A fusion protein binding to CTGF and its application

Abstract

The present invention provides a fusion protein composed of an anti-human CTGF antibody and the extracellular domain of the human type II TGF-β receptor, linked via a linker. This fusion protein can bind both CTGF and TGF-β. This fusion protein can be purified in a single step to obtain a 100% pure target protein. Experiments have shown that even a low dose of 1 mg / kg can significantly reduce the degree of pulmonary fibrosis in experimental animals.

Description

Technical Field

[0001] The present invention belongs to the technical field of proteins, and in particular relates to a fusion protein combined with CTGF and an application thereof. Background Art

[0002] CTGF (Connective Tissue Growth Factor), also known as CCN2 or connective tissue growth factor, is a cytoplasmic protein of the CCN family of extracellular matrix-associated heparin-binding proteins. As a cofactor involved in TGF-β (Transforming Growth Factor-β) signaling, CTGF can induce fibroblasts to become myofibroblasts, which deposit collagen, ultimately leading to organ scarring and dysfunction.

[0003] The level of CTGF in tissues, blood or vitreous humor has been shown to be associated with fibrosis and its severity in many diseases. The two secreted factors, TGF-β and CTGF, are widely considered to be common mediators of fibrosis. TGF-β mainly inhibits cell proliferation and promotes extracellular matrix accumulation through the Smad and mitogen-activated protein kinase (MAPK) pathways. TGF-β1 can induce the synthesis of other cytokines, such as platelet-derived growth factor, tumor necrosis factor, fibroblast growth factor, etc., and further promote the development of myocardial fibrosis through these factors [1]. When fibrosis occurs in the renal tubular interstitium, extracellular matrix deposition increases, which is one of the causes of chronic renal failure. Renal interstitial fibrosis is a common pathway and pathological basis for the development of renal failure in various kidney diseases. Continuous liver damage can lead to large-scale liver fibrosis and cause cirrhosis. TGF-β also plays an important role in renal interstitial fibrosis and liver fibrosis. TGF-β is a potent inducer of CTGF, and most models assume that CTGF acts as a downstream mediator of TGF-β activity, while other studies support an interdependence between the profibrotic activities of TGF-β and CTGF, rather than a sequential relationship.[2] Some existing animal data suggest a synergistic effect of TGF-β and CTGF in the initiation and maintenance of fibrotic responses.

[0004] Patents US20040248206A1, US20140343258A1, US201214367081, US201213385320, CN109402127A, CN109824777A, and CN104011206A describe the use of biological products such as anti-CTGF antibodies or interfering RNA for the treatment of fibrotic diseases such as pulmonary fibrosis, liver fibrosis, cancer, and ophthalmic diseases. Fibrotic diseases are characterized by fibroblast mutation and tissue proliferation mediated by multiple cytokines. For example, in pulmonary fibrosis, TGF-β, PDGF, and IGF-1 are all fibrogenic growth factors. Their increased secretion in the early stages triggers the development of pulmonary fibrosis, leading to irreversible lung damage.

[0005] Currently, this field still needs to provide new molecular entities to further optimize the treatment of diseases.

[0006] [1] (Ding Wenjin et al. "The role of TGF-β1 in myocardial fibrosis caused by different causes." Science and Technology Review 2 (2016): 221-225.)

[0007] [2](José G. Abreu, et al. "Connective-tissue growth factor (CTGF) modulates cell signaling by BMP and TGF-β." Nature Cell Biology 4.8 (2002): 599-604.; Oganesian, A., Y. Zhu, and LJ Sandell. "Type IIA procollagen aminopropeptide is localized in human embryonic tissues." Journal ofHistochemistry&Cytochemistry 45.11(1997):1469-1480. Summary of the Invention

[0008] To address these issues, the present invention provides a novel bifunctional fusion protein that simultaneously binds to CTGF and TGF-β. Surprisingly, this fusion protein can be purified in a single step to obtain a highly purified (100%) target protein. Furthermore, experiments have shown that even a very low dose (1 mg / kg) of this fusion protein can significantly reduce the severity of pulmonary fibrosis in experimental animals.

[0009] Explanation of terms

[0010] In the present invention, unless otherwise specified, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include the singular.

[0011] Exemplary techniques for use in conjunction with recombinant DNA, oligonucleotide synthesis, tissue culture and transformation (e.g., electroporation, lipofection), enzymatic reactions, and purification techniques are known in the art. Many such techniques and procedures are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), and many other places. In addition, exemplary techniques for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and patient treatment are also known in the art.

[0012] In this application, unless otherwise stated, the use of “or” means “and / or.” In the case of multiple dependent claims, use of “or” is used only in the alternative to refer to more than one of the preceding independent or dependent claims.

[0013] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer within the range, and where appropriate, fractions thereof (such as tenths and hundredths of integers), unless otherwise indicated.

[0014] Units, prefixes and symbols are all expressed in their SI (International System of Units) recognized form. Numerical ranges include the numbers defining the ranges. The subheadings provided herein are not limitations of the various aspects of the present disclosure, which can be obtained by reference to this specification as a whole.

[0015] Unless otherwise indicated, as used in accordance with this disclosure, the following terms shall be understood to have the following meanings:

[0016] "Antibody" refers to a molecule comprising at least the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain and at least the CDR1, CDR2, and CDR3 of the light chain, and also comprising the framework regions FR1, FR2, FR3, and FR4, wherein the molecule is capable of binding to an antigen. The term antibody includes, but is not limited to, fragments capable of binding to an antigen, such as Fv, single-chain Fv (scFv), Fab, Fab', and (Fab')2. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species such as mouse, human, cynomolgus monkey, alpaca, etc.

[0017] In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region. In some embodiments, the antibody comprises at least one heavy chain comprising a heavy chain variable region and at least a portion of a heavy chain constant region and at least one light chain comprising a light chain variable region and at least a portion of a light chain constant region. In some embodiments, the antibody comprises two heavy chains and two light chains, wherein each heavy chain comprises a heavy chain variable region and at least a portion of a heavy chain constant region, and, wherein each light chain comprises a light chain variable region and at least a portion of a light chain constant region. Any other antibody comprising a single polypeptide chain comprising, for example, all six CDRs (three heavy chain CDRs and three light chain CDRs) is considered to have a heavy chain and a light chain. In some such embodiments, the heavy chain is an antibody region comprising three heavy chain CDRs, and the light chain is an antibody region comprising three light chain CDRs.

[0018] "Complementarity determining region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics The North CDR definition is based on the IMGT database and affinity propagation clustering using a large number of crystal structures. The CDRs in the present invention are divided according to the definition scheme of Kabat et al.

[0019] In some embodiments, the amino acid changes described herein include amino acid substitutions, insertions, or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0020] In a preferred embodiment, the amino acid changes described herein occur in regions outside of the CDRs (e.g., in the FRs). More preferably, the amino acid changes described herein occur in regions outside of the heavy chain variable region and / or outside of the light chain variable region.

[0021] In some embodiments, the substitution is a conservative substitution. A conservative substitution refers to the substitution of one amino acid with another amino acid within the same class, for example, the substitution of an acidic amino acid with another acidic amino acid, the substitution of a basic amino acid with another basic amino acid, or the substitution of a neutral amino acid with another neutral amino acid. Exemplary substitutions are shown in the following table:

[0022]

[0023]

[0024] "Fc" or "Fc fragment" refers to the "crystallizable fragment" region of an immunoglobulin heavy chain. Generally speaking, an Fc domain can interact with another Fc domain to form a dimeric complex. The Fc domain can bind to cell surface receptors (Fc receptors) and / or proteins of the complement system, or can be modified to weaken or enhance this binding activity. The Fc domain can be derived from IgG, IgA, IgD, IgM or IgE antibodies and produce immune functions, such as opsonization, cell lysis, mast cell degranulation and other Fc receptor-dependent processes. The original immunoglobulin source of natural Fc is preferably human immunoglobulin, preferably IgG1, IgG2 and IgG4.

[0025] In certain embodiments, an "Fc mutant" refers to a molecule or sequence modified from a native Fc that still binds to an Fc receptor. "Fc mutants" include molecules or sequences that have been humanized from a non-human native Fc. "Fc domains" include molecules or sequences of native Fc and Fc variants as described above, including monomeric or multimeric molecules, which can be obtained by decomposing intact antibodies, expressing through genetic recombination, or other means.

[0026] "Host cell" refers to a cell that can be or has been a recipient of a vector or isolated polynucleotide. Host cells can be prokaryotic or eukaryotic. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, and 293 and CHO cells and their derivatives, such as 293-6E and DG44 cells, respectively.

[0027] "Coronavirus" is named for the crown-like spikes on its surface. There are four major subgroups of coronaviruses, called alpha, beta, gamma, and delta. The seven coronaviruses that can infect humans are: 229E (alphacoronavirus), NL63 (alphacoronavirus), OC43 (betacoronavirus), HKU1 (betacoronavirus), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), SARS coronavirus (the betacoronavirus that causes severe acute respiratory syndrome, or SARS, also known as SARS-CoV), and SARS-CoV-2 (the novel coronavirus that causes coronavirus disease 2019, or COVID-19). People around the world are commonly infected with the human coronaviruses 229E, NL63, OC43, and HKU1. Sometimes coronaviruses that infect animals evolve and make people sick, becoming new human coronaviruses. Three recent examples are 2019-nCoV, SARS-CoV, and MERS-CoV. Patients with severe pneumonia and critical illness caused by coronavirus experience a cytokine storm, a severe hyperimmune inflammatory response and tissue destruction in the lung tissue, which may be followed by pulmonary fibrosis.

[0028] "Fibrotic diseases" are connective tissue disorders, such as systemic sclerosis, in which excessive activation of connective tissue cells leads to tissue hardening and scarring within affected organs. In principle, these diseases can affect any organ system and often result in impaired organ function. Fibrosis can occur in a variety of organs or tissues, such as the liver, kidneys, lungs, heart, pancreas, eyes, skin, and joints, and underlies the pathogenesis of many diseases. Fibrotic diseases include hepatic fibrosis, cirrhosis, nonalcoholic steatohepatitis, hepatolenticular degeneration, idiopathic pulmonary fibrosis, renal fibrosis, myocardial fibrosis, scleroderma, toxic oil syndrome, dermatofibromas, keloids, hypertrophic scars, bone fibrosis, intestinal fibrosis, cystic fibrosis (CF), Duchenne muscular dystrophy (DMD), pseudohypertrophic muscular dystrophy, Becker's guanidine dystrophy, Emery-Drcifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, and myotonic dystrophy.

[0029] "Cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.

[0030] "Tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

[0031] "Cancer" is customarily used to refer to all malignant tumors. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific, non-limiting examples of these cancers include squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous cell non-small cell lung cancer), lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, renal cell carcinoma, prostate cancer, vulvar cancer, thyroid cancer, brain cancer, endometrial cancer, testicular cancer, bile duct cancer, gallbladder cancer, gastric cancer, melanoma, and various types of head and neck cancer (including head and neck squamous cell carcinoma).

[0032] "Treatment" refers to therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow down (mitigate) the targeted pathological condition or disorder. In certain embodiments, the term "treatment" covers any administration or application of a disease therapeutic agent in a mammal (including a human), and includes inhibiting or slowing down the disease or disease progression; partially or completely responding to the disease, for example, by causing regression, or restoring or repairing lost, lost or defective function; stimulating an ineffective process; or stabilizing the disease to a reduced severity. The term "treatment" also includes reducing the severity of any phenotypic characteristic and / or reducing the incidence, degree or likelihood of the characteristic. Those in need of treatment include those who already have a disorder as well as those who have a tendency to suffer from the disorder, or those in whom the disorder is to be prevented.

[0033] In one aspect, the present invention provides a fusion protein.

[0034] The fusion protein is formed by connecting an anti-human CTGF antibody and an extracellular segment of a human type II TGF-β receptor via a linker.

[0035] The sequence of the CTGF is SEQ ID NO.1.

[0036] The sequence of the human type II TGF-β receptor is SEQ ID NO.2.

[0037] The extracellular segment of the human type II TGF-β receptor of the fusion protein is selected from the sequence SEQ ID NO.3 or SEQ ID NO.4.

[0038] The light chain variable region CDR of the anti-human CTGF antibody is selected from SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10.

[0039] Preferably, the light chain variable region of the anti-human CTGF antibody is selected from SEQ ID NO. 11 or SEQ ID NO. 12, or an amino acid sequence that is at least 90% identical to SEQ ID NO. 11 or SEQ ID NO. 12. The CDR sequences of the light chain variable region of SEQ ID NO. 11 are SEQ ID NO. 5 (LCDR1), SEQ ID NO. 6 (LCDR2), and SEQ ID NO. 7 (LCDR3), respectively; and the CDR sequences of the light chain variable region of SEQ ID NO. 12 are SEQ ID NO. 8 (LCDR1), SEQ ID NO. 9 (LCDR2), and SEQ ID NO. 10 (LCDR3), respectively.

[0040] Preferably, the light chain constant region of the anti-human CTGF antibody is SEQ ID NO. 21 or an amino acid sequence that is at least 90% identical to SEQ ID NO. 21.

[0041] Or the heavy chain variable region CDR of the anti-human CTGF antibody is selected from SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, and SEQ ID NO.18, wherein the amino acid C in SEQ ID NO.15 can be replaced, and preferably the amino acid C can be replaced with amino acid S, Y, or F.

[0042] Preferably, the heavy chain variable region of the anti-human CTGF antibody is selected from SEQ ID NO. 19 or SEQ ID NO. 20, or an amino acid sequence that is at least 90% identical to SEQ ID NO. 19 or SEQ ID NO. 20. The CDR sequences of the heavy chain variable region of SEQ ID NO. 19 are SEQ ID NO. 13 (HCDR1), SEQ ID NO. 14 (HCDR2), and SEQ ID NO. 15 (HCDR3), respectively; and the CDR sequences of the heavy chain variable region of SEQ ID NO. 20 are SEQ ID NO. 16 (HCDR1), SEQ ID NO. 17 (HCDR2), and SEQ ID NO. 18 (HCDR3), respectively.

[0043] The heavy chain constant region of the anti-human CTGF antibody in the fusion protein is selected from the constant regions of human IgG1, IgG2, and IgG4.

[0044] The linker of the fusion protein is selected from (G x S y ) nLinker, wherein x is 0, 1, 2, 3, 4, 5, y is 0, 1, 2, 3, 4, 5, and n is 0, 1, 2, 3, 4, 5.

[0045] The extracellular segment of the human TGF-β receptor is connected to the N-terminus or C-terminus of the heavy chain of the anti-human CTGF antibody to form a fusion protein, or the extracellular segment of the human TGF-β receptor is connected to the N-terminus or C-terminus of the light chain of the anti-CTGF antibody to form a fusion protein; preferably, the extracellular segment of the human TGF-β receptor is connected to the C-terminus of the heavy chain of the anti-human CTGF antibody to form a fusion protein.

[0046] The fusion protein can completely or partially block the binding of CTGF and TGF-β to their receptors at the same time.

[0047] In another aspect, the present invention provides a nucleic acid molecule.

[0048] The nucleic acid molecule encodes the aforementioned fusion protein.

[0049] In yet another aspect, the present invention provides an expression vector.

[0050] The expression vector is used to transfect mammalian cells, and the expression vector contains the aforementioned nucleic acid molecule.

[0051] In yet another aspect, the present invention provides a host cell.

[0052] The host cell comprises the aforementioned expression vector.

[0053] In yet another aspect, the present invention provides a pharmaceutical composition.

[0054] The pharmaceutical composition comprises the aforementioned fusion protein and / or nucleic acid molecule and / or expression vector and / or host cell.

[0055] The pharmaceutical composition also includes other pharmaceutically acceptable carriers.

[0056] In another aspect, the present invention provides the use of the aforementioned fusion protein and / or nucleic acid molecule and / or expression vector and / or host cell and / or pharmaceutical composition for preventing or treating tissue proliferative diseases, cancers or fibrotic diseases.

[0057] The tissue proliferative disease, cancer or fibrotic disease treatment uses are selected from the following diseases:

[0058] Idiopathic interstitial pneumonia, idiopathic pulmonary fibrosis, common interstitial pneumonia, acute interstitial pneumonia, desquamative interstitial pneumonia, respiratory bronchiolitis, associated interstitial lung disease, nonspecific interstitial pneumonia, lymphocytic interstitial pneumonia, cryptogenic organizing pneumonia, sarcoidosis, lymphangioleiomyomatosis, rheumatoid arthritis, progressive systemic sclerosis, systemic lupus erythematosus, polymyositis and dermatomyositis, Sjögren's syndrome, mixed connective tissue disease, ankylosing spondylitis, alveolar filling disease, Goodpasture syndrome (Goodpasture's syndrome), diffuse alveolar hemorrhage syndrome, pulmonary alveolar proteinosis, chronic eosinophilic pneumonia, lymphocytic interstitial pneumonia, angiocentric lymphoma (lymphomatoid granuloma), familial pulmonary fibrosis, tuberous sclerosis, neurofibromatosis, chronic active hepatitis, primary biliary cirrhosis, Whipple's disease , ulcerative colitis, Crohn's disease, immunoblastic lymphadenopathy, amyloidosis, bronchocentric granuloma, Langerhans histiocytosis, scleroderma, myelofibrosis, renal fibrosis, myocardial fibrosis, hepatic fibrosis, cirrhosis, pneumoconiosis, alcoholic cirrhosis, nonalcoholic fatty liver disease, retinal fibrosis, acute pancreatitis, breast cancer, glioblastoma, thyroid cancer, melanoma, pancreatic cancer, prostate cancer, liver cancer, lung cancer, kidney cancer, gastric cancer, esophageal cancer, progressive muscular dystrophy, Duchenne muscular dystrophy, pseudohypertrophic muscular dystrophy, Becker muscular dystrophy, Emery-Drcifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, myotonic dystrophy, fibrosis in the area of glaucoma filtration surgery, macular degeneration, diabetic retinopathy, choroidal neovascularization, proliferative vitreoretinopathy, and wound healing.

[0059] The disease is pulmonary fibrosis caused by coronavirus or other respiratory virus infection.

[0060] The fusion protein, nucleic acid molecule, expression vector or host cell and the second pharmacologically active compound are used separately, sequentially or simultaneously.

[0061] In another aspect, the present invention provides a method for preparing a fusion protein.

[0062] The fusion protein is the aforementioned fusion protein, and the preparation method comprises the following steps:

[0063] (1) Splicing antibody light and heavy chains separately;

[0064] (2) The amino acid sequences of the light and heavy chains of the above antibodies are cloned into vectors; clones are selected for sequencing, and the bacteria with correct sequencing are selected for seed preservation and expanded culture. The expanded bacteria are used for plasmid extraction;

[0065] (3) Plasmids are transformed into cells and then isolated and purified after culture.

[0066] The benefits of the present invention are:

[0067] 1. The fusion protein of the present invention can be purified in one step to obtain the target protein with extremely high purity (100%).

[0068] 2. Using the fusion protein provided by the present invention, drug treatment at a dose of 1 mg / kg can also greatly reduce the degree of pulmonary fibrosis in experimental animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 This is the SEC-HPLC test result of the fusion protein in Example 2.

[0070] Figure 2 This is the affinity fitting spectrum of the fusion protein in Example 3 and human CTGF.

[0071] Figure 3 This is the affinity fitting spectrum of the fusion protein in Example 3 and human TGF-β.

[0072] Figure 4 This is a pathological section of the lung tissue of the control group mice in the fusion protein animal efficacy experiment.

[0073] Figure 5 This is a pathological section of the lung tissue of untreated mice in the fusion protein animal efficacy experiment.

[0074] Figure 6 These are pathological sections of the lung tissue of mice in the low-dose treatment group in the fusion protein animal efficacy experiment.

[0075] Figure 7 This is a pathological section of the lung tissue of mice in the high-dose treatment group in the fusion protein animal efficacy experiment. DETAILED DESCRIPTION

[0076] The present invention will be further described in detail below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0077] Example 1 Molecular construction of fusion protein

[0078] This part of the experiment was commissioned by Suzhou Genewise Biotechnology Co., Ltd. and included the following steps:

[0079] (1) Splicing SEQ ID NO. 11 and SEQ ID NO. 21 to form the light chain of the antibody;

[0080] (2) splicing SEQ ID NO. 22 and SEQ ID NO. 23, and then connecting to SEQ ID NO. 4 via SEQ ID NO. 24 to form the heavy chain of the antibody;

[0081] (3) The amino acid sequences of the light and heavy chains of the above antibodies were codon-optimized for human host cells and synthesized conventionally. The 5' (EcoRI) and 5' UTR (SEQ ID NO. 25) as well as the 3' UTR (TGATGA) and 3' (HindIII) were added. The genes were cloned into the vector pTT5 (Ampicillin) via 5' EcoRI and 3' HindIII. Clones were selected for sequencing, and the cells with the correct sequence were selected for seed preservation and expanded. The expanded cells were used for plasmid extraction.

[0082] Example 2 Expression and purification of fusion protein

[0083] The extracted plasmid was transfected into cells and the protein was isolated and purified as follows:

[0084] (1) Determine the cell density. The viability should be greater than 95%. Use preheated 293 cell culture medium (purchased from Shanghai Aupuma Biotechnology Co., Ltd., catalog number P82019) to adjust the cell density to 3×10 6 cells / mL, shake gently and aliquot the cells (90% of the transfection system). The volume of cells in the shake flask should not exceed 1 / 3 of the shake flask specifications, and place it in a shaker for use.

[0085] (2) Calculate the volume of transfection buffer Opti-MEM (purchased from Thermo Fisher Scientific (China) Co., Ltd., catalog number 11058021) based on the volume of transfected cells, which is 1 / 10 of the transfection system; calculate the amount of transfection reagent PEI (purchased from Thermo Fisher Scientific (China) Co., Ltd., catalog number BMS1003), the ratio of which is 3 μL / mL transfected cells; calculate the total amount of transfected DNA, the ratio of which is 1 μg / mL transfected cells.

[0086] The specific transfection process is as follows:

[0087] (1) Take a 50 mL centrifuge tube, add 10% Opti-MEM transfection system, add plasmid, mix well, filter, let stand for 5 minutes, add PEI to the DNA suspension, gently mix (gently invert 2-3 times), and let stand for 15-20 minutes. Then, gently add the complex to the aliquoted cells, gently shaking the shaker while adding; place the transfected cells in a 37°C shaker for culture.

[0088] (2) Feeding on the first day: After 20 h, cells were transfected at a dilution of 1:1000 and fed at 2% of the transfected cell volume.

[0089] (3) Determine the expression level of transfected cells on the 4th day: Take 200 μL of the sample to be tested and use a label-free analyzer to detect the protein expression level. 293 culture medium is used as a negative control, and a solution with a known protein concentration is used as a positive control for instrument analysis.

[0090] (4) Collect samples on the 5th to 6th day: Detect cell viability (65-75% or higher is preferred), centrifuge the sample at 3000-5000×g for 20-30 minutes, filter the supernatant with a 0.22μm filter, and separate, purify, and test the supernatant. Figure 1 The SEC-HPLC results of the fusion protein are shown in FIG.

[0091] The purification was carried out according to the product instructions, which is briefly as follows:

[0092] 1) Pre-equilibration: Rinse the HiTrap Protein L (GE Medical Systems (China) Co., Ltd., Catalog No.: 29048665) chromatography column with ddH2O for 5 CV to flush out the protective solution; equilibrate the chromatography column with equilibration solution for 5 CV to maintain a stable UV detection baseline.

[0093] 2) Sample loading: Load the sample with a retention time of 4 minutes. Start collecting the flow-through when the UV detection value rises.

[0094] 3) Post-equilibration: After loading, use equilibration solution for 5-10CV until the UV detection baseline remains stable, and stop collecting flow-through.

[0095] 4) Elution: Elute with eluent and neutralizing solution for 3-5 CV respectively, and collect the eluted fractions respectively.

[0096] 5) Column cleaning: Rinse the column with regeneration buffer for 15 minutes, then flush out the regeneration buffer with ddH2O until the pH is neutral.

[0097] Purity testing is carried out as follows:

[0098] An Agilent liquid chromatography detection system (Agilent Technologies, Inc., Model 1200) coupled with a SEC-300 analytical column was used. After centrifugation, the supernatant was filtered through a 0.22 μm filter, and 20 μL of sample was loaded at a flow rate of 0.26 mL / min. At 10.632 min, the instrument detected a major peak with an area of 2392.859 mAU*s, representing 100% of the peak area.

[0099] Example 3 Detection of Binding of Fusion Protein to Human CTGF and Human TGF-β Protein

[0100] Using Gator TM A label-free bioanalyzer was used to analyze the affinity of the expressed fusion proteins for human CTGF (purchased from ACROBiosystems, Catalog No. CTF-H52H5) and human TGF-β (purchased from ACROBiosystems, Catalog No. TG1-H4212). Protein A biosensors were used to capture the antibody samples, and the captured antibody samples were then subjected to kinetic analysis of binding and dissociation with human CTGF and human TGF-β. Kinetic analysis was performed using a 1:1 binding model. The following table summarizes the steps:

[0101] step time Protein loading 200s Combine 180s dissociation 300s regeneration 30s

[0102] Using Gator TM The affinity data measured by the instrument are shown in the following table:

[0103] Capture samples Combined samples Response value Affinity (M) Dissociation constant (1 / s) Binding constant (1 / Ms) Fusion protein human CTGF 0.0665 5.40E-08 1.23E-02 2.28E+05 Fusion protein human TGF-β 0.142 2.84E-09 1.82E-03 6.40E+05

[0104] The corresponding spectrum of affinity detection of fusion protein with human CTGF and human TGF-β is as follows Figure 2 and Figure 3 shown.

[0105] By testing the affinity of the fusion protein with human CTGF and human TGF-β, it is shown that the fusion protein provided by the present invention can normally bind to human CTGF and human TGF-β, and on this basis can play a blocking effect on the signaling pathways of human CTGF and human TGF-β, thereby inhibiting or slowing down the process of tissue fibrosis.

[0106] Example 4 Animal efficacy experiment of fusion protein

[0107] 4.1 Main experimental materials

[0108] BALB / c mice (purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., strain code 211).

[0109] Bleomycin (purchased from Shanghai Maokang Biotechnology Co., Ltd., product number MZ3504).

[0110] Chloral hydrate (purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number 30037516).

[0111] 4.2 Model establishment

[0112] After BALB / c mice were anesthetized by intraperitoneal injection with 4% chloral hydrate in a volume of 200 μL, 30 μL of bleomycin solution with a concentration of 3.33 mg / mL or 30 μL of PBS solution were administered to the mice by nasal drops using a pipette. The converted mouse modeling dosage was 5 mg / kg. During the administration process, the left hand fixed the mouse under general anesthesia, keeping its body in a vertical position with its head up and its tail down. The right hand held a micropipette and gently dripped 30 μL of bleomycin solution into the mouse nostril. If the mouse breathes normally and gradually inhales the liquid, the nasal drop operation can be continued until all the drugs enter the nasal cavity. On the 7th day of modeling, the mouse tail vein treatment was started. The protein used for treatment was the protein purified by Example 2 and was administered once a week for a total of three times. The administration and treatment groups are shown in the following table:

[0113] Group Modeling reagents Treatment administration control group 30 μL PBS No processing Untreated group 30 μL bleomycin solution PBS Low-dose treatment group 30 μL bleomycin solution 1mg / kg, fusion protein High-dose treatment group 30 μL bleomycin solution 10mg / kg, fusion protein

[0114] After treatment, lung tissue was collected from mice and fixed with 4% paraformaldehyde. Once fixed, the tissue was trimmed, dehydrated, embedded, sliced, stained, and mounted. Finally, qualified samples were examined under a microscope. The sections were viewed under a microscope or digitally, and the tissue sections were carefully observed at different magnifications.

[0115] 4.3 Pathological detection and analysis

[0116] Pathological section staining showed that mice administered with bleomycin developed varying degrees of pulmonary fibrosis, but the lungs of mice modeled with PBS were normal.

[0117] The lungs of mice that were simply administered bleomycin without any treatment showed typical and severe pulmonary fibrosis, which was manifested by the proliferation of a large number of collagen fibers, thickening of the alveolar walls, and indistinguishable alveolar septa covered by fibrotic masses in lung tissue sections.

[0118] At the same time, section staining showed that the fusion protein treatment group showed a reduction in the degree of pulmonary fibrosis, and the low-dose fusion protein treatment group of 1mg / kg also achieved significant improvement in pulmonary fibrosis. The in vivo efficacy tests in animals further demonstrated that the fusion protein of the present invention can effectively treat fibrotic diseases.

[0119]

[0120]

[0121] Example 5-8 Molecular Construction of Fusion Protein

[0122] The fusion protein was prepared by referring to the method of Example 1-2, except that the sequences of the light chain variable region, heavy chain variable region and human type II TGF-β receptor extracellular domain were different. The specific sequence information is as follows:

[0123] Light chain variable region Heavy chain variable region Human TGF-β receptor type II extracellular domain Example 5 SEQ ID NO:11 SEQ ID NO: 19 SEQ ID NO:3 Example 6 SEQ ID NO:12 SEQ ID NO: 19 SEQ ID NO:4 Example 7 SEQ ID NO:11 SEQ ID NO:20 SEQ ID NO:3 Example 8 SEQ ID NO:12 SEQ ID NO:20 SEQ ID NO:3

[0124] The fusion proteins of Examples 5-8 were prepared with reference to the experimental method of Examples 3-4, and the results showed that the properties of the fusion proteins prepared by the method of Examples 1-2 were basically consistent.

[0125] Comparative Example

[0126] Fusion proteins were prepared according to the method of Example 1-2, except that the linker sequences were SEQ ID NO.26, SEQ ID NO.27, and SEQ ID NO.28, respectively, to obtain fusion proteins 1, fusion protein 2, and fusion protein 3.

[0127] The purity of fusion protein 1 was 95.8%.

[0128] The purity of fusion protein 2 was 96.4%.

[0129] The purity of fusion protein 3 was 97.7%.

[0130] After further purification, fusion protein 1, fusion protein 2, and fusion protein 3 were subjected to experiments according to the method of Example 4. The results obtained are as follows:

[0131] Sequence Listing <110> Suzhou Pulekang Pharmaceutical Technology Co., Ltd. <120> A fusion protein binding to CTGF and its application <160> 28 <170> SIPOSequenceListing 1.0 <210> 1 <211> 322 <212> PRT <213> Artificial Sequence <400> 1 Asn Cys Ser Gly Pro Cys Arg Cys Pro Asp Glu Pro Ala Pro Arg Cys 1 5 10 15 Pro Ala Gly Val Ser Leu Val Leu Asp Gly Cys Gly Cys Cys Arg Val 20 25 30 Cys Ala Lys Gln Leu Gly Glu Leu Cys Thr Glu Arg Asp Pro Cys Asp 35 40 45 Pro His Lys Gly Leu Phe Cys His Phe Gly Ser Pro Ala Asn Arg Lys 50 55 60 Ile Gly Val Cys Thr Ala Lys Asp Gly Ala Pro Cys Ile Phe Gly Gly 65 70 75 80 Thr Val Tyr Arg Ser Gly Glu Ser Phe Gln Ser Ser Cys Lys Tyr Gln 85 90 95 Cys Thr Cys Leu Asp Gly Ala Val Gly Cys Met Pro Leu Cys Ser Met 100 105 110 Asp Val Arg Leu Pro Ser Pro Asp Cys Pro Phe Pro Arg Arg Val Lys 115 120 125 Leu Pro Gly Lys Cys Cys Glu Glu Trp Val Cys Asp Glu Pro Lys Asp 130 135 140 Gln Thr Val Val Gly Pro Ala Leu Ala Ala Tyr Arg Leu Glu Asp Thr 145 150 155 160 Phe Gly Pro Asp Pro Thr Met Ile Arg Ala Asn Cys Leu Val Gln Thr 165 170 175 Thr Glu Trp Ser Ala Cys Ser Lys Thr Cys Gly Met Gly Ile Ser Thr 180 185 190 Arg Val Thr Asn Asp Asn Ala Ser Cys Arg Leu Glu Lys Gln Ser Arg 195 200 205 Leu Cys Met Val Arg Pro Cys Glu Ala Asp Leu Glu Glu Asn Ile Lys 210 215 220 Lys Gly Lys Lys Cys Ile Arg Thr Pro Lys Ile Ser Lys Pro Ile Lys 225 230 235 240 Phe Glu Leu Ser Gly Cys Thr Ser Met Lys Thr Tyr Arg Ala Lys Phe 245 250 255 Cys Gly Val Cys Thr Asp Gly Arg Cys Cys Thr Pro His Arg Thr Thr 260 265 270 Thr Leu Pro Val Glu Phe Lys Cys Pro Asp Gly Glu Val Met Lys Lys 275 280 285 Asn Met Met Phe Ile Lys Thr Cys Ala Cys His Tyr Asn Cys Pro Gly 290 295 300 Asp Asn Asp Ile Phe Glu Ser Leu Tyr Tyr Arg Lys Met Tyr Gly Asp 305 310 315 320 Met Ala <210> 2 <211> 112 <212> PRT <213> Artificial Sequence <400> 2 Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser Thr Glu Lys Asn Cys Cys 1 5 10 15 Val Arg Gln Leu Tyr Ile Asp Phe Arg Lys Asp Leu Gly Trp Lys Trp 20 25 30 Ile His Glu Pro Lys Gly Tyr His Ala Asn Phe Cys Leu Gly Pro Cys 35 40 45 Pro Tyr Ile Trp Ser Leu Asp Thr Gln Tyr Ser Lys Val Leu Ala Leu 50 55 60 Tyr Asn Gln His Asn Pro Gly Ala Ser Ala Ala Pro Cys Cys Val Pro 65 70 75 80 Gln Ala Leu Glu Pro Leu Pro Ile Val Tyr Tyr Val Gly Arg Lys Pro 85 90 95 Lys Val Glu Gln Leu Ser Asn Met Ile Val Arg Ser Cys Lys Cys Ser 100 105 110 <210> 3 <211> 136 <212> PRT <213> Artificial Sequence <400> 3 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 4 <211> 117 <212> PRT <213> Artificial Sequence <400> 4 Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe 1 5 10 15 Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr 20 25 30 Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys 35 40 45 Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Glu Pro Lys Leu 50 55 60 Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile 65 70 75 80 Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys 85 90 95 Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn 100 105 110 Thr Ser Asn Pro Asp 115 <210> 5 <211> 11 <212> PRT <213> Artificial Sequence <400> 5 Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala 1 5 10 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <400> 6 Ala Ala Ser Ser Leu Gln Ser 1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <400> 7 Gln Gln Tyr Asn Ser Tyr Pro Pro Thr 1 5 <210> 8 <211> 12 <212> PRT <213> Artificial Sequence <400> 8 Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala 1 5 10 <210> 9 <211> 7 <212> PRT <213> Artificial Sequence <400> 9 Gly Ala Ser Arg Arg Ala Thr 1 5 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <400> 10 Gln Gln Tyr Val Ser Thr Pro Trp Thr 1 5 <210> 11 <211> 107 <212> PRT <213> Artificial Sequence <400> 11 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 12 <211> 108 <212> PRT <213> Artificial Sequence <400> 12 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Val Val Tyr Phe Cys Gln Gln Tyr Val Ser Thr Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 13 <211> 5 <212> PRT <213> Artificial Sequence <400> 13 Ser Tyr Gly Met His 1 5 <210> 14 <211> 16 <212> PRT <213> Artificial Sequence <400> 14 Gly Ile Gly Thr Gly Gly Gly Thr Tyr Ser Thr Asp Ser Val Lys Gly 1 5 10 15 <210> 15 <211> 12 <212> PRT <213> Artificial Sequence <400> 15 Gly Asp Tyr Tyr Gly Ser Gly Ser Phe Phe Asp Cys 1 5 10 <210> 16 <211> 5 <212> PRT <213> Artificial Sequence <400> 16 Gly Tyr Tyr Met Tyr 1 5 <210> 17 <211> 17 <212> PRT <213> Artificial Sequence <400> 17 Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 18 <211> 10 <212> PRT <213> Artificial Sequence <400> 18 Gly Ser Lys Trp Asn Tyr Pro Phe Asp Tyr 1 5 10 <210> 19 <211> 120 <212> PRT <213> Artificial Sequence <400> 19 Glu Gly Gln Leu Val Gln Ser Gly Gly Gly Leu Val His Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Gly Thr Gly Gly Gly Thr Tyr Ser Thr Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Asp Tyr Tyr Gly Ser Gly Ser Phe Phe Asp Cys Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 20 <211> 119 <212> PRT <213> Artificial Sequence <400> 20 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Lys Trp Asn Tyr Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 21 <211> 107 <212> PRT <213> Artificial Sequence <400> 21 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 22 <211> 120 <212> PRT <213> Artificial Sequence <400> 22 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val His Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Gly Thr Gly Gly Gly Thr Tyr Ser Thr Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Asp Tyr Tyr Gly Ser Gly Ser Phe Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 23 <211> 329 <212> PRT <213> Artificial Sequence <400> 23 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 <210> 24 <211> 20 <212> PRT <213> Artificial Sequence <400> 24 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 25 <211> 66 <212> DNA <213> Artificial Sequence <400> 25 gccaccatgg agacagatac cctgctgctg tgggtgctgc tgctgtgggt ccctggcagc 60 accgga 66 <210> 26 <211> 14 <212> PRT <213> Artificial Sequence <400> 26 Gly Ser Ser Ser Ser Ser Ser Gly Ser Ser Ser Ser Ser Ser 1 5 10 <210> 27 <211> 14 <212> PRT <213> Artificial Sequence <400> 27 Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Ser 1 5 10 <210> 28 <211> 24 <212> PRT <213> Artificial Sequence <400> 28 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Ser 20

Claims

1. A fusion protein, characterized in that The fusion protein is formed by connecting an anti-human CTGF antibody and the extracellular segment of the human type II TGF-β receptor through a linker. The fusion protein linker is shown in SEQ ID NO.

24. The light chain variable region of the anti-human CTGF antibody is SEQ ID NO.11, and the heavy chain variable region is SEQ ID NO.

22. The extracellular segment of the human type II TGF-β receptor of the fusion protein is SEQ ID NO.

4.

2. The fusion protein according to claim 1, characterized in that The heavy chain constant region of the anti-human CTGF antibody in the fusion protein is selected from the constant regions of human IgG1, IgG2, and IgG4.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the fusion protein according to any one of claims 1 to 2. An expression vector comprising the nucleic acid molecule according to claim 3 . A host cell comprising the expression vector according to claim 4 .

6. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the fusion protein according to any one of claims 1 to 2.

7. A method for preparing a fusion protein, characterized in that: The fusion protein is the fusion protein according to any one of claims 1 to 2, and the preparation method comprises the following steps: (1) Splicing antibody light and heavy chains separately; (2) The amino acid sequences of the light and heavy chains of the above antibodies are cloned into vectors; clones are selected for sequencing, and the bacteria with correct sequencing are selected for seed preservation and expanded culture. The expanded bacteria are used for plasmid extraction; (3) Plasmids are transformed into cells and then isolated and purified after culture.

Images