FR amino acid fraction of the humanized antibody is modified by reversion mutation. The reversion mutations in the light chain variable region include one or more of 36V, 44F, 46G, or 49G, and the reversion mutations in the heavy chain variable region include one or more of 44G, 49G, 27F, 48L, 67L, 71K, 78V, or 80F (the positions of the reversion mutation sites are determined according to the Kabat numbering rules). This yields the humanized heavy chain and light chain of the mab164 antibody, with the following variable region sequence:

[0244] The specific sequence of the variable region of the mAb164 humanized antibody is as follows:

[0245] The amino acid sequence of Hu164-VL7 is shown in SEQ ID NO:30:

[0246]

[0247] The amino acid sequence of Hu164-VL8 is shown in SEQ ID NO:31:

[0248]

[0249] The amino acid sequence of Hu164-VL9 is shown in SEQ ID NO:32:

[0250]

[0251] The amino acid sequence of Hu164-VH5 is shown in SEQ ID NO:33:

[0252]

[0253] The amino acid sequence of Hu164-VH6 is shown in SEQ ID NO:34:

[0254]

[0255] The amino acid sequence of Hu164-VH7 is shown in SEQ ID NO:35:

[0256]

[0257] The amino acid sequence of Hu164-VH8 is shown in SEQ ID NO:36:

[0258]

[0259]

[0260] Note: In the Hu164 antibody sequence above, the underlined part is the CDR sequence, and the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDR sequence is defined according to the Kabat rule.

[0261] 3. Humanization modifications of mab95

[0262] The humanized light chain templates for the murine antibody mab95 were IGKV3-20*02 and IGKV1-40*01, and the humanized heavy chain template was IGHV1-3*01. The CDR of the murine antibody mab95 was transplanted into its humanized templates. Then, the FR portion of the humanized antibody underwent reverse mutation modification. Reverse mutations in the light chain variable region included one or more of 45P, 46W, 48Y, 69S, or 70Y, and reverse mutations in the heavy chain variable region included one or more of 27F, 38K, 48I, 67K, 68A, 70L, or 72F (the positions of the reverse mutation sites in the light and heavy chain variable regions were determined according to Kabat numbering rules), resulting in light and heavy chain variable regions with different sequences. Thus, a humanized antibody containing the CDR of mab95 was obtained, and its variable region sequence is as follows:

[0263] The specific sequence of the variable region of the mAb95 humanized antibody is as follows:

[0264] The amino acid sequence of Hu95-VL1 is shown in SEQ ID NO:77:

[0265]

[0266] The amino acid sequence of Hu95-VL2 is shown in SEQ ID NO:78:

[0267]

[0268] The amino acid sequence of Hu95-VL3 is shown in SEQ ID NO:79:

[0269]

[0270] The amino acid sequence of Hu95-VL4 is shown in SEQ ID NO:80:

[0271]

[0272] The amino acid sequence of Hu95-VH1 is shown in SEQ ID NO:81:

[0273]

[0274] The amino acid sequence of Hu95-VH2 is shown in SEQ ID NO:82:

[0275]

[0276] The amino acid sequence of Hu95-VH3 is shown in SEQ ID NO:83:

[0277]

[0278]

[0279] The amino acid sequence of Hu95-VH4 is shown in SEQ ID NO:84:

[0280]

[0281] The amino acid sequence of Hu95-VH5 is shown in SEQ ID NO:85:

[0282]

[0283] Note: The underlined part is the CDR sequence defined by the Kabat rule, and the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0284] Among them, the CDR region of Hu95-VH5 has been further modified, with HCDR1 as shown in SEQ ID NO:102(NYAIH), HCDR2 as shown in SEQ ID NO:103(LVYPYTGGTAYNQKFKD), and HCDR3 as shown in SEQ ID NO:104(WGMIPGTNSYFDV).

[0285] 4. Construction and expression of humanized full-length anti-CTGF antibody

[0286] Primers were designed for PCR to construct the VH / VL gene fragments of each humanized antibody, which were then homologously recombinated with the expression vector pHr (containing a signal peptide and a constant region gene (CH / CL) fragment) to construct the full-length antibody expression vector VH-CH-pHr / VL-CL-pHr. The constant region of the humanized antibody can be selected from the light chain constant regions of human κ and λ chains, as well as the heavy chain constant regions selected from IgG1, IgG2, IgG3, or IgG4 or their variants. Non-limiting implementations include optimizing the design of the constant regions of human IgG1, IgG2, and IgG4 to improve antibody function. For example, mutations at the M252Y / S254T / T256E (“YTE”) site in the constant region can prolong the antibody's half-life, and mutations at other constant region sites such as S228P, F234A, and L235A can also be performed.

[0287] An example of a constant region sequence for an anti-CTGF humanized antibody is shown below:

[0288] The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:37 (the name of the formed full-length antibody heavy chain is suffixed with w):

[0289] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0290] The amino acid sequence of the heavy chain constant region of the YTE-modified antibody is shown in SEQ ID NO:38 (the name of the formed full-length antibody heavy chain is suffixed with y):

[0291] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0292] The amino acid sequence of the kappa constant region of the light chain of the anti-CTGF humanized antibody is shown in SEQ ID NO:39 (the name of the formed full-length antibody heavy chain is suffixed with k):

[0293] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0294] The amino acid sequence of the lambda constant region of the light chain of the anti-CTGF humanized antibody is shown in SEQ ID NO:40 (the name of the formed full-length antibody heavy chain is suffixed with l):

[0295] GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC

[0296] The full-length humanized antibody derived from mab147 formed by linking the heavy chain variable region of the humanized antibody to the heavy chain constant region shown in SEQ ID NO:37, and linking the light chain variable region of the humanized antibody to the light chain kappa constant region shown in SEQ ID NO:39, includes:

[0297] Table 5

[0298] Hu147-VH1 Hu147-11wk Hu147-12wk Hu147-13wk Hu147-14wk Hu147-15wk Hu147-VH2 Hu147-21wk Hu147-22wk Hu147-23wk Hu147-24wk Hu147-25wk Hu147-VH3 Hu147-31wk Hu147-32wk Hu147-33wk Hu147-34wk Hu147-35wk

[0299] The full-length humanized antibody derived from mab147 formed by linking the variable region of the heavy chain of the humanized antibody to the YTE variant of the heavy chain constant region shown in SEQ ID NO:38, and linking the variable region of the light chain of the humanized antibody to the kappa constant region of the light chain shown in SEQ ID NO:39, includes:

[0300] Table 6

[0301] Hu147-VH1 Hu147-11yk Hu147-12yk Hu147-13yk Hu147-14yk Hu147-15yk Hu147-VH2 Hu147-21yk Hu147-22yk Hu147-23yk Hu147-24yk Hu147-25yk Hu147-VH3 Hu147-31yk Hu147-32yk Hu147-33yk Hu147-34yk Hu147-35yk

[0302] The full-length humanized antibody derived from mab164 formed by linking the variable region of the humanized antibody heavy chain with the constant region of the heavy chain shown in SEQ ID NO:37, and linking the variable region of the humanized antibody light chain with the constant region of the light chain lambda shown in SEQ ID NO:40, includes:

[0303] Table 7

[0304]

[0305]

[0306] The full-length humanized antibody derived from mab164 formed by linking the variable region of the humanized antibody heavy chain with the YTE variant of the heavy chain constant region shown in SEQ ID NO:38, and linking the variable region of the humanized antibody light chain with the lambda constant region of the light chain shown in SEQ ID NO:40, includes:

[0307] Table 8

[0308] Hu164-VL7 Hu164-57yl Hu164-67yl Hu164-77yl Hu164-87yl Hu164-VL8 Hu164-58yl Hu164-68yl Hu164-78yl Hu164-88yl Hu164-VL9 Hu164-59yl Hu164-69yl Hu164-79yl Hu164-89yl

[0309] The full-length humanized antibody derived from mab95 formed by linking the variable region of the humanized antibody heavy chain with the constant region of the heavy chain shown in SEQ ID NO:37, and linking the variable region of the humanized antibody light chain with the constant region of the light chain kappa shown in SEQ ID NO:39, includes:

[0310] Table 9

[0311] Hu95-VL1 Hu95-11wk Hu95-21wk Hu95-31wk Hu95-41wk Hu95-51wk Hu95-VL2 Hu95-12wk Hu95-22wk Hu95-32wk Hu95-42wk Hu95-52wk Hu95-VL3 Hu95-13wk Hu95-23wk Hu95-33wk Hu95-43wk Hu95-53wk Hu95-VL4 Hu95-14wk Hu95-24wk Hu95-34wk Hu95-44wk Hu95-54wk

[0312] The full-length humanized antibody derived from mab95 formed by linking the variable region of the humanized antibody heavy chain with the YTE variant of the heavy chain constant region shown in SEQ ID NO:38, and linking the variable region of the humanized antibody light chain with the kappa constant region of the light chain shown in SEQ ID NO:39, includes:

[0313] Table 10

[0314] Hu95-VL1 Hu95-11yk Hu95-21yk Hu95-31yk Hu95-41yk Hu95-51yk Hu95-VL2 Hu95-12yk Hu95-22yk Hu95-32yk Hu95-42yk Hu95-52yk Hu95-VL3 Hu95-13yk Hu95-23yk Hu95-33yk Hu95-43yk Hu95-53yk Hu95-VL4 Hu95-14yk Hu95-24yk Hu95-34yk Hu95-44yk Hu95-54yk

[0315] An example full-length amino acid sequence of an anti-CTGF antibody is as follows:

[0316] Table 11 Light or Heavy Chain of Full-Length Antibodies

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325] The sequence of the CTGF antibody mAb1 (derived from CN1829740B, antibody name pamrevlumab, also known as FG-3019) used as a positive control is as follows:

[0326] Heavy chain: (SEQ ID NO:67)

[0327] EGQLVQSGGGLVHPGGSLRLSCAGSGFTFSSYGMHWVRQAPGKGLEWVSSGIGTGGGTYSTDSVKGRFTISRDNAKNSLYLQMNSLRAEDMAVYYCARGDYYGSGSFFDCWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0328] Light chain: (SEQ ID NO:68)

[0329] DIQMTQSPSSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0330] Example 3. Detection of CTGF antibody binding activity

[0331] I. ELISA assay for binding of CTGF antibody to human CTGF protein

[0332] The binding affinity of anti-human CTGF antibodies was detected using an ELISA assay and Biacore assay. Recombinant CTGF protein was directly coated onto 96-well microplates; the signal strength after antibody addition was used to determine the binding activity between the antibody and CTGF. The specific experimental method is as follows:

[0333] Dilute CTGF protein (R&D, Cat No. 9190-CC) to a concentration of 0.2 μg / ml using PBS buffer (Shanghai Yuanpei, Cat No. B320) at pH 7.4. Add 50 μl / well to a 96-well microplate (Corning, Cat No. CLS3590-100EA) and incubate at 37°C for 2 hours or overnight at 4°C (16-18 hours). After discarding the liquid, add 250 μl / well of blocking buffer (5% skim milk, Cat No. 232100) diluted in PBS and incubate at 37°C for 3 hours or overnight at 4°C (16-18 hours) to block the protein. After blocking, discard the blocking solution and wash the plate 5 times with PBST buffer (PBS containing 0.1% Tween-20, pH 7.4). Add 50 μl / well of different concentrations of the antibody to be tested (hybridoma purified antibody or humanized antibody) diluted with sample diluent, and incubate at 37°C for 1 hour. After incubation, wash the plate 3 times with PBST, add 50 μl / well of 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 with sample diluent, and incubate at 37°C for 1 hour. After washing the plate three times with PBST, add 50 μl / well of TMB chromogenic substrate (KPL, Cat No. 52-00-03) and incubate at room temperature for 5-10 min. Stop the reaction by adding 50 μl / well of 1M H2SO4. Read the absorbance at 450 nm using a microplate reader (Molecular Devices, VERSAmax). Analyze the data using a GraphPad Prism 5 and calculate the EC50 value of CTGF antibody binding to human CTGF protein. Experimental results are shown below. Figure 1 As shown in Table 12.

[0334] Table 12. Results of ELISA experiment on CTGF antibody binding to human CTGF protein

[0335]

[0336] Experimental results show that the humanized full-length anti-CTGF antibody disclosed herein has a good binding effect with human CTGF protein.

[0337] II. ELISA assay for CTGF antibody binding to cynomolgus monkey CTGF protein

[0338] The binding affinity of the anti-monkey CTGF antibody was detected by an ELISA assay of the antibody and monkey CTGF protein. Anti-mFc protein was directly coated onto a 96-well microplate, followed by binding of Fc-tagged cyno-CTGF protein to the plate. The intensity of the signal after antibody addition was used to determine the binding activity between the antibody and cyno-CTGF. The specific experimental method is as follows:

[0339] Anti-mFc protein (Sigma, Cat No. M4280) was diluted to a concentration of 2 μg / ml using PBS (Shanghai Yuanpei, Cat No. B320) buffer at pH 7.4. 50 μl of this solution was added to each well of a 96-well microplate (Corning, Cat No. CLS3590-100EA) and incubated at 37°C for 2 hours. After discarding the liquid, 250 μl of blocking buffer (5% skim milk, Cat No. 232100) diluted in PBS was added to each well, and the plate was incubated at 37°C for 3 hours or overnight at 4°C (16-18 hours) for blocking. After blocking, discard the blocking solution and wash the plate three times with PBST buffer (PBS containing 0.1% Tween-20, pH 7.4). Add 50 μl of 1 μg / ml cyno-CTGF-mFc protein to each well and incubate at 37°C for 1 hour. After washing the plate three times with PBST buffer (PBS containing 0.1% Tween-20, pH 7.4), add 50 μl / well of different concentrations of the test antibody (hybridoma purified antibody or humanized antibody) diluted with sample dilution buffer and incubate at 37°C for 1 hour. After incubation, wash the plate three times with PBST, add 50 μl / well of 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 with sample dilution buffer, and incubate at 37°C for 1 hour. After washing the plate five times with PBST, add 50 μl / well of TMB chromogenic substrate (KPL, Cat No. 52-00-03), incubate at room temperature for 5-10 min, and stop the reaction by adding 50 μl / well of 1M H2SO4. Read the absorbance at 450 nm using a microplate reader (Molecular Devices, VERSA max), analyze the data using GraphPad Prism 5, and calculate the EC50 value of CTGF antibody binding to cynomolgus monkey CTGF protein. The experimental results are shown in Table 13.

[0340] Table 13. Results of ELISA experiment on CTGF antibody binding to CTGF protein in cynomolgus monkeys

[0341]

[0342] Experimental results show that the humanized anti-CTGF antibodies disclosed herein all have a good binding effect with monkey CTGF protein.

[0343] III. ELISA assay of CTGF antibody and mouse CTGF protein

[0344] The binding affinity of anti-mouse CTGF antibodies was detected by an ELISA assay of the antibody and mouse CTGF protein. The mouse-CTGF fusion protein was directly coated onto a 96-well microplate, and the intensity of the signal after antibody addition was used to determine the binding activity between the antibody and mouse-CTGF. The specific experimental method is as follows:

[0345] Mouse CTGF-his protein was diluted to a concentration of 0.5 μg / ml with PBS buffer (Shanghai Yuanpei, Cat No. B320) at pH 7.4. 50 μl of this buffer was added to each well of a 96-well microplate (Corning, Cat No. CLS3590-100EA) and incubated at 37°C for 2 hours. After discarding the buffer, 250 μl of blocking buffer (5% skim milk, Cat No. 232100) diluted in PBS was added to each well, and the plate was incubated at 37°C for 3 hours or overnight (16-18 hours) at 4°C for blocking. After blocking, the blocking buffer was discarded, and the plate was washed three times with PBST buffer (pH 7.4 PBS containing 0.1% Tween-20). Then, 50 μl of different concentrations of the antibody (hybridoma purified antibody or humanized antibody) diluted with sample diluent was added to each well, and the plate was incubated at 37°C for 1 hour. After incubation, wash the plate three times with PBST, add 50 μl / well of 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 with sample dilution buffer, and incubate at 37°C for 1 hour. After washing the plate five times with PBST, add 50 μl / well of TMB chromogenic substrate (KPL, Cat No. 52-00-03), incubate at room temperature for 5-10 min, and stop the reaction by adding 50 μl / well of 1MH2SO4. Read the absorbance at 450 nm using a microplate reader (Molecular Devices, VERSA max), analyze the data using GraphPad Prism 5, and calculate the EC50 value of CTGF antibody binding to mouse CTGF protein. The experimental results are shown in Table 14.

[0346] Table 14. Results of CTGF antibody-binding mouse CTGF protein ELISA experiment

[0347]

[0348] Experimental results show that the humanized full-length anti-CTGF antibody disclosed herein has a good binding effect with mouse CTGF protein.

[0349] Example 4. Functional blocking assay of CTGF antibody

[0350] The experiment used a Biacore instrument to detect the blocking activity of the humanized anti-CTGF antibody against human TGF-β1 function.

[0351] First, human CTGF was immobilized onto a CM5 biosensor chip (Cat.#BR-1005-30, GE) via amino-coupling at a fixation level of 1500 RU. Then, a high concentration of CTGF antibody (150 μg / ml) was flowed over the chip surface for 150 s, followed by the injection of 50 nM TGF-β1 protein for 120 s. The reaction signal was monitored in real-time using a Biacore T200 instrument to obtain binding and dissociation curves. After dissociation was completed in each experimental cycle, the biosensor chip was washed and regenerated using regeneration buffer.

[0352] The experimental data were statistically analyzed using GE Biacore T200 Evaluation version 3.0 software.

[0353] Blocking efficiency = [1 - (sample response value / blank control response value)] × 100%. Specific results are shown in Table 15.

[0354] Table 15. Results of CTGF antibody functional blocking assay

[0355]

[0356] The experimental results show that the humanized anti-CTGF antibodies disclosed herein all have high functional blocking activity.

[0357] Biacore assay of anti-CTGF antibody affinity

[0358] The affinity of the humanized anti-CTGF antibody to human CTGF and mouse CTGF was determined using a Biacore instrument in this experiment.

[0359] A certain amount of the target antibody was affinity-captured using a Protein A biosensor chip (Cat.#29127556, GE). Then, human and mouse CTGF antigens of specific concentrations were passed through the chip surface. The reaction signal was detected in real time using a Biacore instrument to obtain binding and dissociation curves. After each cycle of dissociation, the biochip was regenerated by washing with a pH 1.5 glycine-hydrochloric acid regeneration solution (Cat.#BR-1003-54, GE). The experimental run buffer was 1×HBS-EP buffer solution (Cat.#BR-1001-88, GE).

[0360] The experimental data were fitted using the GE Biacore T200 Evaluation version 3.0 software with a (1:1) Langmuir model to obtain affinity values, as shown in Tables 16 and 17.

[0361] Table 16. Reactivity affinity of CTGF antibody with human CTGF protein

[0362]

[0363]

[0364] Table 17. Reactivity affinity of CTGF antibody with mouse CTGF protein

[0365]

[0366]

[0367] The experimental results showed that the chimeric antibodies Ch147 and Ch164 derived from mouse antibodies mAb147 and mAb164, as well as the humanized anti-CTGF antibody, could bind to human CTGF and mouse CTGF with high affinity. The substitution and reversion mutations of the FR region of various human-derived antibody lines did not affect the affinity of the humanized antibody, and some modifications even enhanced the affinity between the antibody and the antigen.

[0368] Example 5. Inhibition of TGFβ-induced C2C12 cell migration in vitro by CTGF antibody.

[0369] C2C12 cells (mouse myoblasts, Chinese Academy of Sciences Cell Bank, #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 at a cell density of 2 × 10⁶ cells / year. 5 / ml, the concentration of recombinant human TGFβ1 (Cellsignaling Technology, #8915LC) was 10ng / ml, and the concentration of the antibody to be tested was 30μg / ml.

[0370] Open For the Disposable Chemotaxis System (Neuro Probe, #106-8), add the top cover and filter membrane to the lower chamber. Add 30 μl of the TGFβ-antibody mixture to each well, with 4 wells per group. Cover with the filter membrane, and add 50 μl of the cell-antibody mixture to each well, with 4 wells per group. Cover with the top cover and place in an incubator (37°C, 5% CO2).

[0371] After 48 hours of incubation, blot the liquid off the filter membrane with a clean paper towel, open the filter membrane, add 10 μl of pre-chilled 0.25% trypsin to each well in the lower chamber, and cover with the filter membrane. After digestion for 5 minutes, centrifuge at 1000 rpm for 1 minute. Open the filter membrane, add 20 μl of Cell Titer-Glo solution (Promega, #G7573) to each well in the lower chamber, and incubate at room temperature for 10 minutes. Transfer the liquid to a 384-well white plate (Thermo Scientific, #267462), and read the plate using a microplate reader (BMG, #PheraStar) with chemiluminescence. Analyze the data using Graphpad Prism 6.

[0372] Inhibition rate = [(average TGFβ - average sample group) / (average TGFβ group - average untreated group)] × 100%.

[0373] The experimental results are shown in Table 18.

[0374] Table 18. Inhibition results of CTGF antibody on the in vitro migration of C2C12 cells.

[0375]

[0376]

[0377] The results showed that the humanized anti-CTGF antibody disclosed in this paper could inhibit the migration ability of C2C12 cells after TGFβ1 induction in vitro, and the inhibition of C2C12 cell migration ability was stronger than that of the positive control antibody mAb1.

[0378] Example 6. Inhibition of TGFβ-induced PANC1 cell migration in vitro by CTGF antibody.

[0379] PANC-1 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 using DMEM medium containing 0.1% fetal bovine serum, and the cell density was 4 x 102. 5 / ml, the concentration of recombinant human TGFβ (Cell signaling Technology, #8915LC) was 10 ng / ml, and the concentration of the antibody to be tested was 30 μg / ml.

[0380] Open For the Disposable Chemotaxis System (Neuro Probe, #106-8), add the top cover and filter membrane to the lower chamber. Add 30 μl of the TGFβ-antibody mixture to each well, with 4 wells per group. Cover with the filter membrane, and add 50 μl of the cell-antibody mixture to each well, with 4 wells per group. Cover with the top cover and place in an incubator (37°C, 5% CO2).

[0381] After 48 hours of incubation, blot the liquid off the filter membrane with a clean paper towel, open the filter membrane, add 10 μl of pre-chilled 0.25% trypsin to each well in the lower chamber, and cover with the filter membrane. After digestion for 5 minutes, centrifuge at 1000 rpm for 1 minute. Count the cells settled at the bottom of the plate under an inverted microscope (Leica, #DMIL LED). Analyze the data using Graphpad Prism 6. Inhibition rate = [(average TGFβ - average sample group) / (average TGFβ - average untreated group)] × 100%.

[0382] The experimental results are shown in Table 19.

[0383] Table 19. Inhibition results of CTGF antibody on PANC1 cell migration in vitro.

[0384]

[0385] The results showed that the humanized anti-CTGF antibodies disclosed herein could inhibit the migration ability of PANC1 cells in vitro after TGFβ1 induction.

[0386] Example 7. CTGF antibody inhibits PANC1 proliferation in vitro on soft agar.

[0387] Deionized water was added to agar (BD, #214220) and heated to prepare a 1.4% gel solution. 2×DMEM medium (Thermo, #12100046) was mixed with the gel solution at a 1:1 volume ratio, and 500 μl was added to each well of a 24-well plate (Costar, #3524). The plate was then incubated at 4°C to allow it to solidify.

[0388] PANC-1 cells (human pancreatic cancer cells, ATCC, #CRL-1469) were digested with 0.25% trypsin (Gibco, #25200-072), centrifuged, and then adjusted to a cell density of 5 × 10⁶ cells / year in DMEM medium (GE, #SH30243.01) containing 4% fetal bovine serum (Gibco, #10099-141). 4 Cells / ml. Antibody was prepared to 2 mg / ml using 2×DMEM medium. Cells, antibody, and 1.4% gel solution were mixed at a volume ratio of 2:1:1, and 200 μl was added to each well of a 24-well plate with the bottom layer of gel. After the top layer of gel solidified, 200 μl of DMEM medium containing 2% fetal bovine serum was added to the 24-well plate. After culturing in an incubator (37℃, 5% CO2) for 28 days, images were taken using a high-content analysis system (Molecular Devices, ImageXpress), and the area of ​​the formed cell colonies was analyzed. Inhibition rate = (1 - average value of sample group / average value of untreated group) × 100%. Experimental results are shown in Table 20.

[0389] Table 20. Inhibition results of CTGF antibody on PANC1 cell proliferation in vitro using soft agar.

[0390] Hu147-11wk 23.76％ Hu147-33wk 22.30％ Hu164-67wl 24.2％ Hu164-67yl 21.7％ Hu95-51yk 20.24％

[0391] The results showed that the humanized antibodies from mAb147 and mAb164 clones significantly inhibited the in vitro proliferation of human pancreatic cancer cells (PANC-1 cells).

[0392] Example 8. Biological activity of CTGF antibody in animals

[0393] I. Inhibition test of CTGF antibody on bleomycin-induced pulmonary fibrosis in mice

[0394] Experimental Methods: Forty mice were randomly divided into four groups according to their body weight: normal control group (PBS, ip, qod), mAb1 group (10 mg / kg, ip, qod), Hu164-67wl (10 mg / kg, ip, qod) group, and Hu164-67yl (10 mg / kg, ip, qod) group. Each group consisted of 10 mice. Specific grouping details are shown in Table 21 below. On day 1, mice were intraperitoneally injected with the appropriate solvent and antibody (10 ml / kg; the normal control and model groups received PBS) according to their body weight. Two hours later, bleomycin was nebulized (50 mg / 8 ml nebulization for 30 min followed by a 5 min resting time) to establish the model. Then, the solvent or antibody was injected intraperitoneally every other day according to the above groups. The experimental period was 21 days, with a total of 11 intraperitoneal administrations. On day 22 of the experiment, mice were anesthetized with 1% sodium pentobarbital (10 ml / kg) via intraperitoneal injection. The mice were then fixed supine on the operating table. A 1 cm incision was made along the midline of the neck, extending to the thoracic cavity entrance. Subcutaneous connective tissue and muscle were bluntly dissected using hemostatic forceps to expose the trachea. The connective tissue on both sides of the trachea and between the trachea and esophagus was separated, and the trachea was freed. An indwelling intravenous catheter was inserted, and surgical sutures were used to ligate and fix the catheter at the tracheal inlet. 0.8 ml of PBS was drawn into a 1 ml syringe and attached to the inlet of the intravenous catheter. The PBS was slowly injected into the trachea, held for 30 seconds, and then slowly aspirated. A milky white, foamy liquid was observed being drawn back. This irrigation was repeated three times. The irrigation fluid was transferred to an EP tube, and then 0.5 ml of PBS was drawn again, and the above steps were repeated. After mixing the two rinsing solutions, centrifuge at 1500 rpm for 5 min, and store the supernatant at -20℃ for testing. Centrifuge the BALF supernatant again at 10000 rpm for 10 min, and then take the supernatant to detect soluble collagen (kit: Biocolor, batch number AA883).

[0395] Table 21. Trial Groups and Drug Administration

[0396] 1 10 PBS - IP Once every other day 2 10 mAb1 10 IP Once every other day 3 10 Hu164-67wl 10 IP Once every other day 4 10 Hu164-67yl 10 IP Once every other day

[0397] Excel statistical software was used: the mean was calculated as avg (Average); the SD (Standard Dividend) value was calculated as STDEV (Standard Dividend); the SEM (Standard Error of Mean) value was calculated as STDEV / SQRT (Square Root) (number of animals per group); GraphPad Prism software was used for graphing; and two-way ANOVA or one-way ANOVA was used for statistical analysis of the data.

[0398] The experimental results are shown in Table 22.

[0399] Table 22. Results of the assay showing the inhibitory effect of CTGF antibody on bleomycin-induced pulmonary fibrosis in mice.

[0400]

[0401] The results showed that Hu164-67wl or Hu164-67yl exhibited a strong inhibitory effect on pulmonary fibrosis in mice.

[0402] II. Inhibition experiment of CTGF antibody on subcutaneous xenografts of Su86.86 human pancreatic cancer cells

[0403] Experimental methods: SU86.86 cells (ATCC, CRL-1837, 3.0×10⁶ cells) were used. 6 200 μl of each tumor was injected subcutaneously into the right rib area of ​​Nu / Nu nude mice. Eleven days after inoculation, when the tumor volume reached ~140 mm², 3 After removing mice by weight, excessively large or small tumors, and other factors, mice were randomly divided into 5 groups of 10 mice each, based on tumor volume. Drug administration began on the same day. Antibodies were administered via intraperitoneal injection twice weekly for 18 days. Tumor volume and body weight were measured 1-2 times weekly, and data were recorded. Grouping and drug administration details are shown in Table 23.

[0404] Table 23. Trial Groups and Drug Administration

[0405]

[0406]

[0407] Data were recorded using Excel statistical software: the mean was calculated as average; the SD value was calculated as STDEV; the SEM value was calculated as STDEV / SQRT (number of animals per group); graphs were created using GraphPad Prism software; and two-way ANOVA or one-way ANOVA was used for statistical analysis of the data.

[0408] The formula for calculating tumor volume (V) is: V = 1 / 2 × L 长 ×L 短 2

[0409] Relative tumor proliferation rate T / C (%) = (T t -T0) / (C t -C0)×100, where T t C t T0 and C0 represent the tumor volumes of the treatment and control groups at the end of the experiment; T0 and C0 represent the tumor volumes at the beginning of the experiment.

[0410] Tumor inhibition rate TGI (%) = 1 - T / C (%).

[0411] Experimental results:

[0412] From day 1 to 18, the drug was administered subcutaneously twice weekly. The inhibitory effect of CTGF antibody on the growth of SU86.86 tumors was then assessed. The results are shown in Table 24 and... Figure 2 As shown in the figure. Compared with the blank control group containing isotype human IgG, the tumor inhibition rates of the mAb1-40mg / kg, Hu164-67yl-40mg / kg, Hu164-67yl-20mg / kg, and Hu147-33wk(40mg / kg) groups were 45%, 53%, 47%, and 54%, respectively. These groups significantly inhibited the growth of Su86.86 tumors (p<0.001), and the inhibitory effect on Su86.86 tumors showed a certain dose-dependent effect. Furthermore, there were no significant changes in the body weight of mice in any of the groups.

[0413] Table 24. Inhibition results of CTGF antibody on xenografts in SU86.86 mice

[0414]

Claims

1. An anti-CTGF antibody comprising a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 with the same sequence as the heavy chain variable region shown in SEQ ID NO: 8, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 with the same sequence as the light chain variable region shown in SEQ ID NO:

9. CDR1, CDR2, and CDR3 are defined according to the Kabat, Chothia, or IMGT numbering system.

2. The anti-CTGF antibody according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region includes 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 includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively.

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

4. The anti-CTGF antibody according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein: (ix) the heavy chain variable region, the amino acid sequence of which has at least 90% sequence identity with SEQ ID NO: 8; and the light chain variable region, the amino acid sequence of which has at least 90% sequence identity with SEQ ID NO: 9; or (x) The heavy chain variable region having an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 33, 34, 35 or 36; and the light chain variable region having an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 30, 31 or 32.

5. The anti-CTGF antibody according to claim 2, wherein the anti-CTGF antibody is a humanized antibody, the humanized antibody comprising a framework region of a human antibody or a variant thereof, the framework region variant having up to 10 reversion mutations in the light chain framework region and / or the heavy chain framework region of the human antibody, respectively.

6. The anti-CTGF antibody according to claim 5, wherein the humanized antibody comprises a light chain variable region and a heavy chain variable region selected from the following: The light chain variable region, which includes: LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively, and one or more reverse mutations selected from 36V, 44F, 46G, or 49G, and Heavy chain variable region, which contains: HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 respectively, and heavy chain variable regions selected from one or more amino acid reversion mutations of 44G, 49G, 27F, 48L, 67L, 71K, 78V or 80F.

7. The anti-CTGF antibody according to claim 6, comprising a heavy chain variable region and a light chain variable region, wherein: (x) the heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO: 33, 34, 35 or 36; and the light chain variable region, the amino acid sequence of which is shown in SEQ ID NO: 30, 31 or 32.

8. The anti-CTGF antibody according to claim 7, comprising a heavy chain variable region and a light chain variable region as shown below: (xiv) The heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO: 34; and the light chain variable region, the amino acid sequence of which is shown in SEQ ID NO:

30.

9. The anti-CTGF antibody according to claim 4, comprising a heavy chain variable region and a light chain variable region as shown below: (ix) the heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO: 8; and the light chain variable region, the amino acid sequence of which is shown in SEQ ID NO:

9.

10. The anti-CTGF antibody of claim 1, wherein the antibody further comprises a heavy chain constant region and a light chain constant region; the heavy chain constant region is selected from the constant regions of human IgG1, IgG2, IgG3 and IgG4 and their variants, and the light chain constant region is selected from the constant regions of human antibody κ and λ chains and their variants.

11. The anti-CTGF antibody according to claim 10, wherein 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:

40.

12. The anti-CTGF antibody according to claim 1, comprising: (d) A heavy chain having at least 85% sequence identity with SEQ ID NO: 54, 56, 57, 58, 59, 60, 61, 62 or 63, and a light chain having at least 85% sequence identity with SEQ ID NO: 55, 64, 65 or 66; or (f) Heavy chains as shown in SEQ ID NO: 54, 56, 57, 58, 59, 60, 61, 62 or 63 and light chains as shown in SEQ ID NO: 55, 64, 65 or 66.

13. The anti-CTGF antibody according to claim 12, comprising: (k) Heavy chains as shown in SEQ ID NO: 61 and light chains as shown in SEQ ID NO:

64.

14. A nucleic acid molecule encoding the anti-CTGF antibody according to any one of claims 1 to 13.

15. A host cell comprising the nucleic acid molecule as described in claim 14.

16. A pharmaceutical composition comprising a therapeutically effective amount of an anti-CTGF antibody according to any one of claims 1 to 13, or a nucleic acid molecule according to claim 14, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.

17. The use of the anti-CTGF antibody according to any one of claims 1 to 13 in the preparation of a kit for the immunoassay or determination of CTGF.

18. A kit comprising an anti-CTGF antibody according to any one of claims 1 to 13.

19. The use of the anti-CTGF antibody of any one of claims 1 to 13, or the nucleic acid molecule of claim 14, or the pharmaceutical composition of claim 16 in the preparation of a medicament for treating or preventing CTGF-related diseases, wherein the CTGF-related diseases are fibrotic diseases, hypertension, diabetes, glaucoma, or cancer. The fibrotic diseases mentioned are selected from: spontaneous pulmonary fibrosis, diabetic nephropathy, diabetic retinopathy, cirrhosis, or renal fibrosis; The cancer mentioned is selected from either ovarian cancer or pancreatic cancer.

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