Anti-connective tissue growth factor antibody and application thereof

Abstract

The invention provides an anti-CTGF antibody containing a light chain variable region and a heavy chain variable region and application of the anti-CTGF antibody as a medicine, the antibody can be used for preparing a medicine antibody for treating CTGF related diseases or symptoms, and the antibody is obtained by screening through the following method: immunogen preparation, animal immunization, positive hybridoma cell screening and strain establishment, ascites preparation and antibody purification. Carrying out specificity and affinity screening on 39 antibodies obtained by purification to obtain 3 antibodies with strong specificity and high affinity; and the neutralizing activity of the three antibodies is detected to finally obtain the antibody capable of inhibiting cancer cell migration in vitro.

Description

[0001] Specification Technical Field

[0002] The present invention relates to the field of biomedical diagnosis. More specifically, the present invention relates to an antibody against connective tissue growth factor (CTGF) that can inhibit cancer cell migration in vitro, and this antibody can be used in the treatment fields of diseases such as pulmonary fibrosis and multiple sclerosis. Background Art

[0003] Connective tissue growth factor (CTGF) is a cysteine-rich secreted protein composed of 349 amino acids with a molecular weight of 34 - 38 kDa, and was initially discovered in the conditioned medium of human umbilical vein endothelial cells. CTGF belongs to the CNN family together with Cyr61, nov, elm1, WISP-1, and WISP-3, and is all induced by transforming growth factor-β (TGFβ) superfamily factors. Many regulators including dexamethasone, thrombin, vascular endothelial growth factor (VEGF), and angiotensin, as well as environmental stimuli including hyperglycemia and hypertension, also induce the expression of CTGF.

[0004] CTGF is up-regulated in various kidney diseases (such as chronic kidney disease, diabetic nephropathy, glomerulosclerosis, IgA nephropathy, focal segmental glomerulosclerosis, ANCA-associated nephritis, rapidly progressive glomerulonephritis, chronic transplant nephropathy, nephrotic syndrome, lupus nephritis, membranoproliferative glomerulonephritis), and the expression level of CTGF is related to the degree of kidney injury. In addition, elevated levels of CTGF are associated with various fibrotic diseases, including scleroderma, interstitial lung disease, idiopathic pulmonary fibrosis and other pulmonary fibrosis diseases, fibrotic diseases caused by chronic hepatitis B or C, radiation-induced fibrotic diseases, fibrotic diseases caused by wound healing, and cardiac hypertrophy and fibrosis. In the above diseases, CTGF can be used as an effective therapeutic target.

[0005] So far, the most detailed research has been conducted on the humanized antibody CLN1, and its effects are clear in models of interstitial pulmonary fibrosis and renal interstitial fibrosis caused by unilateral ureteral ligation. CLN1 is currently in clinical trials (Phase III) as FG-3019, but has not been approved for marketing yet, and new and more effective antibodies with neutralizing inhibitory activity still need to be developed. Summary of the Invention

[0006] .The first object of the present invention is to provide a monoclonal antibody that can specifically recognize human CTGF protein. The light chain variable region thereof consists of the CDR-L1 region of the amino acid sequence shown in SEQ ID NO: 01, the CDR-L2 region of the amino acid sequence shown in SEQ ID NO: 02, and the CDR-L3 region of the amino acid sequence shown in SEQ ID NO: 03. The heavy chain variable region consists of the CDR-H1 region of the amino acid sequence shown in SEQ ID NO: 04, the CDR-H2 region of the amino acid sequence shown in SEQ ID NO: 05, and the CDR-H3 region of the amino acid sequence shown in SEQ ID NO: 06.

[0007] Another object of the invention is to provide a method for preparing and screening the above-mentioned specific antibody. The antibody is obtained by the following method:

[0008] (1) Preparation of immunogen: The VWC region of CTGF protein expressed by Escherichia coli is purified and then mixed with Freund's complete adjuvant, Quick Antibody-Mouse 5W adjuvant, and SuperQuick water-soluble adjuvant respectively to prepare an immunogen for immunizing mice.

[0009] (2) Preparation of positive hybridoma cells: Take the spleen cells of immunized mice and fuse them with SP2 / 0 myeloma cells. Positive screening is carried out with eukaryotic-expressed CTGF protein as the positive antigen, and negative screening is carried out with CYR61, NOV, WISP-1, and WISP-3 negative antigens by indirect ELISA to screen positive hybridoma cells.

[0010] (3) Screening of monoclonal antibodies: Subclone and establish strains of positive hybridoma cells, prepare and purify ascites to obtain 39 monoclonal antibodies. ELISA binding ability screening is carried out on the 39 monoclonal antibodies to obtain 3 antibodies with high affinity and good specificity.

[0011] The present invention obtains a hybridoma cell line secreting anti-CTGF antibody through large-scale cell fusion and screening. The preparation method of the hybridoma cell line includes the following steps:

[0012] 1) Select the recombinant expression of the VWC (Von Willebrand factor type C) region of CTGF protein. The C-terminus is fused with a GST tag to promote the soluble expression of the recombinant protein, and the histidine tag is convenient for purification. After gene synthesis, it is cloned into the expression plasmid vector pET30a. After soluble expression in Escherichia coli, it is purified by nickel column affinity chromatography for immunizing mice.

[0013] 2) Sterilely take the spleen cells of immunologically qualified mice as antigen-sensitized B cells. According to the conventional method, fuse the B cells with the myeloma cell line SP2 / 0, and then screen by the conventional fusion cell HAT screening method to obtain fusion cell growth clones.

[0014] 3) Screening for specific recognition of CTGF protein: CTGF protein belongs to the CNN family and is derived from transforming growth factor β

[0015] The members of this family have highly homologous amino acid sequences. Their protein structures include: the N-terminal insulin-like growth factor binding region, von Wilebrand factor (von Wilebrand

[0016] The four main structural regions are the C-type repeat region of cytoskeletal factor (vWf), the thrombospondin (TSP) type 1 repeat region, and the cysteine-rich C-terminal binding region. In order to obtain monoclonal antibodies that specifically recognize CTGF, in the hybridoma cell clone screening stage, CTGF protein expressed by mammalian cells with a structure similar to that of the natural protein was selected as the screening protein, and Nov, CYR61, WISP-1 and WISP-3 were used for negative screening at the same time.

[0017] The present invention prepares monoclonal antibodies from the above hybridoma cell strain, and the preparation method has the following two methods:

[0018] 1) Hybridoma cells are cultured in vitro in serum-free medium, and the culture supernatant is harvested and purified by immunoaffinity chromatography to obtain the desired monoclonal antibody.

[0019] 2) Inoculate hybridoma cells into the abdominal cavity of animals, harvest the ascites of the animals and separate and purify the desired monoclonal antibodies. The antibodies prepared by the ascites method of the present invention are purified by affinity chromatography on a Protein A / G column to obtain the CTGF monoclonal antibody.

[0020] Advantages and beneficial effects of the present invention

[0021] The anti-CTGF monoclonal antibody obtained by the present invention specifically recognizes human CTGF, but does not recognize other CNN family member proteins Nov, CYR61, WISP-1, WISP-3, etc. In addition, the antibody has a high binding activity with the CTGF protein, with an affinity of pM level, and can specifically block the migration of cancer cells in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 :CTGF-GST fusion protein purification gel image

[0023] In the figure, M is a molecular weight marker, 1 is the original supernatant of Escherichia coli fermentation, 2 is the fermentation supernatant flow-through, 3 is the 15mM imidazole elution product, 4 is the 60mM imidazole elution product, 5, 6 and 7 are 300mM imidazole elution products respectively, and the molecular weight of the fusion protein is about 40kDa, which is separated using 12% SDS-PAGE gel.

[0024] Figure 2 : Coomassie blue staining gel picture of CTGF chimeric antibody expression and purification

[0025] In the figure, M is the molecular weight marker, 1, 2, and 3 are the non-reduced forms of the chimeric antibody (numbered #9-65, #10-18, and

[0026] #12-15), 4, 5, and 6 are the reduced forms of the chimeric antibody (numbered #9-65, #10-18, and #12-15), separated by 4-12% SDS-PAGE gradient gel.

[0027] Figure 3 : ELISA assay for the affinity of chimeric antibody

[0028] Figure 4 : Chimeric antibody inhibits TGFβ-induced migration of PANC-1 cells

[0029] Figure 5 : Chimeric antibody inhibits bleomycin-induced pulmonary fibrosis in mice. The ordinate shows the content of soluble collagen in the lung Detailed implementation mode

[0030] The present invention will be further described below in conjunction with the drawings and specific implementation modes, so that those skilled in the art can more clearly understand the technical solution of the present invention, which is not a limitation to the present invention.

[0031] Example 1 Preparation of recombinant CTGF-VWC protein immunogen

[0032] The VWC region (83-169aa) of the protein with Uniprot number P29279 was synthesized by gene synthesis. A BamHI cleavage site was introduced upstream and an XhoI cleavage site was introduced downstream. After double digestion with BamHI / XhoI and recovery, it was cloned into the linearized fragment of pET30a-GST (BPI modified) digested with the same double enzymes. After sequencing identification, plasmid preparation was carried out, and the recombinant protein was induced to express at low temperature using the Escherichia coli expression strain BL21. Since the recombinant protein carried a histidine tag, nickel column affinity purification was used. After elution with imidazole solutions of different concentrations, each fraction and the flow-through were separately loaded onto SDS-PAGE for separation and detection, attached Figure 1 are the expression and purification results of the recombinant CTGF protein fused with GST tag. The purity of the recombinant CTGF protein is above 90%, and the concentration is about 1-1.5 mg / mL, which can meet the requirements of immunizing animals and screening and identifying antibodies.

[0033] Example 2 Establishment of hybridoma cell line and antibody screening

[0034] I. Animal immunization

[0035] The recombinant CTGF-VWC protein in Example 1 was used to immunize 4 female Balb / c mice aged 4-6 weeks (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) with Freund's complete adjuvant (purchased from Sigma), QuickAntibody-Mouse 5W adjuvant, and SuperQuick water-soluble adjuvant, respectively. They were numbered 31392B#1, 31392B#2, 31392B#3, 31392B#4, 31392B#5, 31392B#6, 31392B#7, 31392B#8, 31392B#9, 31392B#10,

[0036] 31392B#11, and 31392B#12. For immunization with Freund's complete adjuvant, it was injected subcutaneously into the abdomen at a dose of 60 μg / mouse. Booster immunization was performed every 14 days. The antigen was emulsified with Freund's incomplete adjuvant (Sigma) at a dose of 30 μg / mouse, and booster immunization was performed 3 times; for immunization with Antibody-Mouse 5W adjuvant, it was injected intramuscularly. The initial immunization dose was 20 μg / mouse, and a booster immunization of 20 μg / mouse was performed 14 days later; for immunization with SuperQuick water-soluble adjuvant, it was injected intramuscularly. The initial immunization dose was 40 μg / mouse, and a booster immunization of 20 μg / mouse was performed 14 days later. Seven days after booster immunization, the polyclonal antibody titer against the immunogen in the mouse serum was detected by indirect ELISA (wavelength 450 nm). The mouse with the highest titer was given a boost immunization by tail vein injection. The antigen was warmed and homogenized with physiological saline at a dose of 50 μg / mouse.

[0037] II. Cell fusion

[0038] A sterile spleen cell suspension of immunized mice meeting the standard was prepared and mixed with mouse myeloma cells sp2 / 0 (ATCC) at a ratio of 5:1. After centrifugation at 1500 rpm for 5 min, the supernatant was discarded, and the centrifuge tube was placed in a 37°C water bath. 1 mL of PEG1500 (Roche) was slowly added within 1 minute, and the cells were stirred. After standing in warm water for 1 min, 10 mL of serum-free IMDM (Sigma) was added, mixed well, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, 10 mL of serum (PAA) was added, and the cells were carefully pipetted up. 5 mL of thymocytes mixed with 10×HAT (Sigma) was added and mixed well. Then 25 mL of semi-solid medium containing 2.1% nitrocellulose (Sigma) was added and mixed thoroughly, and then evenly poured into 20 cell culture dishes. The cell culture dishes were placed in a humid box and cultured in a 37°C 5% CO2 incubator.

[0039] III. Screening and preservation of positive hybridomas

[0040] Seven days after fusion, the size and density of the cloned cell clusters were moderate. Under a dissecting microscope, round, solid, and large cloned clusters were aspirated and transferred into a 96-well culture plate containing pre-prepared medium, and then cultured in an incubator at 37°C with a CO2 concentration of 5%.

[0041] IV. ELISA Screening of Positive Hybridoma Cells

[0042] Three days after culturing the hybridoma cells, when the cell amount occupied approximately 2 / 3 of the bottom area, 200 μl of complete medium containing feeder cells and 1% HT (Sigma) was added. Two days later, the second ELISA screening was performed, and hybridomas with an indirect ELISA detection OD value higher than 0.5 against the immunogen were obtained as positive clones for subsequent culture. These positive clones were transferred to a 24-well plate containing pre-prepared medium (containing feeder cells and HT) for culture. Five days later, 100 μl of the supernatant was taken for the third ELISA screening. The screening process was as follows: 100 μl of the supernatant was used with CTGF protein (R&D, 9190-CC-050), eukaryotic-expressed Nov protein (Peprotech, 120-26), WISP-1 protein (Peprotech, 120-18), and WISP-3 protein (Peprotech, 120-20) as coating antigens for specific screening by the ELISA method. When screening, the antigen protein was diluted to 2 μg / ml with PBS buffer. 100 μl of the antigen was added to each well and coated at 4°C for 12 hours. The solution was discarded, and the plate was washed 3 times with the washing solution (PBS buffer containing 0.05% Tween) and then dried. 300 μL of 2% (v / w) bovine serum albumin was added to each well as a blocking solution and incubated at 37°C for 3 h. The plate was washed 3 times repeatedly, and the cell culture supernatant to be identified was added. After incubation and screening, and after blocking, the test sample was added to the wells of the enzyme-linked immunosorbent assay (ELISA) plate, incubated at 37°C for 1 h, washed 3 times, 100 μl of HRP-labeled goat anti-mouse antibody diluted 1:5000 times (purchased from) was added to each well, incubated at 37°C for 1 h, then washed 3 times and dried. 100 μLl of TMB chromogenic buffer was added to each well, incubated at room temperature for 8 minutes, and then 50 μl of H2SO4 (2M) was added to each well to terminate the reaction. The absorbance value at 450 nm was read on an enzyme-linked immunosorbent assay (ELISA) reader.

[0043] Two hundred and twenty hybridoma cells that could recognize CTGF protein were obtained from twelve mice through preliminary screening. Taking the 12 hybridomas of 31392B#1 as an example, the screening results are shown in Table 1. After screening, 39 antibodies that only recognized CTGF protein and did not bind to other proteins in the CNN family were obtained.

[0044] Table 1 Specific Screening of Hybridomas Prepared from Mice with the Number 31392B#1

[0045]

[0046]

[0047] Example 3 Preparation of Monoclonal Antibody by Ascites Induction Method

[0048] Cells in the logarithmic growth phase were washed and resuspended with serum-free medium, and counted about 5×10 5 , and 1 ml of the suspended cells was intraperitoneally injected into mice sensitized with paraffin oil in advance. Ascites collection began 7 days later. The collected ascites was centrifuged at 4000 rpm for 10 min at 4°C, and the middle ascites was carefully aspirated and collected in a centrifuge tube for purification. The purification of the antibody was carried out according to the instructions of HiTrap rProtein A FF (purchased from GE Healthcare, catalog number 17-5079-02).

[0049] Example 4 Subclass Identification and Affinity Determination of Monoclonal Antibody

[0050] For subclass identification, goat anti-mouse IgG (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) was diluted to 0.5 μg / ml with 100 mM PBS (pH 7.4), 100 μl was added to each well, and after overnight incubation at 4°C, the liquid was emptied. The wells were washed 3 times with PBS containing 0.05% Tween (PBS-T). 200 μl of blocking solution (PBS containing 2% BSA and 3% sucrose) was added to each well and incubated at 37°C for 1 h. The liquid was emptied and the wells were washed 3 times with PBS-T. 100 μl of hybridoma culture supernatant or purified antibody was added to each well, and after incubation at 37°C for 1 h, the liquid was emptied and the wells were washed 3 times with PBS-T. HRP-labeled goat anti-mouse (κ, λ) antibody diluted 1:1000 with blocking solution or HRP-labeled goat anti-mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibody (Southern Biotech) diluted 1:2000 was added to the wells containing the antibody samples to be tested at a volume of 100 μl each, and incubated at 37°C for 1 h. The liquid was emptied and the wells were washed 3 times with PBS-T. 50 μl of citrate buffer (pH 4.0) containing 0.15% ABTS (Southern Biotech, catalog number 0202-1) and 0.03% H2O2 was added to each well for color development, and the absorbance value at 405 nm was measured within 10 min.

[0051] II. Determination of Affinity Constant

[0052] Coat the recombinant CTGF protein at a concentration of 2 μg / m1, 100 μ1 / well, and coat overnight at 4°C. Wash 3 times with PBS-T. Add 200 μl of blocking solution to each well and block at 37°C. Wash 3 times with PBS-T. The purified monoclonal antibody in Example 4 was serially diluted 4-fold starting from 5 μg / mL, and the last well was left as a blank control. Incubate at 37°C for 1 h and wash 3 times with PBS-T. Dilute the HRP-labeled goat anti-mouse secondary antibody 1:20000, add 100 μL to each well, incubate at 37°C for 1 h, and wash 3 times with PBS-T. Add 100 μl of citrate phosphate buffer containing 0.1% TMB (Sigma) and 0.03% H2O2 to each well for color development for 10 min, and then add 50 μl of 0.5 M sulfuric acid solution to terminate the reaction. Measure the absorbance at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and use data analysis and graphing software GraphPad Prism v8 to plot the antibody dilution concentration and OD 450 corresponding curve, calculate the affinity, and obtain 3 high-affinity antibodies through screening for subsequent experiments, namely #9-65, #10-18, and #12-15.

[0053] Determination of the variable region sequence of the antibody in Example 5

[0054] Perform sequence analysis on the three hybridoma cells with higher affinity, #9-65, #10-18, and #12-15. Culture fresh hybridoma cells, take the supernatant for antigen-binding property verification, and confirm that the cell line used for cloning can indeed secrete the required antibody. After the results are confirmed, centrifuge to collect 10 6 of the above hybridoma cells. Extract the total RNA of hybridoma cells by the Trizol method. Take 9 μl of total RNA, add 2.5 μl of oligo(dT)12–18 primer (10 mM), and 5 μl of dNTPs, mix well, incubate at 70°C for 5 minutes, then place on ice for 5 minutes, or perform denaturation according to the reverse transcriptase used. Subsequently, add 5 μL of RT buffer (5X), 2.5 μL of DTT (0.1 M), and 1 μL of reverse transcriptase, and react at 42°C for 1 h. Incubate at 70°C for 15 minutes to terminate the reaction, and store the obtained cDNA at -20°C. Perform PCR amplification on the obtained first-strand cDNA. Add 25 pmol of each primer to a 50 μL reaction system. The sequences of the primers for amplifying the heavy-chain variable region and the light-chain variable region are shown in Table 3

[0055] Table 2 Primers for amplifying the variable region of the antibody

[0056]

[0057] The remaining dNTPs and buffer were added according to the routine. Finally, 1 μL of cDNA template and 1 U of hot-start Taq DNA polymerase were added. The PCR amplification program was set as 40 seconds at 94 °C, 40 seconds at 52 °C, 40 seconds at 72 °C, and amplified for 25 cycles. Finally, it was extended at 72 °C for 3 minutes. The product could be stored at 4 °C for later use or directly electrophoresed. 20 μL of the PCR product was taken for electrophoretic analysis, separated and gel-extracted on a 1.5% agarose gel. The obtained heavy-chain variable region and light-chain variable region were respectively cloned into the pMD18T plasmid vector (TaKaRa) and then sequenced. The DNA sequence of the antibody heavy-chain variable region was analyzed for its framework region and antigenic determinant (CDR) sequence and deduced amino acid sequence using an online analysis tool. This antibody variable region sequence could be used to add the constant regions of the heavy chain and light chain downstream respectively by gene cloning, and an expression vector capable of recombinantly expressing antibody fragments could be obtained by genetic recombination and expressed in eukaryotic cells to express the recombinant complete antibody or fragment. Alternatively, the heavy-chain and light-chain variable regions could be connected with the coding sequence of a linker peptide (such as GGGGSGGGGSGGGGS) and constructed into a single-chain antibody to express antibody fragments in a suitable host cell.

[0058] Example 6 Chimeric Antibody Expression

[0059] According to the antibody variable region sequence results obtained by sequencing in Example 5, its heavy-chain variable region sequence and light-chain variable region sequence were respectively constructed into the pBPI-RD38 and pBPI-RD39 eukaryotic expression vectors. Among them, the heavy-chain variable region sequence was digested with EcoRI / NheI double enzymes and cloned into the pBPI-RD38 vector digested with the same enzymes, constructing a complete heavy-chain antibody sequence containing the IL2 signal peptide sequence and the constant region sequence of the human antibody IgG1 germline gene; the light-chain variable region sequence was digested with EcoRI / BSiWI double enzymes and cloned into the pBPI-RD39 vector digested with the same enzymes, constructing a complete light-chain antibody sequence containing the IL2 signal peptide sequence and the constant region sequence of the human antibody Kappa chain germline gene. The heavy-chain and light-chain eukaryotic expression vectors were co-transfected into Expi-CHO mammalian cells for chimeric antibody expression. The cell culture supernatant was collected 8 days after plasmid transfection. The chimeric antibody was affinity-purified by Protein A and quantified by Nanodrop for further characterization.

[0060] Example 7 Chimeric Antibody Affinity ELISA

[0061] The antibody affinity was detected by ELISA. Briefly, the eukaryotic expression Human CTGF Protein (R&D) antigen was coated with PBS at 10 ng / well and incubated overnight at 4°C. The liquid in the wells was discarded, and the wells were washed 3 times with 0.1% PBST at 300 μL / well. They were blocked with 1% BSA at 37°C for 2 h. The liquid in the wells was discarded, and the wells were washed 3 times with 0.1% PBST at 300 μL / well. The CTGF antibody was diluted to 0.25 μg / mL with the blocking solution and serially diluted 3-fold, with a total of 7 concentrations. The diluted samples were added to the corresponding wells and incubated at 37°C for 1 h. The liquid in the wells was discarded, and the wells were washed 3 times with 0.1% PBST at 300 μL / well. Goat anti-human-HRP was diluted 1:20,000 and added at 100 μL / well, and incubated at 37°C for 1 h. They were washed 3 times with 0.1% PBST at 300 μL / well, developed with TMB, incubated at room temperature for 6 min, terminated by adding 2N H2SO4, and the OD450nm was read with an ELISA reader. The results are as Figure 3 shown. The EC50 values of the #9-65, #10-18, and #12-15 antibodies were comparable to that of FG3019. Among them, the #12-15 antibody had the highest affinity, with an EC50 of approximately 15 pM, the #10-18 antibody was second with an EC50 of 25 pM, and the #9-65 antibody was slightly weaker, with an EC50 of approximately 79 pM.

[0062] Example 8 Chimeric Antibody Inhibits the Migration Ability of TGFβ-Induced Pancreatic Cancer Cells PANC-1

[0063] PANC-1 cells (human pancreatic cancer cells, ATCC, #CRL-1469) were digested into single cells with trypsin / EDTA (0.25% / 0.04%), terminated with a medium containing 10% FBS, and the cells were collected by centrifugation and resuspended in DMEM medium (Gibco, #11965-092) containing 0.1% fetal bovine serum (Hyclone, #SH30406.02). A cell-antibody mixture and a TGFβ-antibody mixture were prepared with DMEM medium containing 0.1% fetal bovine serum. The cell density was 4x10 3 / mL, the concentration of recombinant human TGFβ (Acrobiosystems, TG1-H4212) was 10 ng / ml, and the concentration of the antibody to be tested was 30 μg / mL. 750 μL of the prepared TGFβ-antibody mixed solution was added to the lower chamber, and TGFβ alone was used as a control, with 3 replicates per group. 500 μL of cells resuspended in the medium containing 0.1% FBS was added to the upper chamber, with a density of 5×10 4 / mL. (2.5×10 4 cells), and incubated at 37°C in a 5% CO2 incubator for 48 h.

[0064] Open the filter membrane, add 10 μL of pre-cooled 0.25% trypsin to each well in the lower chamber, and cover the filter membrane. After digestion for 5 minutes, centrifuge at 1000 rpm for 1 minute, and count the cells that migrated to the lower chamber using a CountStar cell counter. Analyze and process the data using Graphpad Prism 6, and calculate the inhibition rate using the formula: [(number of migrated cells in the positive control - number of migrated cells in the antibody group) / (number of migrated cells in the positive control - number of migrated cells in the negative control group)] * 100%. Representative results are shown as Figure 4 and Table 3. This experiment was repeated three times, and the results showed that all three chimeric antibodies could inhibit the in vitro cell migration of pancreatic cancer cells induced by TGFβ, and their inhibitory ability was better than that of the control antibody FG3019. Among them, #12-15 had the best inhibitory effect.

[0065] Table 3 Experimental results of the inhibitory effect of CTGF antibody on the in vitro migration of PANC-1 cells

[0066]

[0067]

[0068] Example 9 Inhibition of murine pulmonary fibrosis by chimeric antibodies

[0069] Pulmonary fibrosis was induced by intranasal instillation of bleomycin, and the treatment model was evaluated by intraperitoneal injection. Immediately after intranasal instillation of bleomycin into the lungs of C57BL / 6 mice, drug administration was started. Male C57BL / 6 mice were randomly grouped by body weight, anesthetized with isoflurane, and a model was established with bleomycin dissolved in 0.9% saline at a dose of 0.1 unit / 50 μL / mouse. Additionally, a group of mice instilled with only saline was used as a control. Immediately after modeling, the mice were treated with saline or antibody by intraperitoneal (IP) injection, and then treated every other day for a total of 7 doses. The specific grouping and drug administration grouping are shown in Table 4. During the experiment, the body weight was measured once every 2 days, weighed and recorded before sacrifice, and the status of the mice was observed and recorded. Fourteen days after intranasal instillation, the mice were sacrificed, lung tissues were taken, and the soluble collagen content in the lung tissues was measured to evaluate the drug efficacy. The results are shown as Figure 5 shown. After the mice were treated with 0.1 U bleomycin, the average soluble collagen content in the lung tissues of non-modeled mice was about 1.8 μg / section, and after bleomycin-induced pulmonary fibrosis modeling, it was about 2.7 μg / section, with an average increase of about 47.5%. After treatment with the antibody drugs, the soluble collagen content in the lung tissues decreased. Among them, there was no significant difference in the #9-65 group, significant differences in the positive control FG-3019 (p = 0.047) and #12-15 (p = 0.01), and extremely significant differences in #10-18 (p = 0.0006).

[0070] Table 4 Grouping of mice, drug administration doses, and body weight statistics

[0071]

Claims

1. A monoclonal antibody against connective tissue growth factor (CTGF), wherein the light chain variable region consists of a CDR1 region of the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region of the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 region of the amino acid sequence shown in SEQ ID NO:

3. The heavy chain variable region consists of a CDR1 region of the amino acid sequence shown in SEQ ID NO: 4, a CDR2 region of the amino acid sequence shown in SEQ ID NO: 5, and a CDR3 region of the amino acid sequence shown in SEQ ID NO:

6.

2. The anti-CTGF antibody according to claim 1, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO 7, and the light chain variable region comprises the sequence shown in SEQ ID NO 8.

3. The anti-CTGF antibody according to claim 1, wherein The antibody can specifically bind to human CTGF protein.

4. An antigen-binding fragment of the CTGF antibody according to claim 1.

5. The antibody of claim 1, wherein The antibody is a murine monoclonal antibody.

6. The antibody of claim 1, wherein the antibody is an antibody fragment selected from Fab, Fab', Fab'-SH, F(ab')2, Fv and scFv fragments.

7. The antibody of claim 1, wherein the antibody comprises a human IgG1, IgG2, IgG3 or IgG4 constant region.

8. The antibody or antigen-binding fragment of claim 1, wherein the antibody or its fragment has one, two or all three of the following characteristics: (i) Inhibiting PANC1 cell migration with an IC50 of about 0.15 nM or lower. (ii) Binding to a different CTGF epitope from FG-3019.

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